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This is to certify that the

thesis entitled
~IDATHOGENIC VARIATION IN COMMON (XANTHOMONAS
PHASEOLI) AND FUSCOUS (XANTHOMONAS PHASEOLI
VAR. FUSCANS) BACTERIAL BLIGHTS OF BEAN (PHASEOLUS
VULGARIS L.) presented by

Ephraim J. A. Ekpo

has been accepted towards fulfillment
of the requirements for

Ph.D. Plant Pathology

degree in

 

Major professor

Datew S

0-7639 ' " '

 

 

 

 

ABSTRACT

PATHOGENIC VARIATION IN COMMON (XANTHOMONAS PHASEOLI)
AND FUSCOUS (XANTHOMONAS PHASEOLI VAR. FUSCANSI
BACTERIAL BLIGHTS OF BEAN (PHASEOLUS VULGARIS L.)

 

 

BY

Ephraim J. A. Ekpo

Pathogenic variation was examined in isolates of

Xanthomonas phaseoli (Xp) and Xanthomonas phaseoli var.

 

 

fuscans (pr), the causal agents of common and fuscous
blights of beans, respectively. A total of eight isolates
of Xp and seven isolates of pr were selected from different
geographical regions represented by Michigan and Nebraska

in the U.S.A., Uganda in Africa, Guatemala in Central
America, and Colombia in South America. Virulence of
isolates was compared on thirteen commercial bean varieties
(Phaseolus spp.) and two cowpea varieties (Vigna unguiculata)
at different stages of plant growth. The methods of seed
infiltration, multiple needle inoculation, leaf incision,
water soaking of leaves, and excised pod inoculation pro—
cedures were examined in comparative pathogenicity tests
using a standard inoculum concentration of 2.8 x 107 cells/ml.
Results were reproducible with leaf incision, water-soaking
and excised pod techniques. Isolates were separated into
different virulence groups or strains based on qualitative

and quantitative differences in disease reactions.

Ephraim J. A. Ekpo

Plant age was important in disease development; the
more virulent isolates of both bacteria incited necrosis on
both young and old leaves while the less virulent isolates
incited symptoms which were restricted primarily to young
succulent tissues. Some isolates that were slightly
virulent on plants in the vegetative stage of growth became
more virulent when plants entered the reproductive phase of
growth. Active multiplication within the tissue was not
always accompanied by symptom deve10pment. For example,
isolates Xle and Xp23 actively multiplied in leaves of
tolerant G.N. Tara without the production of visible disease
symptoms. Mixed isolates of Xp and pr were inoculated
into Manitou and G.N. Jules leaves and re-isolated from
diseased tissue in varying proportions. Symptom development
with such composite inocula was sometimes more severe than
infection with individual isolates thus indicating compat—
ible co—existence of both bacteria in the same plant tissue.
A new cell phenotype was isolated from tissues inoculated
with either several isolates of the same bacterium or a
mixture of Xp and pr isolates. G.N. Tara and P.I. 207262
which are reported as tolerant to Xp by previous workers
were shown to be susceptible to some isolates of Xp and pr
included in the study. Isolates exhibited serological
variability but such variability was not easily correlated
to the observed pathogenic variation among the isolates.

The implications of the findings are discussed in relation

to bean breeding programs.

PATHOGENIC VARIATION IN COMMON (XANTHOMONAS PHASEOLI)

 

AND FUSCOUS (XANTHOMONAS PHASEOLI VAR. FUSCANS)

 

BACTERIAL BLIGHTS OF BEAN (PHASEOLUS VULGARIS L.)

 

BY

Ephraim J. A. Ekpo

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1975

 

 

To my parents

ii

 

ACKNOWLEDGEMENTS

I wish to express my gratefulness and best personal
regards to Dr. A. W. Saettler for his interest, encourage-
ment and guidance throughout this study. I am also
indebted to him for providing experimental materials and
facilities for the study.

Sincere regards and appreciation are also extended
to Dr. M. W. Adams, Dr. W. J. Hooker and Dr. E. J. Klos who
served on my dissertation committee. Their suggestions and
criticisms were useful in the preparation of the manu-
script. I am grateful to Ms. Sandy Perry and Mr. David
Weller for their encouragement and technical assistance,
especially during field studies. Sincere thanks are also
extended to Professor T. Ajibola Taylor and Dr. 0. F.
Esuruoso for their interest in the field of study.

I am especially indebted to my dear parents, Chief
and Mrs. J. A. Ekpo, and my Uncle, Mr. H. A. Ekpo, and my
brothers, sisters and friends for their patience, love,
understanding and miscellaneous support during this effort.

The entire program was funded through University of
Ibadan/Ford Foundation Staff Development Fellowship. This

fellowship is gratefully acknowledged.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . . . . . .

LIST OF TABLES . . . . . . . .

LIST OF FIGURES . . . . . . . .

INTRODUCTION AND LITERATURE REVIEW

MATERIALS, METHODS, AND PRELIMINARY RESULTS

Origin of isolates . . . . . . . . .

Culture media . . . . . . . . . . .

