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This is to certify that the

dissertation entitled

Physiological and Biochemical Aspects of Potato Scab Disease

Caused by Streptomyces Species
presented by

Frank Richard Spooner, Jr.~

has been accepted towards fulﬁllment
of the requirements for

Doctor of Philosophy degree in Botany & Plant Pathology

mm

Major professor

Date 11-17-94

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PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF POTATO SCAB DISEASE
CAUSED BY STREPTOMYCES SPECIES
. By

FRANK RICHARD SPOONER, Jr.

A DISSERTATION
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

DEPARTMENT OF BOTAN Y AND PLANT PATHOLOGY

1994

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ABSTRACT
PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF POTATO SCAB DISEASE
CAUSED BY STREPTOMYCES SPECIES
By

Frank Richard Spooner, Jr.

Physiological and biochemical aspects of the potato scab disease and pathogens
were studied. A Michigan deep-pitting Streptomyces designated D.P. was isolated from
an infected potato tuber. In culture, D.P. produced reﬂexuous spore chains and lacked
melanin production. In culture the S. scabies isolate RL 232 produced spiral spore
chains and melanin. D.P. caused deep pitted lesions on the potato cultivar Atlantic, and
RL 232 produced common scab lesions on the cultivar Atlantic. D.P. , RL 232, and the
nonpathogen RL 95 produced extracellular pectinases when grown on polygalacturonic
acid or pectin. The culture ﬁltrates of all the isolates exhibited lyase activity in the range
of pH 7-9 on pectin and 8-10 on polygalacturonic acid. The culture ﬁltrates from all
isolates were found to macerate potato tuber tissue. Ultraviolet light generated PL-
deﬁcient mutants of D.P. produced scab lesions on Atlantic tubers. No correlation was
shown between the loss of pectinase production and the ability to produce disease. The
deep-pitting isolate D.P. produced the thaxtomin-like compounds that elicited a browning
response from potato tuber discs within 18 hours after application. Spores of RL 232,
RL 95, and DP. elicited the production of chlorogenic acid (CGA) and lignin from
Russet Burbank and Atlantic tuber discs. D.P. elicited higher amounts of CGA between

48 and 72 hours after inoculation and nearly identical amounts of lignin within 24

hours after inoculation on both Russet Burbank and Atlantic tubers, relative to
uninoculated controls. Both RL 232 and RL 95 elicited lower amounts of CGA and
higher levels of lignin throughout the time course relative to the uninoculated controls.
The physiological and biochemical differences between the pathogenic isolates DP. and

S. scabies (RL 232) suggested that D.P. was a different species of potato scab-causing

Streptomyces.

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ACKNOWLEDGEMENTS

I thank all the members of my guidance committee for their help over the years,
but especially my advisor Dr. Raymond Hammerschmidt for getting me out of several
tight spot and keeping me on track.

To all my numerous friends that I have made of the years, I couldn’t maintain
without you, thanks. i

To all my family down in Louisiana and my dearest Kel, thanks for keeping me

motivated to complete this degree.

iv

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TABLE OF CONTENTS

List of Tables .................................. vii
List of Figures .................................. viii
CHAPTER I. Introduction to Dissertation: Research

Objectives and a Review of Literature ..................... 1
Introduction to Dissertation: Research Objectives .............. 2
Review of Literature ............................... 6
References .................................. 29
CHAPTER II. The Characterization of Cultural, Morphological,
Physiological, Biochemical, and Pathological Properties of

Potato Scab Causing Isolates of Streptomyces ............... 37
Introduction .................................. 38
Materials and Methods ............................. 41
Results .................................. 50
Discussion .................................. 61
References .................................. 64
CHAPTER III. The Role of Extracellular Pectinases Produced by
Streptomyces species Pathogenic on Potato in the Disease Process:

Studies on Extracellular Pectinases Produced in Vitro by Pathogenic

and Nonpathogenic Streptomyces species .................. 69

Introduction .................................. 70

Material and Methods .............................. 74
Results .................................. 83
Discussion ................................. 1 1 1
References ................................. 1 17

CHAPTER IV. The Production of Thaxtomin by a Deep Pitting Form

of Streptomyces ................................ 123
Introduction ................................. 124
Materials and Methods ............................ 125
Results ................................. 128
Discussion ................................. 133
References ................................. 137

CHAPTER V. The Effects of Infection by Streptomyces species on
The Biosynthesis of Chlorogenic Acid and Lignin in Tuber Discs of

A Scab-Resistant and Scab-Susceptible Cultivar of Potato ....... 139
Introduction ................................. 140
Material and Methods ............................. 143
Results ................................. 145
Discussion ................................. 151
References ................................. 157
CONCLUSIONS ................................ 160
References ................................. 166

vi

Table

2.1.

2.2.

2.2.

2.3.

2.3.

3.1.

3.1.

3.2.

4.1.

4.2.

5.1.

LIST OF TABLES

Page
Designation and initial characterization
of Michigan isolates ......................... 45
Appearance of pure cultured isolates on media and
pathogenicity ............................. 52
Continued from the preceding page ................ 53
Characteristics of S. scabies and other Streptomyces
isolates ................................. 56
Continued from the preceding page ................ 57
Characteristics of D.P. wild type and UV. generated
isolates of D.P. ............................ 98
Continued from the preceding page ................ 99

Potato tissue macerating capability of enzyme preparations
from Streptomyces species cultured on pectin and polygalacturonic
acid ................................ 108

The Rfs for the concentrated acetone extracts as determined
by thin layer chromatography in chlorofomI/methanol 9:1 (v/v)
solvent system ............................ 134

Averaged Rf values for extracts from experiments
1 and 2 ................................ 135

The effect of chlorogenic acid on the growth of
Streptomyces ............................. 152

vii

LIST OF FIGURES

Figure Page

2.1. Types of potato scab on Atlantic tubers. A, Common
scab caused by RL 232; B, Deep pitted scab caused by
D.P. ................................. 42

2.2. SDS-PAGE of soluble proteins extracted from 10 day
old Streptomyces cultures. Lane 1, RL 232; lane 2, D.P.;
lane 3, B1; lane 4, 3C; lane 5, RL 95; lane 6, 2A; lane 7, 38;
lane 8, Fi 11; lane 9, molecular weight markers in kiloDaltons
(kD): bovine albumin (66 kD), egg albumin (45 kD), pepsin (34.7 kD),
trypsinogen (24 kD), B-lactoglobin (18.4 kD),
lysozyme (14.3 kD) ......................... 59

2.3. SDS-PAGE of soluble proteins extracted from 10 day old
Streptomyces cultures. Lane 1, RL 232; lane 2, D.P.; lane 3, B1;
lane 4, 3C; lane 5, RL 95; lane 6, 2A; lane 7, S. lividans; lane 8,
Fi II; lane 9, molecular weight markers in kiloDaltons (kD):
bovine albumin (66 kD), egg albumin (45 kD), pepsin (34.7 kD),
trypsinogen (24 kD), B-lactoglobin (18.4 kD),
lysozyme (14.3kD) .......................... 60

3.1. The inﬂuence of pH on the activity of pectolytic
enzymes from culture ﬁltrates of RL 232, D.P., and RL 95
induced on A, PGA; or B, pectin. The reducing groups assay
as used to detect hydrolase activity on PGA substrate, activity
was measured on the basis of micrograms (pg) galacturonic acid units
(gal.) produced per minute (min.) per microgram (pg) protein (pro.)
at 540 nm ............................... 84

viii

3.2.

3.3.

3.4.

The inﬂuence of pH on the activity of pectolytic enzymes

from culture ﬁltrates of RL 232, D.P., and RL 95 induced on
pectin. The reducing groups assay was used to detect hydrolase
activity on pectin substrate, activity was measured on the

basis of micrograms (pg) galacturonic acid units (gal.)

produced per minute (min.) per milliliters (ml) reaction

mixture at 540 nm .......................... 85

The inﬂuence of pH on the activity of pectolytic enzymes

from culture ﬁltrates of RL 232, D.P., and RL 95 induced

on pectin. Lyase activity was detected on pectin substrate

by A, measuring the change in absorbance of a reaction after

2 minutes (min.) at 235 nm; B, the reducing groups assay, where
activity was measured on the basis of micrograms (pg) of galacturonic
acid units (gal.) produced per minute (min.) per microgram (pg)
protein (pro.) at 540 nm; C, thiobarbituric acid assay, where activity
was measured on the basis of the concentration in millimicro

moles (mp M) of colored product (prod.) formed per minute (min.)
per microgram (pg) protein (pro.) at 548 nm .......... 87

The inﬂuence of pH on the activity of pectolytic enzymes

from culture ﬁltrates of RL 232, D.P., and RL 95 induced

on PGA. Lyase activity was detected on pectin substrate

by A, measuring the change in absorbance of a reaction

after 2 minutes (min.) at 235 nm; B, the reducing groups

assay, where activity was measured on the basis of micrograms
(pg) of galacturonic acid units (gal.) produced per minute

(min.) per microgram (pg) protein (pro.) at 540 mm C, thiobarbituric
acid assay, where activity was measured on the basis of

the concentration in millimicro moles (mp M) of colored product
(prod.) formed per minute (min.) per microgram (pg) protein (pro.)
at 548 nm ............................... 88

ix

3.5.

3.6.

3.7.

3.8.

The inﬂuence of pH on the activity of pectolytic enzymes

from culture ﬁltrates of RL 232, D.P., and RL 95 induced

on PGA. Lyase activity was detected on PGA substrate by A,
the reducing groups assay, where activity was measured on

the basis of micrograms (pg) of galacturonic acid units (gal.)
produced per minute (min.) per microgram (pg) protein (pro.)

at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles
(mp M) of colored product (prod.) formed per minute (min.) per
microgram (pg) protein (pro.) at 548 nm ............ 90

The inﬂuence of pH on the activity of pectolytic enzymes
from culture ﬁltrates of RL 232, D.P., and RL 95 induced
on pectin. Lyase activity was detected on PGA substrate by
A, the reducing groups assay, where activity was measured
on the basis of micrograms (pg) of galacturonic acid units
(gal.) produced per minute (min.) per microgram (pg) protein
(pro.) at 540 nm; B, thiobarbituric acid assay, where activity
was measured on the basis of the concentration in millimicro
moles (mp M) of colored product (prod.) formed per

minute (min.) per microgram (pg) protein (pro.)

at 548 nm ............................... 91

Cup plates illustrating zones of clearing corresponding

to lyase activity. A, PGA substrate (pH 8.5) was treated

with l N HCl to precipitate the undegraded substrate,

enzyme activity was indicated by the clear halos which

formed around the wells. B, pectin substrate (pH 8.5)

stained with 0.03 % ruthenium red solution, undegraded
substrate was stained pink or red and enzyme activity

was indicated by the halos (unstained pectin) which formed
around the wells .......................... 92

Detection of pectic enzymes produced by Streptomyces

with 0.1% pectin agarose overlay stained with 0.03%

ruthenium red solution. Each lane shows the pattern

of substrate degradation characteristic to each enzyme
preparation, which came from culture ﬁltrates of the

bacteria induced on pectin or PGA. Lane 1, 38 PGA;

lane 2, RL 232 PGA; lane 3, 3S pectin; lane 4,

RL 232 pectin; lane 5, D.P. pectin; lane 6, RL 95

pectin ................................. 94

3.9.

3.]

 

3.9.

3.10.

3.11.

3.12.

3.13.

A, Silver stained polyacrylamide gel containing electrophoresed
pectolytic enzymes from Streptomyces induced on pectin.

Lane 1, 38; lane 2, RL 232; lane 3, D.P.; and lane 4, RL 95.
B, The pectin agarose overlay stained with ruthenium red

shown in ﬁgure 3.8 (lanes 3-6). Lane 1, 38; lane 2, RL 232;
lane 3, D.P.lane 4, RL 95 ..................... 95

Reverse culture color of the UV. mutants of D.P.,
viewed from the bottom side of the petri dish. A, 2A; B, 3C;
C, D.P. (wild type); D, B, ..................... 97

Deep-pitted scab caused by the UV. mutants of the D.P.
isolate. A, B1; and B, 3C ..................... 100

The inﬂuence of pH on the activity of pectolytic enzymes

from culture ﬁltrates of D.P., B,, 2A, and 3C induced on

A, PGA; or B, pectin. The reducing groups assay was used

to detect hydrolase activity on PGA substrate, activity was
measured on the basis of micrograms (pg) galacturonic acid units
(gal.) produced per minute (min.) per microgram (pg) protein
(pro.) at 540 nm .......................... 102

The inﬂuence of pH on the activity of pectolytic enzymes

from culture ﬁltrates of D.P., B1, 2A, and 3C induced

on pectin. Lyase activity was detected on pectin substrate

by A, the reducing groups assay, where activity was measured
on the basis of micrograms (pg) of galacturonic acid units

(gal.) produced per minute (min.) per microgram (pg) protein
(pro.) at 540 nm; B, thiobarbituric acid assay, where activity
was measured on the basis of the concentration in millimicro
moles (mp M) of colored product (prod.) formed per minute (min.)
per microgram (pg) protein (pro.) at 548 nm; C, measuring the
change in absorbance of a reaction after 2 minutes (min.)

at 235 nm ............................. 103

xi

53

3.14.

3.15.

3.16.

3.17.

3.18.

The inﬂuence of pH on the activity of pectolytic

enzymes from culture ﬁltrates of D.P., B1, 2A,

and 3C induced on PGA. Lyase activity was detected on

PGA substrate by A, the reducing groups assay, where
activity was measured on the basis of micrograms (pg) of
galacturonic acid units (gal.) produced per minute (min.)

per microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric
acid assay, where activity was measured on the basis of

the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg)
protein (pro.) at 548 nm ...................... 104

SDS-PAGE of extracellular pectinases from culture

ﬁltrates of Streptomyces induced on A, PGA or B,

pectin substrates. Lane 1, molecular weight markers

in kiloDaltons (kD): bovine albumin (66 kD), egg albumin

(45 kD), pepsin (34.7 kD), trypsinogen (24 kD), B-lactoglobin
(18.4 kD), lysozyme (14.3 kD); lane 2, pectin lyase (EC 4.2.2.10)
from Aspergillus japonicus; lane 3, RL 232; lane 4, D.P.; lane 5,
RL 95; lane 6, 3S; lane 7, B1; lane 8, 2A; lane 9, 3C; and

lane 10, polygalacturonase (EC 3.2.1.15) from

Aspergillus niger .......................... 106

Rating scale for tissue maceration: 0, no browning or

maceration (comparable to controls); 1, tissue slightly

discolored without maceration; 2, tissue slightly discolored

with minimal visible maceration; 3, brown tissue with moderate
maceration; 4, brown or blackened tissue, at least 90% maceration
(tissue was very soft) ....................... 107

Tuber discs 36 hours after treatment with pectin induced

enzyme preparations from Streptomyces isolates RL 232,

D.P., and RL 95. A, Tissue incubated in Tris-HCl buffer

(pH 8.8); B, Tissue incubated in water ............. 109

Tuber discs 36 hours after treatment with PGA induced

enzyme preparations from Streptomyces isolates RL 232,
D.P., and RL 95. A, Tissue incubated in Tris-HCl buffer
(pH 8.8); B, Tissue incubated in water ............. 110

xii

4.1.

5.1.

5.2.

5.3.

5.4.

Bioassay of thaxtomin from RL 232 and D.P. extracts

on Russet Burbank tuber discs. Sources of toxin A,

Culture ﬁltrate from oatmeal broth (CF), oatmeal agar

(agar), and inoculated tuber discs (tissue). B, Culture

ﬁltrate from oatmeal broth (CF), and oatmeal agar (agar).

C, Tissue treated with water (H20) ............... 132

Three day time course of chlorogenic acid (CGA)

accumulation in tuber discs (cv. Russet Burbank),

where the concentration of CGA was measured in

micro moles (pM) per two tuber discs sample at 328 nm.

Tuber discs were inoculated with A, RL 95 (nonpathogen);

B, RL 232 (common scab pathogen); and C, D.P.

(deep pitting pathogen) ...................... 146

Three day time course of chlorogenic acid (CGA)

accumulation in tuber discs (cv. Atlantic), where

the concentration of CGA was measured in micro moles

(pM) per two tuber discs sample at 328 nm. Tuber discs

were inoculated with A, RL 95 (nonpathogen); B,

RL 232 (common scab pathogen); and C, D.P.

(deep pitting pathogen) ...................... 147

Three day time course of lignin accumulation in tuber

discs (cv. Russet Burbank), where the concentration of

lignin was measured in micrograms (pg) per unit area

discs at 280 nm. Tuber discs were inoculated with A, RL 95
(nonpathogen); B, RL 232 (common scab pathogen); and C,
D.P. (deep pitting pathogen) ................... 149

Three day time course of lignin accumulation in tuber

discs (cv. Atlantic), where the concentration of lignin

was measured in micrograms (pg) per unit area discs at 280 nm.
Tuber discs were inoculated with A, RL 95 (nonpathogen);

B, RL 232 (common scab pathogen); and C, D.P.

(deep pitting pathogen) ...................... 150

xiii

CHAPTER I

INTRODUCTION TO DISSERTATION: RESEARCH OBJECTIVES

AND A

REVIEW OF LITERATURE

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INTRODUCTION TO DISSERTATION: RESEARCH OBJECTIVES

The potato scab disease is caused by Streptomyces scabies, S. acidiscabies, and
several other Streptomyces species. These bacteria cause corky lesions to form in the
tuber periderm of several cultivars of Solanum tuberosum L. The type of lesions formed
range in appearance from superﬁcial corky lesions to deep pitted furrows. All types of
lesions lower the quality of potato tubers.

Potato scab research has focused primarily on ﬁnding effective methods to control
the pathogen. Control of potato scab has been achieved through manipulation of soil
moisture, soil pH, soil mineral composition, and soil microorganisms, which are
antagonistic to S. scabies. Additionally, control of the disease has been partially
achieved through crop rotation and use of resistant cultivars of potato. Several studies
also have been conducted to determine the periods at which the susceptibility of potato
to infection is greatest and the means by which the pathogen gains access to susceptible
tubers.

Establishing the cultural, morphological, and pathological characteristics that are
associated with potato scab—causing bacteria has been the subject of much research.
Several diagnostic methods have been developed to categorize S. scabies. However, the
only criterion which has been accepted is the ability of an isolate to cause scab lesions
on potato tubers. Traditionally, if an isolate of Streptomyces caused potato scab, it was
grouped with S. scabies regardless of whether the isolate ﬁt the given descriptions for
the group. Recent studies using molecular methods have shown that several Streptomyces

spp. grouped with S. scabies, on the basis of pathogenicity, standard cultural, and

 

 

 

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3

physiological test used by Streptomyces researchers were not related to S. scabies
on the basis of DNA homology.

Although more is now known about the taxonomy of S. scabies and other potato
scab-causing Streptomyces spp. , relatively little is known about the sequence of
physiological events that occur during the infection process and how the disease
progresses to the expression of symptoms. In addition, very little is known at the
physiological level regarding the response of potato tissue to infection by Streptomyces.
S. scabies and S. acidiscabies are known to produce phytotoxic compounds in potato
tissue and in culture that are capable of reproducing common scab lesions on
microtubers. However, whether this characteristic is shared by streptomycetes other than
S. scabies and S. acidiscabies has yet to be reported. The mode of action and the nature
of cellular destruction caused by the toxin, called thaxtomin, has also yet to be reported.

S. scabies, S. ipomoea, and several other Streptomyces species are known to
produce cell-wall degrading enzymes of which the pectinase have been implicated in the
infection process. As histological studies have revealed, the hyphal ﬁlaments penetrate
the middle lamellae which is primarily composed of pectic polymers. However, whether
there is a correlation between the ability to synthesize pectolytic enzymes in culture and
the ability to cause disease is not known. With regard to the host defense responses,
histochemical work shows that potato tissue infected by S. scabies deposits suberized
barriers to prevent infection. However there is not a physiological study to support
whether the accumulation of phenolic compounds, lignin, or suberin occurs speciﬁcally

in response to S. scabies.

 

 

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In this dissertation, I have attempted to examine potato scab disease from a
physiological perspective. In the initial phases of my research on potato scab, I was
able to isolate several strains of Streptomyces which were culturally dissimilar to S.
scabies. One isolate, in particular, causes deep-pitted scab, lacks the ability to produce
melanin or colored pigments in culture, and bears its spores in reﬂexuous spore chains.
This isolate was designated D.P. and was used in several of my studies because it was
culturally and morphologically different from S. scabies and produced very deep lesions.
Further, I wanted to determine what physiological and biochemical differences or
similarities existed between the D.P. isolate and S. scabies.

In my study of the physiology of potato scab, I reexamined whether pectolytic
enzymes were produced by pathogenic and nonpathogenic streptomycetes in culture to
determine whether different classes of enzymes were produced by nonpathogens and
pathogens. 1 also attempted to determine whether speciﬁc pectic substrates caused the
induction of pectolytic enzymes. It was also important to determine whether pectolytic
enzymes from Streptomyces species caused plant tissue damage and if this was a
characteristic of exclusive to pathogens. I also attempted to generate pectinase deﬁcient
mutants to determine whether the loss of this function correlated with a subsequent loss
in pathogenicity or virulence.

Streptomyces scabies has been reported to produce a toxin called thaxtomin. I
wanted to determine whether this phytotoxic compound was synthesized by the deep-
pitting scab isolate D.P. Such a ﬁnding would suggest that divergent forms of
pathogenic Streptomyces may posses similar or identical pathogenic mechanisms. I also

attempted to determine how potato tissues responded to Streptomyces infection by

5

monitoring the accumulation of two compounds which have been implicated in the
defense response of potato to several fungal and bacterial pathogens. The compounds
monitored were chlorogenic acid and lignin, both of which may have antimicrobial
activity and/ or prophylactic properties which prevent infection by streptomycetes.

The ultimate objective of my research was to develop starting points for future
investigations into the host-parasite physiology of potato scab disease. Understanding the
physiological dynamics and details of infection and symptom formation should allow for

the design of effective measures to control potato scab.

 

 

 

 

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REVIEW OF LITERATURE

After cereals, potatoes (Solanum tuberosum L.) are the most important food crop
in the world (Bajaj, 1987). Potato has been a staple crop around the world for at least
400 years (Burton, 1989). Potatoes are a versatile food product and are, sold as fresh
produce, potato chips, frozen french fries, and potato starch. The potato provides an
excellent source of protein and contains nearly a full complement of essential amino acids
(Bajaj, 1987). Potatoes are also used in producing ethanol (Burton, 1989). The multiple
uses of potatoes as food, fodder and other purposes account for its dominance among the
"root" crops currently used around the world.

Over the past several hundred years as the potato was introduced around the
world it has encountered a variety of environmental conditions and has been exposed to
numerous pest and pathogens to which it may not have natural resistances. These include
Colorado potato beetle, potato viruses X and Y, late blight, ﬁeld and post harvest tuber
rots, andlseveral environmental conditions (Agrios, 1978; Hooker, 1981; Burton 1989).
The potato has been important in our understanding of several fundamental concepts of
plant pathology (Agrios, 1978). Potato scab is a "classic" plant pathological condition
which ﬁrst appeared in the plant pathology literature during the late 1800’s (Bolley,
1890; and Thaxter, 1891).

Proper identiﬁcation of the causal agent responsible for potato scab has proven
to be difﬁcult for most researchers (Lambert and Loria, 1989 a). Bolley correctly
identiﬁed the causal agent as a bacterium (Bolley, 1890). Thaxter (1891), however, was

able to pure culture the organism, conﬁrm its pathogenicity, and concluded that the

 

 

 

 

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causal agent of the disease was a fungus. It was surprising, considering the detail of
Thaxter’s study of Oospora scabies, that he failed to recognize the organism was a
bacterium. Cunningham (1912) reported that Oospora scabies was a bacterium and
speculated that it was related to the Actinomycetes (Cunningham, 1912; and Lutman and
Cunningham, 1914). Gussow (1914) redesignatcd the species Actinomyces scabies. The
genus Streptomyces later replaced the genus Actinomyces (W aksman and Henrici, 1948).
Streptomyces scabies was the new name designated for the potato scab-causing
streptomycetes (W aksman, 1961).
Potato scab has not been considered an extremely destructive disease of potato.
Potato scab primarily reduces the quality of tubers by producing disﬁguring lesions in
the periderm which may range from superﬁcial pimples or ﬂecks too pits 0.5 cm to 1.0
cm deep in harvested tubers (Millard and Burr, 1926; and Hammerschmidt and Lacy,
1990).

Potato scab occurs in nearly all major potato-growing regions of the world
(Keinath and Loria, 1989). However, there are few accurate assessments of the world-
wide loss that this disease causes potato crops. Potato scab does not usually cause
signiﬁcant yield reductions. However, it is not uncommon to ﬁnd whole ﬁelds of
potatoes deemed unacceptable to be sold in markets (Manzer et al, 1977 ; and
Hammerschmidt and Lacy, 1990). This point becomes apparent when one considers a
situation where pitted scab of potato is prevalent and which causes deformation of tubers

and destruction of immature tubers under soil conditions ideal for the pathogen.

Descriptions of Potato Scab Symptoms and Isolates of Bacteria.

 

 

 

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8

"Scab" a generic term, describes any superﬁcial roughness produced on the
periderm of a potato tuber. Powdery scab, for example, caused by the slime mold
Spongospora subterranea, produces lesions occasionally mistaken for those of
Streptomyces scabies (Agrios, 1978). Various symptomatic lesions have been identiﬁed
as potato scab (Thaxter, 1891; Lutman and Cunningham, 1916; Millard and Burr, 1926;
and Hammerschmidt and Lacy, 1990), the reported types of scab are common, pitted,
russet, and tumulus or upraised scab.

Common scab is identiﬁed by superﬁcial, corky lesions on the tuber surface and
lacks evidence of the pathogen penetrating beneath the periderm (Thaxter, 1891; Lutman
and Cunningham, 1914; Millard and Burr, 1925; Jones, 1931; Lutman , 1941; Lapwood,
197 3; Lambert and Loria, 1989 a; and Hammerschmidt and Lacy, 1990). Streptomyces
scabies is usually identiﬁed as the causal agent of common scab (Lambert and Loria,
1989 a). However the acid tolerant scab pathogen, S. acidiscabies, also causes common
scab lesion identical to those of S. scabies (Manzer et al, 1977; and Lambert and Loria,
1989 b). It has also been, reported that several infective isolates differing in morphology
from S. scabies and S. acidiscabies can be isolated from common scab lesions (Faucher
et a1, 1992).

The minimal phenotypic characters used to identify S. scabies are: the ability to
cause common scab on potato; a grey colored spore mass with spores borne in spiral
spore chains; and use of all the International Streptomyces Project (ISP) sugars (Lambert
and Loria, 1989 a). S. scabies also produces black or deep brown melanoid pigments
in culture, especially on peptone yeast extract iron agar (PYI) and yeast extract malt

extract agar (YEME) (Shirling and Gottlieb, 1966; and Lambert and Loria, 1989 a).

9

There has been considerable revision of the taxonomy of common scab-producing
streptomycetes. This was partly due to the loss of the original type species and
misclassiﬁcation of subsequent isolates pathogenic on S. tuberosum (Thaxter, 1891; and
Lambert and Loria, 1989 a). Lambert and Loria (1989 a) proposed that the name
Streptomyces scabies be revived and that Thaxter’s original description be used for the
type species.

S. acidiscabies, an acid-tolerant form of common scab producing bacterium, has
the ability to cause disease in acid soils with pH as low 4.5 (Manzer et a1, 1977; and
Lambert and Loria, 1989 b). The ability to cause disease in acid soils differentiates S.
acidiscabies from S. scabies which typically causes disease in neutral to alkaline soils
(Lambert and Loria, 1989 b). In culture, S. acidiscabies produces ﬂexuous spore chains,
a pH sensitive diffusible pigment instead of melanoid pigments, can grow on acidiﬁed
medium, and has a spore mass color ranging from white to orange-red and a red or
yellow, and does not use raffmose as a sole carbon source (Lambert and Loria, 1989 b).
To date, S. scabies and S. acidiscabies are the only proper taxonomic designations for
scabocausing Streptomyces species pathogenic on S. tuberosum L. .

