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This is to certify that the

thesis entitled
Use of polyethylene glycol and glycerol

as carriers of antibiotics for reduction
of common blight bacteria in bean seeds

presented by

Lizhe Liang

has been accepted towards fulfillment
of the requirements for

Master's degree in Botany & Plant Pathology

 

 

 

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USE OF POLYETHYLENE GLYCOL AND GLYCEROL AS CARRIERS OF
ANTIBIOTICS FOR REDUCTION OF COMMON BLIGHT BACTERIA

IN BEAN SEEDS

BY

Lizhe Liang

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1991

ABSTRACT

USE OF POLYETHYLENE GLYCOL AND GLYCEROL AS CARRIERS OF
ANTIBIOTICS FOR REDUCTION OF COMMON BLIGHT
BACTERIA IN BEAN SEEDS

BY
Li Zhe Liang

Common bacterial blight of beans caused by Xangngmggas
phagggli is an important disease in Michigan and on a world
wide basis. Internally and externally contaminated seeds are
the main sources.of primary inoculum for the disease. There is
no known acceptable method for eradication of internally borne
blight bacteria. The objectives of this study were to: 1)
determine if "seed priming" with polyethylene glycol (PEG) or
glycerol solutions can be used to introduce antibiotics into
bean seeds without major reduction in seed quality, and 2)
determine if introduced antibiotics can effectively control
internally seed-borne bacteria. Non infected and internally
infected bean seeds were soaked in solutions of PEG and
glycerol, as well as in solutions of these materials
containing the antibiotics streptomycin, tetracycline and
chlorotetracycline. These seeds were evaluated for germination

percentage, seedling vigor, and survival of bacteria.

ii

Prolonged soaking (up to 4 days) in either 25% PEG or 60%

glycerol solutions did not diminish germination while seedling
vigor was slightly reduced. PEG solutions were more effective
than glycerol solutions for introduction of antibiotics into
seeds. Tetracycline and chlorotetracycline in PEG solutions
effectively reduced x&_pna§ggli, but had severe phytotoxic
effects. PEG solutions containing streptomycin reduced, but
did not eradicate internally seed-borne W11 and caused
few phytotoxic effects. Thus, treatment of bean seeds
internally infected with 3M1 with solutions of PEG and
streptomycin provides an effective means of reducing, but not
eradicating primary inoculum of this bacterium. Additional
studies are needed.toiassure: 1) that the suggested treatments
will not reduce the production potential of non infected
seeds, 2) that the treatments can effectively reduce disease
development in the field , and 3) that the treatments do not
provide rapid selection for antibiotic resistant strains of

the bacteria.

iii

ACKNOWLEDGMENTS

I am very grateful to Dr. J. Halloin for his support, his
inspiration, his guidance and his belief in me. I would also
like to acknowledge the members of my guidance committee, Drs.
G.A. de Zoeten, L.P. Hart, and L.0. Copeland.

My thanks to the bean group of United State Department of
Agriculture at Michigan State University, especially Drs. G.L.
Hosfield, J.D. Kelly, J.C. Theurer, and Mr. J. Taylor for the
many forms of help rendered to me.

My thanks to Dr..A.W. Seattler who initiated this project
and passed away during the course of the work.

I am especially grateful to my wife Fang for her love,

patience and support.

iv

TABLE OF CONTENTS

Page
LIST OF TABLES 0.0.0.0.........OOOOOOOOOOOOO0...... Vii
LIST OF FIGURES ....................... ............ ix
INTRODUCTION AND LITERATURE REVIEW ................ 1
Causal agent of disease ...................... 1
Disease description .......................... 3
Disease control .............................. 5
Pathogen eradication from seeds .............. 8
MATERIALS AND METHODS ............. ....... ... ..... . 13
seeds .....OOOOOOOOOOOOO.......OOOOOOOOOOOO0.0 13
media O.......OOOOOOOOOOOOOO0.0.0.000...0....O 13
Effects of PEG and glycerol treatments
on germination .................... ........ 15
Effects of antibiotics in PEG and
glycerol solutions on germination .. ...... . 16
Assay of antibiotics in seeds ....... ......... 17
Production of naturally infected
seeds 0.00.0.0.........OOOIIOOOOOO ........ O 18
Production of artificially infected
seeds I......OOOOOOOOOOOOOOOOOO......0.0... 18
Assay for the presence of internally
seed-borne x‘_pn§§§gli ........... ...... ... 20
The effect of antibiotic residue
on bacterial growth ............. ...... .... 21
Efficacy of eradication of common
blight bacteria ......OOCOOOOOOOOOOOO ...... 22
Prevention of phytotoxicity .......... ........ 23
RESUI‘TS 0.0.0000........OOOOOOOOOOOOO0.0.000....... 24

Effects of PEG and glycerol treatments

on germination ............................ 24
Effects of antibiotics in PEG and

glycerol solutions on germination

and seedling vigor ........................ 29

Effect of PEG and glycerol on

antibiotic uptake .........................
Effect of soaking time on antibiotic

uptake ....................................
Efficacy of seed treatment in

eliminating bacteria from seeds.... ........
Prevention of phytotoxicity ..................

DISCUSSION 0 O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O O O O O O 0 O O O
SUGGESTIONS AND FUTURE STUDIES ....................
APPENDICES O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O ......... O

REFMCES ..........OOOOOOOOOOOOOOOOOO......OOOOOO

vi

33

33

33
39

43

47

48

52

LIST OF TABLES

Table Page

1. Weights of imbibed seeds and seed
germination after soaking in
glycerol and polyethylene glycol
(PEG) solutions for 3 days .......... ........ 30

2. Germination percentages of navy bean
seeds soaked in 60% glycerol or 25%
polyethylene glycol (PEG) mixed with
streptomycin, chlorotetracycline and
tetracycline for up to five days and
seeds soaked only in 60% glycerol
or 25% PEG. Four hundred seeds were
tested in each treatment .................... 31

3. Seedling fresh weights of navy bean
seed soaked in 60% glycerol or 25%
polyethylene glycol (PEG) mixed with
streptomycin, chlorotetracycline and
tetracycline for up to five days
compared with seed soaked only in 60%
glycerol or 25% PEG. Four hundred

seeds were tested in each treatment ......... 32
4. Zones of inhibition in zanthgmgnag

phasegli seeded agar plates around

the embryos and cotyledons of navy

bean seeds soaked in 25%

polyethylene glycol (PEG) or in

60% glycerol mixed with either

400 ppm streptomycin, 400 ppm

chlorotetracycline, or 400 ppm

tetracycline for 1 day ........ ............. 34

vii

10.

Diameters (mm) of inhibition zones in

Xanthomonas phasegli seeded agar
plates around the cotyledons of navy

bean seed soaked in 25% polyethylene

glycol (PEG) or 60% glycerol mixed

with 400 ppm streptomycin (STM), 400

ppm chlorotetracycline (CTC) or 400

ppm tetracycline (TTC) for up to

seventy two hours ................ ...... .....

Effect of application of antibiotics

in 25% polyethylene glycol (PEG) and

60% glycerol on recovery of common

blight bacteria from artificially

infected navy bean seeds. Four

hundred seeds were tested in each

treatment ......................... ..........

Effect of application of antibiotics

in 25% polyethylene glycol (PEG) and

60% glycerol on recovery of common

blight bacteria from naturally

infected seeds. Four hundred seeds

were tested in each treatment ...... ........ .

Effect of streptomycin concentration

in 25% polyethylene glycol on

recovery of Xanthomonas phagggli and

fresh weights of seedlings from

naturally infected seeds soaked for

3 days ............ ..... .......... ...........