Bean differentials . . . . . . . . .

Inoculum preparation . . . . . . . .

Inoculation procedures . . . . . . . .
Seed infiltration . . . . . . . .
Multiple needle technique . . . . .
Leaf incision technique . . . . . .
Excised pod technique . . . . .
Leaf water— soaking techniques . .

EXPERIMENTS AND RESULTS . . . . . . . . . .

Comparative pathogenicity of isolates of Xp
and pr in bean leaves assayed by leaf-
incision technique . .

Effect of humidity on symptom development in
beans inoculated with isolates of common
blight . . .

Effect of inoculum concentration on infection
efficiency of X. phaseoli and X. phaseoli
var. fuscans in a susceptible bean

Effect of leaf and plant age on the develop-
ment of common and fuscous blights of beans

Comparative virulence of Xp and pr blight
isolates in Phaseolus spp. inoculated at
vegetative and reproductive stages of
development . . . . .

Effect of mixed inoculum of Xp and pr on
disease development in bean plants assayed

by leaf-incision and water-soaking techniques.

iv

Page

24

30

34

37

45

 

DISCUSSION . . . . . . .

SUMMARY

LITERATURE CITED . . . . .

 

Comparative virulence of X. phaseoli and

X. phaseoli var. fuscans isolates in
bean pods .

Pathogenic variability in X. phaseoli and
X. phaseoli var. fuscans in field—grown
bean plants . .

Population trends of Xp and pr isolates in
tolerant and susceptible bean cultivars
as related to disease reaction

Pathogenicity of isolates of X. phaseoli and
X. phaseoli var. fuscans in —cowpeas (Vigna

unguiculata) .
Serological studies of Xp and pr isolates

Page

55

61

74

84
85
100
117

121

 

Table

10.

11.

12.

LIST OF TABLES

 

 

Page

Isolates of Xanthomonas phaseoli and

Xanthomonas phaseoli var. fuscans used

in comparative pathogenicity studies . . . 12
Bean varieties and their reported reactions

to Xp and pr . . . . . . . . . . . 13
Infection from soaking seed for 10 minutes . . l7
Pathogenicity of Xp 12 and pr 16 using

multiple needle inoculation technique . . . 19
Comparative virulence of Xp and pr isolates

after incision—inoculation of primary

leaves . . . . . . . . . . . . . 26
Comparative virulence of Xp and pr isolates

after incision—inoculation of lst and 2nd

trifoliolate leaves . . . . . . . . . 27
Comparative Virulence of Xp and pr isolates

after incision-inoculation of primary

leaves . . . . . . . . . . . . . 28
Effect of humidity on time (days) required for

visible host/pathogen interaction following

water-soaking with bacteria . . . . . . 32
Effect of humidity on symptom development in

beans inoculated with isolates of Xp . . . 33
Effect of inoculum concentration on disease

development in lB—day-old Manitou bean . . . 36
Effect of leaf and plant age on the development

of common blight in Manitou bean . . . . . 40
Effect of leaf age on development of fuscous

blight in MCC and Manitou beans . . . . . 42

Vi

Table Page

13. Disease reactions of Phaseolus spp. in
vegetative (V) and reproductive (R)
stages of growth to isolates of

X. phaseoli . . . . . . . . . . . . 47
14. Disease reactions of Phaseolus spp. in

vegetative (V) and reproductive (R)
stages of growth to isolates of
X. phaseoli var. fuscans . . . . . . . 48

15. Effect of mixed inoculum of Xp and pr on
development of symptoms in bean varieties
assayed by leaf—incision technique . . . . 52

16. Effect of mixed inoculum of Xp and pr on
disease reaction in bean varieties
assayed by water-soaking technique . . . . 54

17. Lesion types resulting from inoculation of
bean pods with Xp and pr isolates . . . . 58

18. Lesion size (mm) in bean pods following
infection by Xp isolates . . . . . . . 60

19. Lesion size (mm) in bean pods following
infection by pr isolates . . . . . . . 62

20. Reaction of beans (Phaseolus vulgaris) to
Michigan (XplS), Ugandan (XpU2), Colombian
(Xp21), and Nebraska (Xp23) isolates of

X. phaseoli. . . . . . . . . . . 67
21. Reaction of beans (Phaseolus vulgaris) to

 

Michigan (prl6), Guatemala (pr844),

Nebraska (pr29), and Colombian (pr

Ciat A) isolates of X. phaseoli var.

fuscans . . . . . . . . . . . 69

22. Pod infection (no. lesions/pod) in beans
(Phaseolus vulgaris) inoculated with
isolates of Xp and pr . . . . . . . . 70

 

23. Seed infection in field-grown Sanilac bean
inoculated with isolates of Xp and pr . . . 71

24. Effect of Xp and pr isolates on yield (seed
weight) of field—grown Sanilac and Red
Mexican U.I.#3 beans . . . . . . . . . 73

25. Pathogenicity of X. phaseoli and X. phaseoli
var. fuscans isolates in cowpea “(Vigna
unguiculata) inoculated by seedling
injection technique . . . . . . . . 86

vii

 