Deep-pitted scab, unlike common scab, is identiﬁed by semi-circular concave
lesions which may be up to a 1 cm in diameter and 0.5 cm deep in tuber surfaces (Jones,
1931; Archuleta and Easton 1981; Hammerschmidt and Lacy, 1990). In several cases,
the lesions coalesce to form furrows which disﬁgure tubers (Millard and Burr, 1926; and
Jones, 1931). The problem with deep-pitted scab is the depth which the pathogen
penetrates into a tuber; under severe conditions, tubers may be unsuitable for processing

(Archuleta and Easton, 1981). The pitted symptom has been reported numerous times

10
(Lutman and Cunningham, 1914; and Millard and Burr, 1926), but it is not clear whether

S. scabies is responsible for the symptom (Lutman and Cunningham 1914; Millard and
Burr, 1926; Jones, 1931; and Archuleta and Easton, 1981). Deep-pitted scab may be
caused by several species of Streptomyces (Archuleta and Easton, 1981; and Faucher et
al, 1992). Archuleta and Easton (1981) isolated S. diastatochromogenes, S.
atroolivaceus, S. lydicus, and S. resistomyciﬁcus from deep-pitted lesions and found that
these isolates caused deep-pitted scab both in ﬁeld and greenhouse experiments. One
exception was S. cinerochromogenes that produced common scab lesions in the ﬁeld and
deep-pitted scab in the greenhouse. Greenhouse symptoms may vary from those
Observed in the ﬁeld, and this can further complicate proper identiﬁcation of an isolate.
Identiﬁcation on the basis of phenotypic characteristics has not always been
accurate. Unfortunately, Archuleta and Easton (1981) did not provide information on
cultural morphology in their report however their isolates secreted melanin into culture
medium. Faucher et a1 (1992) isolated a deep-pitting isolate which produces light gold
to brown colonies and white spores borne in ﬂexuous chains, does not produce melanoid
pigments in culture, and only utilizes raffmose as a sole carbon source. A deep-pitting
isolate was discovered in Michigan in 1987 that produces a grey aerial spore mass in
culture, has a golden reverse colony color, does not produce melanoid pigments, bears
spore in ﬂexuous spore chains, uses all ISP sugars, will only grow in media with pH 6
and above, and produces deep pits in the cultivars Atlantic, Shepody, and Conestoga, but
not in Russet Burbank (Spooner and Hammerschmidt, 1989, 1992; and Spooner, this

dissertation). The relatedness of deep-pitting isolates will remain uncertain until

11

molecular techniques can be applied to developing proﬁles on group relatedness of all
isolates.

Russet scab or "scurf" is another form of superﬁcial scab lesion which may cover
the tuber surface and is identiﬁed by browning or roughening of the skin (Harrison,
1962; Lapwood, 1973; Bang, 1979). It is also called "superﬁcial scab" (Millard and
Burr, 1926). Russet scab is not as severe as common or pitted scab, yet it can reduce
tuber quality (Harrison, 1962). Russet scab develops in moist ﬁeld conditions which do
not favor development of common scab (Harrison, 1962). The descriptions of this scab
isolate are some what vague. Millard and Burr (1926) described the growth habit of a
superﬁcial scab isolate on numerous media. They reported color of the spore mass as
grey (depending on the medium), a lack of melanin production in culture, and spores
borne in straight or erect chains. Harrison’s isolates were very similar to Millard and
Burr’s isolate. They were slow growing, produced spores in chains, produced white—grey
aerial mycelium on synthetic sucrose agar, and did not produce melanin in culture
(Harrison, 1962). The russet scab isolate differs from S. scabies by not producing aerial
mycelium on gelatin and being negative for tyrosinase and soluble pigments on egg
albumin and nutrient potato agar (Harrison, 1962). Bang (1979) found three grouping
of russet scab isolates in northern Sweden, all of which infected only the Bintje cultivar,
caused disease in soil moisture and pH conditions that do not typically favor scab
development. Bang’s isolates were also fast growing. Groups 1 and 3 of the isolates
produced heavy aerial mycelium white or grey in color and group 2 lacked aerial

mycelium. All groups were tyrosinase negative (Bang, 197 9).

 

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12

Tumulus (also called upraised or pimple scab) represents a form of lesion which
does not penetrate the periderm but results in swellings over the tuber surface and arises
as a result of hyperplastic cell growth of infected tissue within lenticels (Millard and
Burr, 1926; Jones, 1931; and Hammerschmidt and Lacy, 1990). Millard and Burr
(1926) described, two isolates the tumulus form designated Actinomyces ﬂavus and the
pimple form A. wedmorensis. Both isolates had simple branched spore chains. The
tumulus isolate produced a grey spore mass on potato agar and the pimple isolated did
not. Both isolates were tyrosinase negative (Millard and Burr, 1926). Jones (1931)
histological observations on tumulus scab indicated that cells of the infected lenticel
proliferated causing a swelling of the surrounding tissue which gives rise to the tumulus
lesion, S. scabies however causes cell proliferation in the meristem below the point of
infection. Scab lesion often appear on roots and stolons which give rise to tubers
(Hooker, 1949). S. scabies can cause lesions or necrotic ﬂecks on ﬁbrous roots of
potato, pea, beets, and cucumber roots reducing weight of the roots (Hooker, 1949).

It is apparent that several Streptomyces species are able to infect potato. In the
past the criteria used to classify scab-producing bacteria were superﬁcial and reveal the
lack of interaction between plant pathologist and taxonomist which has caused the
development of our current state of miscategorized scab-causing isolates. Millard and
Burr (1926) recognized that without the use of standardized cultural techniques the "task
of deﬁning any species by its characters on artiﬁcial media is practically hopeless. "

Also, this brings into question whether there is more than one species of potato
scab-causing organism or merely several variant forms. It could also be the case that

genetic material encoding pathogenicity is being transferred among Streptomyces spp.

13

Plasmid transfers among the Streptomyces are possible in soil environments (Raﬁi and
Crawford, 1988; and Wellington et al, 1990).

Researchers have begun to investigate the question of relatedness among plant
pathogenic Streptomyces using DNA homology techniques (Tashiro et al, 1990; Healy
and Lambert, 1991; and Labeda and Lyons, 1992). S. scabies and S. acidiscabies are
39% and 17% respectively related to S. ipomoea causal organism of soil rot or pox of
sweet potato Ipomoea batatas (L.) Lam. (Labeda and Lyons, 1992). Tashiro et a1 (1990)
placed potato and beet scab isolates into two groups on the basis of spore chain
morphology and observed that these groups did not differ signiﬁcantly on the basis of
physiology and biochemistry. Utilizing DNA homology, however, they distinguished
four groups of scab—producing bacteria. Healy and Lambert (1991), also utilizing DNA
homology, found that there was high degree of genetic diversity among strains
phenotypically related to S. scabies. Examination of scab-producing isolates revealed the
following groups of species: S. scabies, S. acidiscabies, S. albidoﬂavus and S.
diastatochromogenes. They found that the level of DNA relatedness of each grouping
of isolates to the type isolate S. scabies ACTCC 49173 was 74% for S. scabies, 17.6%
for S. acidiscabies, 5.5 % for S. albidoﬂavus group and 28% for S. diastatochromogenes
group (Healy and Lambert, 1991). Interestingly, it was observed that DNA relatedness
test did not always differentiate between nonpathogenic and pathogenic strains of S.
scabies which were grouped together on the basis of phenotypic characteristics (Healy
and Lambert, 1991). Doering-Saad et a1 (1992), using a DNA probe from the rRNA
operon of S. coelicolor, detected restriction fragments length polymorphisms (RFLPS)

among 40 pathogenic and nonpathogenic Streptomyces isolates. The resultant southern

14

blots revealed a low degree of relatedness among the pathogenic strains tested, with no
signiﬁcant correlation between the phenotypic tests and RFLP groupings of the strains
tested (Doering—Saad et al, 1992). The low level of relatedness or high degree of genetic
diversity among these isolates suggested to this research group that the genes for coding
for pathogenicity were being transferred among different species by plasmids (Doering-
Saad et al, 1992).

Influence of population density on the incidence potato scab.

Lutman was believed that there was only one species of potato scab- producing
bacterium (Actinomyces scabies) with variable pathogenicity (Lutman, 1941). The
problem with his conclusion is determining what he considered to constitute a species or
population. It is not unusual to isolate numerous pathogenic and nonpathogenic types of
Streptomyces from scab lesions (Archuleta and Easton, 1981; and Faucher et al, 1992).
However, only pathogenic Streptomyces are considered S. scabies.

N oncultivated soil (sod) generally has been shown to contain higher Actinomycetes
populations than cultivated soil as determined by colony counting (Conn, 1916). Conn
(1916) found that the ratio of actinomycetes populations in sod versus cultivated soil on
average was 2.15 : l colony forming units (cfu) per gram of soil. In an exceptional case
the ratio was 6.4 : 1 cfu/g soil. Conn (1916) estimated the average population of
actinomycetes in sod that was stored in the laboratory for several weeks (ie. allowed to
desiccate) was 9.8 x 106 cfu/g soil, in freshly unearthed sod 6.6 x 106 cfu/g soil, and in
cultivated soil 2.9 x 106 cfu/g. Conn’s work suggested that cultivation of soil with
agronomic crops may reduce the resident actinomycete population of the soil. Both Conn

(1916) and Lutman (1945) were under the impression that "Actinomycetes" are normally

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15

active in the decomposition of grass roots or, in general, plant roots. However, what
happens to the populations of pathogenic Streptomyces?

Hooker (1956), monitoring the inﬂuence of crop rotations on the incidence of
potato scab in peat soils, determined that the incidence of potato scab was lower in fallow
soil than in soil that was planted with onion, soybean, corn, or potatoes. Peat soils
planted with potatoes had a similar incidence of potato scab as soil planted with a cover
crop of corn. The incidence of potato scab was found to increase over a three year
period in plots planted to potato. There was no consistent difference in the incidence of
potato scab on plots that had been planted with onion, corn, soybean, or left fallow.
These ﬁndings suggested that crop rotations or leaving ﬁelds fallow reduces the
population of infective Streptomyces over time as revealed by the lower incidence of
potato scab (Hooker, 1956). This implies that Streptomyces pathogenic on potato may
have the capacity to survive in the rhizosphere of crops other than potato. However, this
capacity is limited as the incidence of potato scab steadily declines in the absence of
potatoes.

Lutman (1923), found that potato scab could occur in soil never planted with
potatoes. In these studies a pine wood lot was cleared and planted with surface
disinfected potatoes in isolated hills to prevent inoculum from being transported
throughout the area. The implications were that the resident Streptomyces species, which
may not have been S. scabies, were able to shift from a saprophytic form of subsistence
to a pathogenic mode of subsistence. This may also imply that S. scabies has a very

wide host range and is able to live on many nonagronomic plants.

16

There appears to be some effect on S. scabies populations size associated with a
potato cultivars resistance to potato scab. Keinath and Loria (1988; 1989 a and b), found
that the rhizosphere and tuber surface populations of Streptomyces isolates that were
morphologically and physiologically similar to S. scabies were greater on the scab-
susceptible cultivar Chippewa than the scab-resistant cultivar Superior. The population
of scab-producing bacteria increases during the growing season, and declines between
crops (Keinath and Loria, 1989 a and b). Thus, an important factor for control may be
the inoculum density at the time of planting. Keinath and Loria (1991) were able to
monitor populations rifampin—resistant Strains of S. scabies in soil and observed that the
higher the initial level of infective inoculum, the greater the population of bacteria in the
rhizsophere, rhizsoplane, and on tuber surfaces. Increased severity and incidence of
potato scab were positively correlated with high initial levels of inoculum (Keinath and
Loria, 1991). Keinath and Loria (1989) suggest that knowledge of the population density
of scab-producing streptomycetes may be useful for predicting disease severity.
However, since melanin production is an unstable characteristic in several streptomyces
species and antibiotic resistance genes may be exchanged among various Streptomyces
species by plasmids in soil environments, ﬁnding suitable markers for monitoring the
populations of S. scabies in the soil may be difﬁcult (Hollis, 1952; and Hopwood et al,
1986; Raﬁi and Crawford, 1988; and Wellington et al, 1990).

Conditions favoring the development of potato scab and their control.

It is not clear what effect speciﬁc cultural practices have on populations of potato

scab-causing organisms in the soil. The build up of inoculum or increased virulence Of

pathogenic streptomycetes may be favored by improper cultural practices such as

17

improper management of soil pH and moisture, or use of susceptible cultivars of potato.
Investigations into the effects of cultural practices on the populations of potato scab-
causing streptomycetes have apparently been overlooked and yet, it is this type of
research which may explain how certain cultural control methods succeed.

The effect of soil moisture.

The development of potato scab is favored by several environmental factors. The
most notable of these factors are soil moisture content and soil pH. Increased incidence
of potato scab has been reported to occur during brief periods of low soil moisture
content (Lapwood and Hering, 1968). Thaxter (1891) noted that potato scab proliferates
on light "dry" soil and did not agree with the popular notion that excessive soil moisture
favored the development of the disease. It was thought excessive soil moisnrre stimulated
protuberance or swelling of lenticels and infections could occur through these lenticels.
Thus, it could be reasoned that excessive soil moisture increased the number of
susceptible lenticels (Lutman and Cunningham, 1914). Common and pitted scab,
however, are favored by a low soil moisture content and the severity and incidence of
disease increases in warm and relatively dry soils (Hammerschmidt and Lacy, 1990).
Russet scab of potato, however, is favored by moist soil conditions (Harrison, 1962).
Apparently, the critical period in which a tuber is most susceptible to infection is the ﬁrst
5 weeks after tuber initiation, if soil moisture is adequate during this time, the incidence
of disease will be lowered (Lewis, 1970). Sanford (1923) thought the critical time for
the tuber to be in moist soil was the ﬁrst month of tuber initiation, in order to avoid
infection. Tuber susceptibility to infection appears to be temporal, restricted to a few

intemodes close to the tuber apex, and related to the environmental conditions under

 

 

 

 

 

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18
which tubers form. Lapwood and Hering (1968 and 1970) suggest a tuber is most

susceptible to infection during the third week of tuber initiation when the ﬁrst intemodes
are formed. Infection and disease severity may be suppressed by maintaining sufﬁcient
moisture over the ﬁrst month during tuber initiation (Lapwood and Hering, 1970). Davis
et a1 (1976) conﬁrmed the work of Lapwood and reported that a mean moisture depletion
to -0.65 bars approximates the minimum soil moisture required for scab control and that
in their studies this level of soil moisture provided for the highest yield of potato.

Why infection occurs under low soil moisture is not clear? The anatomy of the
lenticel is inﬂuenced by the amount of soil moisture (Adams and Lapwood, 1978). It has
been suggested that in dry soils the tuber intemodes bearing lenticels are heavily
colonized by actinomycete hyphae and in wet soils the actinomycetes are unable to
proliferate on susceptible tissue (Adams and Lapwood, 1978). This supports the ﬁndings
which indicated that actinomycetes tend to proliferate on periderm under conditions of
low soil moisture (Lewis, 1970). Adams and Lapwood (1978) suggest that microbial
antagonism through competition for sites on the tuber or microbial antibiosis is the
responsible for this effect. Lewis (1970) thought the effect of irrigation was to lower the
population of pathogenic streptomycetes.

The effect of soil pH.

The incidence of common scab increases as the soil pH increases (Schaal, 1940;
Lambert and Loria, 1989 b). Soils pH 6 or above are conducive to the development of
common scab caused by S. scabies and several other the infective potato scab-causing
species. S. acidiscabies, however, may cause potato scab in soils with pH 5.2 or lower

(Manzer et a1, 1977; Loria et al, 1986; and Lambert and Loria, 1989 b). Schaal (1940)

19

demonstrated isolates causing acid scab were tolerant of low pH by growth on acidiﬁed
potato dextrose agar. The pH effect has been studied primarily from the aspect of its
potential as a control measure (Schaal, 1940; Houghland and Cash, 1956; and Goto,
1985). In regard to controlling potato scab, proper management of soil pH is important
and it seems to follow that knowing the disease history of an area would go along ways
to prevent favoring the build up of potato scab bacteria that prefer alkaline or acid soils.
The effect of liming and calcium.

Calcium-containing compounds, when applied to acidic soils, improve yield and
increases the incidence of potato scab (Blodgett and Cowan, 1935; Odland and Allbritten,
1949; Horsefall et al, 1954; Doyle and MacLean, 1960; and Goto, 1985). The effect of
calcium may be to increase soil pH (Blodgett and Cowan, 1935; Odland and Allbritten,
1949; and Doyle and MacLean, 1960). However, the effect of calcium may work
independently of the pH effect (Horsefall et al, 1954). The reasoning for this is as
follows: lime reduces soil acidity, and this appears to increase the pathogen’s ability to
penetrate tubers and induce scab lesions. Horsefall et al (1954) observed that calcium
levels in soil inﬂuence accumulation of calcium in tubers.

The level of exchangeable calcium in soil may be a more reliable indicator for
anticipating the incidence of potato scab, associated with liming, than soil pH (Goto,
1985). When the level of a CaO (exchangeable calcium) in the soil exceeds 150 mg/ 100
g soil, the incidence of scab was shown to exceed 20% of the scab index. A scab index
of 30% is critical for market quality tubers (Goto, 1985).

There appears to be a physiological basis for the effect of calcium on increasing

the incidence of potato scab. Calcium has been shown to enhance the germination of

20

S. viridochromogenes spores and regulate the formation of aerial mycelium (Eaton and
Ensign, 1980; and Natsume et al, 1989). Elevated calcium levels in the tuber periderm
or the rhizosphere could possibly trigger germination of dormant spore of pathogenic
Streptomyces in the soil. Arteca (1982) suggested that calcium accumulates in the tuber
periderm. In this study 45Ca was inﬁltrated into whole tubers, it was observed that most
of the 45Ca sampled in the tuber was initially found between 0-5mm from the tuber
surface. After several days of storage the amount of radioactive calcium in the surface
region decreased and the amount of calcium in the middle section of the tuber increased
(Arteca, 1982). Therefore it may be that a tuber is most susceptible to infection when
the amount of calcium in the periderm was elevated, which could account for increased
incidence of potato scab after lime is applied to ﬁelds. To test this idea, it may be
necessary to lime the soil at various times during the development of the tubers, and then
determine whether high calcium levels in periderm favors susceptibility to potato scab.
The role of calcium in potato scab development may be to initiate a series of
physiological events occurring in or near the tuber periderm that result in increased rates
of infection.

To control the level of potato scab which results from increased liming it seems
reasonable to monitor the rate of liming. Also use of sources of calcium that do not
elevate the level of exchangeable calcium in the soil.

Control measures: the effect of mineral amendments.

The chemicals that are used to control potato scab either lower soil pH or may

be toxic to the pathogenic streptomycetes. Manganese-containing compounds have been

successful in control of common scab and function by lowering soil pH (Mortvedt, et al,

21
1961; and McGregor and Wilson, 1964). McGregor and Wilson (1964) observed that

acid soils generally have higher levels of soluble manganese than alkaline soils, and this
could explain the effect acid soils have on common scab. Sulfur was observed to reduce
the incidence of potato scab by lowering the soil pH, but not all sulfur—containing
compounds were effective at controlling potato scab (Hooker and Kent, 1950).
Aluminum compounds were also effective against potato scab and worked by lowering
soil pH (Houghland and Cash, 1956). Bordeaux mixture, a copper-containing fungicide,
also reduced the incidence of scab on potatoes. However, the reason Bordeaux mixture
is effective against potato scab is not clear (Mader and Mader, 1937).

Additional methods of controlling potato scab.

Crop rotation has been effective at managing potato scab (Hooker, 1949 and
1956). Hooker’s work demonstrated that pathogenic Streptomyces spp. survive in the
rhizosphere of crops other than potato but to a limited extent. Rotations of three or more
years between potato crops was shown to reduce the incidence of potato scab in peat soils
(Hooker, 1956).

Menzies (1959) found that ﬁelds in Washington state exerted a suppressive effect
on potato scab-causing bacteria. The suppressive activity of this soil was biological in
nature and could be deactivated through autoclaving. Soil containing pathogenic
Streptomyces when mixed with equal amounts of suppressive soil did not yield scabby
tubers. The effect of suppressive soil on potato scab was enhanced by additions of alfalfa
meal. The use of green manures have been recommended over the years to control

potato scab but this biological control has inconsistent results in the ﬁeld and although

22

it may have future uses it requires more study (Oswald and Lorenz, 1956; and Hanson,
1990).

F oliar applications of 3 ,5-dichlorophenoxyacetic acid have been Shown to reduce
the incidence of potato scab, and this compound was. readily translocated to tubers after
foliar application (Burrell, 1982 and 1984). It was suggested that 3,5-
dichlorophenoxyacetic acid modiﬁed potato metabolism by inhibiting the synthesis of
certain phenolic compounds (Burrell, 1984). McIntosh et a1 (1988) found that foliar
sprays of certain benzoic and picolinic acids also reduced the severity of common scab.
Finally, a lipoglycoprotein elicitor from Phytopthora infestans was shown to induced
resistance to a wide variety of potato pathogens including S. scabies (Metlitskii et al,
1978).

Resistance of potato to S. scabies.

The basis for resistance of potatoes to S. scabies and other pathogenic
Streptomyces is not known. Some researchers suggest that resistance to potato scab
resides in some special properties of the periderm (Cooper et al, 1954; Emilsson and
Heiken, 1956; Hooker and Page, 1960). It has been observed that S. scabies can only
infect newly formed lenticels of the apical end of a tuber (Fellows, 1926). Cooper et al
(1954) found that resistance or susceptibility to potato scab correlated with the presence
or absence of nuclei in the cells of the outer cell layer of the periderm, respectively.
This correlation was uniform for the different cultivars of potato examined. Emilsson
and Heiken (1956) conﬁrmed the ﬁndings of Cooper et al (1954). However, some
cultivars of potato did not correspond to either of Cooper’s periderm categorizes.

Emilsson and Heiken (1956) therefore concluded that periderm structure was not the only

23

factor regulating resistance to potato scab. Hooker and Page (1960) could not obtain
growth of Streptomyces on intact periderm surfaces, nor could the bacterium grow on
autoclaved or propylene-oxide treated periderm. The periderm therefore, primarily
functions as a barrier to infection, but this view of the periderm is too simplistic and
some other factors must be involved. Richardson (1952) demonstrated that there was a
noncharacterized type of resistance in potato tubers, regardless of cultivar resistance, that
is related to the growth rate of the tuber.

Resistance to potato scab has been correlated with greater amounts of chlorogenic
acid (CGA) in the periderm and outer layers of resistant tubers when compared to
susceptible tubers (Johnson and Schaal, 1952). Chlorogenic acid is thought inhibit or
poison pathogenic organisms (Nicholson and Hammerschmidt, 1992).

Jones (1931) observed that suberized barriers are deposited in response to
infection of potato tissue. Spooner and Hammershcmidt (1992) observed the rate of
ligniﬁcation is greater on tuber discs inoculated with Streptomyces spores, than
uninoculated controls. The initial ligniﬁcation response to a deep-pitting streptomycetes
was much lower than S. scabies or the nonpathogenic isolates of Streptomyces. Thus
there is an interaction at the physiological level between potato tissue and pathogenic
Streptomyces the contribution of phenolic compounds and ligniﬁcation in resistance is not
clearly deﬁned.

The infection process.

Infection has been thought to occur primarily through lenticels (Adams and

Lapwood, 1978). The lenticels are the sites of gas exchange in the periderm and arise

from stomata of growing tubers as the periderm forms (Adams, 1975). According to

24

several investigators, the infection of potato tubers by many soil- borne pathogens
proceeds through the developing lenticel at the time in which the stomata are
differentiating into lenticels (Lumran, 1913 and 1941; Fellows, 1926; Jones, 1931; Smith
and Ramsey, 1947; Adams, 1975; and Stein et al, 1994). This is also true of potato

scab-causing streptomycetes (Lutman, 1913 and 1941; Jones, 1931; Adams, 1975; and
Adams and Lapwood, 1978). Light and electron microscopy indicate that the bacterium
may also enter through stomata or penetrate directly through ligniﬁed surfaces (Lutman,
1941; and Stein et al, 1994). It is interesting to note that mature lenticels are resistant
to infections and the period of susceptibility to infection via lenticels is only 5-10 days
(Smith and Ramsey, 1947; Lapwood and Adams, 1973; Adams, 1975; and Adams and
Lapwood; 1978). Scab lesions appear to form in the region of the tuber where the most
rapid growth is occurring (the apical end of the tuber where stomata are rapidly
differentiating into lenticels) (Fellows, 1926). As the tissues beneath the stomata that
develop into the lenticel pushes through the stomata, the exposed tissue may become
susceptible to infection (Fellows, 1926).

The series of events following penetration of S. scabies into susceptible tissue are
not too clear. It has been observed that the bacterial hyphal ﬁlaments grow
intercelluarly, (i.e., in the pectic materials of the middle larnella) (Lutman, 1941;
Richards, 1943; Shoemaker and Riddell, 1953; and Stein et al, 1994). S. ipomoea, the
causal organism of soil rot or pox of sweet potato, was observed using scanning and
transmission electron microscopy growing both intracellularly and intercelluarly in
infected root tissues (Clark and Matthews, 1987). Clark and Matthews (1987)

observations suggested the hyphal ﬁlaments penetrated cell walls by an enzymatic

25

process, as there was not evidence that mechanical force was used to penetrate cell walls.
Stein et al (1994) reported that ﬁlaments of S. scabies peneuate cell walls of potato by
what appeared to be enzymatic action.

Once the bacterium gains access to the host tissue it stimulates meristematic
tissues to undergo hyperplasia and the newly divided cells then undergo hypertrophy
(Lutman, 1913; and Jones, 1931). The invaded tissue eventually collapses and turns to
a brown color characteristic of a newly formed scab lesion (Jones, 1931). Beneath the
disrupted tissue the healthy meristem tissue is induced to deposit a layer of suberin. The
suberin may be six cell layers thick (Jones, 1931). At this point the pathogen may be
completely localized or its hyphal ﬁlaments may penetrate through incompletely suberized
cells in the wound cork barrier. Jones (1931) observed that in deep—pitted scabs the
wound cork barrier was penetrated by hyphal ﬁlaments, which induced another round of
cell division and enlargement followed by the formation of another wound cork barrier.
No more than three wound barriers were ever observed in pitted lesions. Jones (1931)
speculated that deposition of more wound cork was possible but was terminated as active
growth of the tuber ceased. Lutman (1913) never observed hyphal ﬁlaments growing
within cells of infected tissues and also noticed that starch grains underlying a scab spot
were remained unaffected. S. scabies has been observed to only infect and cause scab
lesions on actively growing tubers within the actively growing apical region (Fellows,
1926; and Jones, 1931). This correlates with the observation that small slowly growing
tubers and nongrowing mature tubers are practically immune to disease development

(Fellows, 1926).

26

Histological studies have determined where the bacterium grows in the host tissue
and that its presence elicits a wound response in the healthy meristem tissue. What has
been lacking, however, was physiological studies to determine the effect of the pathogen
on the host tissues. Lutman (1941 and 1945) speculated that the organism was deriving
nutrition from the pectic materials in the middle lamella. Jones (1931) thought S. scabies
created alkaline conditions in the tissue which resulted in hyperplasia and hypertrophy
of the infected tissues. Fellows (1926) could not detect hyphal ﬁlaments in infected host
tissues but speculated that hyperplasia and hypertrophy were caused by diffusible
enzymes or toxin of bacterial origin. Electron microscopy supports a role for the
involvement of enzymes in the infection process and does not suggest that S. scabies
utilizes mechanical pressure to invade host tissues (Clark and Matthews, 1987 and Stein
et al, 1994).