Effect of soaking time on recovery of

common blight bacteria and fresh

weights of seedlings from naturally

infected seeds when seeds were soaked

in solutions of 25% polyethylene

glycol and 1600 ppm streptomycin

solutions ........................... ........

Effects on germination percentages and

seedling fresh weights of resoaking

seeds in NaOCl with different soaking

time following presoaking seeds in

800 ppm chlorotetracycline and

tetracycline or 25% polyethylene

glycol for 24 hours ............ .............

viii

35

37

38

4O

41

42

LIST OF FIGURES

Figures Page

1. Germination percentages of navy bean
seeds soaked in different
concentrations of polyethylene
glycol for up to 5 days. Non soaked
seeds were used as controls. Four
hundred seeds were tested in each
treatment ......................... ...... .... 25

2. Germination percentages of navy bean
seeds soaked in different
concentrations of glycerol for up
to 5 days. Non soaked seeds were
used as controls. Four hundred seeds
were tested in each treatment ............... 27

ix

INTRODUCTION AND LITERATURE REVIEW

Common bacterial blight is one of the most serious seed-
borne diseases of dry, edible beans (Ehaggglug yglggnis L.)
throughout the world (Saettler, 1989; Sutton and Wallen, 1970;
Zaunmeyer and Thomas, 1957) . The major sources of primary
inoculum for the disease are internally and externally
contaminated seeds (Saettler and Perry, 1972). Certification
programs for the production and testing of pathogen-free seed
are helpful; however, blight outbreaks persist because many
growers plant seed sources other than those considered to be
pathogen-free. Effective treatments to eradicate internally
borne blight bacteria from.bean seed would contribute greatly

to an integrated strategy for disease management.

usla o 'sas

Common blight incited by Wag phasegli (E. F.
Smith) Dowson and fuscous blight incited by W

phaseoli var. fuscans (Burkh.) Starr and Burkh. are important

diseases of most commercial bean (Phaseolus w L.)
cultivars. Common blight was first reported by Beach in 1892.

At about the same time, Halsted (1892) reported a similar

2
disease of bean pods and seeds. Smith described the etiology
of common blight in 1897 and named the causal bacterium
Bacillus phaseoli E. F. Smith (1393, 1901). Smith (1905)
further characterized the pathogen and transferred it into the
genus Egegggmonas in 1901, then into the genus Bacterigm in

1905. Bergey's ZManual ed. II (1925) renamed the blight

bacteria Ehytgmgngg phaseoli (E. F. Smith) Bergey e; 31.. In

1938 Dowson placed the bacterium in the currently accepted
genus zenthgmensso

Fuscous blightuwasidiscovered.by‘Burkholder in 1924 on an
undetermined bean cultivar from.Switzer1and. Disease symptoms
were identical to common. blight and the only' means of
distinguishing the diseases was by culturing the causal
bacterium. zgnthgmgnag phaseoli var. figgggns was initially
known as Phytomogas phasegli var. figscans but later was placed
in the currently recognized genus Xanthgmgggs.

zanthgncnas phesegli and 3. 22999911 var. fussed: are
obligately aerobic, gram negative, straight rods, which
produce a yellow, nondiffusible pigment and are motile by a
single polar flagellum. Both bacteria produce hydrogen
sulfide, liquefy gelatin, proteolize milk, hydrolyze starch
and Tween 80 and produce an alkaline reaction in phenol red
dextrose agar (Buchanan and Gibbons, 1974). The bacteria are
differentiated by production of a brown diffusible pigment
produced by z. pnggggli var. fugggns (Basu and Wallen, 1967;

Basu, 1974; Hayward and Waterston, 1965a, 1965b), but are

3
otherwise physiologically and biochemically identical.
Bergey's Manual 8th ed. currently recognizes x. phasegli and

2(- ptheQLi var. fusgags as nomen species of mm
gampggtgig (Buchanan and Gibbons, 1974).

e s t' n
W phaseoli and X- p_h_a_§_e_9_].i var. fiuscans produce

visible disease symptoms on all plant parts except the roots;
symptomatology of the two diseases is essentially identical.
However, Zaumeyer and Thomas (1957) reported that z. phggggli
var. fiusgans may cause a slight hypertrophy in tissue around
a stem lesion and, in seedlings, a darkening of the stem
around the point of inoculation.

Leaf symptoms are the most diagnostic features of the
blights. Disease expression is first visible as minute water-
soaked spots on the abaxial surface of the leaf with yellow
discoloration opposite the spot on the adaxial side. Lesions
then enlarge irregularly, dry' out and, become brown. and
brittle; occasionally a slight crust of bacterial exudate is
present. The necrotic area is surrounded by a distinctive
yellowish halo-like zone. Several lesions on a leaf may
coalesce, and eventually occupy most of the leaf area and
cause premature leaf drop (Burkholder, 1930; Zaumeyer, 1932).

Stem lesions begin as water-soaked dark green spots which

eventually become dry, sunken and reddish brown in color. The

4
lesions usually extend longitudinally up the stem but show
little downward movement. Stem symptoms are most common on
seedlings and less evident as the plant matures (Burkholder,
1930; Wallen and Jackson, 1975).

Pod symptoms appear as dark green, water-soaked spots
which later become dry and sunken. Drying begins at the outer
edge of a lesion and extends inward; a yellow crustation of
bacterial exudate may cover the lesion (Burkholder, 1930) . Pod
infection can result in seed infection. 0n white-seeded bean
cultivars infection is indicated by a shiny yellow spotting of
the seed coat, the extent of which varies from small blotches
to complete seed discoloration. Infection of dark seeded bean
cultivars is harder to detect due to seed coat pigmentation;
symptoms appear as a darkening of the seed coat. Seeds which
are infected early in development may be completely shrivelled
or badly wrinkled, whereas those infected near pod maturity
are darkened only at the hilum (Burkholder, 1930) . Seed
infection is most probable when the dorsal pod suture is
infected. The bacteria invade the funiculus, pass through the
raphe, and finally into the seed coat (Zaumeyer, 1929) . Seeds
also may be infected through the micropyle if the bacteria
invade the pod cavity (Zaumeyer 1930).

Common and fuscous blights are major bacterial diseases
of dry beans (Saettler, 1989). This disease is found in most
countries where beans are grown (Hayward and Waterston, 1965a)

and yield losses can be heavy. In 1967, at least 75% of

5

Michigan's 650,000 acres of navy beans were damaged by common
blight, with 10-20% yield reduction (Anonymous, 1971) . Wallen
and Jackson (1975) reported a 38% yield loss in Ontario,
Canada due to common blight in two years of field trials.
Aerial infrared photographic surveys suggested that losses for
the bean crop grown in Ontario ranged from 1252 tons in 1970
to 218 tons in 1972 (Jackson and Wallen, 1975; Wallen and
Jackson, 1975). Yield losses estimated at 22% and 45% have
been obtained by natural and artificial infections,
respectively, in Colombia (Yoshii g; a1" 1976). Economic
surveys, based upon field observations in the same region,
estimated yield losses of 13% due to common blight bacteria
(Pinstrup-Andersen gt g1. , 1976) .

Establishment of the disease in crops usually originates
from infected seeds in which the bacteria can survive for many
years. Thus, seed treatments to eradicate common blight
bacteria could provide important means of disease control,

provided such treatments are effective and ecologically sound.