Table Page
26. Cross—agglutination relationships of isolates

of Xp and pr . . . . . . . . . . . 90
27. Serological relationships of Xp and pr

isolates by slide agglutinationxabsorption

technique . . . . . . . . . . . . 92
28. Serological reactions of isolates of Xp and

pr by immunodiffusion technique . . . . 95

viii

LIST OF FIGURES

Figure Page

1. Typical blight symptoms on (a) primary and

(b) trifoliolate leaves of MCC bean 14

days after inoculation with.Xp24 . . . . . 5
2. Symptoms on primary leaves of MCC bean 16

days after incision—inoculation with

Xp15, Xp22, Xp24, and Xp26 . . . . . . . 22
3. Symptom types on green pods of P.I. 207262;

(A) = 'W' type; (B) = 'N' and 'D' types;

and (C) = 'WC' type . . . . . . . . . 22
4. Effect of leaf age (A) and plant age (B) on

symptom development in Manitou bean
inoculated at 10, 20, and 30 days after
planting . . . . . . . . . . . . . 43

5. Disease reaction classes in field—grown beans
inoculated with blight bacteria; T =
tolerant, $1 = slightly susceptible; Mod =
moderately susceptible, Se = severely

susceptible . . . . . . . . . . . . 64
6. Population trends of Xp isolates in trifoliolate

leaves of G.N. Tara inoculated with 2.8x107

cells/ml . . . . . . . . . . . . . 78
7. Dilution plate used for isolating bacteria from

G.N. Tara leaves 12 days after inoculation

with a mixture of Xle, Xp23, Xp24, and

XpU2; (A) = large colonies of Xp; (B) =

small colonies of orange—yellow new pheno—

type (XpE); and (C) = grayish white colonies

of resident bacteria . . . . . . . . . 78

8. Population trends of Xp isolates in primary
leaves of Manitou bean inoculated with
2.8x104 cells/ml . . . . . . . . . . 81

9. Effect of mixed infection on the number of
viable Xp and pr cells in diseased
Manitou leaves . . . . . . . . . . . 82

ix

 

Figure Page

10. Effect of mixed infection on the number of

viable Xp and pr cells in diseased

G.N. Jules leaves . . . . . . . . . . 82
ll. Immunodiffusion patterns obtained from the

reaction of pr28 antiserum (central well)

with pr isolates before (A), and after

(B) homologous absorption of antiserum

and, reaction of pr28 antiserum (central

well) with Xp isolates before (C), and

after (D) absorption with pr28 . . . . . 96

12. Immunodiffusion patterns obtained frOm the
reaction of pr16 antiserum (central well)
with Xp isolates before (A) and after (B)
homologous absorption of antiserum with
pr16 . . . . . . . . . . . . . . 97

13. Immunodiffusion patterns obtained from the
reaction of XplS antiserum (central well)
with Xp isolates before (C) and after (D)
homologous absorption of antiserum with

XplS . . . . . . . . . . . . . . 97
14. Disease reaction (A) and reaction categories

(B) of P.I.207262 inoculated with pr

isolates . . . . . . . . . . . . . 99

INTRODUCTION AND LITERATURE REVIEW

Common blight caused by Xanthomonas phaseoli (E.F.S.)

 

Dows and fuscous blight caused by Xanthomonas phaseoli var.

 

fuscans (Burkh.) Starr & Burkh. are bacterial diseases of

major importance in bean production. Plant pathologists

 

consider common and fuscous blights to be two of the most
devastating seed-borne diseases of dry edible and green
beans in many production areas in the world (48, 50, 69).
The prevalence of common blight in most cases parallels
that of fuscous blight and both have been reported from
Australia (4, 40), Russia (20), Yugoslavia (61, 62),
Michigan, U.S.A. (3), and Uganda (34). The causal agents
can be recovered from pods, seeds, and vegetative tissue of
bean (69).

Incidence and severity of blight on bean may vary
from year to year depending on weather conditions, locality,
and cultural practices. In some cases, bean seed production
is restricted to dry areas where the blight problem is less
severe. In Nebraska, for example, most of the planted bean
seed is produced in disease-free, semi—arid areas of Idaho
to eliminate seed-borne infection (14). Occasionally,

however, crop losses do occur when environmental conditions

are favorable for bacterial development and spread.

X. phaseoli (Xp) and X. phaseoli var. fuscans (pr) are
known to overwinter on infected bean straw which is

source of inoculum that can infect the new bean crop (69).

Widespread destruction of beans by bacterial
blights often necessitates the use of chemical protectants
to prevent spread of the disease organisms. Oshima and
Dickens (44) report appreciable control (88.6—91.5%) of
secondary spread of common blight in Colorado snap and
pinto beans using copper sprays. In navy bean production,
however, there are no satisfactory chemical control measures.
Growers have used copper bactericides for several years as
protective sprays, but the control of secondary spread is
not always satisfactory (16, 45). Chemical control by seed
treatment (25) is also reported to be generally ineffective
for control of internally-borne blight pathogens.