The production of extracellular enzymes and/ or toxins is currently thought to
explain how S. scabies and other pathogenic Streptomyces invade and colonize potato
tissue. The ability of Streptomyces species to produce degradative enzymes is well
known, and several streptomycetes degrade substrates such as lignin, cellulose, and
pectin which are often inaccessible to many bacteria (Knosel, 1970; Sato and Kaji,
1973,1975,1977 a and b; Antai and Crawford, 1981; Borgmeyer and Crawford, 1985;
Pometto and Crawford, 1986; and MacKenzie et al, 1987; Kom-Wendisch and Kutzner,
1992). Potato scab—causing bacteria are thought to utilize pectolytic enzymes to grow
within the middle lamella of plant tissues (Richard, 1943; Shoemaker and Riddell, 1953;
and Knosel, 1970; Spooner and Hammerschmidt, 1992). Knosel (1970) found that S.

scabies and some nonpathogenic Streptomyces produced pectate lyase when induced on

27

a medium consisting pectin and cellulose. The nonpathogens S. ﬁadiae and S.
nitrosporeus produce pectate lyases in culture ﬁltrate that cause tissue maceration (Sato
and Kaji, 1973, 1975, 1977 a and b). Knosel (1970) found that there was no correlation
between synthesis of pectolytic enzymes by streptomyces and virulence. Considering that
the enzymes were bioassayed in pea leaf, we cannot be certain of the validity of his
conclusion. To date no one has ever investigated the possible involvement of lignin
degrading enzymes in potato scab disease, however, some studies indicate lignin is
degraded by streptomycetes (Jones, 1931; and Pometto and Crawford, 1986).
Production of toxin by S. scabies has been investigated as a pathogenicity factor.
The phytotoxin produced by S. scabies and S. acidiscabies in scabby tissues, culture
ﬁltrates and agar medium was named thaxtomin (King et al, 1989 and 1991; Lawrence
et al, 1989; and Eckwall et al, 1992). Thaxtomin occurs in two major forms (A and B)
and is characterized as unique 4—nitroindol-3-yl containing 2,5-dioxopiperazines. The A
form occurs at 20 fold higher concentration than the B form (Lawerence et al, 1990).
Thaxtomin induces scab-like lesions on minitubers. Thaxtomin also elicits rapid
browning of tuber discs within 24 hours after applications (Spooner, in this dissertation).
Doering-Saad et al (1992) suggests that genes coding for thaxtomin production be used
to determine whether S. scabies pathogenicity genes occur on plasmids and to develop
DNA probes for detection of potato scab-causing streptomycetes, without having to resort
to traditional pathogenicity screening methods. However, the validity of using such a
DNA probe depends on whether thaxtomin is necessary for Streptomyces to cause potato

scab.

28
Many of the fundamental questions related to pathogenicity of S. scabies and other

potato scab-causing streptomycetes may be addressed by obtaining a greater
understanding of the physiology of the infection process. Obtaining physiologically based
information on the factors involved with spore germination, infection, and spread of the
pathogen within the host tissue, as well as the physiology of the host responses to
infection, would tie together, in a meaningful fashion, the numerous observations made
on this disease over the past 103 years since Thaxter’s (1891) initial report on potato

scab.

29

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33

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35

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36

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CHAPTER H
THE CHARACTERIZATION OF CULTURAL, MORPHOLOGICAL,
PHYSIOLOGICAL, BIOCHEMICAL, AND PATHOLOGICAL PROPERTIES OF

POTATO SCAB CAUSING ISOLATES OF STREPTOMYCES

37

38

INTRODUCTION

Potato scab, a superﬁcial roughening of potato periderm reduces tuber quality and
marketability (Lapwood, 1973). Potato scab lesions may appear as superﬁcial ﬂecking,
russeting, corky surface lesions, deep-pitted lesions, or upraised lesions on tubers of
several cultivars of potato (Millard and Burr, 1926; Lapwood, 1973; and
Hammerschmidt and Lacy, 1990). All varieties of potato scab lesions or symptoms were
thought to be caused by a single species of streptomycetes, namely Streptomyces scabies
(Waksman, 1961; Kutzner, 1981; Lambert and Loria, 1989 a; and Healy and Lambert,
1991). Lutman (1913) thought the causal agent of common scab, was exclusively
responsible for all forms of potato scab lesions and that the symptom variation associated
with the disease was due to variation in the degree of pathogenic virulence of S. scabies.
This was a reasonable conclusion, when one considers that the primary characteristic
used to conﬁrm the identify S. scabies was lesion formation on any susceptible cultivar
of potato. The S. scabies type-isolate discovered by Thaxter (1891), produced dark
pigments when grown in agar culture (the pigment color varied according to the culture
media used), produced spiral spore chains or ﬁlaments consisting of rod-like bodies of
various lengths and the ﬁlaments gave rise to the grey colored spore mass. The
description given by Thaxter for Oospora scabies (S. scabies) became the standard by
which all other potato scab-causing streptomycetes were compared (Lambert and Loria,
1989 a; Healy and Lambert, 1991). The taxonomy of the potato scab-causing
Streptomyces became uncertain for two reasons. First, the Thaxter type-isolate was not

maintained and became unavailable for the purpose of comparison (Lambert and Loria,

39
1989 a). Finally, there has not been an attempt to standardize the methods used to study

potato scab disease or potato scab-causing Streptomyces until quite recently (Kutzner,
1981; Lambert and Loria, 1989 a; and Korn-Wendisch and Kutzner, 1992). This has
resulted in improper classiﬁcation of several potato scab-causing Streptomyces.

Millard and Burr (1926) analyzed 24 strains of potato scab-causing actinomycetes,
and found several isolates that were morphologically and physiologically different from
A. scabies (S. scabies). Subsequent reports have suggested that potato scab could be
caused by several species of Streptomyces other than S. scabies (Jones 1931; Harrison,
1962; Manzer, et al, 1977; Bang, 1979; Archuleta and Easton, 1981; Loria et al, 1986;
Lambert and Loria, 1989 b; and Faucher et al, 1992).

One of the most enterprising attempts to standardize the study of Streptomyces
was the International Streptomyces Project (ISP) (Shirling and Gottlieb, 1966, 1968 a and
b, 1969, and 1972). The criteria used for the ISP were: spore chain morphology; spore
ornamentation; color of the mature sporulating aerial mycelium; production of melanoid
pigments; color of the vegetative mycelium as seen from the reverse side of growth;
color in agar medium as caused by diffusible pigments; the effect of pH on pigment
color; and utilization of ISP carbon sources (Shirling and Gottlieb, 1966 and 1976).
However, researchers have not relied entirely upon the criteria set forth by the ISP.
Several other techniques have been developed that have proven useful to study
Streptomyces. Paper chromatography of whole cell hydrolysates has been used to
distinguish between species of Nocardia and Streptomyces (Becker, et al, 1964).
Polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE of culture ﬁltrates and

whole-cell proteins have been used to supplement the taxonomic study of streptomycetes

40
(Gottlieb and Hepden, 1966; Hughes et al, 1971; Valu et al, 1984; Gardes and Lalonde,

1987; and Dietz, 1988). Molecular techniques have also been used to study the
taxonomy of Streptomyces (Tashiro et al, 1990; Healy and Lambert, 1991; Doering-Saad
et al, 1992; and Labeda and Lyons, 1992). On the basis of DNA homology, the potato
scab-causing Streptomyces have been found to represent a genetically diverse group
(Tashiro et al, 1990; Healy and Lambert, 1991; and Doering-Saad et al, 1992). Species
grouped with S. scabies on the basis of phenotypic similarities, were found to posses low
relatedness to the S. scabies group on the basis of DNA homology (Healy and Lambert,
1991).

The taxonomy of S. scabies has been complicated by the seemingly ubiquitous
distribution of the pathogen. Potato scab-causing bacteria have been reported to occur
in most potato-producing regions around the world (Keinath and Loria, 1989). Potato
scab-causing bacteria have been reported to survive in the rhizospheres of several
agronomic crops other than potato (Hooker, 1949 and 1956). Also, potato scab-causing
bacteria have been reported to survive for several years in fallow ﬁelds usually planted
with potatoes (Hooker, 1945). Potato scab has been reported to occur in soils which
have never been planted to potato or any other agronomic crop (Conn, 1916; and
Lutman, 1923). Researchers have suggested that Streptomyces, as a group and potato
scab-causing streptomycetes in particular, survive in the absence of a suitable host plant
by degrading complex polymers in plant debris (Lutman and Cunningham, 1914; Conn,
1916; and Lutman, 1945).

We must expand our current deﬁnition of S. scabies or develOp an alternative

system of classiﬁcation for the potato scab-causing streptomycetes. To control potato

41

scab we must understand more about the potato scab-causing streptomycetes interactions
with agronomic crops and alternate hosts, their distribution and survival, as well as their
interaction with nonpathogenic streptomycetes. To properly identify scab-causing
species, standard methods of identiﬁcation must be developed.

The purpose of this study was to characterize the isolates of Streptomyces,
especially the Michigan isolates, that would be used in the other aspects of my research.
My purpose was not to determine whether the isolates were S. scabies, but to develop
a proﬁle of morphological, biochemical, physiological, and pathogenic characteristics for
each isolate. Between the summers of 1987 and 1988, isolations were made from
potatoes with scab lesions. Some of the resultant Streptomyces cultures appeared to be
morphological and culturally distinct from S. scabies. It appeared to be necessary to
establish the similarity of Michigan isolates to S. scabies, because variation in the
patterns of infection could possibly be related to variations in the physiology and
biochemistry of the individual isolates.

MATERIALS AND METHODS
Reference isolates. Reference isolates were provided by Dr. Rose Loria of Cornell
University, New York. The isolates RL 84-01-232 (RL 232) and RL 83-01-03 (RL 03)
pathogenic strains of S. scabies. RL 232 and RL 03 cause common scab lesions on
several different cultivars of potato (Fig. 2.1) The nonpathogenic isolates RL 84-01-95
(RL 95) and RL 84-01-108 (RL 108) saprophytic forms of Streptomyces appeared
morphologically similar to S. scabies. The isolates RL 95 and 108 were virtually

indistinguishable from one another on the basis of cultural morphology. RL 232 and

42

 

Fig. 2.1. Types of potato scab on Atlantic tubers. A, Common scab caused by RL 232;
B, Deep pitted scab caused by D.P.

43

RL 95 were selected as isolates to be studied in the majority of the physiological studies
and pathogenicity screening. The isolate of S. lividans (S.l.) was donated by Dr. Wendy
Champness (Department of Microbiology, Michigan State University). S. lividans has
been studied by antibiotic researchers and geneticists and was selected for its comparison
to S. scabies. The Wallace and Hammerschmidt isolate 38 was selected as a reference
isolate. 38, was isolated from potato and was similar in appearance to S. scabies. 38
produced a deep blue pigment on agar medium, had a blue-grey colored spore mass, and
was not pathogenic on any cultivars of potato.

Isolation of Streptomyces from potatoes. Infected ﬁeld tubers were rinsed for several
minutes in tap water and scrubbed with a soft bristle brush to remove heavy particles of
soil adhering to the tuber surface. After washing, the tubers were disinfected in 10%
commercial Chlorox solution for 10 minutes. The lesions were excised from the
periderm and soaked in sterile distilled water to leach out excess disinfectant.
Alternatively, infected tissue was excised from the tuber after washing and disinfected
directly for 5 minutes followed by several rinses of the tissue in sterile distilled water.
When heavily infected tubers were available, only small amounts of periderm were
required for isolations. With smaller tubers or tubers with sparse infection it was
necessary to remove all of the infected periderm for adequate isolations.

Disinfected lesion tissue was ground in a mortar and pestle ﬂamed with 95%
ethanol. After the tissue was macerated, 2 ml to 3 ml of sterile distilled water was added
to produce a suspension of tissue. The suspension was used to inoculate 2 % water agar.
The inoculated plates were incubated in the dark at 27 C. Small, isolated, powdery grey

colonies formed within 10 days. Water agar provided the minimal nutrients required for

44

sporulation, but growth of mycelium was not detected. It was usually necessary to
subculture the isolated bacteria on water agar for two or three rounds until fungal and
bacterial contaminants were no longer present in culture. Within 30 and 60 days the
streptomycetes were pure cultured on yeast malt extract agar (YEME) (Shirling and
Gottlieb, 1966). The Michigan isolates were pure cultured during the summer of 1987
and 1988. The summers were particularly hot, dry and favored the development of
potato scab (Table 2.1).

Maintenance of pure cultured Streptomyces isolates. The pure cultured Streptomyces
isolates were grown on YEME agar. Cultures were maintained in the incubator for at
least three weeks prior to subculture. A culture was not transferred more than three
times. Short-term cultures were maintained at 4 C on YEME slants and plates for
several months. Several methods were used for long-term storage of inoculum:
desiccated cultures stored at 4 C and -20 C, the spore were rehydrated to start new
cultures. Cultures were also maintained at -20 C on YEME slants overlaid with 20%
glycerol. These methods kept the inoculum viable for several months to years. The
preferred method of inoculum storage was freezing spores at -20 C in 20%
glycerol/water solution (Hopwood et al, 1985). Frozen inoculum remained viable for
several years. YEME agar was inoculated with stocks of frozen spores and incubated
10 to 14 days at 27 C. The mature spore could be subcultured or used to inoculate other
substrates for physiological testing.

Naming of the isolates. The isolates were named on the basis of the type of lesions it

produced on the sample tuber, the cultivar of the sample tuber, or according to the label

TABLE 2.1. Designation and initial characterization of Michigan isolates

45

 

 

 

Designation Abbr.‘ Origin” Year‘ Lesion‘I Cultivar
Conestoga 1 Corr. 1 Samariae 87 common Cum
Conestoga 2 Con. 2 Samaria 87 common Conestoga
Conestoga 3 Con. 3 Samaria 87 common Correstoga
Plant residue' P.R. 1 Samaria 87 NA' NA
Farmer’s Stuff F.S. MI 87 ?" ?‘

Red Pontiac 1 R.P. 1 MI 87 common/deep Minnie
Red Pontiac 2 R.P. 2 MI 87 common/deep MW
Onaway 9/25 same MI 87 ? Onaway
Field II Fi II MI 87 common ?
Atlantic 9/25 AT MI 87 deep Atlantic
Deep Pit 1 D.P. MI 87 deep ?

Deep Pit 2 D.P(W) MI 87 deep ?
Onaway Large Lesion same Montcalm 87 deep Onaway
Onaway Little Lesion same Montcalm 87 common Onaway
F945 same MI 87 deep F945
88-001 same MI 87 common Onaway
88-002J same MI 88 NA NA
88-003 same Newbury 88 deep Atlantic
88-004 same Newbury 88 deep Atlantic

 

' Abbreviated designation within text.

” Site where sample was collected.
‘ Year sample was collected.

" Type of Lesion.

‘ City. If unknown MI (Michigan).

' Isolated from plant debris in potato ﬁeld.

' Not applicable

" Type of lesion on the sample was not recorded.
' Cultivar of sample unknown.

1 A contaminant on an Erwinia culture.

 

 

matL

solu

1L

46
on the bags of tubers brought into the laboratory for inspection (Table 2.1). The 1988

isolates were assigned a numerical code designation.
Propagation of plants. The isolates pathogenicity were conﬁrmed with greenhouse
cultivated potatoes. Tubers of scab-susceptible Atlantic and scab-resistant Russet
Burbank were propagated for pathogenicity screenings. Single-eye cuttings from mature
tubers were usually used to propagate whole plants.
Preparation of inoculum for pathogenicity screening. Inoculum was produced from
mature sporulating cultures grown on YEME agar or from mycelium cultured in nutrient
solution .20 g sucrose, 10 g yeast extract, 1.2 g L-asparagine, 0.6 g KZHPO4 diluted to
1 L with water, adjusted to pH 7.5 and autoclaved for 20 minutes (Loria and Davis,
1988).
Design and preparation of pots for greenhouse screening of isolates for
pathogenicity. The inoculum was mixed by hand with Bacto-mix potting mixture.
Twelve to twenty sporulating YEME culture plates were mixed with the approximately
7-10 full pots of Bacto-mix. When the nutrient solution was used to produce inoculum,
1 L of the solution was mixed with Bacto-mix until the consistency was similar to
ordinary Bacto-mix (Loria and Davis, 1988). Bacto-mix/inoculum mixture was applied
to the upper 6 inches of the pot since tubers were usually formed in this region of a pot.
This method of soil preparation allowed for high inoculum densities in a conﬁned area.
An additional layer of potting soil was applied over the inoculum-zone to prevent
dispersal of bacteria by splashing from over head watering.

The plants were watered heavily twice a week and additional water was provided

on hot days. To favor disease development the soil was kept relatively dry, while

47

preventing the plants from wilting. Normal sized tubers and mini-tubers were usually
produced within 8 to 12 weeks after planting.

Initial characterization of pure culture isolates. Cultures of the isolates were
examined for aerial spore-mass color, reverse color of mycelium, production of diffusible
colored pigments and melanin in culture on YEME and oatmeal agar (OA). Peptone
yeast extract iron agar (PYI) was used to detect melanin production (Shirling and
Gottlieb, 1966). These methods of isolate characterization were adapted from the ISP
methods for examination of Streptomyces (Shirling and Gottlieb, 1966).

Cultural, physiological and biochemical characterization of Streptomyces isolates.
Various physiological and biochemical tests were used to characterize the Michigan
isolates and determine their similarity to RL 232 (S. scabies) and the other reference
isolates.

The biochemical and physiological test used were a combination of the ISP
criteria (Shirling and Gottlieb, 1966), and methods recommended by Kutzner (1981), also
tests of my own design. Kutzner’s methods of characterization were developed by
various Streptomyces researchers and also included some ISP criteria. The Kutzner
(1981) tests originally came from the following sources: 1) Resistance to lysozyme
(Kutzner et al, 1978); 2) Melanin formation on PYI (Shirling and Gottlieb, 1966); 3)
Utilization of ISP carbohydrates D-glucose, fructose, L-arabinose, D-xylose, rhamnose,
sucrose, raffmose, D-mannitol, and i-inositol (Shirling and Gottlieb, 1966); 4)
Hydrolysis of esculin (Gordon and Horan, 1968); 5) Utilization of organic acids
gluconate, citrate, malate, lactate, malonate and, oxalate (Nitsch and Kutzner, 1969 a);

6) Formation of organic acids (Cochrane, 1947 and Ziegler and Kutzner, 1973); 7)

48
Hydrolysis of urea (Nitsch and Kutzner, 1969 b); 8) Hydrolysis of hippuric acid (Ziegler

and Kutzner, 1973 b); 9) Iceiﬂrovitellin—reaction on egg yolk agar (Nitsch and Kutzner,
1969 c); 10) Resistance toward sodium chloride (Tresner et al, 1969). Spore chain
morphology, spore color, and growth at different temperatures (25 C and 37 C) were
also determined as recommended by the ISP (Shirling and Gottlieb, 1966).

Tolerance of low pH was determined by growing isolates in acidiﬁed YEME
broth, dispensed in 5 ml aliquots to test tubes. The cultures were incubated at room
temperature on a utilization rotary shaker. Pectin and polygalacturonic acid was
determined on a modiﬁed Czapek-Dox medium: 5 g of pectin or polygalacturonate, 2 g
NaNO,, 1 g KZHPO4, 0.5 g MgSO,.7H20, 0.5 g KCL, 0.01 g FeSO4, 0.15 g CaClz, and
15 g agar adjusted to pH 8.5 with NaOH and diluted to 1 liter (Mann, 1962; and Tuite,
1969). The cultures were incubated at 25 C and growth was assessed weekly. The
growth ratings were based on two and three week old cultures.

The ability of the streptomyces isolates to grow in the presence of antibiotics was
also determined. Filter sterilized antibiotic solutions were incorporated into 300 ml of
YEME agar in the following formulations (Maniatis, 1982): ampicillin 45 pg/ml,
chloramphenicol 10 pg/ml, kanamycin 50 pg/ml, streptomycin 25 pg/ml, and tetracycline
14 pg/ml. Penicillin was also used at the concentration of 28 pg/ml. The medium was
dispensed in 25 ml aliquots to sterile plastic petri plates. Five replicates were used for
each treatment for each bacterial isolate tested. The plates were inoculated with mature
spores and incubated at 25 C for 3 weeks. Growth was assessed weekly and growth

ratings were based on two and three old week cultures.

49

SDS-PAGE of water soluble extracts from whole cells. The purpose of this experiment
was to detect whether discemable differences exist between the electrophoretic patterns
of water soluble protein from the various bacterial isolates.

The isolates were grown in glucose-asparagine (GA) medium: 1 g asparagine, 4
g KZHPO4.3HZO, 0.7 g KHZPO4, 2 g MgSO4.7HZO, 10 g glucose, 5 g glycine, 0.1 g
yeast extract, 1000 ml dHZO adjusted to pH 7 (Gottlieb and Hepden, 1966). This
medium was modiﬁed with yeast extract to provide additional growth factors and
nutrients, and glycine to produce mycelium predisposed to easy extraction. The medium
was dispensed in 25 ml aliquots in 250 ml erlenmeyer ﬂask and autoclaved for 20
minutes. The ﬂasks of the medium were inoculated with mature spores and incubated
at room temperature on a rotary shaker for 10 days. The mycelium was recovered and
rinsed with sterile distilled water. The mycelium was frozen with liquid nitrogen, and
ground with sterile sand in a mortar until a powder was produced. The powdered
mycelium was rehydrated with sterile water. The suspension was centrifuged and the
supernatant containing water soluble proteins was retained and concentrated by
lyOphilization. Lyophilized extracts were dissolved in 2 ml of sterile distilled water and
the protein content of each sample was determined by the Bradford method (Bradford,
1976).

Electrophoresis was carried out on slab gels (Studier, 1973). The formulations
of the stock solutions used for casting SDS-polyacrylamide gels were adapted Keleti and
Lederer (1974). The formulations for the gel buffer and electrode buffers were adapted
from Suelter (1985). Formulations for the gel stock solutions were: acrylamide solution

22.2 g acrylamide and 0.6 g methylene-bisacrylamide diluted to 100 ml with water; APS

soluti
contz

dilut

con
but
deg
ele

of

50

solution, 1.5 g ammoniumpersulfate diluted to 100 ml with water. The gel buffer
contained 18.3 g Tris, 6 ml concentrated HCl, 4 ml of 10% SDS in water, the solution
diluted with water to 100 ml.

A single 12.5 % acrlyamide Slab gel of 1.5 mm thickness was cast with solutions
combined as follows: 22.81 mi acrylamide solution, 2.56 nrl APS solution, 14.82 ml gel
buffer, and 30 ul of N,N ,N ’ ,N’-tetramethylethylenediamine (TEMED). The solution was
degassed for approximately 2 minutes and the gel was cast without a stacking gel. The
electrode buffer (pH 8.3 Tris-glycine-SDS) consisted of 3 g Tris, 14.4 g glycine, 10 ml
of 10% SDS in water, with the entire solution diluted with water to 1 L.

The protein samples were denatured and diluted to the desire concentration with
solutions recommended by Suelter (1985). The denaturing solution consisted of: 3 ml
0.5 M Tris-Hcl buffer (pH 6.8), 5 ml of a 10% SDS in water, 10 ml glycerol, and 2 ml
2-mercaptoethanol. Protein samples were mixed with the denaturing solution in a test
tube, boiled for 2 minutes, and removed an immediately placed in a ice-bath. The
protein samples were diluted to a concentration of 15 pg/ sample with the diluting solution
which consisted of 1.5 ml of 0.5 M Tris-HCl buffer (pH 6.8), 2.5 ml 10% SDS in
water, 5 ml glycerol, 4 ml of 0.005% bromophenol blue, and 7 ml water.
Electrophoresis was carried out at 120 V for approximately 7 hours. Following
electrophoresis the gels were ﬁxed and silver stained by the Bonnen (1988) method,
which was modiﬁed from Morrissey (1981).

RESULTS
Explanation of the origin of selected isolates.. Twenty isolates were pure cultured

including the 3S isolate (Wallace and Hammerschmidt, unpublished data) (Tables 2.1

5 1
and 2.2). All the isolates came from potato, except for (P.R. 1) plant residue and 88-002.

P.R.l came from plant debris in a potato ﬁeld in Samaria, Michigan and 88-002 was
found growing as a contaminant of an Erwinia culture. P.R.l and 88-002 were PYI
negative and produced no diffusible pigments on YEME or OA. 88-002 to slightly
darken YEME medium which suggested melanin production, but this was not conﬁrmed
by the PYI test (Table 2.2). The Conestoga isolates (Con.1, Con.2, and Con.3) were
isolated from tubers with common scab lesions, from Samaria, MI (Table 2.1). Con.1
and Con.3 appeared to be similar on the basis of cultural morphology, however the
reverse color of the culture on YEME was slightly different for Con.2 (Table 2.2). The
Red Pontiac isolates (R.P.l and R.P.2) were isolated from a single Red Pontiac tuber
with common and deep pitted lesions (Table 2.1). When grown on YEME R.P.l and
R.P.2 produced melanin of slightly different shades of brown. 88-003 and 88-004 were
isolated from an Atlantic tuber with deep-pitted lesions from Newbury, Michigan (Table
2.1). The pure cultured 88-003 was PYI negative and 88-004 was PYI positive (Table
2.2). Isolations from other single tubers never produced streptomycetes isolates that
were PYI and positive and negative. The Onaway large lesion and Onaway little lesion
isolates were isolated from Onaway tubers grown in Montcalm County, Michigan (Table
2.1). The large lesion isolate came from a tuber with deep pitted lesions and the little
lesion isolate came from a tuber with common scab lesions (Table 2.1). Neither isolate
produced pigment on PYI, YEME, or CA (Table 2.2). Onaway little lesion was the only
common scab isolate that was PYI negative (Table 2.2). The deep pitting isolates (D.P.l
and D.P.(W)) were isolated from tubers of an unidentiﬁed cultivar (Table 2.1). D.P.l

and D.P.(W) were PYI negative (Table 2.2). D.P (D.P.l) was used in other phases of

Mn

'I

52

TABLE 2.2. Apperance of pure cultured isolates on media and pathogencity

 

 

 

Isolate' Pigments“ Spore Color Reverse Color
PYI YEME CA on YEME‘ on YEME‘

Con. l + - - Grey Golden Brown

Con. 2 + +/-’ - Grey Light Brown

Corr. 3 + - - Light Grey Golden Brown

P.R. l - - - Yellowish White Golden Brown

F.S. + + - Grey Light Brown

R.P. l + + + Grey Light Brown

R.P. 2 + + - Grey Chocolate Brown

Onaway 9/25 + + - Grey Golden Brown

Fi II + + - Grey Brown

A.T. + + - Grey Brown

D.P. - - - Grey Brown

D.P.(W) - - - Grey Golden Brown

Onaway Large

Lesion - - - Grey Golden Brown

Onaway Little

Lesion + N.R.' - Grey Golden Brown

F945 + + - Grey Brown

88-001 + + - Grey Light Brown

88-002 - +/- - Greenish White Brown

88-003 + + - Grey Brown

88-004 - - - Grey Golden Yellow

38‘ - +" + Grey Blue' Deep Blue

RI. belliesI

84-01-232 + + - Grey Brown

83-01-03 + + - Grey Yellow Brown

84-01-95 -/ + + - Olive Grey Black Brown

84-01-108 -/+ + - Olive Grey Black Brown

Chum” halite“

S. iividans(S.l.) - +/-' - Whitish Grey Golden Brown

 

' Abbreviated form of names (Table 1).

" Melanin (chromogenic) and diffusible (colored) pigment formation in media.
PYI (peptone yeast iron agar): Melanin.
YEME (yeast extract malt extract agar): Melanin and/or colored pigment.
OA (oatmeal agar): Melanin and/or colored pigment.

‘ Color of the spore mass.

‘ Color of culture as seen from the botton of the petri plate in full light.

' +/- The ability to produce pigment is variable.

' N.R. No result was obtained.

' Wallace and Hammerschmidt isolate from potato.

" 38 produced a deep blue pigment on YEME and CA. No melanin observed.

' The blue pigment diffused into the medium and spore mass.

’ Rose Loria’s isolates also written as RL 232, RL 03, RL 95, RL 108.

" Wendy Champness’ isolate S. iividans.

' Exhibited weak to moderate production of diffusible pigment on YEME. No melanin observed.