'ses cot

Strategies to control common blight bacteria on beans
include, chemical control in the field, genetic control using
resistant and tolerant cultivars, cultivation practices, and
use of pathogen-free seed obtained either by eradicative

treatment or certification schemes.

6

Various chemical controls that have been found effective
include: sprays of 50% copper hydroxide and 40% potassium
(hydroxymethyl) methyl dithiocarbamate, Bordeaux mixture, 5%
puratized spray and cuprox dust (copper oxychloride) , and
sprays of 1000 ppm streptomycin sulfate (Burke and Starr,
1948; Edgerton and Moreland, 1913; Weller and Saettler, 1976) .
However, Marlatt (1955), found that streptomycin failed to
control common blight when used as a spray. The following year
Gray (1956) demonstrated that the addition of a wetting agent,
Lg. , glycerol, greatly increased the effectiveness of
streptomycin sprays in controlling common blight in greenhouse
plants. Generally, chemical field treatments for the control
of the disease have not proven to be sufficiently effective to
be commercially useful.

Long term hope for controlling bean blights rests in the
development of resistant and tolerant commercial cultivars of
mm spp. (Ekpo, 1975). Some such cultivars do exist,
however, their wide-spread use is by no means assured,
especially with reports that current tolerant and resistant
cultivars support the epiphytic growth of the bacteria on
plant parts and may produce infected seed (Cafati and
Saettler, 1980a, 1980b; Schuster gt 11., 1979).

Cultural practices such as remaining out of bean fields
when plants are wet, and proper sanitation of farm machinery,
are helpful in minimizing the spread of the disease. Crop

rotation, weed control, and plowing refuse under after harvest

7
are recommended practices (Andersen gt al., 1970; Hart and
Saettler, 1981).

Planting of pathogen-free seed is widely recognized as
being of fundamental importance in the control of many seed-
borne diseases (Baker and Smith, 1966; Neergaard, 1979). The
principle of controlling bean blight through the use of
pathogen-free seed has been recognized and applied in many
production areas of the United States and Canada (Copeland gt
al., 1975; Sheppard, 1983; Sheppard, 1983).

Bacterial plant pathogens have been demonstrated to
survive in various ways: in plant residues, associated with
perennial hosts, as epiphytes on nonhosts, in the soil, or in
association with insects (Leben, 1974, 1981; Schuster and
Coyne, 1974, 1975). However, in the case of X- phagggli in
Michigan, contaminated seeds are the major source of primary
inoculum (Weller and Saettler, 1980).

The inability of 3- Meg]; to survive in plant residues
to any significant degree has been demonstrated in New York
(Burkhold, 1930), Ontario (Wallen and Galway, 1979) and
Australia (Wimalajeewa and Nancarrow, 1980) . Saettler and
coworkers (1986) provided evidence that the primary inoculum
of common blight in Michigan is contaminated seed. Several
attempts were made to recover 20 isolates of X- phaseoli from
ten different bean genotypes ranging in susceptibility from
resistant to susceptible, over a 10-year period. Infected bean

plant samples were either left standing, were buried in the

8
soil, or were left on the soil surface at several sites in
Michigan over several different winters. Bean blight bacteria
never were recovered in any of the 191 trials. 3- phggggli
has, however, been shown to survive from one growing season to
the next in the field in some regions with less severe winters
(Schuster, 1955; Thomas and Graham, 1952).

The inability of foliar pathogens to survive long periods
in the absence of a living host is often explained by a low
"competitive saprophytic ability" (Garrett, 1950), i.g., the
lack of physiological attributes that enable successful
colonization of dead organic substances (Gray, 1976; Hirano
and Upper, 1983; Lenon, 1981) . Also, reduced pathogenicity and
lowering of numbers of recoverable antibiotic-resistant x.
phagggli were demonstrated when infested leaves were
repeatedly exposed to freezing and thawing (Lopez, 1984). No

explanation as to why this occurred was provided.
di ' s 5

External contamination with x. phagegli on bean seeds is
easily controlled with streptomycin sulfate (Saettler and
Anderson, 1978) and sodium. hypochlorite (Weller, 1978).
However there are no known acceptable methods for eradicating
internally borne blight bacteria from the seed. Thus, improved
seed treatments are needed. to reduce or eradicate. this

pathogen internally. Among the important conditions in

9

development of eradication protocols are 1) localization of
blight bacteria, i.g internal or external, 2) physiological
state of the bacteria, Lg. hypobiotic resting state or
actively growing and 3) the sensitivity of the seeds, in terms
of germination and vigor, to potential treatment. If indeed,
blight bacteria within the seeds are in a hypobiotic state,
they may be resistant to treatments normally effective on
viable growing bacteria.

Treatments to eliminate the pathogen from seed have
historically been useful (Adimihardja, 1981; Marlatt, 1955;
Neergaard, 1979). A slurry treatment containing streptomycin
sulfate is routinely used on.many Michigan seedlots (Saettler
and Anderson 1978). It is generally thought that this slurry
treatment is helpful against external bacterial pathogens. The
most widely used method to surface disinfest seeds is
submersion in 1-5% NaOCl followed by rinsing with sterile
water (Abdul-Baki, 1974) . Weller (1978) demonstrated that
shaking infested seeds in 1:1 (v:v) commercial bleach:
distilled water solution for 30 seconds consistently
eliminated all surface-borne blight bacteria, and 45 seconds
usually eliminated all surface microorganisms.

Seed treatment that completely eradicates internal blight
bacteria without significantly affecting seedling emergence,
is not available. Still there are many seed treatments that
are helpful. Dihydrostreptomycin has been shown to act

similarly to streptomycin sulfate. These two antibiotics were

10
absorbed by the stem of bean seedlings and translocated upward
to the primary leaves. Within three to four days the chemical
accumulated in sufficient amounts to inhibit the growth and
development of halo and common blight organisms in seeds
(Mitchell g; a;,, 1952, 1953, 1954). Kreitlow (1940) found
that sequential immersion in four solutions reduced blight
incidence in bean seeds from 23.6% to 0.2%: 1) 1:500 mercury
bichloride in di-ethyl ether 2) 1:20,000 brilliant green in
50% ethyl alcohol plus 3% acetic acid 3) 1:500 mercury
bichloride in 70% ethyl alcohol plus 3% acetic acid 4)
1:20,000 gentian violet in 50% ethyl alcohol plus 3% acetic
acid. Person and Edgerton (1939) found that seeds treated for
12-14 minutes in a solution of 1:500 HgCl2 in 70% ethyl
alcohol plus 2% acetic acid provided the best eradicative
treatment for blight. Treatment of seed with dry heat at 80%:
for 35 minutes followed by New Improved Ceresan for 24 hours
was helpful in reducing the number of diseased plants (Burke
and Starr, 1948). Adimihardja (1981) demonstrated that
treatment. with 'tetracycline (800 ppm) in :methanol under
partial vacuum eradicated seed-borne blight bacteria
completely from bean seed. The treatment also reduced
germination and field emergence. Seed immersion in 800 ppm
‘tetracycline and chlortetracycline in methanol for 30 minutes
can help by reducing numbers of bacteria in the seeds. Soaking
longer than 30 minutes reduced germination as a result of

phytotoxicity due to the antibiotics.

11

Many reports indicate that soaking seeds in polyethylene
glycol (PEG) solutions does not impair germination and can
increase seedling resistance to adverse effects of environment
(Fu gt g;., 1988; Heydecker, 1973/1974; Khan gt al., 1978;
Muhyaddin and Wiebe, 1989) . This treatment called osmotic
conditioning may thus be an efficient means to introduce
antibacterial materials into bean seeds infected with
pathogenic bacteria.