Absence of immunity to common and fuscous blights
further underscores the role of blight in bean production;
extensive screening for disease resistance in greenhouse and
field tests revealed no immunity among commercial bean cul-
tivars (69). Tolerant commercial cultivars have only
recently been made available with the introduction of late
maturing Great Northern Tara (G.N. Tara) and Great Northern
Jules (G.N. Jules), in Nebraska (5, 10, 11). The future of
world bean production depends largely on the discovery and
successful incorporation of tolerant germ-plasm into

acceptable high yielding blight-susceptible bean varieties.

 

 

The literature is rather scanty on estimates of
economic damage caused by Xp and pr. Seventy—five percent
of the fields in New York were affected and serious losses
occurred in 1918 (69). During the same period, Burkholder
(7) estimated losses due to common blight at 3—8% and in
1930, Zaumeyer (68) recorded an average loss of 10% for the
entire U.S. In 1936, the U.S. suffered an estimated loss
of 3,400,000 pounds of snap beans and about 34,700,000

pounds of dry beans (69). Andersen (3) estimated a blight

 

loss of $3.5 million to growers in three Michigan counties
in 1951, and Zaumeyer et_al. (69) reported a loss of about
$1.0 million in Nebraska in 1953. Yield reduction in Russia
was 37% in 1958 and 65% in 1959 (33). In similar studies,
Zaumeyer et_§1. (70) estimated losses due to common and
halo blight diseases at about 22—28% in garden beans and
about 25—30% in field beans. Reductions of about 30—40% in
total yields were attributed to common and fuscous blights
in Michigan (3). Recently, Coyne et_al. (12) have reported
that common blight and bean wilt will often cause large
reductions in yields and quality of beans grown in Nebraska.
Andersen (2) also reported that 75% of the seed from Colorado,
Montana, Nebraska, and Wyoming was infected with fuscous
blight. The importance of bacterial blight in commercial
bean production remains to be established for developing
countries where protein—bean diet is being emphasized in
nutrition studies.

The pathological histology of Xp and pr is similar;

under natural conditions the bacteria enters leaves through

 

stomata or wounds and then invade the intercellular spaces
(30), causing a gradual dissolution of the middle lamella
(69). Later the cells begin to disintegrate with the forma—
tion of bacterial pockets. Stem infection occurs either
through the stomata of the hypocotyl and epicotyl, through
the vascular connections leading from the leaf, through
mechanical wounds and insect damage, or from infected
cotyledons (34, 69). In severe cases, bacteria and the dead
disintegrating tissue in the xylem vessels may cause a wilt—
ing of the plant, either by plugging the vessels or by the
disintegration of the cell wall. The pathogen is harbored
below the seed coat (69); it enters the pod sutures from
the vascular system of the pedicel and then passes into the
funiculus and through the raphe leading into the seed coat.
The micropyle also serves as a portal of entry into the
seed.

Studies suggest a great deal of similarity in
sympotomatology between common (Xp) and fuscous (pr)
blights. This similarity often makes their separation in
the field impossible. Typical symptoms consist of necrotic
lesions bordered by narrow bands of yellow tissue or chlorotic
halos (Figure 1). In greenhouse and field inoculations,
however, Zaumeyer (69) reported more severe symptoms on
plants infected with pr than on plants infected with Xp.

In culture, the two pathogens are easily separated on the
basis of pigment production; Xp produces a non-diffusible

yellow pigment on certain media whereas pr produces a

 

 

Xp24
MCC

 

Figure l.——Typical blight symptoms on (a) primary and (b)
trifoliolate leaves of MCC bean 14 days after
inoculation with Xp24.

diffusible brown pigment. The primary role of these melanin—
like pigments in pathogenicity has not been conclusively
established but Burkholder (8) in a comparative study of the
bacterial diseases of the bean suggested that pigment produc-
tion was a factor in the severity of stem splitting on sus—
ceptible hosts. Leakey (34) also suggests that there is

perhaps a tendency for pigment-producing Xanthomonas spp.

 

to be of greater virulence than non—pigment producing types.
Separation of Xp and pr based on phage typing differenti-
ates the two pathogens into two lysotypes. Klement et_a1,
(31) isolated a bacteriophage specific for pr from infected
bean seed and Katznelson and others (27) isolated the
Specific phage for Xp. These phages only lyse their homolo-
gous hosts, thus indicating a high degree of specificity.
Evidence regarding the serology of Xp and pr is fragmentary.
Elrod and Braun (18) point out that the two groups of
pathogen may belong to the same serotype.