 

 

Con. 1
Con. 2
Con. 3
P.R. l
F.S.
R.P. l
R.P.lYi
Onaway
Fr ll
A.T.
D.P.
D.P.m
Onaw:
Lesion
Onaw
Lesior
T945
88-00
88-00

 

53

TABLE 2.2. (continued from the preceding page)

 

 

 

Isolate Spore Color Reverse Color Pathogenicity
on OAc on OA‘I on potato"
Con. 1 Light Grey Light Grey +
Con. 2 Grey White +
Con. 3 Grey White +
P.R. 1 Greenish White" Greenish Yellow -
F.S. Dark Grey Grey N.R.'
R.P. 1 Grey Brown +
R.P.(F) Grey Grey +
Onaway 9/25 Grey Light Grey N.R.
Fi II Grey Light Grey +
A.T. Grey Light Grey +
D.P. Grey Light Grey +
D.P.(W) Grey Light Grey +
Onaway Large
Lesion Grey Light Grey +
Onaway Little
Lesion Grey Light Grey 4-
F945 Grey Light Grey +
88-001 Grey Light Grey +
88-002 Greenish White Greenish White N.R.
88-003 Grey Light Grey +
88-004 Grey Grey N.R
38" Purple Grey' Blue Purple" -
RI. Isolates
84-01-232 Grey Light Grey +
83-01-03 Grey Light Grey +
84-01-95 Grey Light Grey -
84-01-108 Grey Light Grey -
Clnmpaels bone
S. lividans(S.l.) Grey White' -

 

° Pathogenecity as exhibited on potatoes when cultivated in potting soil inoculated
with the pure cultured isolate.
" The greenish cast was not due to the production of diffusible pigments.

I
l

andb.

my re

a tube

 

of S. .‘
Grom
pure c
isolat
PYI :

prod:

0n ‘
diff
pur
“n:

the

54

my research because this isolate was PYI negative, culturally different from S. scabies
and because it caused deep pitted scab (Fig 2.1 B). Fi II (Field II) was isolated from
a tuber with common scab lesion. Pure cultured Fi II had the characteristic appearance
of S. scabies (Table 2.2).

Growth of cultures on PYI, YEME, and 0A and pathogenicity testing. Most of the
pure cultured streptomycetes were PYI positive (Table 2.2). Several of the PYI negative
isolates were nonpathogenic on potato (Table 2.2). RL 95 and RL 108 produced a weak
PYI reaction after a minimum of 14 days of growth. On YEME RL 95 and RL 108
produced melanin (Table 2.2).

Except for the Conestoga isolates, most PYI positive isolates produced melanin
on YEME (Table 2.2). S. lividans (8.1.) and 3S were PYI negative, but produced
diffusible or colored pigments in YEME (Table 2.2). 3S produced a deep blue to
purplish pigment in YEME and OA. S]. to produced colored pigment in YEME but was
unable to produce pigments in OA. Shirling and Gottlieb (1966) recommend comparing
the characteristics of cultures grown on YEME to cultures grown on OA. Several
isolates failed to produce melanin or colored pigments when grown on OA. However,
R.P.l produced a brown melanin-like pigment on OA and 38 produced a blue pigment
on CA (Table 2.2).

Spore mass color varied from whitish grey or very light grey to dark grey for
most isolates grown on OA. However, 3S produced blue-grey spore, P.R.l produced
light yellow spores, and 88-002 produced greenish spores on oatmeal agar (Table 2.2).
Isolates generally produced the typical grey S. scabies type spore mass when grown on

YEME. The spore masses of RL 95 and RL 108 were grey with an olive cast (Table

was
char

golc

for
Ru:

Ph;

55
2.2). On CA the spore mass color of RL 95 and RL 108 were light grey to dark grey.

The spore mass color of P.R.l and 88-002 on CA were light green or a greenish-white
(Table 2.2). The spore mass color of 38 on OA was blue or purple grey spore (Table
2.2).

The reverse color of the cultures, as viewed from through bottom of a petri dish,
was also used as a cultural characteristic (Shirling and Gottlieb, 1966). This method of
characterizing cultures was very subjective. The reverse colors were usually yellowish,
golden brown, brown, or chocolate brown for cultures grown on YEME (Table 2.2).

In greenhouse screenings most of the isolates were pathogenic on potato except
for P.R.l, 3S, RL 95, RL 108, and S. lividans. In greenhouse screenings the cultivar
Russet Burbank was resistant to infection by all of the Streptomyces isolates.
Physiological and biochemical testing of isolates and further pathogenicity screening.

Physiological and biochemical characterization were performed on RL 232, RL
95, D.P., Fi II, and to a lesser extent 3S and 8.1. (Table 2.2). The pathogenic D.P. and
Pi II were compared to RL 232 and RL 95. All the isolates had typical spiral spore
chain morphology, except for the D.P. which had reﬂexuous spore chains. RL 232 and
Pi II were PYI positive, RL 95 gave a weak positive PYI reaction, and D.P., 3S, and
8.1. were PYI negative. Colored pigments were produced by 38 on YEME and OA.
S.l. weakly produced colored pigments on YEME.

RL 232 and Fi 11 produced common scab on Atlantic and D.P. produced deep
pitted scab on Atlantic (Table 2.3). On the basis of the above mentioned characteristics
RL 232 and Pi H appeared S. scabies. D.P. lacked several S. scabies characteristics, but

most notably it produced deep-pitted scab. The isolate RL 95 was culturally similar to

56

TABLE 2.3. Characteristics of S. scabies and other Streptomyces isolates

 

 

 

 

Isolates

RL 232 D.P. Fi II RL 95 38 SJ.
Characteristics'
Spore color” G G G G BG G
Chain morphology‘ S F S S S S
Pigment on PYI +d - + +l-e - -
Colored pigments on ':
YEME - - - - +' +\-h
OA - - - - +‘ -
Carbon usage
Glucose + + + + + N.R.'
Fructose + + + + + N.R.
Sucrose + + + + + N.R.
Mannitol + + + + + N.R.
Arabinose + + + + + N.R.
Xylose + + + + + N.R.
Raffinose + + + + + N.R.
Rhamnose + + + + + N.R.
myo-Inositol + + + + + N.R.
Degradation of:
Pectin + + + + + +
Polygalacturonate + + + + + +
Minimum growth pH" 5 0 6 0 4.5 4 5 6 0 6.0
Salt tolerance'
0% NaCl + + + + + +
4% NaCl + + + + + +
7% NaCl - - - - + +
10% NaCl - - - - + -
l3% NaCl - - - - - -
Lysozyme resistance”
0 pg/ml + + + + + +
10 pg/ml +\-" +\- +\- + + -
50 pglml - +\- +\- +\- + -
100 pg/ml - - - - - -
Hydrolysis of:
Esculin + + + + + +
Urea + + + + + +
Hippuric acid - - - - + +
LV reaction - - - - + +
Antibiotic resistance °:
Ampicillin 45 pg/ml + + + + N.R. N.R.
Chloramphenicol 10 pg/ml - - - + N.R. N.R.
Kanamycin 50 pg/ml - - - - N.R. N.R.
Penicillin" 28 pg/ml + + + - N.R. N.R.
Streptomycin 25 pg/ml - - + - N.R. N.R.
Tetracycline 14 pg/ml - - + + N.R. N.R.

 

' Test were taken from ISP methods (Shirling and Gottlieb, 1966) and Kutzner (1981) and our own assays.
" G, Grey and BG, Blue-Grey.

‘ 8, Spiral and F, Flexuous as determined by electronmicroscopy.

‘ Positive(+) and Negative(-).

57

TABLE 2.3. (continued from ﬁre preceding page)

 

 

 

 

Isolates

RL 232 D.P. Fi II RL 95 38 SJ.
Characteristics
Growth at ‘:
25 C + + + + + +
37 C - - - + \- + +
Utilization of organic acids
Citrate + + + + + +
Malate + + + + + +
Malonate + + + + + +
Lactate + + + + + +
Succinate + + + + N .R. N.R.
Gluconate + + + + + +
Oxalate + + + + + + \.
Production of organic acids - - + \- + \- + +
Pathogenlcity on potato ':
Russet Burbank - - - - - -
Atlantic CM' DP CM - - -

 

‘ +/- Variable reaction quite often th'm was a weak reaction.

' Diffusible colored pigment on Yeast Extract Malt Extract(YEME) and Oatmeal Agar(OA).

' 3S produced a blue colored pigment on both YEME and OA.

' Weak reaction on YEME.

' N.R. no result.

’ Czapek medium with 0.5% pectin or polygalacturonic acid as the sole carbon source in 1.5% agar.

" Acidiﬁed YEME solution.

' NaCl incorporated into YEME agar at 40 g/L, 70 g/L, 100 g/L, and 130 g/L.

" Liquid broth with lysozyme. Determines the ability of the organkm to grow in presence of lysozyme.

' Either no growth or very weak growth.

' Antibiotic mixed with YEME agar. Rate at which antibiotics are incorporated into the medium are from
Maniatis (1982).

" This concentration of antibiotic was of our own design.

' The effect of growing bacteria at two temperatures.

' Infection on greenhouse grown tubers. The lesion type was noted.

' CM, common scab; DP, deep pitted scab.

 

3pp€2

to pr«

snni

char

that

sun

acic

to l

“tn

58

RL 232 but was not pathogenic and therefore not S. scabies. Isolates 3S and 8.1.,
appeared similar in regard to basic cultural appearance although not identical. S.l. tended
to produce less pigment in culture.

The six isolates appeared to ﬁt into four groups. RL 232 and Pi II formed the
S. scabies group, RL 95 formed a nonpathogenic group which bore superﬁcial
similarities to S. scabies, D.P. formed a pathogenic group that lacks many cultural
characteristics ascribed to S. scabies, and 3S and 8.]. formed an nonpathogenic group
that formed colored pigments in culture.

The utilization of ISP carbon sources, pectic substrates, and organic acids was
similar for all isolates studied (Table 2.2). The hydrolysis of esculin, urea, hippuric
acid, and lecithovitellin (LV) reaction, tolerance toward sodium chloride, and resistance
to lysozyme was identical for RL 232, RL 95, D.P. and Pi II. The isolates 38 and 8.1.
were both positive for hydrolysis of hippuric acid and LV reaction, both isolates
exhibited high tolerance for sodium chloride. The 3S isolate was more resistant to
lysozyme than 8.1., The D.P., 3S, and SI. would only grow at pH 6 and above, and
RL 232; RL 95 and Fi II were able to grow in YEME broth pH 4.5 which was
uncharacteristic of S. scabies (Lambert and Loria, 1989 a and b). RL 232 and D.P. had
Similar resistance to antibiotics, and RL 95 and Pi II had additional antibiotic resistance
lacked by RL 232 and D.P. (Table 2.2).

SDS-PAGE of soluble proteins extracted from Streptomyces isolates.
The electrophoretic patterns of water soluble proteins, extracted from mycelium, were
compared between isolates cultured under identical conditions (Fig. 2.2 and 2.3). From

experiment to experiment this method of characterization lacked consistent results. The

 

Fig, ;
Cumin
2A: la
bOVim
B‘lactt

59

‘10‘

q
“t 5' Txm.i‘§i’."‘.“
Immense“ 'u J.
.- :1.’ .‘

 

Fig. 2.2. SDS-PAGE of soluble proteins extracted from 10 day old Streptomyces
cultures. Lane 1, RL 232; lane 2, D.P.; lane 3, Bl; lane 4, 3C; lane 5, RL 95; lane 6,
2A; lane 7, 3S; lane 8, Pi H; lane 9, molecular weight markers in kiloDaltons (kD):
bovine albumin (66 kD), egg albumin (45 kD), pepsin (34.7 kD), trypsinogen (24 kD),
B-lactoglobin (18.4 kD), lysozyme (14.3 kD).

lli

 

Fig. 2.3. SDS-PAGE of soluble proteins extracted from 10 day old Streptomyces
cultures. Lane 1, RL 232; lane 2, D.P.; lane 3, B1; lane 4, 3C; lane 5, RL 95; lane 6,
2A; lane 7, S. lividans; lane 8, Pi II; lane 9, molecular weight markers in kiloDaltons
(kD): bovine albumin (66 kD), egg albumin (45 kD), pepsin (34.7 kD), trypsinogen
(24 kD), B-lactoglobin (18.4 kD), lysozyme (14.3 kD).

variatit
form (

scabie

inbei

frorr
iron

pati

cul'
stu.
0n
ﬁe
SCE
Ph
Prt
Ch.

big

61

variation in the bands were probably due to the highly variable growth conditions. This
form of characterization would best be used for streptomycetes that grow faster than S.
scabies.

Heavy staining bands of protein appeared with the approximate MW of 45 kD and
in between 18.4 kD and 14.3 kD for the isolates RL 232, D.P., Fi H, RL 95, 3S, and
SJ. (Fig. 2.2 and 2.3). The banding patterns for RL 232, DP, and Pi H were similar
(Fig. 2.2 and 2.3). However, D.P. had a triplet of protein bands between 18.4 kD and
14.3 kD, where as RL 232 and Pi H had a doublet of protein bands (Fig. 2.2). Both 38
(Fig. 2.2) and 8.1. (Fig. 2.3) had protein patterns that were similar. The protein patterns
from 3S and SJ. appeared different from RL 232, D.P., and Pi H. The protein pattern
from RL 95 was similar to 38 and 8.1., but also resembled the protein patterns from the
pathogens (Fig. 2.2 and 2.3).

DISCUSSION

The purpose of the work in this chapter was to catalog the morphological,
cultural, and physiological characteristics of the Streptomyces isolates used in these
studies. Nearly all of the isolates that came from potato scab lesions were pathogenic
on potato. Deep-pitted and common scab lesions were most frequently observed on the
ﬁeld grown tuber samples from which isolations were made (Fig 2.1). The common
scab isolates RL 232 and Pi H produced melanin and spiral spore chains, had similar
physiological and biochemical characteristics. The deep-pitting isolate D.P. , did not
produce melanin, had reﬂexuous spore chains, and thus on the basis of commonly used
characteristics could not be a strain of S. scabies. However, the physiological and

biochemical characteristics of D.P. suggested similarity to RL 232 and Pi H (S. scabies).

Thep
nor e
bioeii
to inie
the p
Strep
elect
Hos
Phi

the

he

62

The physiological and biochemical methods used to categorize the S. scabies isolates was
not entirely conclusive. On the basis of culture morphology, physiology, and
biochemical tests RL 95 could have been placed in the S. scabies group, but was unable
to infect potato. Since it was not my purpose to identify the isolates to the species level,
the physiological tests served to determine similarity of the potato scab-causing
Streptomyces used in these studies were to one another. The value of using
electrophoresis to differentiate between streptomyces species was not speciﬁc.
However, electrophoresis clearly showed differences between isolates that had major
physiological and biochemical differences from the potato scab-causing bacteria, namely
the nonpathogenic isolates 38 and SJ.

Finding easily detectable characters that are correlatable with pathogenicity would
help to overcome our reliance upon pathogenicity testing to conﬁrm the identity of S.
scabies and other potato-scab causing streptomycetes. Doering-Saad et a1 (1992)
suggested developing a DNA probe for thaxtomin genes, the production of thaxtomin has
been positively correlated with pathogenicity. Traditionally, melanin production was
thought to be correlated with pathogenicity for potato scab-causing streptomycetes
(Hollis, 1952). However, my research and Faucher et al (1992) demonstrated that
melanin-negative pathogenic Streptomyces occur. Molecular techniques such as DNA
homology may overcome the limitations of the physiological tests used in taxonomic
studies of S. scabies and other potato scab-causing streptomycetes.

A ﬁnding of major importance in this study was the deep-pitting isolate D.P. The
characteristics which distinguished it from S. scabies, ultimately made this isolate idea

to study in comparison to the S. scabies isolate RL 232. D.P. was similar to the deep

 

pitiin
by gt
pigm’
soure
sensiti
D.P.

Archt

strept.
isolati
World

pitting

63

pitting isolate found in Canada by Faucher et al (1992). This isolate was characterized
by gold to light brown colonies, white spores borne in ﬂexuous chains, lack of melanoid
pigment production, resistance to streptomycin, and utilization of raffmose as sole carbon
source (Faucher et al, 1992). D.P. had to have a grey colored spore mass, streptomycin
sensitivity, and utilized all ISP carbon sources. The Faucher et a1 (1992) isolate and the
D.P. (Spooner and Hammerschmidt) isolates differ from the deep pitting isolates of
Archuleta and Easton (1981) by being unable to produce melanin agar media.
Considering the powerful molecular tools currently available for the Study of
streptomyces taxonomy it would be worth while to determine how related the Michigan
isolates, particularly D.P. , are to S. scabies and other deep-pitting isolates around the
world. This could be useful in determining the distribution of S. scabies versus deep-

pitting isolates in the United States.

 

 

 

Arch
POIai

Bang
north

Beck
differ
cell h

Bonn
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State

Bradf
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72:24é

COChI:
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Conn.

64
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Hughes, C., McCrum, RC, and Manzer, RE. 1971. Protein and esterase
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in th
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Jeljas

Label
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Appl

[amt
Syst.

laml
Baete

lapv
Aetii
eds.

Lori
Guic
Pres

Lori
Cans

66

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Kutzner, H.J., Bottiger, V., and Heitzer, RD. 1978. The use of physiological criteria
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-i- .4

CHAPTERIII

THE ROLE OF EXTRACELLULAR PECTINASES PRODUCED BY

STREPTOMYCES SPECIES PATHOGENIC ON POTATO IN THE DISEASE

PROCESS: STUDIES ON EXTRACELLULAR PECTINASES PRODUCED in

WW0 BY PATHOGENIC AND NONPATHOGENIC STREPTOMYCES SPECIES

69

70

INTRODUCTION

Streptomycetes produce a wide array of extracellular enzymes which degrade
several polymers found in plant and animal debris (Kom-Wendisch and Kutzner, 1992).
Many Streptomyces species enzymatically degrade persistent plant polymers such as
lignin, cellulose, and xylan (Antai and Crawford, 1981; Crawford et a1, 1982 and 1983;
McCarthy and Broda, 1984; Brogmeyer and Crawford, 1985; Pometto and Crawford,
1986; and MacKenzie et al, 1987). Chitin (N-acetyl-D-glucosamine residues linked by
B 1—>4 glycosidic linkages), a polymer of fungal and animal origin, can be degraded by
some Streptomyces species (Lingappa and Lockwood, 1961 and 1962; Hsu and
Lockwood, 1975; Trudel and Asselin, 1989). Most Streptomyces are relatively slow-
growing soil inhabitants and have evolved the life strategy of degrading complex
biopolymers which are inaccessible to many rapidly-growing soil bacteria and fungi.
Lutman (1945) thought that S. scabies survived on plant debris, in fallow potato ﬁelds.
This may explain why the potato scab-causing streptomycetes are able to degrade
complex biopolymers.

The mode of pathogenesis used by S. scabies and other Streptomyces species
pathogenic on Solanum tuberosum has been a subject of much speculation. How the
infection occurs has not been established. The presence of hyphal ﬁlaments within
infected potato tissues suggests that the bacterium possesses means to actively degrade
tissues. Light microscopy has revealed hyphal ﬁlaments of S. scabies growing in the
middle lamellae of potato tissue (Richards, 1943; and Shoemaker and Riddell, 1954).

Electron microscopy has revealed hyphae growing between cells and penetrating cell

wall:

and

71

walls directly (Stein et al, 1994). The pattern of infection demonstrated by S. scabies
and other plant pathogenic streptomycetes that suggests enzymatic degradation of the
middle lamella and cell walls occurs during the infection process (Jones, 1931; Lutman,
1941; and Clark and Matthews, 1987; and Stein et al, 1994). Lutman (1923, 1941, and
1945) has speculated that S. scabies utilizes pectin as a primary carbon source both in
the pathogenic and saprophytic phases of its life cycle.

The major plant cell wall components are cellulose, hemicellulose, pectic
polysaccharide, structural proteins, and lignin (Talmadge et al, 1973). Plant cells are
held together by polymeric carbohydrates, of which pectin is a major component
(Rombouts and Pilnik, 1980). Pectin occurs in most plant species and functions to
maintain the structural integrity of plant tissues. Pectin is a complex mixture of acidic
and neutral polymers which are characterized by chains of (ll-’4 linked galacturonosyl
residues in which 2-linked rhamnosyl residues are interspersed (Talmadge et al, 1973).
The carboxyl groups of some galacturonosyl residues are methyl esteriﬁed and the pectic
fraction usually possess a neutral polysaccharide which may or may not be covalently
attached to the acidic rhamnogalacturonan main chain (Talmadge et al, 1973).

The pectolytic enzymes can be divided into two general classes: the desterifying
enzymes, also called pectinesterases, and chain—splitting enzymes, the depolymerases
(Rombouts and Pilnik, 1980). The depolymerases cause the greatest damage to plant
tissue (Bateman and Millar, 1966; Collmer and Keen, 1986). There are two categories
Of depolymerases, the hydrolases and lyases, which respectively degrade pectin by
hydrolysis and B-elimination (Rombouts and Pilnik, 1980). These two classes of

enzymes are particularly destructive and usually cleave the pectic substrate in a random

lash

0ft

72

fashion. In plant tissues, this results in rapid loss of tissue integrity and eventual death
of plant tissue (Garibaldi and Bateman, 1971; Quantick et al, 1983; Bashan et al, 1985;
Keon, 1985; and Movahedi and Heale, 1990).

Exopolygalacturonase and endopolygalacturonase are two examples of hydrolases.
The endopolygalacturonases have been frequently cited for their ability to macerate plant
tissue (Collmer and Keen, 1986; Rombouts and Pilnik, 1980; and Talmadge et al, 1973).
Several plant pathogenic fungi and bacteria have been shown to produce
polygalacturonases in culture (Collmer and Keen, 1986). Similarly, pectolytic activity
has been noted for saprophytic bacteria and fungi (Rombouts and Pilnik, 1980). The
puriﬁed polygalacturonases (PG) exhibit high afﬁnity for polygalacturonic acid and
exhibit optimal activity between pH 4 and pH 7 (Rombouts and Pilnik, 1980). The
endoPGs do not require calcium for optimal activity (Rombouts and Pilnik, 1980).
Endopolygalacturonases cleave endopolygalacturonic acid internally or randomly to create
large fragments of galacturonides, whereas the exopolygalacturonases cleave the pectic
polymer terminally releasing only the D-galacturonide residue (Rombouts and Pilnik,
1980).

Lyases or transeliminases are the most extensively studied pectic enzymes
implicated in plant disease (Keon, 1985; and Collmer and Keen, 1986). Three classes
of lyases have been reported: endopectin lyases (which Show preference for methylated
pectin substrate), exopectate lyases and endopectate lyase (both of which prefer
polygalacturonic acid as a substrate) (Rombouts and Pilnik, 1980). The lyases attack the
pectic polymer by introducing a double bond between carbons 4 and 5 of the galacturonic

acid residue, in transelirninative fashion, which results in the formation of a free reducing

 

giOL
diva
poly
spec
ruir
a at
an
5P

in

73

group. Lyases activity is stimulated by millimolar concentrations of calcium or other
divalent ions (Rombouts and Pilnik, 1980). The endolytic lyases rapidly degrade pectic
polymers in plant tissues which results in tissue maceration. Several Streptomyces
species produce extracellular pectolytic enzymes in culture. S. fradiae and S.
nitrosporeus produce pectate lyase in shake culture (Sato and Kaji 197 3, 1975, and 1977
a and b). The culture ﬁltrate from S. fradiae was observed to macerate plant tissue (Sato
and Kaji 197 3). Neither S. fradiae or S. nitrosporeus are pathogenic toward any plant
species. Knosel (1970) observed that S. scabies produced endopectate lyase and cellulase
in shake culture when supplied a medium consisting of citrus pectin and cellulose.
Knosel (1970) and Sato and Kaji (1973, 1975, 1977 a and b) demonstrated that puriﬁed
transeliminases produced by streptomyces in shake culture exhibit optimal activity at pH
9 or higher. Knosel (1970) demonstrated that S. scabies produced pectic enzymes that
macerate plant tissue. However, no correlation was found between the ability to
produce pectolytic enzymes in culture and the virulence of the isolates studied.

My objective was to determine whether there was a correlation between
pathogenicity of potato scab-causing streptomycetes and their ability to degrade pectic
substrates. It was necessary to determine whether pathogenic and nonpathogenic
Streptomycetes grew on puriﬁed pectic substrates, as a sole carbon source and produced
extracellular pectinase in culture. Secondly, I wanted to establish which classes of
pectolytic enzymes were induced by pectin or polygalacturonic acid in shake culture and
establish pH optimums for the enzyme preparation from crude culture ﬁltrates on pectin
and polygalacturonic acid substrates. This would indicate the types of pectolytic

enzymes that were being induced. Thirdly, I wanted to determine whether pectolytic

enzy
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74

enzymes from pathogenic and nonpathogenic streptomyces were capable of causing
damage to potato tuber tissue. Finally, an attempt was made to determine whether the
loss of pectin degrading ability would affect the pathogenecity of isolate D.P. This was
examined with ultraviolet light mutants of D.P., the deep-pitting scab isolate.
MATERIALS AND METHODS
Bacterial isolates. The isolates RL 232, RL 95, D.P., 3S, and Pi II were used in the
experiments.
Maintenance of the bacteria and cultivation of inoculum. The bacterial stocks were
maintained as spores at -20 C in 20% glycerol/water solution (Hopwood et al, 1985).
Spores were used to inoculate plates of yeast extract malt extract agar (YEME) (Shirling
and Gottlieb, 1966). The cultures were incubated at 25 C for 14 days or until they
produced mature spores which served as the inoculum for other media.
Plant materials. The potato cultivars Russet Burbank and Atlantic were used for
greenhouse pathogenicity screening. Tissue maceration assays were performed on tuber
Russet Burbank tubers discs.
Induction of pectolytic enzymes in shake cultures. Bacteria were cultured in a
modiﬁed Czapek-Dox medium (Mann, 1962; and Tuite, 1969) containing: 5 g pectin or
polygalacturonic acid, 2 g NaNO,, 1 g KZI-IPO,, 0.5 g MgSO4.7H20, 0.5 g KCl, 0.01 g
FeSO,, 0.15 g CaCl2 /L adjusted with 0.5 M NaOH to pH 8.5. Since pectin has low
solubility in water, it was mixed into 500 ml boiling distilled water with a Waring
blender. The pectin solution was diluted with 400 ml of salts solution. Polygalacturonic
acid (PGA) is very soluble in water but forms an insoluble precipitate when CaCl2 is

added to solution. Thus, CaCl2 was slowly added as an aqueous solution to the PGA

med

 

31in
(hot

5pc

W61

Pr.

di.

CC

ﬁl

Wt

75

medium. The pectin or PGA media was dispensed into 1 L erlenmeyer ﬂasks in 250 ml
aliquots and autoclaved for 30 minutes. The pH of the pectin medium occasionally
dropped as low as pH 7 after autoclaving, without adverse effects on bacterial growth.
Spores scraped from the surface of mature cultures were used to inoculated the liquid
pectin medium. The cultures were incubated on a rotary shaker at 23 C for 3 to 5
weeks.

Preparation of the culture ﬁltrates. After the culture period the medium was ﬁltered
through two layer cheesecloth, centrifuged at 4000 rpm for 45 minutes at 4 C, and
dialyzed against distilled water for 48 hours at 4 C. The culture ﬁltrates were
concentrated by freeze drying to powder form. The powders were maintained at -20 C
until assayed.

Preparation of concentrated culture filtrates for enzyme assays. In general, 200 ml
of culture ﬁltrate produced less than 0.5 g of dry powdered material. The powders were
suspended in 1 to 2 ml of distilled water. The undegraded substrate in the powders
gelled and the insoluble material was removed by centrifugation. The protein content
of the enzyme preparations was determined by the method of Bradford (1976). Enzyme
preparations containing little protein were reconcentrated by lyophilization, rehydrated
in less water, and measured for protein concentration. Culture ﬁltrates stored at -20 C
for long-terrn usage or maintained at 4 C for 14 days.

Enzyme assays. Characterization of the pectolytic activities in the concentrated culture
ﬁltrates was conducted on pectin and PGA substrates. The pH of the pectic substrates

were maintained with the following buffering systems: (pH 4 - 5.5) 0.05 M acetate

but

 

 

76
buffer; (pH 6 - 7) 0.05 M citrate phosphate buffer; (pH 7.5 - 9) 0.05 M Tris-HCl buffer;

and (pH 9.5 - 10.5) 0.05 M glycine-NaOH buffer.

Hydrolase and lyase activity were detected by Nelson’s (1944) assay for
reducing groups, viscometry (Cooper and Wood, 1975), and the cup plate assay (Dingle
et al, 1953; Mann, 1962; and Movahedi and Heale, 1990). Lyase activity was detected
by the thiobarbituric acid assay (TBA) (W eissbach and Hurwitz, 1959; and Bateman,
1966) and measuring the change in absorbance of a reaction mixture after 2 nrinutes at
A23, (Edstrom and Phaff, 1964; and Nasuno and Starr, 1966). Lyase activity was
detected with pectin and PGA agarose gels overlaid onto polyacrylamide gels containing
electrophoresed pectolytic enzymes (MacKenzie and Williams, 1984; Ried and Collmer,
1985 and 1986).