Some research has been devoted to using osmotic
conditioning as an aid in eradication of microorganisms from
the seeds. Saettler (unpublished) showed that seeds incubated
up to 5 days in a 65% glycerol solution containing 800 ppm
chlorotetracycline retained normal viability. Antibiotic
activity was found inside the seeds using standard bioassay
procedures. Hepperly and Sinclair (1977) used.PEGrsolutions to
introduce penicillin G and streptomycin into soybean seeds.
Antibiotic activity was detected in all seed parts after 15
weeks of storage. Thus, simultaneous osmotic conditioning of
seeds and application of bactericides may provide a means for
delivery of the bactericides into seeds without deleteriously
affecting germination potential.

This study assesses the potential of osmotic conditioning
as an aid in eradicating or reducing the incidence of X-
pnggggli from bean seeds. The objectives are to: (1) assess
the impact of concentration and time of immersion in PEG and

glycerol solutions on navy bean seed germination, (2) test.the

12
efficacy of PEG and glycerol solutions for introducing
antibiotics into seeds, (3) test whether PEG or glycerol can
cause phytotoxicity when mixed with antibiotics, and (4)
determine if these treatments are effective_in eliminating

common blight bacteria from internally contaminated seeds.

MATERIALS AND METHODS

M5

Seeds of commercial cultivar C-20, a white-seeded navy
bean (Phggggltgflyglggtig L.), were obtained from the Michigan
Foundation Seed Association. Seeds were stored dry at room

temperature for 9 months before use in this study.

Mgiié

All chemicals, unless stated otherwise, were purchased
from Sigma Chemical Corporation, St. Louis, MO. Media were
autoclaved for 20 minutes before pouring into 9 cm diameter
disposable plastic petri plates. Agar plates were allowed to
cool to room temperature overnight before use.

Bacto yeast extract (BYE) agar: BYE agar was prepared by
dissolving 10 grams of Bacto yeast extract and 15 grams of
Bacto agar in 1000 ml of 0.01 M sodium phosphate buffer (pH
7.2), and autoclaving. BYE agar seeded with X- phgseoli was
used for assaying of antibiotic activity in seeds.

Bacto yeast extract (BYE) liquid medium: BYE liquid media

was prepared by dissolving 1 gram Bacto yeast extract in 1000

13

14
ml of 0.01 M sodium phosphate buffer (pH 7.2) , and
autoclaving. This medium was used for increasing Wt;
for the production of artificially infected seeds.

Yeast extract calcium carbonate agar (YCA): YCA was used
as a standard medium for the recovery of z. phaseoli . It
contained 10.0 g yeast extract, 2.5 g calcium carbonate, and
15.0 g Bacto-agar per liter of distilled water. Colonies of L.
W on YCA were not visible until 24 hours of incubation
at 27°C. mm; colonies first appeared as small yellow,
circular and convex colonies with entire margins. After 3 days
of incubation, isolates of M var. Luggagg were
differentiated by the presence of a brown, water soluble
diffusible pigment on this medium.

Semi-selective medium (SSM) : SSM was used to isolate and
confirm the identity x._p_hg§_e_9_li. The SSM was prepared by
dissolving 1.0 g of yeast extract, 15.0 g Bacto-agar, and 8.0
g soluble potato starch in 970 ml 0.01 M sodium phosphate
buffer (pH 7.2). Six ul of a 1% aqueous solution of methyl
green and three ul of a 1% solution of methyl violet 2B in 20%
ethanol were added. The protocols for preparation of stock
solutions were as outlined by Trujillo and Saettler (1980).
Stock solutions of antibiotics were filter-sterilized and
stored at 4°C. the final amount of antibiotics per liter of
SSM were: 25.0 mg cycloheximide, 2.0 mg nitrofurantoin, 1.0 mg

nalidixic acid, and 0.5 mg gentamycin sulfate. Some non L.

my; bacteria grow on SSM, however, the x._phg_s_e_g;1 are

15

clearly differentiated on the basis of colony morphology.
Colonies of zt_png§ggli and.zt_phg§ggli var. tugggng appeared
as light yellow, convex, slimy colonies with entire margins
and a zone of starch hydrolysis. With further incubation the
regions of starch hydrolysis expand and coalesce. Isolates
that are fuscans variants do not produce the brown pigment in
SSM.

Liquid semi-selective medium (LSSM): LSSM was used for
selectively enhancing growth of K- phasggli and xt_phg§gg;i
var. Luggagg while inhibiting the growth of contaminating
bacteria. LSSM was prepared by dissolving 1.0 g yeast extract
in 970 ml 0.01 M sodium phosphate buffer (pH 7.2) , sterilizing
by autoclaving 20 minutes, allowing to cool to 25°C, and
aseptically adding following antibiotics: 25 mg cycloheximide,
2 mg nitrofurantoin, 1.0 mg nalidixic acid, and 0.5 mg
gentamicin. Preparation of stock solutions of the four
inhibitors was outlined by Trujillo and Saettler (1980). LSSM
was dispensed aseptically into sterilized test tubes for

recovery of x. phgsgoli from contaminated seeds.

8 0 f G -1d 0, C’ O 1‘: "‘1 : 01 "Qu.!- '0

Polyethylene glycol 8000 powder (Formerly PEG 6000) was
dissolved in distilled water at room temperature. Samples of
400 hundred seeds were soaked in 100 ml solutions with 10%,

15%, 20%, 25% and 30% PEG, or in solutions containing 50%,

16
60%, and 70% glycerol. The seeds were soaked at 20°C for one
.to five days (Fermentation occurs when seeds are soaked for up
to 4 days at 25°C) . After soaking, seeds were washed for 20
seconds in tap water and dried in a laminar flow hood for 16
hours at 25°C. Germination tests were performed by using the
between paper (BP) method according to the International Rules
for Seed Testing (ISTA, 1985). Seeds from each treatment were
divided into four equal portions and placed in rolled towels.
The towels were wrapped in plastic bags and kept in an upright
position at a constant temperature of 25°C. Germination
percentage and fresh weights of seedlings were determined
after 7 days. Numbers of normal and abnormal seedlings were
noted during the counting (ISTA, 1979). Nontreated seeds were

germinated in the same manner as controls.

s 9- a ' ot'cs i 'G .. . . eo s-u '03 on
germination

Samples of 400 seeds were soaked in 100 m1 solutions of
25% PEG or 60% glycerol mixed with 400 ppm or 800 ppm
streptomycin sulfate (Sigma Chemical Co. , ST Louis, MO 63178) ,
400 ppm or 800 ppm chlortetracycline hydrochloride (United
States Biochemical Co., Cleveland, Ohio 44128), or 400 ppm or
800 ppm tetracycline hydrochloride (United States Biochemical
Co., Cleveland, Ohio 44128) for one, three or five days. After

soaking, seeds were washed in tap water, dried in a laminar

17
flow hood for 16 hours at 25°C and germinated as described
previously. For testing antibiotic-induced reduction in
seedling vigor, both the germination percentages and fresh
weights of seedlings were recorded on the seventh day of
germination. Seeds soaked only in 25% PEG or 60% glycerol were

used as controls.