Xp and pr share a number of diagnostic character-
istics. According to Dye (17), the cells are non—sporing,
gram-negative, strictly aerobic, monotrichous rods. The
cells are medium sized with rounded ends occurring singly
or in pairs. They measure on the average about 1.9—2.3u
x 0.87—0.98u (8); they are non-capsulated. Nutritionally
there is considerable homology between them as well; in
weakly-buffered media, acid is produced in small amounts
from many carbohydrates and glucose metabolism is strictly

oxidative. They are generally characterized as

high-temperature pathogens in contrast to Pseudomonas
phaseolicola, the causal agent of halo blight of beans (34).
They are seed—borne, both internally and externally (34,69)
and are capable of being transmitted long distances. Basu
§E_a1. (6), in a study of survival of Xanthomonas in bean
seed, demonstrated ability of pr to survive in seed for
three years at 20—35 C. Survival of Xp in dry leaf thrash
for up to 18 months was demonstrated by Sabet and Ishag (49).
Thus survival of the blight pathogens between crops may
either occur in or on seeds, or through crop debris, par-
ticularly in areas with mild weather.

Xp as a group has been studied more extensively
than pr. In most cases, studies of the two groups have
emphasized host range and varietal resistance rather than
pathogenic variation among isolates within the groups. The
existence of pathogenic variation among populations of
phytopathogenic bacteria may be a common phenomenon. Schroth
et_a1. (52) described the occurrence of pathogenic and
nutritional variation in the bean halo blight group of
fluorescent pseudomonads. Thyr (64) in a study of virulence

of Corynebacterium michiganense from six geographical areas,

 

reported significant differences among seven isolates which
differed in degrees of aggressiveness. He suggested the
inclusion of highly virulent pathotypes in tomato breeding
programs to maintain an acceptable level of resistance.

In a related study, Strider and Lucas (60) emphasized the

need for a specialized inoculation technique in order to

 

demonstrate the existence of variation in virulence. They
used a knife stabbing procedure (59) and demonstrated that

variation in Virulence did exist in C. michiganense.

 

Gerarda Perlasca (47) in a study of California isolates of

Pseudomonas syringae pathogenic on stone fruit trees,

 

reported considerable differences in isolate pathogenicity
or virulence on different hosts. In more recent studies,
Shuster and Coyne (55) reported new, highly virulent strains
of common blight bacteria in isolates from Colombian dry
bean seed. This finding suggests that additional patho«
genic variation may be discovered as other blight isolates
from diverse origins are studied. Little or no information
is available regarding the existence of pathogenic variation
in pr. Because of the economic importance of common and
fuscous bacterial blights, it was decided to study compara—
tive virulence in order to forestall possible resistance—
breeding—complications involving pathogenic variations in
isolates of both bacterial pathogens.

The present study has focused on determining whether
Xp and pr isolates from different geographical origins
(labeled as different isolates in the present system of
numerical nomenclature) possess the same pathogenicity or
whether they are heterogeneous in their pathogenic spectrum.
It is hoped that the information gained from such study
will be of immediate practical importance in meaningful

bean breeding endeavors.

Previous studies of pathogenic variation have been
limited to foliage infection based on one method of
inoculation, a water—soaking technique (54). Additional
inoculation procedures could be useful for scoring
virulence on both leaves and pods, and they have been
included in this study. The host plant is a complex
diagnostic medium and disease reactions, which result after
it is inoculated with a blight isolate, reflect the physio-
logical properties (genetic potential) of the isolate.
Isolates that have a similar host range and produce iden-
tical host reactions are usually placed in the same viru—
1ence class. However, we recoqnize that it is difficult
to differentiate isolates on the basis of "complete“ host
range. Accordingly, the pathoqenicity spectrum and tissue
reaction (symptom development) on an arbitrary sample of
commercial bean varieties have been used as an index of
virulence. Qualitative and quantitative aspects of symptom
development serve to differentiate the isolates of both Xp
and pr into virulence groups or strains. Mixed—infection
studies with a combination of several isolates of each group
and combinations of both Xp and pr in appropriate ratios
have been included to investigate the hitherto unreported
interactions or co—existence of both bacteria, in the same
tissue, in relation to intensity of disease.

The objectives of the present investigation, then,
were: (a) to compare different inoculation techniques in

order to develop a useful system(s) for comparative

lO

pathogenicity studies; (b) to compare qualitative and
quantitative aspects of symptom development of Xp and pr
isolates from different geographical regions; (c) to follow
population trends in tolerant and susceptible bean tissues;
and (d) to relate the implications of the findings to
breeding programs to develop blight-resistant bean

varieties.

MATERIALS, METHODS, AND PRELIMINARY RESULTS

Origin of Isolates: Pathogenicity tests were con—

 

ducted with 8 isolates of Xp and 7 isolates of pr using

different cultivars of Phaseolus vulgaris, L (common bean),

 

P. coccineus (Scarlet Runner), P: acutifolius (Tepary bean),

and Vigna unguiculata (cowpeas). Isolates from infected

 

bean seeds were selected from bacterial cultures maintained
by Dr. A. W. Saettler (Department of Botany and Plant
Pathology, Michigan State University) and by Dr. M. L.
Schuster (Nebraska Agricultural Experimental Station,
Nebraska) (Table l) and their pathogenic behavior on bean

varieties has been recorded by various workers (Table 2).