For the Nelson’s (1944) assay, the volume of the reaction mixture was initially
1.25 ml but was later scaled down by half the volume to conserve the enzyme, therefore
the total volume was 0.625 ml. A 1.25 ml reaction mixture contained 0.75 ml of
buffered 1.2% pectin or PGA subsuate, 0.25 ml enzyme preparation or 0.25 ml buffer
for controls, and 0.25 ml of 0.05 M CaCl2 to stimulate lyase activity. The reaction
proceeded for 30 minutes at 27 C after which time the reaction was stopped by the
addition of 1 ml Nelson’s reagent (copper solution) and boiled for 20 minutes. The
reaction was stopped by immersion of test tubes in a cold water bath, and 1 ml of
aresenomolybdate solution (Nelson, 1944) was added with shaking, and the reaction was
diluted to 7 ml with distilled water. The absorbance of the solution was read at A540.
Estimates of the amount of reducing groups per reaction were based on a standard curve

developed from D-galacturonic acid. The results were expressed as micrograms of

 

 

moi.

COllt

mil

77

galacturonic acid units released per minute per microgram of protein in each reaction
mixture.

The thiobarbituric acid assay reaction mixture consisted of 0.5 ml of 1.2%
buffered pectic substrate (0.05 M Tris-HCl or 0.05 M glycine-NaOH), 0.17 ml of 0.05
M CaClz, 0.17 ml distilled water, 0.17 ml of the enzyme preparation or buffer for
controls. The reaction proceeded for 30 minutes until stopped with the addition of 3.33
ml of 0.01 M TBA reagent and 0.83 ml 1 N HCL, then was boiled for 30 minutes and
cooled to room temperature. The absorbance of the solution was read at A548. The
molar extinction coefﬁcient 1.55 X' 105 M" was used to determine the molar
concentration of product formed per reaction. The results were expressed in terms of
millirnicromoles (mp moles) of product formed per minute per microgram of protein.

Lyase activity was detected by the measuring the change in absorbance (A235) of
pectic substrate over time. The reaction contained 1 ml of 0.5 % buffered substrate (0.05
M Tris-HCI or 0.05 M glycine-NaOH), and a volume of enzyme preparation which
provided 2.5 pg of protein. To stimulate lyase activity the substrate was formulated
with CaCl2 to a concentration of 0.002 M. The change in absorbance of the substrate
was measured while the reaction proceeded for 2 minutes at 27 C.

IRG 200 viscometers (International Research Glass Ware, Kenilworth, NJ) were
used for viscometry assays which were performed by the Hancock et al (1964) method.
The reaction mixture consisted of 2 ml enzyme preparation and 4 ml of 1.2% buffered
pectic substrate, and 1 ml of 0.05 M CaCl2 to stimulate lyase activity. The reduction in
viscosity of the substrate was measured at 28 C. Rapid loss of viscosity was usually

indicative of endolyase or endopolygalacturonase activity.

78

Cup plate assays were utilized for detection of hydrolase and lyase activity. The
cup plate medium consisted of 50 ml of the desired buffer solution (0.05 M buffer
solution mentioned above), 0.25 g of pectic substrate, 1.5 g agar, 5 ml of 0.02 M CaCl2
to stimulate lyase activity. The medium was adjusted to the desired pH and diluted to
100 ml. The solution was steamed until the agar liquiﬁed and approximately 20 ml of
the medium was poured into sterile plastic peu’i dishes. Wells were cut out of the
solidiﬁed medium with a cork borer. The enzyme preparations were adjusted to similar
protein concentrations and pipetted into the wells. The cup plates were incubated for 20
hours at 27 C. After incubation the cups were rinsed with distilled water. The cup
plates containing PGA were ﬂooded with l N HCl (Dingle, et al 1953; and Puhalla and
Howell, 1975). The cup plates containing pectin were stained with 0.03 % ruthenium red
(Sterling, 1970; Ried and Collmer, 1985; and Movahedi and Heale, 1990). Evidence of
enzyme activity was detected by zones of clearing around the wells.

Polyacrylamide gel electrophoresis of concentrated culture ﬁltrates. Polyacrylamide
gel electrophoresis was performed under native conditions at pH 9.3 (anionic gel system)
using the Keleti and Lederer (1974) method for preparation of tube gels, which was
modiﬁed for slab gel electrophoresis (Studier, 1973). A gel was typically 1.5 mm thick
and consisted of a 7.5% acrylamide resolving gel and a 2.5% acrylamide stacking gel.
Enzyme samples to be loaded for electrophoresis contained 20 pg to 60 pg of protein in
a 40 pl aliquot, 20 pl of glycerol, and 10 p1 of bromophenol blue tracking dye.
Electrophoresis was conducted at room temperature under constant voltage using 60 V
through the stacking gel for 1 hour and 120 V through the resolving gel for

approximately 6 to 7 hours. After electrophoresis, the gels were ﬁxed for silver staining

79

as described by Bonnen (1988), and silver staining was carried out according to Bonnen
(1988) which was a modiﬁcation of Morrissey (1981).

SDS-PAGE of pectic enzymes. The SDS-polyacrylamide gels were cast according to the
methods outlined in Chapter 2 of this dissertation. The formulations for stock solutions
and procedures for casting gels were adapted from Keleti and Lederer (1974) and Seulter
(1985). The SDS polyacrylamide gel contained of 12.5% acrylamide, was 1.5 mm thick
and cast without a stacking gel. Samples for electrophoresis were prepared according
to the method of Suelter (1985) as outlined in Chapter 2 of this dissertation. The ﬁnal
concentration of the sample loaded in each well was 12 pg of protein. The enzymes
(Sigma) polygalacturonase EC 3.2.1.15 from Aspergillus niger and pectin lyase EC
4.2.2.10 from Aspergillus japonicus were electrophoresed for comparison with the
electrophoretic patterns of Streptomyces culture ﬁltrates. The samples were
electrophoresed under constant voltage (120 V) for approximately 7 hours. After
electrophoresis the gels were ﬁxed and silver stained by the Bonnen (1988) method,
which was a modiﬁcation of the Morrissey (1981) method.

Agarose pectin/pectate overlay of polyacrylamide gels for detection of pectolytic
activity. The pectic enzymes electrophoresed in native polyacrylamide gels were
detected with pectic substrate gels (Ried and Collmer, 1985 and 1986; and Collmer et
al, 1988). The pectin agarose overlays were cast in a Studier electrophoresis apparatus
to a thickness of 1.5 mm. The glass plates were heated with boiling water prior to
casting a gel. Initially the overlays were formulated as two solutions: solution (1)
consisted of 100 ml of 0.2 M Tris-HCl, 20 ml of a 1% pectin or PGA solution, and 10

ml of 0.02 M CaCl2 solution; solution (2) consisted of 100 ml of 2% agarose. Both

79

as described by Bonnen (1988), and silver staining was carried out according to Bonnen
(1988) which was a modiﬁcation of Morrissey (1981).

SDS-PAGE of pectic enzymes. The SDS-polyacrylamide gels were cast according to the
methods outlined in Chapter 2 of this dissertation. The formulations for stock solutions
and procedures for casting gels were adapted from Keleti and Lederer (1974) and Seulter
(1985). The SDS polyacrylamide gel contained of 12.5 % acrylamide, was 1.5 mm thick
and cast without a stacking gel. Samples for electrophoresis were prepared according
to the method of Suelter (1985) as outlined in Chapter 2 of this dissertation. The ﬁnal
concentration of the sample loaded in each well was 12 pg of protein. The enzymes
(Sigma) polygalacturonase EC 3.2.1.15 from Aspergillus niger and pectin lyase EC
4.2.2.10 from Aspergillus japonicus were electrophoresed for comparison with the
electrophoretic patterns of Streptomyces culture ﬁltrates. The samples were
electrophoresed under constant voltage (120 V) for approximately 7 hours. After
electrophoresis the gels were ﬁxed and silver stained by the Bonnen ( 1988) method,
which was a modiﬁcation of the Morrissey (1981) method.

Agarose pectin/pectate overlay of polyacrylamide gels for detection of pectolytic
activity. The pectic enzymes electrophoresed in native polyacrylamide gels were
detected with pectic substrate gels (Ried and Collmer, 1985 and 1986; and Collmer et
al, 1988). The pectin agarose overlays were cast in a Studier electrophoresis apparatus
to a thickness of 1.5 mm. The glass plates were heated with boiling water prior to
casting a gel. Initially the overlays were formulated as two solutions: solution (1)
consisted of 100 ml of 0.2 M Tris-HCl, 20 ml of a 1% pectin or PGA solution, and 10

ml of 0.02 M CaCl, solution; solution (2) consisted of 100 ml of 2% agarose. Both

sol
res
was

prie

dupi

enzi

 

“'an
was
(her

inet

80

solutions heated by Steaming and 75 ml of each solution was mixed together. The
resultant solution was pipetted into the gel casting apparatus. After the gel solidiﬁed it
was wrapped in plastic ﬁlm and stored at 4 C. A gel was adjusted to room temperature
prior to use.

Enzyme samples for PAGE were loaded so that each half of a gel contained a
duplicate sample. After the polyacrylamide gel was electrophoresed it was cut in half.
Half of the gel was silver stained and the other half was overlaid to detect pectic
enzymes. A polyacrylamide gel to be assayed for pectic enzymes was rinsed in distilled
water, and soaked in three changes of buffer, to adjust the gel pH. The agarose overlay
was placed on a glass plate and the polyacrylamide gel was afﬁxed on top of the agarose
overlay. The two gels were wrapped in plastic ﬁlm to minimize loss of moisture and
incubated for 4 to 7 hours at 28 C. After incubation the overlay was trimmed to the size
of the polyacrylamide gel and the two gels were separated. The agarose overlay was
stained with 0.03% ruthenium red and destained by soaking in distilled water.
Enzymatic activity was indicated by clear bands in the pink or red stained pectin agarose
overlay.

Tissue maceration assay. Potato tuber discs were used to assess the tissue macerating
ability of the enzyme preparations. Tuber discs were prepared according to
Hammerschmidt (1984). A cork borer was used to remove a single cylinder of tissue
from the internal medullary region of a tuber, which was sliced into individual tuber
discs 2 cm in diameter and 0.5 cm in thickness. The discs were placed into a 1%
sodium chloride solution to prevent browning and rinsed twice with sterile distilled

water. The discs were transferred to sterile plastic petri dishes containing sterile distilled

81
water or sterilized 0.05 M Tris-HCl buffer pH 8.8. The tuber discs were treated with

a volume enzyme preparation which contained 5 pg of protein, and the controls were
treated with sterile distilled water. Tuber discs were incubated in 0.1 M Tris-HCl pH
8.8 or water to determine the effect of pH on the maceration. The discs were incubated
at 27 C in darkness and observed every 8 hours for 48 hours. The extent of the tissue
damage was visually assessed and expressed qualitatively in terms of tissue browning or
blackness and loss of tissue hardness.
Mutagenesis of D.P. with ultraviolet light. Mutagenesis by ultraviolet light has been
used to create mutants of fungi and Streptomyces (Mann, 1962; Puhalla, 1973; Puhalla
and Howell, 1975; Hopwood et al, 1985; and Baltz, 1986). Attempts were made to
produce pectinase deﬁcient streptomyces mutants by ultraviolet light mutagenesis to
determine whether pathogenicity and production of pectinases were correlated. The D.P.
isolate was chosen for this experiment because of its marked virulence and ability to
produce deep pitted lesions on potato tubers.

The method of mutagenesis was a modiﬁcation of Hopwood et al (1985). D.P.
spores were maintained at -20 C as a stock in 20% glycerol/ water solution (v/v) at a
concentration of 2.8 X 105 cfu/ml. One ml of the stock was diluted to 100 ml with
sterile distilled water and this solution was transferred in 20 ml aliquots to sterile petri
dishes. An ultraviolet lamp (General Electric germicidal lamp 254 nm) was used to
irradiate the spore suspension. To reduce contamination prior to carrying out the
mutagenesis, the work area was irradiated with UV. light for 40 minutes. The UV.
light source was placed approximately 22.5 cm from the surface of the spore suspension.

The spore suspension was exposed to the UV. light for approximately 40 seconds. The

 

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82

Spore suspension was gently agitated by a bench top rotary Shaker or a sterile magnetic
stir bar placed in the solution, while being exposed to the UV. light. One ml of the
irradiated spores were plated to a modiﬁed Czapek-Dox medium containing 0.5 % pectin
or polygalacturonic acid medium adjusted to pH 8.5 (Mann, 1962; and Tuite, 1969) and
to YEME agar (Shirling and Gottlieb, 1966). To verify that the UV. light was killing
the spores 1 ml of untreated spore suspension was also plated on pectin media and
YEME agar. The inoculated plates were wrapped in aluminum foil to avoid photo repair
of the DNA and incubated at 25 C for 14 days. Plates with untreated spores usually
produced 200 or more colonies. The UV. dose on average resulted in the recovery of
10 to 40 colonies per plate.

Sporulating colonies, resulting from U.V. irradiated spores, were transferred from
pectin or PGA medium to YEME agar. The original pectin or PGA plate from which
the spores were selected was then ﬂooded with 1 N HCl (Puhalla and Howell, 1975).
Colonies lacking distinct zones of clearing around them were selected, as this indicated
that the colony was unable to degrade the surrounding pectic substrate. Colonies were
not selected that had zones of clearing around them as this indicated the substrate was
enzymatically degraded. This screening method worked best when PGA was used as a
substrate, thus PGA deﬁcient mutants were selected for in my experiments. The
screening medium (PGA) was adjusted to a pH between pH 8 and 9.5, to favor selection
of PGA lyase-deﬁcient mutants. The isolates selected were physiological and

biochemically characterized and compared to the wild-type D.P. isolate.

83
RESULTS

Growth of Streptomyces on pectic substrates. The isolates studied were capable of
utilizing pectin or polygalacturonic acid (PGA), as a sole carbon source in agar culture
or shake culture. The size of streptomyces grown colonies on pectin agar medium were
usually smaller than colonies grown on YEME agar. The colonies grown on pectic
substrates would not always sporulate and bald (nonsporulating) colonies occurred
frequently. Most isolates grew well in shake culture and produced bands of sporulating
mycelium. Growth was not usually visible for at least one week, but heavy growth
normally occurred after 2 to 3 weeks. The 3S isolate (a nonpathogen) grew more rapidly
than the isolates RL 232, D.P., and RL 95. The pectolytic activity found in the 38
culture ﬁltrates were compared to RL 232, D.P. , and RL 95 enzyme preparations in
enzyme assays.

Detection of hydrolase activity in concentrated culture ﬁltrates. The reducing groups
assay detected a peak of activity for pectin and PGA induced enzyme preparations from
RL 232, D.P., and RL 95, when assayed on PGA substrate at pH 5 (Fig. 3.1 A and B).
Viscometry suggested that the enzyme activity was exolytic, as PGA and pectin induced
enzyme preparations caused minimal reduction in the viscosity of PGA substrate at pH
4.5, 4.8, 5, and 5.2. Zones of clearing were not detected in PGA substrate (pH 5) for
pectin or PGA induced enzyme preparations assayed by the cup plate method. This
ﬁnding suggested that at low pH on PGA substrate the enzyme preparations exhibited
exolytic activity. An example of an assayed PGA cup plate after ﬂooding with 1 N HCL

is shown in ﬁgure 3.7 A.

 

 

 

 

 

 

 

 

7
2' II
6 6
a 5.
g . I ntzsz
E. "' . "0P.
5 . A ates
\. 3‘
a 4
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Fig. 3.1. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on A, PGA; or B, pectin. The reducing groups
assay was used to detect hydrolase activity on PGA substrate, activity was measured on
the basis of micrograms (pg) galacturonic acid units (gal.) produced per minute (min.)
per microgram (pg) protein (pro.) at 540 nm.

85

 

7

G u
I III. 232
O D.P.
A Ill 95

ug galJmanml

 

 

 

 

Fig. 3.2. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on pectin. The reducing groups assay was used to
detect hydrolase activity on pectin substrate, activity was measured on the basis of
micrograms (pg) galacturonic acid units (gal.) produced per minute (min.) per milliliters
(ml) reaction mixture at 540 nm.

85

 

I Ill 232
O D.P.
A Ill 95

ug galJmanml

 

 

 

 

 

Fig. 3.2. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on pectin. The reducing groups assay was used to
detect hydrolase activity on pectin substrate, activity was measured on the basis of
micrograms (pg) galacturonic acid units (gal.) produced per minute (min.) per milliliters
(ml) reaction mixture at 540 nm.

86

Zones of clearing were detected in pectin substrate (pH 5) for pectin and PGA
induced enzyme preparations from RL 232, D.P. , and RL 95 by the cup plate assay. An
example of an assayed pectin cup plate after ﬂooding with ruthenium red solution is
shown in ﬁgure 3.7 B. Hydrolases (polygalacturonases) generally Show higher afﬁnity
for PGA substrate. However, the reducing groups assay detected peaks of activity for
pectin induced enzyme preparations, from RL 232, D.P. , and RL 95, when assayed on
pectin substrate at pH 4.8 and 7 (Fig. 3.2). Viscometry suggested that the enzyme
activity was exolytic as the pectin induced enzyme preparations caused minimal reduction
in the viscosity of pectin substrate at pH 4.8. These results suggested that RL 232, D.P.
and RL 95 produced exopolygalacturonase when grown on pectin or PGA substrate.
Detection of lyase activity in culture filtrates assayed on pectin substrate. Pectin
induced enzyme preparations, from RL 232, D.P. , and RL 95 caused peak increases in
the absorbance (235 nm) of pectin substrate at pH 6.5, 7.5, and 8.5 (Fig. 3.3 A). This
suggested that the isolates produced pectin lyase when grown on pectin. The reducing
groups assay detected peaks of activity for pectin induced enzyme preparation, from RL
232, D.P., and RL 95, when assayed on pectin assayed on pectin substrate at pH 8.5,
and 9.5 (Fig. 3.3 B). This also suggested lyase activity, but the activity occurred at a
pH much higher than reported for most pectin lyases to function (Rombouts and Pilnik,
1 980). The TBA assay detected low activity for the enzyme preparations which were
assayed on pectin substrate, however enzyme activity increased at pH 9.5 (Fig. 3.3 C).

Cup plate assays conﬁrmed that pectin induced enzymes, from RL 232, D.P., and RL
95, degraded pectin substrate at pH 7, 8.5, 9, and 9.5. This suggested that

S treptomyces produce pectin lyases when grown on pectin substrate. The cup plate assay

87

 

 

 

 

 

 

 

 

 

 

 

 

 

0.3
E II
I 1
In
In
N
g I It 232
a . '0'.
g A ll 95
a
II
a
I
8
U
9
o'
In
I
9 I It 232
\
:- O D.P.
E A ll 95
‘.
'3
D
I
I
a e 10 ' 11
pll
:- so
I C
a so -
. d
\
.- 40‘
E . I It 232
\. 3°: . .I'I
: - A ll 95
'i zo-
.1
2 1o~
I 1
E o . r
7 s e 10
pl

Fig. 3.3. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on pectin. Lyase activity was detected on pectin
substrate by A, measuring the change in absorbance of a reaction after 2 minutes (min.)
at 235 nm; B, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; C, thiobarbituric acid assay, where activity
was measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

88

 

 

 

 

 

 

 

 

 

 

 

 

0.3
E II
I
In
'0
N 0.2"
03 I II. 232
3 e 0.9.
e 0.1- A II. as
I
I
I 1
I
I
U
0.0 ﬂ
5 0 7 0 0
pl
0
.- I
i
.1
3' I ll 232
x
I. I D.P.
- "‘
E A ll 95
‘.
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=’ J
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7 8 0 10 11
. "'
00
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'3 50‘
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g 40-
E , I IL 232
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E . A Ill 95
I 20..
I ‘ HM
. 10
E 4
0 ﬁ 1
7 0 0 10

Fig. 3.4. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on PGA. Lyase activity was detected on pectin
substrate by A, measuring the change in absorbance of a reaction after 2 minutes (min.)
at 235 nm; B, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; C, thiobarbituric acid assay, where activity
was measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

88

 

 

 

 

 

 

 

 

 

 

 

 

0.3
E II
a
in
in
N 0.2"
mi I Ill 232
3 e as.
.5 0.11 A Ill 95
I
a
B r
I
a
u
0.0 ﬂ
5
8
.- B
L
I.
6'!
3’ I Ill 232
‘.
I: O I.l’.
- ""
E A II. 95
‘.
TI 2
U 2 ‘
I)
a
0 v . . .
7 0 0 10 11
. P“
O 00
h l c
a) 50‘
3 .
‘.
5 40‘
E . I III 232
\
6 30d . 0.,-
E i . A III 95
I 20"
I . W
a 10‘
E i
0 f ' I '

 

 

7 8 9 10

Fig- 3.4. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on PGA. Lyase activity was detected on pectin
Substrate by A, measuring the change in absorbance of a reaction after 2 minutes (min.)
at 235 mm B, the reducing groups assay, where activity was measured on the basis of
In.iC-trograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
mlcrogram (pg) protein (pro.) at 540 nm; C, thiobarbituric acid assay, where activity
Was measured on the basis of the concentration in millimicro moles (mp M) of colored
pr Och—let (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

89

could not detect activity for pectin induced enzyme preparation when assayed on pectin
at pH 10.5. An example of an assayed pectin cup plate is shown in ﬁgure 3.7 B.
PGA induced enzyme preparations, from D.P. caused an increase in absorbance
(235 nm) of pectin substrate at pH 7. However, no increase in absorbance of pectin
substrate (pH 7) was observed when PGA induced enzyme preparations from RL 232 and
EL 95 were assayed (Fig. 3.4 A) The reducing groups assay detected peaks of activity
for PGA induced enzyme preparations, from RL 232, D.P. , and RL 95, when assayed
on pectin substrate at pH 8.5 and 9.5 (Fig. 3.4 B). The TBA assay detected peak
activity for PGA induced enzyme preparations, from RL 232 and D.P. , when assayed
on pectin substrate at pH 9.5. However, no peak of activity was observed at pH 9.5
for the RL 95 enzyme preparation when assayed on pectin substrate (Fig. 3.4 C). These
findings suggested that the PGA induced enzyme preparations contained pectin lyase.
Cup plate assays conﬁrmed that PGA induced enzymes, from RL 232, D.P. , and RL 95,
degraded pectin substrate at pH 7, 8.5, 9, and 9.5 (Fig. 3.7 B). Viscometry suggested
that the PGA induced enzyme preparations contained endolyase as the enzyme
Preparations from RL 232, D.P., and RL 95 reduced the viscosity of pectin substrate at
PH 8.4.
Detection of lyase activity in culture ﬁltrates assayed on polygalacturonic acid
su bstrate. The reducing groups and TBA assays detected peak activity for the PGA
induced enzyme preparations, from RL 232, D.P. , and RL 95, when assayed on PGA
Suhstrate at pH 8.5 and 9.5 (Fig 3.5 A and B). These ﬁndings suggested that
Streptomyces produce lyases when supplied PGA as a substrate. The PGA lyases

genﬁl‘ally exhibit optimal activity at pH 8 or higher. PGA induced enzyme preparation,

 

 

 

 

 

 

 

 

 

4
a n
L
" i
5' I In. 232
.25 o D.P.
E A II. 95
\.
3
I!
D
I

11

- 00
i * '

50"
a 1
I
} oo-
'5' . I II. 232
\. 30.7 . 'I'.
E ‘ A III. 95
‘ 201
I ‘0:
. 1
E o - .

7 0 0 10

Fig. 3.5. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on PGA. Lyase activity was detected on PGA
substrate by A, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

90

 

 

 

 

 

 

 

 

 

4
I a
'i
i
E I II. 232
5- o D.P.
E A ll 95
‘.
T:
I!
U
I
11
.- 00
Ii 4 I
50‘
gr .
§ 40-
'E' r I In. 232
~: 30* . D.P.
E ‘ A ll. 95
-_ zo-
I 101
g i
0 v
7 0 0 10

Fig. 3.5. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on PGA. Lyase activity was detected on PGA
substrate by A, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

91

 

 

 

 

 

 

 

. , II

I

L

3 8-1

3’ I 111232
\

5- ‘_ O D.P.
E A It 95
“.

3. 2‘

U

I

0‘ LF '
s 9 1o 11
all

3 so
‘ I
on so-

I
\. 4
.5 40‘

E 4 I 01.232
3 ,0. o I”.
g . A 01.95
9 20-
t 1

=,104

E

o
7 1o

 

 

Fig. 3.6. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of RL 232, D.P., and RL 95 induced on pectin. Lyase activity was detected on PGA
substrate by A, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

92

 

Fig. 3.7. Cup plates illustrating zones of clearing corresponding to lyase activity. A,
PGA substrate (pH 8.5) was treated with 1 N HCl to precipitate the undegraded
substrate, enzyme activity was indicated by the clear halos which formed around the
Wells. B, pectin substrate (pH 8.5) stained with 0.03% ruthenium red solution,
undegraded substrate was stained pink or red and enzyme activity was indicated by the

halos (unstained pectin) which formed around the wells.

93
from RL 232, D.P. , and RL 95 reduced the viscosity of PGA substrate at pH 8.4, which

suggest that the enzyme preparations contained endolyases. Zones of clearing were
detected in PGA substrate at pH 7, 8, 8.5, for PGA induced enzyme preparations assayed
by the cup plate method (Fig. 3.7 A). The PGA induced enzyme preparation could not
degrade PGA substrate buffered to pH 10.5. The above ﬁndings suggested that
streptomyces produced lyases when grown on PGA substrate.

The reducing groups and TBA assays detected peak activity for the pectin induced
enzyme preparations, from RL 232, D.P., and RL 95, when assayed on PGA substrate
between pH 8 and 9.5 (Fig. 3.6 A and B). Pectin induced enzyme preparations degraded
PGA substrate at pH 7, 8, and 8.5, as detected by the cup plate method (Fig. 3.7 A).
Viscometry also suggested that this activity was endolyase as the pectin induced enzyme
preparations reduced the viscosity of PGA substrate at pH 8.4. All these ﬁndings
suggested that the streptomycetes produced PGA lyase when grown on pectin substrate.
Detection of pectolytic enzyme activity with pectic agarose overlays. Preliminary
studies revealed that the 3S isolate produced lyases in shake culture when grown on
pectin or PGA substrates. When the 3S enzyme preparations were analyzed using the
pectin agarose overlay method, highly visible bands of clearing appeared, which
corresponded to the electrophoresed enzymes in the overlaid polyacrylamide gel. The
bands of clearing produced by electrophoresed 3S enzymes preparations were usually
more intense than those produced by enzyme preparations from the other isolates studied.
Therefore the 3S isolate was used in all PAGE-overlay work.

Lanes 1 and 2 of the PAGE-overlay gel (Fig. 3.8) showed that PGA induced

enzymes from 38 and RL 232 produced dissimilar patterns of enzyme degradation at pH

94

ll)

 

Fig. 3.8. Detection of pectic enzymes produced by Streptomyces with 0.1% pectin
agarose overlay stained with 0.03% ruthenium red solution. Each lane shows the pattern
of substrate degradation characteristic to each enzyme preparation, which came from
culture ﬁltrates of the bacteria induced on pectin or PGA. Lane 1, 38 PGA; lane 2, RL
232 PGA; lane 3, 3S pectin; lane 4, RL 232 pectin; lane 5, D.P. pectin; lane 6, RL 95

pectm.

95

A123481234

raw-K

 

In

Fig. 3.9. A, Silver stained polyacrylamide gel containing electrophoresed pectolytic
enzymes from Streptomyces induced on pectin. Lane 1, 3S; lane 2, RL 232; lane 3,
D.P.; and lane 4, RL 95. B, The pectin agarose overlay stained with ruthenium red

shown in ﬁgure 3.8 (lanes 3-6). Lane 1, 3S; lane 2, RL 232; lane 3, D.P.; lane 4, RL
95.

96

8.4. The pectin induced enzymes from RL 232, and D.P. appeared to produce similar
types of pectic enzymes in culture, and caused similar type clearing on the overlay (Fig.
3.8, lanes 4-5). Pectin induced enzymes from RL 95 did not produce bands of clearing
in the overlay similar to RL 232, D.P., or 3S isolates (Fig 3.8, lane 6).