Antibiotic activity in seeds was measured by using a BYE
agar. Petri plates of BYE agar seeded with x. phggggli were
prepared by mixing 900 ml BYE agar at 40°C with 100 ml of 109
cells per ml of x. phagggli suspension in 0.01 M phosphate
buffer, pH 7.2, giving a final concentration of 10° bacterial
cells per ml. Twenty ml of the seeded medium were pipetted
into 90 mm diameter petri plates, which then were stored at
4°C prior to use. Seeds soaked for 1, 3, 5, 7, 24, and 72
hours respectively in PEG or glycerol, mixed with either 400
ppm streptomycin sulphate, 400 ppm chlortetracycline
hydrochloride or 400 ppm tetracycline hydrochloride were
assayed. Treated seeds were rinsed three times in distilled
water, halved longitudinally, and seed coats were removed.
Cotyledons and embryos were separated, dried overnight in a
laminar flow hood, and placed on bacteria-seeded BYE agar
plates. Plates were placed in a 28°C incubator for 24 hours

and zones of inhibition were measured from the edge of the

18
assayed piece to the farthest point of clearing. Cotyledons
and embryos of seeds which were soaked only in PEG or glycerol
served as controls. Forty eight seeds were used for each

treatment.

Ezggggtign g: naturally infggtgg §gggs

Naturally infected seeds were produced in field plots at
the Botany and Plant Pathology Farm, Michigan State
University, East Lansing, Michigan. Two isolates of
W mu, and two isolates of X- phggggli var.
figgggng_were used to inoculate navy bean plants. X- phggggli-
susceptible cultivar (C-20) of navy bean was used for the
experiment. A bacterial suspension containing about 105 colony
forming units per ml was prepared by washing a 24 hour-old
culture of X..- 211.346.99.11 from YCA plates and sprayed onto
foliage at 15, 30 and 45 days after planting. One hundred
pounds of seeds were harvested mechanically and assayed for
the presence of internally seed-borne blight bacteria. Seeds
with visible symptoms were selected manually to provide a
sample with 24% internal infection. This sample was used for

the eradication treatments throughout this study.

Ezgdggtign of artificially infgcted seeds

To produce large quantities of seed internally infected

with M w, seeds were inoculated by vacuum

19
infiltration as described by Goth (1966), with the following
modifications. An isolate of z. w var. fts_cgn§ was
grown on YCA plates for 48 hours. Bacteria then. were
transferred into BYE liquid medium. with 18 hours shaking at
25°C. The bacterial suspension used for inoculation was
adjusted to an optical density 0.2 at 620 nm, and contained
approximately 10‘5 colony forming units per ml. Seeds were
surface-disinfected by rinsing in a 1:1 dilution of commercial
bleach (2.6% NaOCl) for two minutes, rinsed three times in
sterile distilled water, blotted with sterile paper towels,
and dried at room temperature in laminar flow hood for 24
hours. Seeds then were submerged in the bacterial suspension
and exposed to a vacuum of approximately 500 mm of Hg for two
minutes. Air bubbles were allowed to escape from the seeds by
shaking the suction flask lightly. At the end of the vacuum
infiltration period the suction was released abruptly, and the
seeds were allowed stand for an additional 1 minute. Seeds
then were blotted dry with sterile paper toweling and left in
trays in a laminar flow hood for 4 hours, and dried at 25%:
for another 48 hours before use. Seeds that appeared to have
broken or cracked seed coats were removed. This procedure
produced seeds with 43% internal infection, which were used

for eradication treatments in this study.

20
ss 0 se e of ' te nal eed- on , z. phagggli

Assays for internally seed-borne X- mu were done as
follows. Seeds were surface disinfected by shaking in a 1:1
commercial bleach-distilled water solution for 1 minute. This
method has been demonstrated to eliminate all surface-borne
blight bacteria (Weller, 1978). Seeds then were rinsed in
sterile distilled water and incubated individually in test
tubes with 3 ml of a liquid, semiselective medium. Seeds were
placed on a shaker at 20°C for up to ten days. Samples of
liquid from tubes showing turbidity were streaked on YCA. Pale
yellow colonies were transferred onto semiselective media
(SSM) . Pale yellow, convex, shining colonies surrounded by
zones of starch hydrolysis were diagnostic for the occurrence
of X- phaseoli. Pathogenicity of isolated bacteria was tested
by infiltrating a bacterial suspension (108 cfu/ml) into 15
day-old Pinto bean seedlings. Pathogenicity was expressed by
development of bacterial lesions within 10 days after
inoculation.

Results from 46 positively identified fuscous blight
bacteria isolations showed that the production of brown
pigmentation on YCA accompanied by a pale yellow colony was
100% correlated with a positive reaction on semiselective
media and with pathogenicity. This showed that accurate
identification of fuscous blight isolates can be obtained by

only examining them on YCA. Results from 154 positively

21
identified non fuscous blight bacteria isolations showed that
use of YCA yielded 22% false positives. The chances for
positive identification using both semiselective media and the
leaf pathogenicity test were 100% with both groups of
bacteria. Thus, accurate identification of non fuscous blight
bacteria can be achieved by examining them both on YCA and on
SSM. Based upon the above observation, the use of YCA for
identifying fuscous isolates and the use of YCA together with
SSM for identifying non fuscous isolates were applied

throughout this study.

t of ' 'ot' es' e

To determine if antibiotic residue in the seeds can
affect bacterial growth in semiselective liquid media, seeds
were soaked in 25% PEG mixed with 800 ppm streptomycin, 800
ppm chlorotetracycline and 800 ppm tetracycline respectively
for 3 days. Seeds then were rinsed in sterile distilled water
and incubated individually in test tubes with 1, 2, 3, 4 or 5
ml of semiselective liquid medium. Seeds then were soaked for
1 day at 5°C to allow antibiotic to diffuse into the liquid
medium. Then, 5 ul of a suspension of blight bacteria with a
concentration of 102 cfu/ml was delivered into each tube.
These tubes were incubated for 7 days on a shaker at 25°C.
Samples of liquid from each.tube were streaked on YCA.and SSM.

Blight bacteria were detected in all tubes, except that those

22
tubes that contain only 1 ml of liquid medium showed less
bacterial colony forming units (Less than 0.7 x 102/m1) . Tubes
which contained from 2 to 5 ml of liquid medium all had more
than 0.7 x 103 colony forming units (Appendix 1). Use of tubes
with 3 ml of liquid medium was selected as a standard

throughout the study.

For eradication treatment, artificially and naturally
infected seeds were soaked for one and three days at 20°C in
solutions containing 25% PEG or 60% glycerol mixed with 400
ppm or 800 ppm streptomycin, 400 ppm or 800 ppm
chlorotetracycline and 400 ppm or 800 ppm tetracycline
separately for one and three days at 20°C. After soaking seeds
were rinsed in sterile distilled water for 15 seconds and
dried at room temperature in a laminar flow hood for 24 hours.
Treated seeds were incubated individually in test tubes with
3 ml of semiselective liquid medium on a shaker for 10 days.
Treated seeds were surface-disinfected with 1:10 commercial
bleach-distilled water for 1 minute immediately before the
incubation to avoid decreased )5. phgseoli recovery due to
interference by saprophytic bacteria. Turbid tubes were noted
and liquid samples were streaked on YCA and SSM plates. Four

hundred seeds were tested in each treatment for artificially

23
infected seeds. Eight hundred seeds were tested in each

treatment for naturally infected seeds.
'0 o t “cit

Seeds treated with tetracycline and chlorotetracycline in
this study showed signs of phytotoxicity, both etiolated
seedlings and reduced seedling fresh.weights. Humaydan gt g1.
(1979) demonstrated that phytotoxicity caused by
oxytetracycline and chlorotetracycline could be overcome by
resoaking seeds 30 minutes in 0.5% NaOCl in the case of
firgssicg. The method thus was applied for prevention of
phytotoxicity in bean seeds. Seeds that were soaked in PEG
solution containing tetracycline for one day were rinsed in
tap water, dried, and immediately resoaked in a freshly
prepared solution of 1:1 and 1:10 commercial bleach (2.6%
NaOCl) for various times. After the final soak, seeds were
rinsed in running tap water and dried in a laminar flow hood

for 24 hours before germination tests.