Culture Media: Yeast extract—calcium carbonate—agar
(YCA) (1000 ml distilled water, 10 gm yeast extract (Difco),
15 gm bacto agar, and 2.5 gm CaCO3) was used throughout the
study. There was no observable change in pathogenicity of
individual isolates on this medium during the period of
investigation as confirmed by disease reactions in suscepti—
ble Manitou bean periodically inoculated with transferred
cultures. Re—isolates from infected leaves were not used

for comparative studies since, according to M. Goto (23),

ll

12

TABLE 1.——Isolates of Xanthomonas phaseoli and Xanthomonas
phaseoli var. fuscans used in comparative
pathogenicity studies.

 

 

Isolate Isolate

(Xp) Origin (pr) Origin
12 (879—2) Michigan 16 (988-2) Michigan
15 (1205—2) Michigan 18 (1101—2) Michigan
21 (C7) Colombia 19 (1253—1) Michigan
22 (Pinto) Colorado 28 Idaho
23 (S) Nebraska 29 Nebraska
24 (C6) Colombia 844 Guatemala
25 (BBL-25) Canada Ciat A Colombia
U2 Uganda

 

isolates with relatively low virulence show high Virulence
when re—isolated from infected leaves, and re«inoculated
into healthy tissues.

Bean Differentials: The bean varieties were selected

 

on the basis of their reported susceptibility or toleranceto
isolates of Xp and pr (Table 2). Test plants for foliage
inoculations were grown under field, greenhouse and growth
chamber conditions. Plants for pod inoculations were grown
in the field using commercial production practices. In the
growth chamber, plants were grown in vermiculite medium in
4.5—inch (11.5 cm) diameter, 32 oz wax—lined cardboard
cartons while greenhouse plants were grown in soil contained
in either 10—inch earthen pots or 4.5 inch cartons. During

November—May, daylight was supplemented l4 hr/day with

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14

fluorescent bulbs. Temperature was maintained at 27 i 2 C
and plants were watered with nutrient solution (sequestrene,
Fe as metallic 1.8 ppm and Rapid-Gro, 1 teaspoon/2 liters

of water) alternated with tap water. Disease—free seed was
used for all plantings. At inoculation, plants were

examined and selected for uniformity of growth and freedom
from damage. Plant age at inoculation varied with individual
experiment but was uniform for all isolates in any parti-

cular study.

Inoculum Preparation: Bacterial suspensions were

 

prepared from 48 hour cultures grown at room temperature
(25 i 1 C). Cells were scraped from agar surface into
sterile distilled water and mixed thoroughly to produce a
homogeneous suspension. Unless otherwise stated, all
isolates were used at a concentration of 2.8 x 107 cells/ml
as determined by standard turbidimetric and dilution plate

techniques.

Inoculation Procedures: Pathogenic variation among
isolates of the same species is common and its analysis
demands standardized, reliable, and convenient devices for
scoring virulence. Pathogenic variation is not unique among
isolates of Xanthomonas pathogens. It has been reported for
the bean halo blight group of fluorescent pseudomonads (53).
Different investigators have used various inoculation
techniques which make it difficult to directly compare their

results. Most of the previous work on Xp and pr has been

15

based on the use of intact plants (55, 56). This is a
useful approach when screening for field tolerance but
intact plants sometimes have limited utility in the green—
house and growth chambers where space is a factor. Under
these conditions, intact plants are not only inconvenient
and tedious to use in the numbers required for genetic
analysis, but they are also variable in infectivity (sus-
ceptibility) to a degree that large replications are
necessary to ensure statistical reliability. To avoid the
problem of using intact plants, it is necessary that obser-
vations be obtained from the same plant tissue(s) each time.
Additionally, it would be necessary to compare results in
tissues of the same age since plant age is an important
factor in disease development.

Comparative pathogenicity in these studies required
an inoculation procedure that circumvented morphological
barriers, such as cuticle, by placing the bacteria directly
in the tissue where they may encounter host resistance
factors such as phytoalexins or naturally—occurring com“
pounds that may be toxic to the pathogen. Such an inocula-
tion technique also reveals which bean varieties exhibit
"true" genetic resistance or tolerance to invading pathogens.
The use of excised plant tissues for the study of bacterial
disease reactions has been reported. Klement et_g1. (32)
used excised bean pods while studying the defense reactions
induced by phytopathogenic bacteria. Perlasca (47) also

used excised bean pods in his study of relationships among

16

isolates of Pseudomonas syringae pathogenic on stone fruit

 

trees. More recently, Starr and Douglas (58) have reported
the use of excised bean pods as a sufficiently reliable,
sensitive and convenient technique for scoring virulence

of phytopathogenic bacteria. Until the present study, the
utility of this assay technique has not been employed in
the study of pathogenic variation among isolates of common
and fuscous blight bacteria. Previous workers (11, 55, 56)
have maintained strict adherence to the use of intact plants
purported to simulate natural situations in the field.
However, neither the use of intact plants nor excised
material actually simulates natural infection since both
methods involve artificial inoculation.

In the present study, my interest was not in the
ability of the bacterial isolates to overcome external
morphological barriers but in whether an isolate was able
to multiply and cause measurable disease within host tissue.
The following inoculation procedures were therefore
examined as possible techniques for scoring the pathogenic

potential of the isolates.