Pectin and PGA induced enzymes from 3S culture ﬁltrates were predominately
acidic in nature, whereas pectin induced enzymes from RL 232 and D.P. culture ﬁltrates
were basic in nature (Fig. 3.9 A and B, lane 1-4). In general, pectin substrate gels gave
the best evidence of clearing with sharp bands of activity at pH 8.4 for the isolates
tested. PGA substrate gels also gave sharp bands of clearing for the 3S enzyme
preparations induced on pectin or PGA substrates. The enzyme preparations for RL 232,
D.P. , and RL 95 induced on pectin or PGA, usually gave very faint and diffused zones
of clearing at pH 8.4 and 9.1 on PGA substrate gels (not shown).

Recovery and screening of pectinase deﬁcient mutants of D.P. The UV. light
dosage killed approximately 99.9% of the irradiated D.P. spores, between 10 to 40
colonies per plate were recovered on the PGA medium. Three isolates were selected to
study, which appeared to have loss the ability to degrade PGA agar medium. The three
isolates designated B1, 2A, and 3C were able to grow on pectin and PGA medium in
shake culture. Accurate screening for mutants lacking the ability to produce pectinases
was difﬁcult, the D.P. isolate would not pit PGA medium, as do many Erwinia species.
Also, the Puhalla and Howell (1975) method of screening for pectinase deﬁcient mutants
on agar gave unreliable results with streptomyces. This may indicate that streptomyces
fail to export large amounts of enzyme into the surrounding agar medium. Additionally

the colonies, resulting from irradiated spores, on PGA medium were slow-growing and

97

 

Fig 3

. ' . 10. Reverse culture color of th U -

31d _ . e .V. mutants of D.P.

e of the petrr drsh. A, 2A; B, 3C; C, D.P. (wild type) D ’ {sewed from the honor“

98

TABLE 3.1. Characteristics of D.P. wild type and U.V. generated isolates of D.P.

 

 

 

 

Isolates‘

D.P. BI 3C 2A
Characteristics”
Spore colorc G G G G
Chain morphology“ F F F F
Pigment on PYI - - - -
Colored pigments on ’:
YEME - - - -
OA - - - -
Carbon usage
Glucose + + + +
Fructose + + + +
Sucrose + + + +
Mannitol + + + +
Arabinose + + + +
Xylose + + - +
Raff'mose + + - +
Rhamnose + + + +
myo-Inositol + + - +
Degradation of ':
Pectin + + + +
Polygalacturonate + + + +
Salt tolerance'
0% NaCl + + + +
4% NaCl + + + +
7% NaCl - - - -
10% NaCl - - - -
13% NaCl - - - -
Lysozyme resistanceh
0 pg/ml + + + +
10 pg/ml +\-' - - -
50 pg/ml +\- - - -
100 pg/ml - - - -
Hydrolysis of:
Esculin + + + +
Urea + + + +
Hippuric acid - - - -
LV reaction - - +\- -
Antibiotic resistance’:
Ampicillin 45 pg/ml + + + +

Chloramphenicol 10 pg/ml - - - -
Kanamycin 50 pg/ml - - -
Penicillin" 28 pg/ml + + + +
Streptomycin 25 pg/ml - - -

Tetracycline 14 pg/ml - - +

 

' Isolates from D.P. spores irradiated with U.V. 254 nm light, recovered on PGA medium.

" Test were taken from ISP methods (Shirling and Gottlieb, 1966) and Kutzner (1981) and our own assays.
° G, Grey

" F, Flexuous

99

TABLE 3.1. (continued from the preceding page)

 

 

 

 

Isolates

D.P. B, 3C 2A
Characteristics
Growth at ':
25 C + + + +
37 C - + - +
Utilization of organic acids
Citrate + + + +
Malate + + - +
Malonate + + - +
Lactate + + - +
Succinate + + + +
Gluconate + N.R.“I N.R. N.R.
Oxalate + + - +
Production of organic acids - - - + \-
Pathogenicity on potato II:
Russet Burbank - - - -
Atlantic DP‘ DP DP DP

 

‘ Diffusible colored pigment on Yeast Extract Malt Extract (YEME).

' Czapek medium with 0.5% pectin or polygalacturonic acid as the sole carbon source in 1.5% agar.

' NaCl incorporated into YEME agar at 40 g/L, 70 glL, 100 glL, and 130 g/L.

" Liquid broth with lysozyme. Determines the ability of the organism to grow in presence of lysozyme.
' +\-, either no growth or very weak growth.

’ Antibiotic mixed with YEME agar. Rate at which antibiotics are incorporated into the medium are from
Maniatis (1982).

" This concentration of antibiotic was of our own design.

' The effect of growing bacteria at two temperatures.

" N.R., no result

" Infection on greenhouse grown tubers. The lesion type was noted.

' DP, deep pitted lesions, the isolates D.P. and 3C were more virulent than the isolates B. and 2A.

 

Fig. 3.11. Deep—pitted scab caused by the U.V. mutants of the D.P. isolate. A, B,; and
B, 3C.

101

nonsporulating which made replica plating of the colonies difﬁcult. Therefore, screening
of D.P. mutants lacking the ability to degrade PGA substrate was discontinued and the
three isolates were further characterized and studied.

Characterization of the U.V. irradiated isolates of D.P. The isolates B1, 2A, and 3C
were physiologically characterized and compared to D.P. (Table 3.1). When grown on
YEME agar the U.V. generated isolates were nearly indistinguishable from D.P. , except
the 2A culture had a darker reverse color (Fig. 3.10 A). The 3C isolate was unable to
utilize several substrates (xylose, raffmose, myo-inositol, malate, malonate, lactate, and
oxalate) utilized by D.P. 3C also acquired the ability to grow in presence of tetracycline.
However, the level of growth was very poor. The isolate 3C’s utilization of pectin and
PGA and its ability to cause deep-pitted scab lesions were unaffected by mutagenesis
(Table 3.1; and Fig. 3.11 B). The isolates B1 and 2A grew poorly at 37 C and formed
bald colonies, however, this may not be the result of a mutation. The isolates B1 and 2A
produced deep pitted scab on Atlantic tubers, but the lesions appeared to be shallower
than those caused by D.P. This lowered virulence was not correlated with a subsequent
loss of pectinase degrading ability.

Detection of pectolytic activity in culture ﬁltrates of the U.V.-irradiated isolates of
D.P. The reducing groups assay detected activity for pectin and PGA induced enzyme
preparations from B,, 2A, and 3C, when assayed on PGA and pectin substrate (between
pH 5-6) (Fig. 3.12 A and B). This suggested that the isolates produced hydrolase in
culture as was observed for RL 232, D.P., and RL 95. However, the activity was not

conﬁrmed by cup plate assays on PGA substrate at pH 5. Viscometry suggested that the

102

 

 

 

 

 

 

 

 

 

7
a s- "
L
1:

5.-
g 4 . [J.P.
\. 4- 0 El
.5 ‘ A 211
E 3 A31:
3 2‘
a
51‘
:I

o a .

3 4 5 s 7
all

7
e‘ 6‘
h-
a

5.1
g! 0 D.P.
5 A 20
E 3j Ass
3 2-
3! .
a 14
a

o I I r

3 4 5 s 7

Fig. 3.12. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of D.P., B1, 2A, and 3C induced on A, PGA; or B, pectin. The reducing groups assay
was used to detect hydrolase activity on PGA substrate, activity was measured on the
basis of micrograms (pg) galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm.

103

 

 

 

 

 

 

 

 

 

 

6

b

I

9 O D.P.
\

,5 O or
E A 2|
3‘ A 31:
I

D

I

3' so

. I

5 “l e or
5: 0 II
E so-

\. a as
' .
2 zo- A 3‘:
I

x 10.1

I J

E 0

7 10
0.3

g 1:

R

"f o as.
g 0 II
B A 211
" A as
I

I

I

I

8

U

 

 

 

Fig. 3.13. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of D.P., Bl, 2A, and 3C induced on pectin. Lyase activity was detected on pectin
substrate by A, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm;
C, measuring the change in absorbance of a reaction after 2 minutes (min.) at 235 nm.

103

 

 

 

 

 

 

 

 

 

 

 

e'

b

B

a O D.P.
2

'3. O or
E A 211
3' A 31:
I!

I!

I

s so

I a I

I! .

E. ‘o e or.
5 O or
E so-

\. A 211
‘ .
E 20- A 3‘:
I

z 10¢:

I

E 0

7 10
0.3

g c

R

“f °"‘ 0 ll.l’.
3 O In
E

g A 211
" 0-1 A 31:
I

I)

I

I

8

"’ o.o

 

 

 

Fig. 3.13. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of D.P., B1, 2A, and 3C induced on pectin. Lyase activity was detected on pectin
substrate by A, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm;
C, measuring the change in absorbance of a reaction after 2 minutes (min.) at 235 nm.

104

 

 

 

 

 

 

 

 

 

4
9' II
I-
. J
D
: O D.P.
5 2 0 II
E l A 211
‘.
.5 A 31:
a t
D
3
o .
7 s 9 10
all
2- 50
I i I
I1 40‘
E .
=' O 0.?
E 30‘ 0 II
\. i A 2|
3 ‘20- A 3:
I.
I.
2 1o-
. l
E o . . . T .
7 a 9 10

Fig. 3.14. The inﬂuence of pH on the activity of pectolytic enzymes from culture ﬁltrates
of D.P. , B1, 2A, and 3C induced on PGA. Lyase activity was detected on PGA substrate
by A, the reducing groups assay, where activity was measured on the basis of
micrograms (pg) of galacturonic acid units (gal.) produced per minute (min.) per
microgram (pg) protein (pro.) at 540 nm; B, thiobarbituric acid assay, where activity was
measured on the basis of the concentration in millimicro moles (mp M) of colored
product (prod.) formed per minute (min.) per microgram (pg) protein (pro.) at 548 nm.

105

enzyme activity was exolytic as the PGA induced enzyme preparation from 2A caused
minimal reduction in the viscosity of PGA substrate at pH 5.

The pectin induced enzyme preparations from B,, 2A, and 3C generally exhibited
the lyase activity that was associated with enzyme preparations from D.P. , when assayed
on pectin substrate (Fig. 3.13 A,B, and C). Pectin induced enzymes from B,, 2A, and
3C when assayed by the cup plate method, exhibited activity on pectin substrate at pH
7, 8, 8.5, 9, and 9.5. This suggested that the mutants were still producing lyases in
culture when supplied pectin as a substrate. The PGA induced enzyme preparations from
B,, 2A, and 3C also exhibited lyase activity which was associated with enzyme
preparations from D.P. , when assayed on PGA substrate (Fig. 3.14 A and B). The PGA
induced enzyme preparations from B, , 2A, and 3C when assayed by the cup plate method
on PGA substrate exhibited activity at pH 7, 8, and 8.5. Viscometry suggested that
endopectate lyase was produced in culture, as the PGA induced enzyme preparation from
B, rapidly reduced the viscosity of PGA substrate at pH 8.4.

SDS-PAGE of enzyme preparations from Streptomyces isolates. SDS polyacrylamide
gel electrophoresis of culture ﬁltrates has been used to determine the similarity of
different strains of actinomycetes (Dietz, 1988). The electrophoretic patterns of the
proteins in the enzyme preparations from the streptomyces shake cultures induced on
pectin and PGA were compared for similarity. The electrophoretic patterns of proteins
in from PGA induced cultures (Fig. 3.15 A) were different for each isolate tested, which
was also the case for enzyme preparations from pectin induced culture (Fig. 3.15 B).
Electrophoretic protein patterns of the enzyme preparations differed for a single isolate

when grown on pectin or PGA substrates. For example, the protein pattern for RL 232

45kD --—_ '
34.7 kD ----
24 kD --- ,1
18.4kD ---- ’

14.3kD ---- ‘ '

66kD—--

45 kD --
34.7 kb an

24 kD .... in
18.4 kD ----

14.3 kD -..-

 

Fig. 3.15. SDS-PAGE of extracellular pectinases from culture ﬁltrates of Streptomyces
induced on A, PGA or B, pectin substrates. Lane 1, molecular weight markers in
kiloDaltons (kD): bovine albumin (66 kD), egg albumin (45 kD), pepsin (34.7 kD),
trypsinogen (24 kD), B-lactoglobin (18.4 kD), lysozyme (14.3 kD); lane 2, pectin lyase
(EC 4.2.2.10) from Aspergillusjaponicus; lane 3, RL 232; lane 4, D.P.; lane 5, RL 95;
lane 6, 3S; lane 7, B,; lane 8, 2A; lane 9, 3C; and lane 10, polygalacturonase (EC
3.2.1.15) from Aspergillus niger.

107

 

Fig. 3.16. Rating scale for tissue maceration: 0, no browning or maceration (comparable
to controls); 1, tissue slightly discolored without maceration; 2, tissue slightly discolored
with minimal visible maceration; 3, brown tissue with moderate maceration; 4, brown
or blackened tissue, at least 90% maceration (tissue was very soft).

108

TABLE 3.2. Potato tissue macerating capability of enzyme preparations from Streptomyces species cultured on
pectin and polygalacturonic acid‘

 

 

Enzyme induced on”:

 

 

 

PGA Pectin
Tris“ 11,0c Tris 11,0

Enzyme Prep.d

RL 232 3' 3 3 3
RL 95 l 0 2 2
D.P. 2 2 3 2
B,’ 2 2 3 2
2Ar l 0 l 2
3C' 2 2 2 2
3S 3 2 2 2
PG‘ 3 3 - -
PL' 4 2 - ..

 

' Rating scale: 0, no browning or maceration; 1, slight browning and no maceration; 2, slight browning and
slight maceration; 3, moderate browning and moderate maceration; 4, tissue is brown or black with 90% or
more maceration.

" Streptomyces isolates were induced to produce enzymes on polygalacturonic acid (PGA) or pectin substrates.
‘ Tuber discs were incubated in sterile Tris-HC1 buffer pH 8.8 and distilled H20.

" The isolate of Streptomyces from which the enzyme preparation was obtained.

° Numerical values corresponding to the level of tissue maceration.

' U.V. generated isolates of the D.P. (Chapter 3, thk dissertation).

' Commercial enzyme preparations. PG, polygalacturonase from Aspergillus niger; PL, pectin lyase from

Aspergillus japonicus.

[are ' .
r13

—eee
i115

., f

EEG
Emacs
ImOOO
matte
-@OO
HIE

 

Fig. 3.17. Tuber discs 36 hours after treatment with pectin induced enzyme preparations
from Streptomyces isolates RL 232, D.P., and RL 95. A, Tissue incubated in Tris-HCl
buffer (pH 8.8); B, Tissue incubated in water.

110

 

Fig. 3.18. Tuber discs 36 hours after treatment with PGA induced enzyme preparations
from Streptomyces isolates RL 232, D.P., and RL 95. A, Tissue incubated in Tris-HCl
buffer (pH 8.8); B, Tissue incubated in water.

110

-1333

E36119.
:13 OO’
.

 

Fig. 3.18. Tuber discs 36 hours after treatment with PGA induced enzyme preparations
from Streptomyces isolates RL 232, D.P., and RL 95. A, Tissue incubated in Tris-HCl
buffer (pH 8.8); B, Tissue incubated in water.

111
induced on PGA was different from that of RL 232 enzyme preparation induced on

pectin. The wild type isolate D.P. had different electrophoretic protein patterns than its
U.V. generated mutants B,, 2A, and 3C, which suggested that the isolates were possibly
mutated in their ability to synthesize pectinases, or the isolates synthesized different
extracellular proteins than D.P., while growing on pectin and PGA.

Tissue maceration assay. Tissue macerating ability of the enzyme preparations were
Observed on potato tuber discs. Browning and loss of tissue integrity (hardness) were
visually rated. The ratings ranged from 0 to 4 where: 0, no browning or maceration
(comparable to the water inoculated controls); 1, tissue slightly discolored without
maceration; 2, tissue slight discolored with minimal visible maceration; 3, brown tissue
with a moderate level of maceration; 4, brown or blackened tissue with nearly complete
maceration, tissue was very soft (Fig. 3.16).

Pectin and PGA induced enzyme preparations from RL 232, RL 95, and D.P.
cultures caused tissue maceration approximately 24-36 hours after treatment (Fig. 3.17
and 3.18). Controls browned slightly after 36 hours without loss of hardness, even after
48 hours. Maceration occurred on tissue incubated in Tris-HCl buffer or water (Fig.
3.17, 3.18 and Table 3.2). PGA induced culture ﬁltrates ofRL 95 and 2A (U.V mutant
of D.P.) did not cause maceration of tissue incubated in water (Table 3.2). Commercial
preparations of polygalacturonase EC 3.2.1.15 from A. niger and pectin lyase EC
4.2.2.10 from A. japonicus caused maceration of tuber discs incubated in Tris-HCl buffer
or water (Table 3.2).

DISCUSSION

The isolates RL 232, D.P., RL 95, and 38 were induced on citrus pectin or

112

sodium polypectate (PGA) to produce pectolytic enzymes in shake culture. Additionally,
the isolates RL 232, D.P., and RL 95 were not found to produce pectolytic enzymes
when supplied glucose as a sole carbon source. The pectin and PGA enzyme
preparations, from the culture ﬁltrates of each isolate, exhibited hydrolase and lyase
activity when assayed on pectin or PGA substrates. Using the reducing groups assay
hydrolase activity was detected in enzyme preparations from the various streptomyces
isolates used in this study. Hydrolases exhibited activity on PGA substrate at pH 5.
However, hydrolase activity was not detected with viscometry or the cup plate assay on
PGA substrate at pH 5, which suggested that possibly exopolygalacturonases were
induced in culture. The reducing groups assay detected hydrolase activity in pectin
induced enzyme preparations assayed on pectin substrate at pH 4.8 (Fig. 3.2). Pectin
and PGA induced enzyme preparations, however, exhibited activity on pectin substrate
(pH 5) when assayed by the cup plate method. This activity however, was not conﬁrmed
by viscometry on pectin substrate at pH 5. This also suggested that
exopolygalacturonases were induced in culture. The production of hydrolases by
Streptomyces species in culture has not been reported. However, Seguin and Lalonde
(1989) reported that culture ﬁltrates from Frankia species grown on pectin showed
activity on pectin substrate at pH 5.2. Frankia species are symbionts of Alnus and are
mildly pathogenic to the many Alnus species (Lalonde and Knowles, 1975).

All the streptomyces isolates examined produced lyases in shake culture when
grown on pectin or PGA substrates. Pectin and PGA induced enzyme preparations
exhibited activity on pectin and PGA substrates at the characteristic pH optimums for

endopectin lyase and endopolygalacturonate lyase. Lyases activity was detected readily

113

with all the enzyme assays used in this study. Lyase activity was usually optimal near
pH 9.5 for both pectin and PGA induced enzyme preparations, assayed on PGA
substrate. Pectin and PGA induced enzyme preparations, assayed on pectin substrate
were active between pH 6-8, but also exhibited activity between pH 8-9.5. The
production of lyases by Streptomyces species in culture was reported by Knosel (1970)
for S. scabies and S. ipomoea, and by Sato and Kaji (1973, 1975, 1977 a and b) for S.
nitrosporeus and S. fradiae. Knosel puriﬁed pectate lyases, which exhibited optimal
activity at pH 9, from culture ﬁltrates of streptomycetes grown on a medium consisting
of pectin and cellulose. Sato and Kaji (1973) detected pectate lyase in the crude culture
ﬁltrate of S. fradiae, which exhibited optimal activity on PGA substrate at pH 9.5. Upon
puriﬁcation the enzyme was determined to be an endopectate lyase which exhibited
Optimal activity between pH 9-9.2 (Sato and Kaji, 1975). S. nitrosporeus produced a
pectate lyase in culture, which exhibited optimal activity at pH 9.3 and 9.5 on pectin
substrate, however, upon puriﬁcation the enzyme was found to be more active on PGA
substrate at pH 9.5 (Sato and Kaji, 1977 b). The concentrated culture ﬁltrates of
RL 232, D.P. , and RL 95 also exhibited activity on pectin and PGA substrate at pH 9.5.

Thus, streptomycetes produce several types of pectic enzymes that may be
induced by pectin or PGA substrates. Further puriﬁcation of the culture ﬁltrates from
RL 232, D.P. , and RL 95 induced on pectin and PGA substrates, would help to conﬁrm
which types lyases were produced in shake culture.

Sato and Kaji (1973) found that pectic enzymes from S. fradiae macerated potato
and several other types of plant tissues when incubated in Tris-HCl buffer (pH 8). Pectin

and PGA induced enzymes, from RL 232, D.P. , and RL 95 macerated potato tuber discs

114
incubated in Tris-HCl buffer at pH 8.8 or water pH 7 (Figs. 3.17 and 3.18). The pH

at which the tissue was incubated had no effect on the ability of the enzyme preparations
to cause tissue maceration. Neither S. fradiae or S. nitrosporeus are plant pathogens and
yet produce enzymes that degrade plant tissue. RL 95 and 38 were not pathogenic on
potato, but both isolates produced pectolytic enzymes in culture that macerated potato
tuber discs (Table 3.2). This ﬁnding suggested the ability to produce pectolytic enzymes
was not exclusively restricted to S. scabies or other potato scab-causing streptomycetes.
However, this ﬁnding does not negatively implicate the involvement of pectolytic
enzymes in the infection process.

Production of pectinase deﬁcient S. scabies mutants would determine the
importance of pectolytic enzymes for infection. Attempts to produce endopectate lyase
deﬁcient mutants from the D.P. isolate were not successful. Screening for mutants was
not speciﬁc enough to determine whether certain pectinase functions were inactivated.
U.V. mutagenesis causes nonspeciﬁc base shifts and does not inactivate speciﬁc genes
as does transposon mutagenesis, and therefore has limited uses in the study of pectinases.
Transposon mutagenesis has been used to speciﬁcally inactivate pel genes in Erwinia
species (Collmer and Keen, 1986). The U.V. generated isolates of D.P. (B,, 2A, 3C)
which were screened for the loss of pectinases grew on both PGA and pectin as sole
carbon sources. The pectin and PGA induced enzymes from the mutants had hydrolase
and lyase activity similar to the wild-type D.P. (Figs. 3.12, 3.13, 3.14) and caused tissue
maceration (Table 3.2). The isolates 8,, 2A, and 3C remained pathogenic on Atlantic.
U.V. mutagenesis appeared to alter the isolates physiology (Table 3.1 and Fig. 3.10),

apparently without affecting utilization of pectic substrates. .Pel genes in Erwinia Species

115

occur in multiple copies and inactivating one gene does not prevent the bacterium for
producing pectolytic enzymes (Collmer and Keen, 1986). Therefore it is possible that
Streptomyces species have a similar type of arrangement of pectinase genes in multiple
forms. This would account for the apparent loss of pectinase production by 8,, 2A, and
3C during the mutant screening process on agar plates, and allow for the production of
pectinases in shake culture.

The pectin agarose overlay experiments (Figs. 3.8 and 3.9) suggested that RL 232
and D.P. produced similar types of pectinases, which were different from the
nonpathogen 38. The PGA and pectin induced enzyme preparations from 38 produced
identical bands of clearing on the pectic substrate overlays (Fig. 3.8), which suggested
PGA and pectin substrates induced production of similar forms pectolytic enzymes from
streptomyces in shake culture. It was not possible to distinguish pectolytic enzymes from
other proteins by SDS-PAGE of culture ﬁltrates (Fig. 3.15 A and B). However the
varied protein patterns on the SDS-PAGE gels suggested that PGA and pectin substrates
induced different proteins in culture for the streptomyces used in these studies. Also the
SDS-PAGE gels demonstrated that U.V. light generated isolates were physiologically
altered, as none of the isolates produced a protein pattern to identical to the D.P. isolate
(Fig. 3.15 A and B). The pattern of proteins extracted from whole cells of Bl, 2A, and
3C grown on glucose-asparagine medium (Gottlieb and Hepden, 1966), were different
from those of D.P. This also suggested that the U.V. generated D.P. isolates were
altered by the mutagenic agent (Fig. 2.2 and 2.3).

The next series of studies should begin with the puriﬁcation of the pectolytic

enzymes induced on potato tissue. As the isolates RL 232, D.P., RL 95, and 3S grew

116

on a medium which consisted of potato tissue. Using the cup plate assay, it was possible
to detect pectolytic activity on pectin substrate (pH 5 and 7) and PGA substrate (pH 7
and 9) for all isolates induced on potato tissue. This ﬁnding was signiﬁcant because it
demonstrated that the streptomycetes produced pectolytic enzymes even in the presence
of the more accessible carbon sources which should have been provided by the potato
tissue. Using the potato based medium should provide a more comprehensive view of
the array of cell wall degrading enzymes produced by potato scab-causing streptomycetes
during the infection process. My studies indicate that Streptomyces species have the
capacity to degrade pectic substrates and tend produce lyases in shake culture. As
endopectate lyases are known to cause tissue maceration, production of these enzymes
by streptomycetes would facilitate invasion of potato tissue by streptomyces. However,
S. scabies and other potato scab-causing streptomyces do not produce any type of tissue
rot. Therefore pectinases cannot be considered a pathogenicity factor in potato scab
disease, and should be considered enhancers of the pathogens virulence. However, to
test this idea mutants need to be produced which deﬁnitely lack the ability to synthesize
pectinases. Also these mutants would have to be tested for the loss of virulence on
potato. Conversely it may be possible to create hypovirulent mutants of S. scabies and
assess whether the ability to produce various cell wall degrading enzymes is subsequently

loss.

1 17
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CHAPTER IV

THE PRODUCTION OF THAXTOMIN BY A DEEP PITTING FORM OF

STREPT 0M Y CES

123

124
INTRODUCTION

Many species of Streptomyces synthesize secondary metabolites which allow them
to survive in soil environments (Kutzner, 1981). The streptomycetes are well known for
their ability to synthesize antibiotics which inhibit the growth of other microorganisms
(Kom-Wendisch and Kutzner, 1992).

S. scabies and S. acidiscabies (Lambert and Loria, 1989 a and b), the causal
agents of common scab and acid scab of potato (Solanum tuberosum L.), synthesize the
phytotoxic secondary metabolites designated as thaxtomins (Lawrence et al, 1990 and
King et al, 1991). Thaxtomins constitute a unique group of 2,5-dioxopiperazines that
have a 4-nitroindol group attached to the 3rd carbon of a 2,5- dioxopiperazine structure
(King et al, 1992). Two forms of thaxtomin have been isolated: Form A, contains a
hydroxyl group at carbon 20 of the 2,5-dioxopiperazine ring; and Form B has a hydrogen
group at carbon 20 of the 2,5-dioxopiperazine ring (King et al, 1989, 1991, and 1992;
and Lawrence et al, 1991). The production of thaxtomin A normally exceeds the B form
by the ratio of 20:1 in potato tissue (King et al, 1991).

Thaxtomin reproduces the symptoms of common scab on mini-potato tubers
(Lawrence et al, 1990). Since S. scabies and S. acidiscabies both synthesize thaxtomin
it is possible that other species of potato scab-causing bacteria produce thaxtomin. The
purpose of this study was to determine whether D.P. , a deep-pitting scab isolate,

produces thaxtomin or thaxtomin-like compounds.

125
MATERIALS AND METHODS

Streptomyces isolates. The Streptomyces isolates RL 232, D.P. , and 3S (Tables 2.1 and
2.3) were selected for their contrasting morphologies and differing pathogenic abilities.
Production of inoculum. The bacteria were grown for inoculum production on yeast
extract malt extract agar (YEME) (Shirling and Gottlieb, 1966). YEME was inoculated
with spores from stocks stored frozen in 20 % glycerol/water at -20 C Glopwood et al,
1985). The cultures were usually incubated at 25 C for at least 14 days. The spores of
mature cultures were used to inoculate all other media and plant tissue.

Induction of thaxtomin in culture. Oatmeal agar and broth (Shirling and Gottlieb,
1966) both support the production of thaxtomin A and B by S. scabies (Eckwall et al,
1992). Oatmeal agar was inoculated with mature spores and incubated at 25 C for up
to 22 days. Thaxtomin was, however, detected in 9 day old oatmeal agar cultures and
the induction period was reduced to 9 days.