RESULTS

cts P G c o t tme s o e in 'o

The effect of PEG concentration and soaking time on navy
bean seed germination percentage is presented in Figure 1. The
untreated control seeds had a germination percentage of 85%.
The germination percentage of treated seeds increased with
increasing PEG content of the soaking solution, but decreased
with increasing soaking times. Seeds soaked in 25% and 30% PEG
solutions for one to four days had germination percentages
equal to or greater than that of the control. Seeds soaked
longer than one day in solutions with 20% or lower PEG content
had lower germination percentages than the control.

Figure 2 shows the effect of glycerol concentration and
soaking time on navy bean seed germination. Seeds soaked in
50% glycerol had a lower germination percentage than the
control, and germination percentage decreased with increased
soaking time. Seeds soaked in 60% or 70% glycerol solutions
had germination percentages comparable with the control (85%
germination) except for those soaked in 60% glycerol solution

for five days.

24

25

Figure 1. Germination percentages of
navy bean seeds soaked in different
concentrations of polyethelene glycol
for up to 5 days. Non soaked seeds
were used as controls. Four hundred

seeds were tested in each treatment.

26

A963 mE: mc_.v_oom

 

 

 

I l I T fl l I T

o o o o o o o o 0

Ch 00 rx no LO <1- m (\J <—
uoqouaneb aficwawed

m .V m, N _ o
O
A u 306"“: own 1
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xomdlld
0 man— OIIO -
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4 o°o -
l/Illd.
B/mff‘ 1:11.", ..
/fl’/ 1
fl

 

 

00w

27

Figure 2. Germination percentages of
navy bean seeds soaked in different
concentrations of glycerol for up to
5 days. Non soaked seeds were used
as controls. Four hundred seeds were

tested in each treatment.

28

nmxocv mE: mcioom
m . .w m N e

 

 

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O m n 306an emu

 

 

 

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29
Several of the above treatments were repeated with
weighing of seeds before and after soaking, to determine if
the solutions were absorbed into the seeds. Results of this
experiment presented in Table 1 demonstrate that the 60%
glycerol solution was not taken up by seeds, whereas the other
solutions were. This explains the absence of germination

reduction with 60% glycerol.

- ts . ... '..' 'cs '1' on. . - . s- 09:01}

No consistent reduction in germination percentages (Table
2) or seedling fresh weights (Table 3) were found between
seeds treated with 60% glycerol mixed with streptomycin,
chlorotetracycline or tetracycline and those soaked only in
60% glycerol. Seeds treated with PEG mixed with both
chlorotetracycline and tetracycline showed significant
reduction in seedling fresh weights (Table 3), but no
reduction in seed germination compared.with seeds soaked only
in 25% PEG (Table 2). No significant difference in germination
percentage (Table 2) or seedling fresh weights (Table 3) was
observed between seeds soaked in PEG with streptomycin and
seeds soaked only in 25% PEG. Albinism was observed on the
seedlings which received treatment of PEG and tetracycline
(400 ppm) or chlorotetracycline (400 ppm) when these seedlings

were grown in a green house. No albinism was observed on the

30

Table 1. Weights of imbibed seeds and seed germination
after soaking in glycerol and polyethylene glycol (PEG)
solutions for 3 days.

 

 

Treatment Imbibed seed weight Germination
(g/100 seeds) (g)

Dry seed 16 85

Glycerol 60% 16.8 87

Glycerol 50% 22.7 22

PEG 25% 24.9 91

PEG 20% 26.1 78

LSD (0.05) 2.14 5.7

 

31

 

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ov.o m¢.o m¢.o m¢.o mo.o 66.0 m>.o m
m¢.o «m.o m¢.o m¢.o ms.o ms.o mm.o m
om.o «6.0 ~m.o om.o om.o sm.o om.o a

0mm

-..----:-------------wmum-.u.-mm-o“-mm ..........................
mn.o 65.0 Hm.o om.o eb.o mm.o mn.o m
om.o mn.o om.o nm.o mm.o mm.o mm.o m
mm.o mm.o em.o wo.a um.o mm.o mo.H H

Houooaau

smdoom addooe adooom scoooe sauces adaoov

-mmmmmmmwm wmmmmmmmwmmmm -mmmmmmmm .020 05.2

........ ---------..-:..--Mm-m..:mmmmm-mm-.u.mmm...-m.mm-mmwm mum

 

.pcosummuu comm cw ooummu 0Hm3 mommm
compass usom .Omm wmm no Houmomam «om cw maso cosmom 600m sues
concafioo mast m>flu ou as you mafiaoaoouuou can mcflHO>omuumuouoHno
.cfioaaoummugm Baez cmxwfi Aummv Hooaao msoaasum>aom wmm uo Houmo>am

woo cfi cmxoom comm coon >>oc no musmflmz smoum oceacomm

.m OHQMB

32

 

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mm mm «m em mm mm wm n
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. 0mm

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«m om «m mm mm «m mm m
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Houmoaau
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-mmmmmmwm mmmwmmmwmmmmmm -mmwmmmmmwm ..85 Eng
.............................................. mmmwmmmmm “Mm.

 

.ucmeummuu comm ca cmummu
mum3 mcowm coupes: usom .Umm «mm no Houmo>am wow cw aaco cmxoom
mooom can mmmo o>flu on a: you ocwHoaoouuou can mcflaomomuumuouoHno
.sfloafioummuum :uw3 boxes Awmmv Hooaam msm~>cum>aom wmm no Houmowao
woo as cmxoom mcmom coon >>mc mo mommucmoumm cowuoCHEumO .m manna

33
seedlings which received treatment with PEG and streptomycin

up to 1600 ppm) under the same conditions.

e f ce 0 o t' 'ot'c ake

Antibiotic activity as determined by zones of inhibition
on bacteria-seeded BYE agar was detected in cotyledons and
embryos when seeds were soaked either in PEG or in glycerol
solutions containing streptomycin, chlorotetracycline or
tetracycline. Less antibiotic activity was detected for seeds
soaked only in PEG or glycerol (Table 4). More antibiotic
activity was detected in seeds soaked in PEG than in glycerol.
Small zones of inhibition were observed around embryos and

cotyledons soaked only in glycerol or PEG.

Antibiotic activity in cotyledons gradually increased
between.1 and 24 hours of soaking in PEG or glycerol solutions
(Table 5). There were no further increases in antibiotic

activity when the seeds were soaked longer than 24 hours.

Efficagy a: seeg tzgatmgnt in eliminating bactetia gram sgggs

The blight bacteria were completely eliminated from

artificially infected seeds by immersion for either 1 or 3

34

Table 4. Zones of inhibition in Xanthgmgaaa

seeded agar plates around the embryos and cotyledons
of navy bean seeds soaked in 25% polyethylene glycol
(PEG) or in 60% glycerol mixed with either 400 ppm
streptomycin, 400 ppm chlorotetracycline, or 400 ppm

tetracycline for 1 day.