(a) Seed Infiltration: Although there is no study

 

regarding the actual biology of seed resistance to infec—
tion in nature, it became desirable, in the present study,
to explore the utility of seed infiltration in comparative
pathogenicity study in the greenhouse. Disease-free seeds

selected for uniformity of size and absence of cracks were

l7

pre—soaked in bacterial suspensions for different periods
of time and dried on paper towels before planting in
vermiculite medium. If germination and emergence occurred,
plants were scored for presence of lesions on cotyledons,
cotyledonary node and primary leaves. The result of pre—
soaking seeds for 10 minutes is given in Table 3. In

some cases there was 100% seedling emergence with little

or no lesion development while in other cases pre—emergence
rotting complicated the results. Because of the great
variability in results associated with the same isolate/
seed combinationjn three trials, this inoculation technique

was not employed in succeeding experiments.

 

 

 

 

TABLE 3.--Infection from soaking seed for 10 minutes.a'b
Xp 12 pr l6

Bean # # # #
Variety Expt.# Germinated Blighted Germinated Blighted
Tara I 14 0 15 12

II 15 4 12 8

III 15 0 10 0
MCC I 15 O 10 0

II 11 5 14 10

III 9 1 7 0

aReadings were taken 16 days after planting

b15 seeds were planted in each test

 

18

(b) Multiple Needle Technique: Leaves were

 

supported on a Sponge saturated with bacterial suspension
and pierced with a multiple needle inoculator (flower
arranging frog containing 66 number 1 pins) previously
wetted with bacterial suspension. By applying a small
pressure to the inoculated area, bacterial suspension was
forced through the puncture and the inoculum was spread
over the punctured surface by the forefinger. Controls
consisted of leaves punctured dry and leaves punctured and
'inoculated" with sterile distilled water.

The utility of this technique was limited mainly to
primary leaves and the results (Table 4) were not repro—
ducible in preliminary studies using Xp 12 and pr 16. The
inadequacy of the method was caused primarily by the diffi-
culty of controlling the amount of inoculum coming into
contact with the punctured surface. This method also
required a large number of plants for sufficient replication.
Needle punture was too severe on small trifoliolate leaves
which sometimes collapsed following inoculation with water.
This method was therefore considered unsuitable for

reliable comparison of isolates.

(c) Leaf Incision Technique: This was a modifica-

 

tion of a leaf-clipping technique designed to evaluate

resistance of rice varieties to Xanthomonas oryzae (28).

 

The procedure used in the present study consisted of

incising bean leaf lamina with a pair of dissecting

19

TABLE 4.——Pathogenicity of Xp 12 and pr 16 using multiple
needle inoculation technique.

 

% Necrosisb

 

 

Bean Varietya Expt.# Xp 12 pr 16

Tara I 0.0 25.3
II 10.7 15.0
III 0.0 45.1

MCC I 15.0 15.8
II 0.0 60.5
III 40.3 25.0

 

aPlants were 20 days old at inoculation

bReadings are averages of 6 replicates

scissors previously dipped in bacterial suspension. Disease
symptoms developed within 5+7 days after inoculation. In
susceptible reactions, water—soaked lesions readily developed
from the cut surface and advanced away from the incision.
The tolerant or resistant reaction was characterized by

mere browning of the cut edges, but no yellowing. Quantita—
tive and qualitative disease—reaction-differences were
easily demonstrated in terms of lesion size and rate of
spread of chlorotic halo which preceeded the advancing
necrotic lesion (Figure 2). Consistent results in lesion
size were obtained with inoculations performed at any point
along the leaf blade. However, the position of the leaf

was critical during the incision operation. Unless the

blade was held horizontally, excess inoculum accumulated at

 

20

the far end of the incision and resulted in varying lesion
shapes and sizes making quantitative measurements diffi—
cult. Therefore, leaves were held in a horizontal position
to achieve uniform inoculum distribution along the incised
surface. The incision method was useful for comparative
studies since (a) more than one isolate could be studied
on the same leaf or leaflet (Figure 2), (b) this method
reduced the number of plants required for replication, and
(0) less inoculum was required for inoculation (as many as
three incisions could be effected with a single dip of the
scissors into the inoculum suspension). The main dis~
advantage of the incision method was the possibility of
predisposition of wounded tissue to other low grade para—
sites. The utility of the procedure was therefore limited

to greenhouse and growth chamber studies.