Oatmeal broth was dispensed in 25 ml aliquots to 125 ml ﬂask and autoclaved
under standard conditions. The ﬂasks were inoculated with a suspension of mature
spores and placed on a rotary shaker at 23 C. Twelve and 18 day incubation periods
were initially used to induces thaxtomin in oatmeal broth culture. However, thaxtomin
was detected in 7 day old oatmeal broth cultures and the induction time was reduced to
7 days. Uninoculated media served as controls.

Plant materials for induction of thaxtomin and bioassay of toxin. Katahdin and
Onaway tubers were used to induce thaxtomin. Onaway was selected for its marked
susceptibility to the D.P. isolate in greenhouse pathogenicity screenings. Katahdin has

been reported to be susceptible D.P. and S. scabies (Ludlam, 1991). Russet Burbank

126

tuber discs were used to bioassay the toxin extracted from tissue and artiﬁcial media.
Russet Burbank was resistant to infection by RL 232, D.P., and 38 in greenhouse
pathogenicity tests.
Induction of toxin on tuber disks. Thaxtomin was induced on tuber discs. A single
cylinder of internal medullary tissue was removed from each tuber, sliced into individual
discs 2 cm in diameter and 0.5 cm thick (Hammerschmidt, 1984). After slicing the discs
were placed into 1% sodium chloride solution to prevent browning. The discs were
rinsed in several changes of sterile distilled water prior to inoculation. Inoculum was
applied to the discs by pressing them directly onto the surface of a sporulating culture.
Only RL 232 and D.P. were used to induce toxin in tissue. The discs were transferred
to sterile petri dishes lined with moist ﬁlter paper and incubated in darkness at 25 C for
9 days. Uninoculated discs were used as controls.
Initial acetone extraction of agar and culture filtrate. The extraction of toxin was
performed according to the procedure of Lawrence et al (1990) and King et al (1991).
Six sporulating cultures, grown on oatmeal agar, were combined to form single sample
per treatment in the ﬁrst experiment. For subsequent experiments, four plates were used
per sample per treatment. Two ﬂasks of oatmeal broth culture ﬁltrate were combined
to form a single sample per treatment in the ﬁrst experiment. For subsequent
experiments the sample size was increased to four ﬂasks of oat meal broth culture ﬁltrate
per treatment.

A sample of oatmeal agar culture was extracted with 200 ml of cold acetone by
high speed blending with a homogenizer. The resultant slurry was ﬁltered through

Whatman No. 1 ﬁlter paper and the acetone extract was retained for further extraction.

127

A sample of broth culture was ﬁltered through cheesecloth followed by high speed
centrifugation. The ﬁltrate was extracted with an equal volume of acetone and ﬁltered
through Whatman No. 1 ﬁlter paper.

Initial acetone extraction of tuber discs. Inoculated tuber discs were necrotic and
showed sporulation on all surfaces. Seven tuber discs were extracted by homogenization
in cold acetone. The resultant slurry was ﬁltered through Whatman No. 1 ﬁlter paper
and the acetone extract was retained for further extraction.

Extraction of the media and tissue acetone extracts. Acetone extracts from artiﬁcial
media or tissue were treated identically. The extracts were evaporated under reduced
pressure at 35 C and the remaining residue was dissolved in 25 ml of distilled water.
The residue was extracted twice with equal volumes of chloroform. The chloroform
fractions were combined and concentrated under reduced pressure at 35 C. The resultant
residue was dissolved in 1 ml of acetone and concentrated by evaporation to
approximately 0.25 ml, and stored at -20 C or immediately assayed for the presence of
toxin by thin layer chromatography (TLC).

Thin layer chromatography (TLC) of crude extracts. The crude extracts were applied
to silica gel G thin layer plates in three 5 p1 aliquots. The plates were chromatographed
in a chloroform/methanol 9:1 (v/v) solvent system (Lawrence et al, 1990 and King et al,
1991). A sample containing toxin generally resolved two bright yellow bands.
Pooling of the crude extracts to concentrate toxin for bioassaying. To maximize the
bioassay efﬁciency of the toxin, the ﬁnal acetone extracts (from each type of induction

media or tissue) for each streptomyces isolate (crude extract) were pooled together.

128

These ﬁnal acetone extracts were reextracted with chloroform as previously mentioned
and the concentrated residue was solubilized in 0.5 ml acetone.

The concentrated acetone extracts from tissue, agar medium, or culture ﬁltrate
for RL 232, D.P., and 38 were handed onto TLC plates in 100 pl aliquots. The plates
were chromatographed in a chloroform/methanol 9:1 (v/v) solvent system. The bands
that resolved (visibly corresponding to the yellow spots associated with thaxtomin), were
scraped directly from the plates. The silica powders for each spot were extracted with
ethyl-acetate. The ethyl-acetate was evaporated under vacuum at 35 C and the remaining
residue was redissolved in acetone. The concentrated extracts were maintained at -20 C
until bioassayed.

Bioassay of the concentrated extracts for toxicity. The acetone was evaporated from
the extracts and the residue was suspended in 200 [1.1 of distilled water. Twenty
microliters of the toxin suspension was applied to the upper surface of tubers discs,
which were maintained in a petri dishes containing moist ﬁlter paper. Six tuber discs
were bioassayed per treatment. The bioassay was repeated twice for each toxin sample
to verify the result.

RESULTS

Induction of thaxtomin in agar culture. In the ﬁrst experiment, 22 day old OA
cultures of RL 232 and D.P. were extracted. The ﬁnal acetone extract was yellowish-
green for both samples. When chromatographed, each sample produced a single yellow
band with a Rf of 0.34 for the RL 232 sample and a Rf of 0.17 for the D.P. sample.
Control samples did not reveal any yellow bands. When these samples were reextracted

and concentrated, the RL 232 sample resolved into two bands with R,s of 0.32 and 0.98.

129
The D.P. sample resolved into two bands with R,s of 0.40 and 0.44, the second band

appeared very faint. In each case the concentrated samples retained their yellow color
through the second round of extractions.

The RL 232 acetone extracts were yellowish-green in color, the D.P. samples
were faintly yellowish. The 38 acetone extract was pink. When the samples were
chromatographed, only the RL 232 extracts resolved into single yellow bands in each
lane. The average Rf for all the RL 232 samples was 0.30. The D.P., 3S, and control
samples produced no visible bands.

The pooled extracts of RL 232 and D.P. were yellowish-green when concentrated.
The concentrated extract of 38 was a dark pink color. When chromatographed the RL
232 sample produced a single yellow band with an Rf of 0.27. The D.P. sample
produced a single yellow band with an Rf of 0.32. The 38 and control samples produced
no bands when chromatographed.

Induction of thaxtomin in culture ﬁltrates. In the ﬁrst experiment, oatmeal broth
cultures were allowed to incubate for 12 or 18 days. Two separate batches of oatmeal
broth were prepared for this experiment and RL 232 and D.P. were used to inoculate the
medium. Two ﬂasks containing 25 ml of culture medium (50 ml total) were sampled for
extraction. When the 12-day and 18—day old D.P. cultures were extracted, one batch of
the extracted medium lacked the characteristic yellow-green tint associated with toxin
production. After chromatography, only those extracts of yellowish-green color
produced yellow bands characteristic of thaxtomin. The 12 day D.P. extracts which
produced a single yellow band upon being chromatographed had an average Rf of 0.24,

and the 18 day extracts had an average Rf of 0.29. RL 232 produced thaxtomin in both

130

batches of oatmeal agar broth as indicated by the yellowish-green acetone extracts for
each sample. The majority of the acetone extracts for the 12 day cultures revealed two
yellow bands after chromatography. The average R, of the ﬁrst resolving band was R,
0.35, the second resolving band had an average R, of 0.83. After chromatography the
extracts of 18 day cultures of RL 232 resolved into single yellow bands for each sample.
The average Rf for the extracts of 18 day cultures of RL 232 was Rf 0.23.

The extracts from 12 day and 18 day cultures of RL 232 or D.P. were pooled to
form a sample for each of isolate. The sample was extracted and concentrated under
vacuum, the intensity of the yellow-green color in each acetone extract increased. After
chromatography the RL 232 sample yielded a single yellow band with a Rf of 0.29, the
D.P. sample produced a single yellow band with an R, of 0.32.

Oatmeal broth cultures were incubated for 7 days in a second experiment, which
included the isolate 38 in addition to RL 232 and D.P. Approximately 100 ml of culture
ﬁltrate (four ﬂask containing 25 ml of culture medium) was extracted for toxin. Only
one D.P. sample extract resolved a single yellow band after chromatography with a Rf
of 0.19. All RL 232 sample extracts produced single yellow bands after
chromatography, with an average Rf of 0.27 for all samples. Bands corresponding to
thaxtomin were not detected for 3S and control treatments.

Pooling the extracts intensiﬁed the yellow-green color of the ﬁnal acetone extracts
for both RL 232 and D.P. The ﬁnal acetone extract for 3S was a dark pink color. After
chromatography, the RL 232 extract resolved a single yellow band with a R{ of 0.30 and
the D.P. extract resolved 3 single yellow band with a R{ of 0.24. Bands were not

detected for 38 or control pooled extracts after chromatography.

13 1
Induction of thaxtomin in potato tuber discs. A seven tuber disc sample was extracted

for toxin. The acetone extracts from D.P. infected Onaway tuber discs produced no
detectable bands corresponding to thaxtomin after chromatography. The acetone extracts
from D.P. infected Katahdin tuber discs produced detectable bands corresponding to
thaxtomin after chromatography. The average R, of the material from crude extracts was
R, 0.28. The acetone extracts RL 232 infected Onaway tuber discs resolved into single
yellow bands for each sample after chromatography. The average R, for all the acetone
extracts was R, 0.23. The acetone extracts of RL 232 infected Katahdin tuber discs
resolved a single yellow band for each sample tested after chromatography. The average
R, for all the acetone extracts was R, 0.20. Bands corresponding to thaxtomin were not
detected in the acetone extracts of uninoculated tuber discs after chromatography.

The acetone extracts from both Katahdin and Onaway samples for either RL 232
and D.P. were concentrated by pooling and reextracted. The concentrated extracts for
both RL 232 and D.P. were yellow-green in color. The pooled D.P. extract after
chromatography, resolved into a single yellow band with a R, of 0.38. The pooled RL
232 extract, after chromatography resolved into a single yellow band with a R, of 0.29.
Bands were not detected in the pooled extracts of uninoculated tuber discs after
chromatography.

Bioassay of the RL 232 and D.P extracts. The bioassays were conducted with the
concentrated, pooled extracts from the tissue, oatmeal agar cultures, or oatmeal broth
cultures. When applied to tuber discs the aqueous toxin suspension resulted in rapid
browning (by 18 hours) over most of the discs surface. The compound extracted from

artiﬁcial media or tissue for RL 232 and D.P. produced the phytotoxic compound

132

Em

KEEP (3.351
TISSUE 3a ( a,

 

Fig. 4.1. Bioassay of thaxtomin from RL 232 and D.P. extracts on Russet Burbank tuber
discs. Sources of toxin A, Culture ﬁltrate from oatmeal broth (CF), oatmeal agar (agar),
and inoculated tuber discs (tissue). B, Culture ﬁltrate from oatmeal broth (CF), and
oatmeal agar (agar). C, Tissue treated with water (I120).

133
thaxtomin (Fig. 4.1 A). RL 232 and D.P. produced thaxtomin in oatmeal broth and

oatmeal agar (Fig. 4.1 B). The browning caused by the toxin extracted from liquid

culture was more rapid and intense than toxin extracted from agar culture (Fig. 4.1 B).

Browning was not detected on discs treated with water (Fig. 4.1 C). The toxin caused

browning response on tuber discs approximately 18 hours after application.
DISCUSSION

This investigation demonstrated, that the deep pitting Streptomyces isolate D.P.
produced a thaxtomin-like compound, and conﬁrmed that RL 232 (S. scabies) and D.P.
produced the phytotoxin under similar cultural conditions. The nonpathogenic
Streptomyces 38, produced no detectable thaxtomin-like compounds in culture, which
suggested that synthesis of thaxtomin was correlated with pathogenicity. This also
demonstrated that tuber discs could be used to simply conﬁrm the phytotoxicity of
thaxtomin without growing potato tubers.

Concentrating the toxin by pooling acetone extracts made it possible to
approximate which forms of thaxtomin were being synthesized (Table 4.1). Based upon
the R, values for crude acetone extracts, it appears that RL 232 produced thaxtomin A
(Table 4.1). D.P. possibly produced both thaxtomin A and B or an intermediate of the
two forms of toxin (Table 4.1). The averaged R, values from experiments 1 and 2,
showed that RL 232 produced thaxtomin A R, 0.27 (Tables 4.1 and 4.2). The reported
R,s for thaxtomin A and B are 0.27 and 0.41 respectively (Lawrence et al, 1990; and
King et al, 1991). The predominance of form A over B of the toxin may explain the
inability to detect the B form in any of the RL 232 extracts. All the concentrated

extracts from RL 232 and D.P. exhibited phytotoxicity on tuber discs regardless of the

134

TABLE 4. 1 . The R,s for the concentrated acetone extracts as determined
by thin layer chromatography in chloroform/methanol 9:1 (v/v) solvent system

 

 

 

 

 

 

Isolates
RL 232b D.P.c 3Sd Control‘

Source of toxin

Experiment 1'

Tuber discs‘ 0.29 0.38 -" -
Agar' 0.32 0.40 - -
Culture filtratcsj 0.29 0.32 - -
Experiment 2'

Agar 0.27 0.32 - -
Culture filtrates 0.30 0.24 - -

 

' The characteristics of these isolates are listed in chapter 2 tables 2.2 and 2.3 this
dissertation.

" RL 232 S. scabies from R. Loria, causes common scab.

° D.P. deep pitting isolate from Michigan.

" 3S nonpathogen Wallace and Hammerschmidt.

’ Extracts from uninoculated tuber discs or media.

' Two experiments performed. Induction of thaxtomin on tuber discs was examined once.
' Discs from Russet Burbank tubers.

" 3S was not used in the first experiment.

' Extracts from oatmeal agar (Eckwall et al, 1992).

’ Culture filtrate from oatmeal broth (Eckwall et al, 1992).

135

Table 4.2. Averaged R, values for extracts from experiments 1 and 2'

 

 

 

 

Isolates”
RL 232 D P
Source of toxin
Tuber discsc 0.29 0.38
Agar 0.29 0.36
Culture ﬁltrates 0.29 0.28

 

‘ The data from table 4.1 averaged together.

" Chromatography from the extracts of 3S and the controls did not resolve into discernable
spots.

‘ The R, values are from one experiment.

136

form of thaxtomin produced in culture. The extracts from RL 232 and D.P. were toxic
to Russet Burbank tuber discs. In greenhouse tests, RL 232 and D.P. were incapable of
infecting this cultivar. However, thaxtomin produced by RL 232 induced lesions on
tissue cultured mini-tubers of Russet Burbank (Stein and Hammerschmidt, unpublished
data).

Doering-Saad et a1 (1992) suggests that a plasmid may actually encode thaxtomin
synthesis by streptomycetes and this characteristic may be common to several species of
potato scab-causing Streptomyces. One implication of this hypothesis is a nonpathogenic
streptomyces species may acquire the plasmid and the ability to produce thaxtomin, thus
potentially become pathogenic. Such a phenomenon would further complicate proper

identiﬁcation of the streptomycete species which cause potato scab.

137
REFERENCES

Doering-Saad, C., Kampfer, P., Manulis, S., Kritzman, G., Schneider, J ., Zakrzewska-
Czerwinska, J. , Schrempf, H. , and Barash, I. 1992. Diversity among Streptomyces
strains causing potato scab. Appl. Environ. Microbiol. 58:3932-3940.

Eckwall, E.C. , Babcock, M.J. , and Schottel, J .L. 1992. Production of the common scab
inducing phytotoxin thaxtomin A, by Streptomyces scabies. Phytopathology 82: 1153.

Hammerschmidt, R. , 1984 Rapid deposition of liginin in potato tuber tissue as a response
to fungi non-pathogenic on potato. Physiol. Plant Pathol. 70:775-780.

Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Burton, C.J., Kieser, H.M.,
Lydiate, D.J., Smith, C.P., Ward, J .M., and Schrempf, H. 1985. Genetic manipulation
of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich, United
Kingdom.

King, R.R., Lawrence, C.H., Clark, M.C., and Calhoun, LA. 1989. Isolation and
characterization of phytotoxins associated with Streptomyces scabies. Chem. Commun.
13:849-850.

King, R.R., Lawrence, CH, and Clark, MC. 1991. Correlation of phytotoxin
production with pathogenicity of Streptomyces scabies isolates from scab infected potato
tubers. Am. Potato J. 68:675-680.

King, R.R., Lawrence, CH, and Calhoun, LA. 1992. Chemistry of phytotoxins

associated with Streptomyces scabies, the causal organism of potato common scab. J.
Agric. Food Chem. 40:834-837.

Kom-Wendisch, F ., and Kutzner, H.J. 1992. The family Streptomycetaceae. Pages 921-
995 in: The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology,
Isolation, Identiﬁcation, Applications, Vol. 1, A. Balows, H.G. Truper, M. Dworkin,
W. Harder, and K.H. Schleifer, eds. Springer Verlag, Berlin.

Kutzner, H.J. 1981. The family Streptomycetaceae, pages 2028-2090 in: The
Prokaryotes, Vol II. M.P. Starr, H. Stolp, H.G. Truper, A. Balows, and HG. Schlegel,

eds. Springer-Verlag, Berlin.

Lambert, DH, and Loria R. 1989 a. Streptomyces scabies sp. nov., nom rev. Int. J.
Syst. Bacteriol. 392387-392.

Lambert, DH, and Loria, R. 1989 b. Streptomyces acidiscabies sp. nov. Int. J. Syst.
Bacteriol. 39:393-396.

138

Lawrence, C.H., Clark, M.C., and King, RR. 1990. Induction of common scab
symptoms in aseptically cultured potato tubers by the vivotoxin thaxtomin.
Phytopathology 80:606-608.

Ludlam, K. 1991. Factors inﬂuencing the resistance of the potato to common scab. M.S.
thesis. Michigan State University, East Lansing. 91 pp.

Shirling, E.B. , and Gottlieb. 1966. Methods for characterization of Streptomyces species.
Int. J. Syst. Bacteriol. 16: 313-340.

CHAPTER V

THE EFFECTS OF INFECTION BY STREPTOMYCES SPECIES ON THE

BIOSYNTHESIS OF CHLOROGENIC ACID AND LIGNIN IN TUBER DISCS OF

A SCAB-RESISTANT AND SCAB-SUSCEPTIBLE CULTIVAR OF POTATO

139

140
INTRODUCTION

Little is known about the infection process or symptom development in potato
scab disease. Potato scab-causing bacteria apparently infect through stomata and through
stomata differentiating into lenticels (Jones, 1931; Lutman, 1941; C00per et al,1954; and
Adams, 1975). The host’s responses following infection by streptomycetes are not
known. Light microscopic observations reveal that cells several layers beneath the
advancing bacterial ﬁlaments, respond by undergoing cell division and becoming
suberized (Jones, 1931). The suberized cells function as a barrier and are thought to
prevent the bacterium from invading the unaffected tissue beneath the suberized zone of
cells (Jones, 1931). In the case of deep-pitted scab, the suberized barrier is either
breached or develops cracks as the tuber expands. These openings may allow the hyphal
ﬁlaments to continue through to the healthy tissue. Tuber expansion combined with
repeated formation and breaching of suberized cell layers may ultimately lead to the
formation of deep-pitted scab lesions (Jones, 1931). The formation of suberized barriers
in response to infection may constitute all or part of the defense responses to
streptomycetes.

The physiological basis for resistance of potato to S. scabies has not been
determined. Resistance was thought to be a function of the physical and chemical
properties inherent to the periderm of some cultivars of potato (Fellows, 1926; Johnson
and Schaal, 1952; Cooper et al, 1954; Emilsson and Heiken, 1956; and Johnson and
Schaal, 1957). Hooker and Page (1960) observed that S. scabies could not grow on
living periderm, or grow on heat or chemically-killed periderm. This demonstrated that

periderm was possibly a barrier to infection. Cooper et a1 (1954) observed that the

141

periderm of scab-resistant potato cultivars possess a greater number of living nucleated
cells than scab-susceptible potato cultivars. However, Emilsson and Heiken (1956)
observed that not all cultivars of potato ﬁt the categories for resistant and susceptible
types of periderm described by Cooper et a1 (1954).

It seems that periderm is not solely responsible for resistance of potato to
infection by streptomycetes. Hooker and Page ( 1960) observed that S. scabies grew
poorly on living tuber discs. When tuber discs were killed by autoclaving or chemical
treatment prior to inoculation, the bacterium was able to grow. This suggested that
freshly wounded potato tissue had active wound responses that may have inhibited the
growth of S. scabies. Emilsson and Heiken (1956) indicated that the physical barrier
provided by periderm in combination with active host responses possibly constitutes the
sources of potato scab resistance.

Wounded and infected plant tissues have been shown to synthesize phenolic
compounds, which are thought to play an integral role in resistance responses that include
the synthesis of chlorogenic acid, lignin, or suberin (Hammerschmidt, 1985; and
Nicholson and Hammerschmidt, 1992). Plant phenolics appear to possess antimicrobial
activity (Schaal and Johnson, 1955; Friend et al, 1973; Ride, 1978; and Nicholson and
Hammerschmidt, 1992). Wounded and infected potato tissue has been reported to
accumulate chlorogenic acid (CGA) which inhibits growth of several fungal pathogens
of potato and S. scabies (Johnson and Schaal, 1952; Schaal and Johnson, 1955; Patil
et al, 1966; Friend et al, 1973; and Nicholson and Hammerschmidt, 1992). Johnson and
Schaal (1952 and 1957) found that highly scab-resistant cultivars of potato possessed high

levels of CGA in the peripheral layers of their tubers. CGA was hypothesized to either

142

lower the host tissue pH thus creating an unfavorable environment for growth of the
pathogen, or contributed to the formation of suberized periderm (Johnson and Schaal,
1952).

Accumulation of phenolics as a result of infection in plant tissues not only
functions to inhibit pathogen growth, but phenolics also polymerize to form lignin which
functions as a barrier to penetration by plant pathogens (Ride, 1978; Vance et al, 1980).
Ride (1978) proposed several functions for ligniﬁcation. Lignin may reinforce cell walls,
making them resistant to mechanical penetration. Ligniﬁcation may make cell walls
resistant to degradation by pathogen enzymes. A ligniﬁed barrier may prevent diffusion
of pathogen enzymes or toxins into host tissue and also prevent nutrients and water from
reaching the pathogen. Ligniﬁcation of host tissue, may encase the pathogen and prevent
its growth. Low molecular weight phenolic precursors of lignin and free radicals
produced during polymerization may render pathogen membranes, enzymes, toxins, and
elicitors non-functional.

Ligniﬁcation constitutes a measurable resistance response in potato
(Hammerschmidt 1984). Several studies suggest that suberin prevents the invasion of
host tissues by S. scabies (Lutman, 1913 and 1941; and Jones, 1931). Suberin is a
complex heteropolymer consisting of aliphatic and phenolic domains in association with
waxes (Hammerschmidt, 1985). The structure of the phenolic domains of suberin and
lignin are similar (Cottle and Kolattukudy, 1982). Wounded plant tissues accumulate
precursors of lignin which eventually contribute to the formation of suberin, which in

turn prevents infection and loss of moisture from tissues (Kolattukudy and Agrawal,

143
1974). Fellows ( 1926) suggested that hyphal ﬁlaments of S. scabies gain access to

host tissues through the unsuberized lenticels.

The objective of this study was to assess the physiological responses of potato
tuber discs to inoculation with pathogenic and nonpathogenic streptomycetes. I attempted
to measure whether differential patterns of CGA and lignin accumulation could be
detected in resistant and susceptible potato cultivars in response to inoculation with
Streptomyces. Attempts were also made to determine whether chlorogenic acid
functions as an inhibitor of Streptomyces growth.

MATERIALS AND METHODS
Streptomyces isolates. The isolates used were RL 232 and RL 95 (R. Loria) and the
deep-pitting Michigan isolate D.P. RL 232 (S. scabies) caused common scab and
D.P. (unclassiﬁed) caused deep-pitted scab on the cultivar Atlantic. RL 232, D.P. ,
and RL 95 were not pathogenic on Russet Burbank. RL 95 was not pathogenic on
Atlantic.
Plant materials. Tubers of the cultivars Russet Burbank and Atlantic were used in these
studies and stored at 4 C. Russet Burbank exhibited resistance to several isolates of
potato scab-causing bacteria in greenhouse pathogenicity screenings. The cultivar
Atlantic was found to be susceptible to several isolates of potato scab-causing bacteria.
Tubers were held at room temperature for at least 3 days prior to use. The tubers were
washed, surface sterilized in 10 % commercial Chlorox solution, and ﬂamed with 95 %
ethanol before preparation of the tuber discs.
Production of inoculum. Spore of the isolates were frozen in 20 % glycerol/ water

solution (v/v) at -20 C (Hopwood et al, 1985). The spores were used to inoculate yeast

144

extract malt extract agar (YEME) (Shirling and Gottlieb, 1966). The cultures were
incubated at 25 C for 14 days or until the cultures were sporulating and mature.

Two to three inoculating loops of spores were dislodged from the surface of a sporulating
culture and suspended in approximately 5 ml of sterile distilled water, and 100 pl of the
spore suspension was used to inoculate single tuber discs.

Preparation and inoculation of tuber discs. Tuber discs were prepared according to
Hammerschmidt (1984). A cork borer was used to remove a single cylinder of tuber
tissue from the internal medullary region and sliced into discs 2 cm in diameter and 0.5
cm thick. The discs were placed 1 % sodium chloride solution to prevent tissue
browning and rinsed twice with sterile distilled water prior to being placed in petri
dishes. Five to six tuber discs were placed in sterile petri dishes lined with moistened
ﬁlter paper and each disc was inoculated with 100 pl of spore suspension. Tuber discs
treated with sterile distilled water served as controls. The inoculated discs were
incubated at 25 C in complete darkness and sampled daily for 3 days.

Sampling of tuber discs. Disc samples for each treatment were selected from petri
dishes at random. The upper 1 to 2 mm of the inoculated disc surface was excised and
placed into a vial containing 5 ml of absolute methanol. A single disc sample or
replication consisted of two excised discs. The samples were stored in this fashion for
2 to 6 weeks.

Chlorogenic acid (CGA) assay. Prior to measuring the CGA content, the methanol
extract for each sample was diluted to 10 ml with absolute methanol. The absorbance
of the diluted methanol extract was measured at Am. The concentration of CGA per 2

discs sample was calculated using the molar extinction coefﬁcient (19,200 M“). Each

145

treatment was replicated a minimum of 3 times, and the experiment was repeated three
times.
Lignin assay. After the methanol extract was removed, the tissue sample remaining in
each vial was dried under vacuum. Lignin was extracted from the samples by the
thioglycolic procedure (Hammerschmidt, 1984). The resultant ligninthioglycolic acid
(LGTA) precipitate was solubilized in 5 ml of 0.5 N NaOH. High yields of the LGTA
precipitate were diluted to a volume of 10 ml to 15 ml with 0.5 N NaOH. The
absorbance of the solution was read at Am. The concentration of lignin was estimated
from a standard curve for lignin extracted from potato tuber periderm and expressed in
terms of pg of lignin per area of disc. Each treatment was replicated a minimum of 3
times and the experiment was repeated three times.
Inhibitory activity of chlorogenic acid (CGA) on the growth of Streptomyces. The
inhibitory effect of CGA on the growth of streptomycetes was tested by incorporating
CGA into YEME agar. The CGA was dissolved in water, ﬁltered sterilized, and poured
into cool YEME to achieve the ﬁnal concentrations of 0.005, 0.01, 0.1, 0.5, and 1 mg
CGA/ml medium. The bacterial isolates were streaked into quadrants on a single dish
of media such that 4 isolates were grown in the same dish. Each treatment was
replicated a minimum of 5 times per isolate. The cultures were incubated 14 days at
25 C and growth was checked weekly.