 

 

Treatment Inhibition zone (mm)*
3.3;; """ c ”.1213;
Untreated 0.0 0.0
PEG only 0.3 1.2
PEG+streptomycin 1.3 5.2
PEG+chlorotetracycline 1.3 5.9
PEG+tetracycline 1.3 6.1
Glycerol only 0.3 1.2
Glycerol+streptomycin 0.5 1.6
Glycerol+chlorotetracycline 0.6 1.6
Glycerol+tetracyc1ine 0.5 1.6
LSD (0.05) 0.16 0.55

 

*Each value is the mean of 48 embryos or cotyledons.

35

Table 5. Diameters (mm) of inhibition zones in
KQDLDQEQDQS phaseoii seeded agar plates around the
cotyledons of navy bean seed soaked in 25%
polyethylene glycol (PEG) or 60% glycerol mixed with
400 ppm streptomycin (STR), 400 ppm chlorotetracycline
(CTE) or 400 ppm tetracycline (TET) for up to seventy
two hours.

 

 

Treatment Soaking time (hours)
I""'§"'"§ """ J """ ETTS'
PEG+STR 1.8* 2.7 3.1 4.3 5.2 5.3
PEG+CTE 2.1 2.9 4.2 5.1 6.1 6.0
PEG+TET 2.3 3.3 4.3 5.0 6.0 6.2
Glycerol+STR 0.8 1.1 1.4 1.5 1.6 1.6
Glycerol+CTE 1.0 1.2 1.4 1.5 1.6 1.7
Glycerol+TET 1.0 1.2 1.5 1.5 1.6 1.6

LSD (0.05) = 0.36

 

*Each value is the mean of 48 cotyledons.

36

days in a 25% PEG solution containing 1600 ppm streptomycin,
400 ppm tetracycline or 400 ppm chlortetracycline (Table 6).

igher concentrations of streptomycin than of tetracycline or
chlorotetracycline were required for elimination of blight

bacteria. With all treatments, increases in. either’ the
antibiotic concentration or the soak time aided in reducing

the recovery of bacteria from seeds. None of the treatments
involving application of antibiotics in glycerol solutions
were effective in eliminating common blight bacteria from the
artificially infected seeds.

The most promising treatments from the artificially
infected seeds were repeated using naturally infected seeds.
Results of this experiment are summarized in Table 7. None of
the treatments involving application of antibiotics with
glycerol provided useful reductions in recovery of blight
bacteria from :naturally infected seeds as they did in
artificially infected seeds. Conversely, all treatments with
mixtures of antibiotics in 25% PEG solutions provided large
reductions in the recovery of blight bacteria from naturally
infected seeds, especially with 3 days of soaking. Because
bacteria within naturally infected seeds were not eradicated
by treatments with PEG and streptomycin, the experiment was
partly repeated. An additional treatment was included,
doubling the maximum streptomycin concentration to 3200 ppm,
and a group of seeds were soaked for one to five days in 25%

PEG and 1600 ppm streptomycin. Additionally, a.portion of the

37

Table 6. Effect of application of antibiotics in 25%
polyethylene glycol (PEG) and 60% glycerol on recovery of
common blight bacteria from artificially infected navy bean
seeds. Four hundred seeds were tested in each treatment.

 

Treatment Bacterial recovery percentage

1 day soak 3 day soak

 

Untreated 43.0 43.0
Glycerol only 27.8 27.0
Glycerol+streptomycin 400* 25.6 10.4
Glycerol+streptomycin 800 24.4 8.2
Glycerol+streptomycin 1600 17.8 7.4
Glycerol+tetracycline 400 16.7 9.6
Glycerol+tetracycline 800 5.2 1.2
Glycerol+chlortetracycline 400 13.9 7.4
Glycerol+chlortetracycline 800 4.6 1.6
PEG only 20.0 19.0
PEG+streptomycin 400 18.0 3.3
PEG+streptomycin 800 8.9 0.6
PEG+streptomycin 1600 0 0

PEG+tetracycline 400 0 0

PEG+chlortetracycline 400 0 0

LSD (0.05) = 3.18

 

'Antibiotic concentrations in parts per million.

38

Table 7. Effect of antibiotics in 25% polyethylene:glycol
(PEG) and 60% glycerol on recovery of common blight
bacteria from naturally infected seeds. Eight hundred
seeds were tested in each treatment.

 

Treatment Bacteria recovery percentage

1 day soak 3 day soak

 

Untreated 23.0 23.0
Glycerol only 22.3 21.5
Glycerol+tetracycline 800* 18.9 15.8
Glycerol+chlortetracycline 800 20.6 16.1
Glycerol+streptomycin 1600 21.8 20.6
PEG only 19.4 18.6
PEG+tetracycline 400 0.6 0.1
PEG+chlortetracycline 400 0.8 0.2
PEG+streptpmycin 1600 2.4 0.4

LSD (0.05) = 2.32

 

*Antibiotic concentrations in parts per million.

 

39
seeds were germinated for fresh weight determinations.
Results of these experiments are presented in Tables 8 and
9. None of these treatments eradicated.the blight bacteria
from naturally infected seeds. Slight reduction in
seedling fresh weight was
apparently caused by the highest concentrations of

streptomycin.

Egeveatign gf phytotgxicity

The efficacy of NaOCl in reducing the phytotoxic
effects of tetracycline and chlorotetracycline is
presented in Table 10. Seedling fresh weights improved by
NaOCl soaking. Three to ten minutes resoaking in NaOCl
provided the best results among the partially recovered
seedlings. Seedling vigor was reduced either by prolonging
the resoaking time or by increasing the concentration of
NaOCl. However, seedlings from the resoaked

seeds still were severely etiolated.

40

Table 8. Effect of streptomycin concentration in 25%
polyethylene glycol on recovery of Xaathomgaas

and fresh weights of seedlings from
naturally infected seeds soaked for 3 days.

 

 

Streptomycin Bacteria Fresh

content recovery weight
(ppm) (%) (9)
Untreated 23.0 1.2
0 18.4 0.9
400 6.8 0.8
800 2.3 0.8
1600 0.4 0.7
3200 0.4 0.6

LSD (0.05) 1.42 0.09

 

 

41

Table 9. Effect of soaking time on recovery of
common blight bacteria and fresh weights of
seedlings from naturally infected seeds when

seeds were soaked in solutions of 25% polyethylene
glycol and 1600 ppm streptomycin solutions.

 

 

Soaking time Bacteria recovery Fresh weight
(DAY) (M (9)
0 23.0 1.2
1 2.4 0.8
2 2.2 0.8
3 0.4 0.7
4 0.6 0.7
5 0.3 0.6

LSD (0.05) 0.82 0.09

 

42

Table 10. Effects on germination percentages and seedling
fresh weights of resoaking seeds in NaOCl with different
soaking times following presoaking seeds in 800 ppm
chlortetracycline or tetracycline or 25% polyethylene
glycol for 24 hours.

 

Resoak time Germination (%) Fresh weight (g)

 

(min) -------------------------------
CTC TTC CTC TTC
1: 1 NaOCl:HZO
0 91 91 0.50 0.50
3 79 74 0.73 0.71
10 51 32 0.61 0.60
20 36 16 0.48 0.40
30 17 12 0.45 0.41
1: 10 NaOCl:HZO
0 91 91 0.50 0.50
3 92 94 0.74 0.72
10 89 90 0.73 0.70
20 87 90 0.66 0.68
30 83 87 0.61 0.60

LSD (0.05)

 

DISCUSSION

This study showed that PEG solutions can be used as an
antibiotic carrier for reduction but not eradication of
internally seed-borne common blight bacteria without affecting
seed germination percentage. A PEG concentration of 25% showed
the best result in facilitating movement of the antibiotics
into the seeds and reducing 2g. ptagggi within internally
contaminated seeds. However, seedling vigor was expressed by
fresh weights was slightly reduced in this treatment. This
problem could be overcome by increasing the PEG concentration.
Unfortunately, percentage reduction of X- 212559.011 was
impaired by increasing the concentration of PEG solution
(Appendix 2). Quite probably, increased viscosity of the PEG
solution resulted in less antibiotic infiltration. Another
problem noticed was that PEG treatment is only good for seeds
that are less than one year old. Germination percentage was
reduced by 18% with seeds that were one and a half years old,
and by 54% with seeds that were two years old (Appendix 3).