RESULTS
Accumulation of CGA in response to inoculation. The amounts of extractable CGA
increased in inoculated Russet Burbank and Atlantic tuber discs and uninoculated controls

over the three day time course (Figs. 5.1 A-C and 5.2 A-C). The general patterns of

146

 

. —o— RL as A
o.s-J —0— Control

ul CON 2 dlscs

 

 

 

 

Days After Inoculation

 

* + RI. 232 B
o.s- —0'— Control

III CON 2 dlaca

 

 

 

 

0.0 T r u w u v v w

1
Days After lnoculatlon

 

. + D.P. C
0...] —O-— COHIfO|

ul CON 2 dlsos

 

 

 

 

o . 1 2 2: ' 4
Days After Inoculation

Fig. 5.1. Three day time course of chlorogenic acid (CGA) accumulation in tuber discs
(cv. Russet Burbank), where the concentration of CGA was measured in micro moles
(pM) per two tuber discs sample at 328 nm. Tuber discs were inoculated with A, RL 95
(nonpathogen); B, RL 232 (common scab pathogen); and C, D.P. (deep pitting
pathogen).

147

 

 

 

 

 

  
 
  

 

 

 

 

 

 

 

 

1.0
. A
+ RL 95
. 0-1‘ —0— Control
3 1
" o.s«
N 1
2
o 0.4‘
o
g 0.2-
o.oT ~ . . , - , .
o 1 2 s 4
Days After Inoculatlon
1.0
.—0— RL 232 3
—0— Control
8 0.0-1
3
u
N o.s-
2
o 0.4-
o
3 0.2
0.0 Y ' I ' I v 1 v
0 1 2 3 4
Days After lnoculatlon
1.0 .
. + D.P. C
. o.s- —0— Control
3
1s
a
2
0
o
I
a
o 1 2 s . 4

Days After lnoculatlon

Fig. 5.2. Three day time course of chlorogenic acid (CGA) accumulation in tuber discs
(cv. Atlantic), where the concentration of CGA was measured in micro moles (pM) per
two tuber discs sample at 328 nm. Tuber discs were inoculated with A, RL 95
(nonpathogen); B, RL 232 (common scab pathogen); and C, D.P. (deep pitting
pathogen).

148

CGA accumulation were not speciﬁc for either cultivar of potato. After 3 days, a two
disc sample of Atlantic tissue on average accumulated 0.572 p moles of extractable CGA
and uninoculated controls 0.576 p moles of extractable CGA. After 3 days, a two disc
sample of Russet Burbank tissue on average accumulated 0.640 p moles of extractable
CGA and the uninoculated controls 0.578 p moles of extractable CGA.

The amounts of CGA extracted from Russet Burbank tuber discs were similar for
all isolates over the 3 day time course (Fig 5.1 A-C). The amounts of CGA extracted
from Russet Burbank tuber discs inoculated with RL 95 and RL 232 were not
signiﬁcantly different from the uninoculated controls (Fig. 5.1 A and B). Higher
amounts of CGA were extracted from Russet Burbank tuber discs inoculated with D.P. ,
than uninoculated controls at day 2 and 3 of the time course (Fig. 5.1 C). The amounts
of CGA extracted from Atlantic tuber discs inoculated with RL 232 and RL 95 were
lower than the uninoculated controls over the 3 day time course (Fig. 5.2 A and B). The
amounts of CGA extracted from Atlantic tuber discs inoculated with D.P. were generally
higher than the uninoculated controls (Fig 5.2 A), and higher than the amounts of CGA
extracted from RL 232 and RL 95 inoculated tuber discs at day 2 and 3 of the time
course (Fig. 5.2 A-C).

Deposition of lignin in response to inoculation. Lignin deposition increased steadily
for 3 days after inoculation of Russet Burbank and Atlantic tuber discs (Figs. 5.3 A-C
and 5.4 A-C). The amounts of lignin deposited on Russet Burbank discs inoculated with
RL 232, RL 95, and D.P. were similar to uninoculated controls during the ﬁrst 2 days
of the time course (Fig. 5.3 A-C). Uninoculated Russet Burbank controls produced

nearly identical amounts of extractable lignin as tuber discs inoculated with D.P. , and

149

 

too
+ RL 95 A
‘ —0— Control

200 '

1ooJ

ug Lignin! area disc

 

 

 

 

Days After Inoculation

 

300

—-0— RL 232 B
‘ —0— Control

    

200 "

1 j
, C
1- v r ﬁr v

o 1 2 s 4
Days After inoculation

ug Lignin! area disc

 

 

 

 

—e— D.P. C
‘ —0— Control

ug Lignin! area disc

 

 

 

 

Days After Inoculation

Fig. 5.3. Three day time course of lignin accumulation in tuber discs (cv. Russet
Burbank), where the concentration of lignin was measured in micrograms (pg) per unit
area discs at 280 nm. Tuber discs were inoculated with A, RL 95 (nonpathogen); B, RL
232 (common scab pathogen); and C, D.P. (deep pitting pathogen).

 

150

 

300

+ RL 95 A
‘ —'0-— Control

200 'I

100‘

an Lignin! area disc

 

 

 

 

0 1 2 3 4
Days After Inoculation

 

—e— 121.232 3
‘ —0— Control

 

ug Lignin! area disc

 

 

 

Days After Inoculation

 

too

3 + D.P. C
; ‘ —0— Control
C

2

C

3

c

a

:1

o

:

 

 

 

 

Days After inoculation

Fig. 5.4. Three day time course of lignin accumulation in tuber discs (cv. Atlantic),
where the concentration of lignin was measured in micrograms (pg) per unit area discs
at 280 nm. Tuber discs were inoculated with A, RL 95 (nonpathogen); B, RL 232
(common scab pathogen); and C, D.P. (deep pitting pathogen).

 

151

less lignin was extracted from the D.P. inoculated discs than Russet Burbank tissue
inoculated with RL 232 and RL 95 after three days (Fig. 5 .3 A-C).

Generally, less lignin was deposited on uninoculated Atlantic controls, than on
Atlantic tuber discs inoculated with RL 232, RL 95, and D.P. (Fig. 5.4 A-C). After,
24 hours nearly identical amounts of lignin were extracted from uninoculated Atlantic
tuber discs and discs inoculated with D.P. (Fig. 5.4 A-C). However, less lignin was
extracted from Atlantic tuber discs inoculated with D.P. than from Atlantic tuber discs
inoculated with RL 232 and RL 95 (Fig. 5.4 A-C).

Inhibitory effect of CGA on Streptomyces growth. The isolates RL 232, RL 95, and
D.P. were able to grow normally in the presence of CGA at all concentrations (Table
5.1). The isolate RL 232 was slightly inhibited at 1 mg/ml CGA. The nonpathogenic
isolates 38 (Wallace and Hammerschmidt) and Streptomyces lividans (donated by
W. Champness) were able to grow in the presence of CGA, but at 1 mg CGA/ml the
isolates produced scant sporeless mycelium (Table 5.1).

DISCUSSION

The physiological basis for resistance of potato to S. scabies and other potato
scab-causing streptomycetes is not understood. The periderm appears to facilitate
resistance by acting as a physical barrier to infection (Cooper et al, 1954; Emilsson and
Heiken, 1956; and Hooker and Page, 1960). However, Hooker and Page (1960)
illustrated that periderm was not entirely responsible for resistance to infection by
showing that spores were unable to grow on living potato tuber discs. It was difﬁcult
to detect Streptomyces growth on Russet Burbank or Atlantic tuber discs in my

experiments, even after 3 days. However, electron microscopy has revealed that RL

152

TABLE 5 .1. The effect of chlorogenic acid on the growth of Streptomyces

 

 

 

 

 

Isolates'
RL 232 D.P. RL 95 38 SJ.
Concentration”
of

CGA (mg/ml)

0.005 4c 4 4 4 4
0.01 4 4 4 4 4
0.1 ' 4 4 4 4 4
0.5 4 4 4 3 3
1.0 3 4 4 l l

 

a Characteristics of the isolates shown in Tables 2.2 and 2.3 this dissertation.

b Yeast extract malt extract agar (YEME) supplemented with varying concentrations (mg/ml) of
chlorogenic acid (CGA).

c Visual rating of growth based on comparison to growth of bacteria on unsupplemented (YEME),
Bestgrowth = 4andnogrowth = 0.

153

232, D.P. and RL 95 spores germinate and grow over the surface of tubers (Stein and
Hammerschmidt, unpublished data). It may be assumed that after spore germination
mycelia spreads over the disc surface (this is true for nonpathogenic strains as well), but
only pathogenic strains infect the tissue. By using very high concentrations of spores,
I have induced visible growth of aerial mycelium on living tuber discs.

Hooker and Page’s (1960) study suggests that resistance to potato scab is coupled
to the natural wound healing responses. Phenolic compounds such as CGA and
precursors of lignin and suberin accumulate in wounded and infected potato tissues
(Vance et al, 1980; and Hammerschmidt, 1984 and 1985). Johnson and Schaal (1952)
suggest that CGA may be a source of resistance to S. scabies infection. Russet
Burbank a particularly scab-resistant cultivar was found to have much higher amounts of
CGA in the peel than the scab-susceptible cultivar Triumph (Johnson and Schaal, 1957).
Johnson and Schaal (1957) observed that CGA levels in potato periderm were highest
when tubers undergo their most rapid growth, which oddly coincides with the period in
which stomata differentiate into the unsuberized lenticels that appear to provide the
natural portals for infections to occur.

My study showed the amounts of extractable CGA in freshly cut tuber discs of
Atlantic and Russet Burbank were roughly similar (Figs. 5.1 and 5.2). Therefore the
constitutive levels of CGA in the medullary tissues for both cultivars were similar. Also
similar amounts of CGA were extracted from uninoculated controls of Russet Burbank
and Atlantic at day 3 of the time course (Figs. 5.1 and 5.2). The amounts of CGA
extracted from Russet Burbank tuber discs inoculated with RL 232 or RL 95 were nearly

identical to the amounts of CGA extracted from the uninoculated controls over the course

154

of three days. However, D.P. inoculated discs accumulated signiﬁcantly higher levels
of extractable CGA than the controls two days after inoculation (Fig. 5.1 A-C). The
amounts of CGA extracted from Atlantic tuber discs inoculated with RL 232 and RL 95
were generally lower than the uninoculated controls (Fig 5.2 A and B). However, the
amounts of CGA extracted from D.P. inoculated Atlantic tuber discs were generally
higher than the uninoculated controls at days 2 and 3 of the time course (Fig 5.2 C).
When incorporated in YEME medium, chlorogenic acid appeared to exert
minimal inhibition on the growth of RL 232, RL 95, and D.P. (Table 5.1). CGA at the
concentration of 1 mg/ml approximately corresponds to 2.8 p mole CGA/ml of media.
This concentration (1 mg/ml) approximated the average amount of CGA/ml extracted
from a single inoculated tuber disc, which was roughly 3 p mole CGA/ml/ disc of Russet
Burbank or Atlantic. The isolates 38 and 8.1. were inhibited by the highest concentration
of CGA incorporated into YEME. Schaal and Johnson (1955) demonstrated that CGA
and other phenolic compounds inhibited growth of pathogenic streptomycetes. When
chlorogenic acid was applied to ﬁlter discs it exerted the greatest inhibitory effect at
concentrations of 4 mg/disc and 6 mg/disc at pH 8.5 (Schaal and Johnson, 1955).
However, my experimental design was such that the CGA was incorporated directly into
the medium thus effectively lowering the concentration of CGA in any region.
Therefore, the inhibitory effect demonstrated by Schaal and Johnson’s study appears to
result from the CGA concentration being much higher in the region immediately
surrounding a ﬁlter disc. As the photographs accompanying their article illustrated,

inhibition of growth occurred only a few millimeters away from the CGA saturated disc

155
(Schaal and Johnson, 1955). From my experiments it appears that CGA has minimal

effect on inhibiting the growth of pathogenic Streptomyces.

Ligniﬁcation of tissues is thought to be part of the host defense responses (Vance
et a1, 1980). Hammerschmidt (1984) demonstrated that the rate of ligniﬁcation of potato
tuber discs was higher in response to a nonpathogenic fungal species than to a pathogenic
fungal species. The pattern of lignin deposition in response to streptomyces infection
was similar to that reported by Hammerschmidt (1984). Tuber discs of Atlantic and
Russet Burbank inoculated with RL 232 and RL 95 accumulated greater amounts of
extractable lignin than discs inoculated with D.P., 24 after inoculation (Figs. 5.3 and
5.4). The response of tuber discs to D.P. was similar to that of potato toward the
pathogen Fusan'um sambucinum, within 1 day (Fig. 5.4) and 2 days (Fig. 5.3). It
appears as though F. sambucinum was suppressing the ligniﬁcation response or
inhibiting the conversion of phenolics into lignin (Hammerschmidt, 1984). The ﬁndings
of my study were consistent with those of Hammerschmidt (1984), because less lignin
was extracted at days 1 and 2 from Russet Burbank discs (Fig. 5.3 B and C) and at day
1 from Atlantic discs (Fig. 5.4 B and C) inoculated with D.P., the most virulent isolate,
than tuber discs inoculated with RL 232. This could possibly explain how deep-pitted
lesions are produced by D.P. It may be possible that D.P. prevents the conversion of
phenolics into lignin thus becomes able to penetrate much further into susceptible tissues
than RL 232 and RL 95.

Inoculation of tuber discs with pathogenic streptomycetes may serve as a rapid
and simple method to screen for potato scab resistance. The isolates RL 232 and D.P.

grew on tuber discs of Atlantic and produced superﬁcial damage 7 days after inoculation.

156

None of the tested isolates were able to infect Russet Burbank or cause observable
damage even after 7 days of growth. Thus a screening program may be developed in
which scab resistant potato cultivars are selected on the basis of their ability to rapidly
synthesize high levels of lignin in response to inoculation with highly virulent
streptomyces isolates. Also potential resistance to potato scab could be indicated by the
inability of streptomyces to grow on potato tuber discs.

The contribution of chlorogenic acid and lignin synthesis in resistance of potato
toward S. scabies infection was not clearly demonstrated by these experiments.
However, chlorogenic acid and lignin apparently accumulated in tuber discs inoculated
with Streptomyces. My experiments suggested that the patterns of CGA and lignin
accumulation in response to Streptomyces were analogous to the types of CGA and
lignin responses elicited by fungal pathogens of potato. Based on these observations,
D . P. expressed a compatible-type interaction with the susceptible Atlantic cultivar, where
the pathogen grew without eliciting a hypersensitive response from the host. While, RL
232 and RL 95 possibly induced incompatible responses and were recognized as
pathogens and caused rapid synthesis of the previously mentioned resistance related

compounds.

 

157
REFERENCES

Adams, M.J. 1975. Potato tuber lenticels: development and structure. Ann. App. Biol.
79: 265-273.

Archuleta, J .G. , and Easton, GD. 1981. The cause of deep-pitted scab of potatoes. Am.
Potato J. 58:385-392.

Cooper, D.C. Stokes, G.W., and Rieman, G.H. 1954. Periderm development of the
potato tuber and its relationship to scab resistance. Am. Potato J. 31:58-66.

Cottle, W., and Kolattukudy, PE. 1982. Biosynthesis, deposition, and partial
characterization of potato suberin phenolics. Plant Physiol. 69:393-399.

Doering-Saad, C., Kampfer, P., Manulis, S., Kritzman, G., Schneider, J ., Zakrzewska-
Czerwirrska, J ., Schrempf, H., and Barash, I. 1992. Diversity among Streptomyces
strains causing potato scab. Appl. Environ. Microbiol. 58:3932-3940.

Emilsson, B., and Heiken, A. 1956. Studies on the development and structure of the
periderm of the potato tuber in relation to scab resistance. Acta Agric. Scand. 4:229 242.

Fellows, H. 1926. Relation of growth in the potato tuber to the potato-scab disease. J.
Agr. Res. 32:757-781.

Friend, J ., Reynolds, SB, and Aveyard, M.A. 1973. Phenylalanine ammonia lyase,
chlorogenic acid and lignin in potato tuber tissue inoculated with Phytophthora infestans.
Physiol. Plant Pathol. 3:495-507.

Hammerschmidt, R., 1984 Rapid deposition of lignin in potato tuber tissue as a response
to fungi non-pathogenic on potato. Physiol. Plant Pathol. 24:33-42.

Hammerschmidt, R. 1985. Determination of natural and wound-induced potato tuber
suberin phenolics by thioglycolic acid derivatization and cupric oxide oxidation. Potato.
Res. 28:123-127.

Hammerschmidt, R., and Lacy, M.L. 1990. Disease of potato: common scab. in: Best
Management Practices for Potatoes. Cooperative Extension Service, Michigan State
University.

Healy, F.G. , and Lambert, DH. 1991. Relationships among Streptomyces spp. causing
potato scab. Int. J. Syst. Bacteriol. 41:479-482.

158

Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Burton, C.J., Kieser, H.M.,
Lydiate, D.J.,Smith, C.P., Ward, J .M., and Schrempf, H. 1985. Genetic manipulation
of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich, United
Kingdom.

Hooker, W.J., and Page, OT. 1960. Relation of potato tuber growth and skin maturity
to infection by common scab. Streptomyces scabies. Am. Potato J. 37:414-423.

Johnson, G., and Schaal, LA. 1952. Relation of chlorogenic acid to scab resistance in
potatoes. Science 115:627-629.

Johnson, G., and Schaal, LA. 1957. Chlorogenic acid and other orthodihydricphenols
in scab-resistant russet burbank and scab-susceptible triumph potato tubers of different
maturities. Phytopathology 47:253-255.

Jones, A.P. 1931. The histogeny of potato scab. Ann. Appl. Biol. 18:313-333.

Kolattukudy, PE, and Agrawal, VP. 1974. Structure and composition of aliphatic
constituents of potato tuber skin (suberin). Lipids 9:682-691.

Lambert, DH, and Loria R. 1989 a. Streptomyces scabies sp. nov., nom. rev. Int. J.
Syst. Bacteriol. 39:387-392.

Loria, R. , and Kempter, BA. 1986. Relative resistance of potato tubers produced from
stem cuttings and seed-piece-propagated plants to Streptomyces scabies. Plant Dis.
70:1146-1148.

Lutman, B.F . 1913. The pathological anatomy of potato scab. Phytopathology 3:255-264.
Lutman, B.F. 1941. Actinomyces in potato tubers. Phytopathology. 31:702-717.

Millard, W.A., and Burr, S. 1926. A study of twenty-four strains of Actinomyces and
their relation to types of common scab of potato. Ann. Appl. Biol. 13:580-644.

Nicholson, R.L., and Hammerschmidt, R. 1992. Phenolic compounds and their role in
disease resistance. Annu. Rev. Phytopathol. 30:369-389.

Patil, S.S., Zucker, M., and Dirnond, A.E. 1966. Biosynthesis of chlorogenic acid in
potato roots resistant and susceptible to Verticillium albo-atrum. Phytopathology 56:971-
974.

Ride, J .P. 1978. The role of cell wall alterations in resistance to fungi. Ann. Appl. Biol.
89:302-306.

 

159

Schaal, L.A., and Johnson, G., 1955. The inhibitory effect of phenolic compounds on
the growth of Streptomyces scabies as related to the mechanism of scab resistance.
Phytopathology 45:626-628.

Shirling, E. B., and Gottlieb, D. 1966. Methods for characterization of Streptomyces
species. Int. J. Syst. Bacteriol. 16: 313- 340.

Vance, C.P., Kirk, T.K. , Sherwood, RT. 1980. Ligniﬁcation as a mechanism of disease
resistance. Annu. Rev. Phytopathol. 18:259-288.

CONCLUSIONS

160

161

From my research it has been possible to draw several conclusions about the
physiology of potato scab disease. The isolate D.P. , a strain of Streptomyces with the
ability to produce deep-pitted scab on potato, bears many physiological similarities to the
isolate RL 232 which causes common scab of potato. Both isolates degraded several of
the same substrates, had similar tolerances for antibiotics, produced thaxtomin in culture,
and the electrophoretic patterns of the soluble proteins extracted from whole cells were
very similar although not identical. Inspite of their physiological similarities D.P. and
RL 232 should not be considered to belong to a single species of Streptomyces. The
facts that D.P. does not produce melanoid pigments in culture, bears its spores in
ﬂexuous chains, and causes deep-pitted scab constitutes signiﬁcant variations from the
typical physiology associated with S. scabies. The D.P. isolate also differed from the
deep-pitting isolates of Archuleta and Easton (1981) which produced melanoid pigments
in culture, and from the Faucher et al (1992) deep-pitting isolate which only used
raffmose as a sole carbon source. Physiological characterization of the potato scab-
causing streptomycetes has not established the phylogeny of this group. Therefore, the
relatedness of D.P. to S. scabies and other potato scab-causing streptomycetes should be
veriﬁed by DNA homology or ﬁnger printing by the polymerase chain reaction method.

The ability to degrade pectin has been cited as a possible means by which S.
scabies and other pathogenic streptomycetes invade potato tissues or survive as
saprophytes on plant debris (Lutman, 1923 and 1945). From my work, it appears that
the ability to degrade pectic substrates was not restricted to the pathogenic
streptomycetes, as several nonpathogens produced pectic enzymes in culture. Pectolytic

enzymes were induced in culture by both pectin and sodium polypectate for pathogenic

162

and nonpathogenic streptomyces. The enzyme activities detected in culture ﬁltrates
suggested that endopectate or endopectin lyases were induced in culture, as well as an
enzyme which could degrade pectin between pH 5 and 6, which could have been an
exopectin lyase or an exopolygalacturonase. The literature however does not indicate
that polygalacturonases (PG) degrade pectin (Rombouts and Pilnik, 1985). Other
researchers have found that Streptomyces species produce endopectate lyases which
exhibit activity on pectin substrate (Sato and Kaji, 1973).

Knosel (1970) could not establish a correlation between the ability to produce
pectinase in culture and virulence of S. scabies toward potato. My work concurred with
Knosel’s, as I was unable to ﬁnd any correlation between the ability to produce
pectinases in culture and pathogenicity. This was illustrated by the fact that pectinase
preparations from nonpathogenic streptomyces which were capable of degrading pure
pectic substrates also caused maceration of potato tuber discs. Sato and Kaji (1973)
demonstrated that culture ﬁltrates from S. fradiae were able to cause extensive tissue
maceration of potato and several other plants. However, S. fradie has not been reported
to be pathogenic toward potato. Thus, I regard the ability of streptomycetes to degrade
pectin as a factor that enhances the infection process, rather than constituting the primary
factor in pathogenicity. To test this hypothesis it would be necessary to generate
pectinase deﬁcient mutants from a pathogenic streptomyces isolate and determine whether
the loss of pectin degrading ability results in the loss of pathogenicity or lowered
virulence. The method of mutagenesis used in my research did not produce true

pectinase deﬁcient mutants, the mutants apparently remained able to degraded pectic

 

163

substrates. Interestingly, a mutant was recovered that loss the ability to degrade several
sugars and organic acids without loss of pathogenicity.

Preliminary research showed that nonpathogenic and pathogenic isolates of
Streptomyces, when grown on a dilute slurry of potato tissue, produced extracellular
pectolytic enzymes capable of degrading both pectin and sodium polypectate. I was able
to detect pectolytic activity in the concentrated culture ﬁltrates of RL 232, RL 95, D.P. ,
and 3S grown on the potato slurry medium, using the cup plate assay (Dingle et al,
1953). The culture ﬁltrates of these isolates exhibited activity on polygalacturonic acid
at pH 7 and 9 but not at pH 5, and on pectin at pH 5, 7, 8.5, and 9. Therefore similar
types of pectolytic enzymes were produced by both pathogenic and nonpathogenic
streptomycetes grown on potato tissue, citrus pectin or sodium polypectate. This ﬁnding
suggests that pathogenic Streptomyces species, synthesize pectolytic enzymes while
invading living potato tissue, however it must noted that the potato slurry medium was
autoclaved and the tissue was killed. It was amazing that pectolytic enzymes were
induced at all, the cells of the potato tissue should have been disrupted by autoclaving
making available several substrates more easily utilized than pectin for bacterial growth.
The potato tissue would have supplied numerous sugars, starches, celluloses and other
readily accessible substrates. Bearing this in mind, it would be very interesting to
determine whether other cell wall degrading enzymes and thaxtomin were induced on
potato slurry medium as well.

Synthesis of thaxtomin appears to be a characteristic unique to pathogenic
Streptomyces species (King et al, 1992). Production of thaxtomin by D.P. was

signiﬁcant because this represented a pathogenic factor which could possibly be identiﬁed

164

as a common characteristic among all potato scab-causing streptomycetes. However,
further research needs to done in this area to establish whether species other than S.
scabies, S. acidiscabies, and the deep-pitting isolate D.P. synthesize the phytotoxin. It
would be interesting to determine whether Streptomyces species that infect beets, carrots,
and other root crops produce thaxtomin as well.

The deep-pitting isolate D.P. , elicited a different pattern of host defense responses
from potato tuber discs than RL 232 and RL 95. My ﬁndings supports those of Jones
(1931) which suggested that deep-pitting streptomycetes affect the ligniﬁcation response
in potato tissue in a manner that differs from the common scab-producing streptomyces.
Lowered lignin accumulation in potato tissues infected with deep-pitting streptomyces,
may possibly explain how the D.P. isolate is able to penetrate further into potato
periderm than common scab streptomyces, thus producing deep lesions. High level
lignin deposition in response to infection by common scab-producing streptomyces, may
explain why these streptomyces are conﬁned to the outer layers of the periderm, thus
producing in superﬁcial lesions. However, further work must be done to establish how
early in the infection process the potato tissue recognizes the bacterium and responds to
the infection. Dr. Barry Stein has observed streptomyces spores germinating as early as
6 hours following inoculation of in vitro grown potato tubers and producing an extensive
hyphal network on the tuber surface within 48 hours (Stein et al, 1994). It may be
reasonable then to assume that the pathogenic interactions between streptomycetes and
potatoes are established within 24 hours.

Ultimately the signiﬁcance of my research is found in that it examines both the

infection process and host responses in potato scab disease from a physiological

165

perspective. In this regard, we can see that potato scab-causing streptomycetes
synthesize cell wall degrading enzymes and phytotoxin, which may facilitate the infection
process. Furthermore, potato scab-causing streptomycetes may in fact be necrotrophs
or facultative parasites. Their ability to synthesize cell wall degrading enzymes and
phytotoxin appear to be necessary for them to produce the necrotic tissue required to
establish an infection. This hypothesis appears to be consistent with two observations.
First, the streptomycetes are hardly ever observed to growing in living tissue, and second
streptomycetes seem to cause potato scab in an opportunistic fashion, which appears to
be dependent on the occurrence of several environmental factors. To ultimately develop
effective control measures for potato scab disease the physiological impact of the
interactions between the environment, potato, and the species of streptomyces must be

understood.

166
REFERENCES

Archuleta, J .G., and Easton, GD. 1981. The cause of deep-pitted scab. Am. Potato J.
58:385-393.

Dingle, J. , Reid, W.W. , and Solomons, G.L. 1953. The enzymatic degradation of pectin
and other polysaccharides. 11‘. Application of the cup-plate assay to the estimation of
enzymes. J. Sci. Food Agric. 4:149-155.

Faucher, E. , Savard, T. , and Beaulieu, C. 1992. Characterization of actinomycetes
isolated from common scab lesions on potato tubers. Can. J. Plant Pathol. 14:197-202.

Jones, A.P. 1931. The histogeny of potato scab. Ann. Appl. Biol. 18:313-333.

King, R.R., Lawrence, CH, and Calhoun, LA. 1992. Chemistry of phytotoxins
associated with Streptomyces scabies, the causal organism of potato common scab. J.
Agric. Food Chem. 40:834-837.

Knosel, D. 1970. Utersuchugen zur cellulolytischen und pektolytischen Aktivitat
pﬂanzenschadlicher Actinomyceten. Phytopath. Z. 67:205-213.

Lutman, BR 1923. Potato scab in new land. Phytopathology 13:241-244.

Lutrrran, BE 1945. the spread of potato scab in soil by plant humus. Vermont Agric.
Expt. Sta. Bull. 528:1—40.

Rombouts, F.M., and Pilnik, W. 1980. Pectic enzymes. Pages 227-282 in: Economic
Microbiology: Microbial Enzymes and Bioconversions. Vol. 5, A.H. Rose, ed.
Academic Press, Inc. , New York.

Sato, M. , and Kaji, A. 1973. Macerating activity of Streptomyces fradiae IFO 3439.
Agr. Biol. Chem. 37:2219-2225.

Stein, B. , Hammerschmidt, R. , and Douches, D. 1994. Ultrastructure of Streptomyces
scabies infection of in vitro cultured potato tubers. Phytopathology 83:1414.

 

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