The results demonstrated that glycerol is not suitable as
an antibiotic carrier for the reduction or eradication of
internally seed-borne bacteria. Glycerol solutions with

concentrations greater than 60% were not absorbed by the

43

44

seeds, and lower concentrations reduced seed germination. This
phenomenon explains why no phytotoxicity was observed when
seeds were soaked in 60% glycerol solutions containing
tetracycline and chlorotetracycline, although these two
antibiotics induced phytotoxicity in PEG solutions.

Tetracycline and chlorotetracycline in PEG solutions
proved effective in reduction of internally seed-borne x.
piiagegii. However, phytotoxicity rendered these treatments
unacceptable. Streptomycin appears to be the most promising
antibiotic for reduction of K- phaaggli. A concentration of
1600 ppm in a 25% PEG solution provided the most effective
reduction of z. phasegli infection in this study. Further
increases in streptomycin concentration did not provide
increased reduction in z. phasegii, and seedling fresh weight
was reduced. The inability to completely eliminate the
bacteria from naturally infected seeds appeared to be
partially due to the occurrence of seeds with hard coats.
These seeds did not imbibe the PEG solution within the soaking
period. Seeds with.hard.coats accounted for 10.2, 8.5, 6.9 5.5
and 4.5% of naturally infected seeds of cultivar C-20 which
were soaked for 1, 2, 3, 4 and 5 days, respectively.
Investigation showed that 72% of the surviving X. phagggii
were recovered from seeds with hard coats soaked for one day
and 67% from seeds with hard coats soaked for three days

(Appendix 4).

45

Antibiotics such as streptomycin (Klisiewiecz and.Pound,
1961) and.chlorotetracycline (Lockhart at al., 1976) have been
used to eradicate zanthgmgaaglgampggttig from §£Q§§i§§ seeds,
but these treatments often resulted in reduced germination and
seedling vigor (Huber and Gould, 1949). Humayadan at al.
(1980) overcame this disadvantage with a thirty minute soak in
a solution of 0.5% sodium hypochlorite following a one hour
soak in 500 ppm of either oxytetracycline or streptomycin.
Seed lots that received the combined treatment were unaffected
either in germination or seedling vigor. However, this method
was not effective in our study. Resoaking seeds in NaOCl only
partially overcame the phytotoxicity induced by tetracycline
or chlorotetracycline. The seedlings nevertheless were still
severely etiolated. Success in eliminating cm s
gampestzis pv. gampggtgig from Brassica seeds was probably
due to the fact that this pathogen is an.externally seed-borne
bacterium on Brasgiga, and could be eliminated by conventional
surface type treatments. For example, one hour soaking with
antibiotics in water proved effective in eliminating the
bacteria (Humaydan g a1. , 1980) . However, such a short period
of time did not allow the antibiotics to penetrate into the
embryo and.cotyledon.‘Thus the antibioticsion.the seed surface
and in the seed coat were easily oxidized by NaOCl. Failure of
NaOCl to completely overcome phytotoxicity of the

tetracyclines in bean seeds was probably due to the

46
localization of the antibiotics inside the seeds, where the
oxidant failed to penetrate.

Blight bacteria in artificially infected seeds are more
sensitive to antibiotic treatment than those in naturally
infected seeds. Clearly, the vacuum inoculation technique did
not completely duplicate field infection. Field inoculation
with common blight bacteria was more realistic for providing
internally infected seed for this study. Bacteria in
artificially infected seeds probably are localized around.the
outside of the cotyledon, whereas bacteria in naturally
infected seeds may be localized within the cotyledons and

embryo tissues.

SUGGESTIONS AND FUTURE STUDIES

Several additional, related lines of investigation seem
warranted prior to potential commercial application of methods
developed in this investigation. 1) Determine if the treatment
(1600 ppm streptomycin in 25% PEG solution) could have an
effect on seed production and crop yield. 2) Determine if the
treatment effectively reduces disease development under field
conditions. 3) Ascertain whether application of the method
affords the hazard of selecting for pathogenic strains of z.
phaseoii that are resistant to streptomycin. One previous
study demonstrated the occurrence of such strains in.Michigan
(Weller, 1978). 4) Determine if the antibiotic can be
systemically transported by the plant following seed
germination, and the time that the antibiotic persists in the
plant in sufficient. concentration. to retard blight
development. This may provide evidence on the potential effect
of the antibiotic on bacteria which were not eliminated. 5)
Investigate whether other antibiotics that are lethal to z.
phasgoli but have no phytotoxicity on bean are available. 6)
Use the same technique to eliminate other seed-borne bacteria

from beans and other crops.

47

APPENDICES

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adamOADQomo can .namm on Hooo on mcfizoaam .mmuscfifi on mcfi>maoouso
E 653203.“ .3; so: 0363 mumfimofi 5:68 z 8.6 d: 08
Ca uomuuxw ammo» e o.H mcw>aommwc an omuommum mm3 amends cwsvflq.

 

 

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nOH x h.o A ooa mcaao>omuume as m on m
noa x 5.0 A ooa cflomaoudmuum
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~oa x 6.0 v ooH ocflHOhoouuma as H
noa x n.o A ooa cfioxsouemuum
Auv 00:»
pass ocHsuou >u0>oomu ummu CH «sowcms
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.mflumuoon unmaan coesoo mo cpaoum on» so mOwuownwuco mo uumumm

H XHszmmd

48

APPENDIX 2

Effect of polyethylene glycol (PEG) concentration on seed
germination, seedling fresh weight and blight bacteria
recovery percentage of naturally infected seeds, when
these PEG solutions were mixed with 1600 ppm streptomycin
and seeds were soaked for three days.

 

 

PEG Germination Mean fresh % Bacteria
concentration percentage weight (g) recovery
25% 88 0.84 0.4

30% 89 1.03 5.6

35% 92 1.09 13.1

40% 96 1.12 13.8

45% 96 1.20 14.4
Untreated 86 1.20 23.0

LSD (0.05) 5.6 0.118 1.94

 

49

APPENDIX 3

Germination of navy bean seeds soaked in 25% polyethylene
glycol solutions for 3 days following different times of
storage after harvesting.

 

Seed storage time Germination percentage

(months) ..................................
Untreated (Check) PEG soaking

 

9 85 91
18 81 75
24 72 56

LSD (0.05) = 5.8

 

Note: Harvested seeds were stored in polyethelene plastic
bags at room temperature (ca. 25°C) .

50

APPENDIX 4

Common blight bacteria recovered from impermeable
(hard coat) and permeable (soft coat) seeds when the
naturally infected seeds were soaked for 1 and 3 days
in solutions of 25% polyethylene glycol and 1600 ppm
streptomycin.

 

Soaking time Bacteria recovery percentage
(days) ----------------------------
Hard coat Soft coat
1 1.7 0.7
3 0.3 0.1
Untreated 23.0

 

51

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