(d) Excised Pod Technique: Green pods were

 

harvested from field-grown plants when the seeds were
approximately 50% of full size. Pods were surface—
decontaminated by washing in running tap water for 10
minutes and then by rinsing in sterile distilled water.
Pods were examined for freedom from injury and selected for
uniformity in size and maturity. To prevent cross“
infection during the inoculation procedure, 6—10 pods were
randomly distributed into sterile 14.5 cm diameter petri
dish moist-chambers lined with moist paper towel. Pods

were pricked while still immersed in bacterial suspension

21

contained in steam sterilized plastic containers. Depend-
ing on pod size (variable with individual bean variety),
5-10 points were pricked to a depth of 2 mm using a flame—
sterilized dissecting needle. Approximately 6.1 x 104
cells were introduced into the pod at each needle prick

as determined by serial dilution plate method. Inocula-
tions were restricted to one side of the pod. The pods
were then placed side by side in the moist chambers with
the inoculated side up and incubated at laboratory tempera—
ture (25 i 2 C). Pods were examined after 2w7 days, and
observations made of lesion types. Lesion classes were
quite discrete so that no difficulty was encountered in

classifying them (Figure 3). Quantitative measurements of

lesion sizes were also made.

(e) Leaf Water Soaking Techniques: Attempts were

 

made to simulate natural conditions of infection by gently
spraying inoculum to the point of run off from leaf sur—
faces. This treatment caused neither internal soaking of
the tissues nor wounding of leaf surfaces so that bacteria
would be required to enter through natural opening such as
stomata. Though this procedure seems desirable, consistent
results were not obtained in repeated trials and the method
was therefore rejected. A more conventional water—soaking
technique as described by Schuster (54) was then examined
with several modifications. Plants were inspected to

eliminate abnormal plants not in the desired physiological

22

 

 

Figure 2.-«Symptoms on primary leaves of MCC bean 16 days
after incisionninoculation with Xpl5, Xp22,
Xp24, and Xp 26.

 

Figure 3.-—Symptom types on green pods of P. I. 207262;
(A) = 'W' type; (B) = ‘N' and ‘D' types; and
(C) = 'WC' type.

23

stage of development. Generally, plants were either in the
vegetative stage, or in the reproductive stage at inocula—
tion. ’The leaf stage of the plants was noted and disease
reactions were scored on only those tissues present at the
time of inoculation. Although time consuming, it was
necessary, for purposes of uniformity, to individually
spray-inoculate the leaves. Leaves were held against the
palm and inoculated on the lower surface from a distance

of about 2 cm using a sprayer attached to a compressed air
line at 17 p.s.i. This treatment produced visible water—
soaked spots (approx. 0.5—1.0 mm diam.). In early studies,
plants were given 48 hours of 100% humidity treatment after
inoculation but this practice was discontinued in subsequent
experiments as discussed elsewhere. Typical blight symptoms
(Figure 1) were obtained in repeated trials. The main dis—
advantage of this inoculation procedure was related to the
length of time required for full symptom expression. It
usually took 14-21 days for optimum symptom development.
However, the method proved very efficient and reliable for
testing the reaction of bean varieties to blight bacteria

under field and greenhouse conditions.

EXPERIMENTS AND RESULTS

Comparative pathogenicity of isolates
of Xp and pr in bean leaves assayed
by leaf—incision technique

 

 

 

The pathogenicity of Xp and pr isolates was studied
under growth chamber and greenhouse conditions using the
leaf-incision method. The experimental design involved a
half-leaf inoculation procedure which permitted up to four
isolates to be tested on a single primary leaf. This
procedure eliminated plant to plant and leaf to leaf varia—

tions in susceptibility.

Growth chamber inoculation. Inoculations with

 

standard inoculum were generally performed within two hours
of inoculum preparation between 2:00 p.m. and 4:00 p.m.
After 10 days, leaves were scored for disease reactions.
Further information was obtained by similar inoculations

on the first and second trifoliolate leaves. An incomplete
block design allowed comparison of three isolates on the
same trifoliolate leaf at different leaflet positions.
Disease readings were taken on trifoliolate leaves seven

days after inoculation.

24

25

Greenhouse Inoculation. Primary leaves were inocu-

 

lated and plants were incubated at 100% R.H. for 48 hours.
Disease reactions were recorded seven days later. In all
cases, plants were 14 days old when primary leaves were
inoculated and 28 days old when trifoliolate leaves were
studied. Each experiment was repeated twice and the mean
readings were calculated for each isolate/tissue combina—
tion.

The results of growth chamber studies are given in
Tables 5 and 6. Disease reactions were scored in terms of
lesion size and symptom type. No isolate incited the same
symptom type and all tested host differentials; symptom
type was dependent upon the specific isolate/host combina—
tion. None of the host systems gave a uniform reaction
with all isolates. In general, the more pathogenic
(virulent) isolates incited 'D‘ and 'E‘ symptom types while
less virulent isolates incited 'A,‘ 'C' and 'D' types. The
'B' type of reaction was observed primarily in Idaho
Refugee and G.N. #1 Sel. 27 varieties (Table 6). Lesion
size varied with isolates from 0«l.7 mm in MCC, 0—2.1 mm
in Tara, 0-2.4 mm in Idaho Refugee, to 0-3.2 mm in G.N.

Sel 27.

In the greenhouse studies (Table 7), disease reac—
tion different qualitatively (lesion type) and quantitatively
(lesion size and number). Percent infection based on the
proportion of total inoculations showing pathogenic

symptoms varied from 0—100% in most isolate/host

26

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30

combinations. Lesion sizes and symptom types varied with
individual isolates on any particular host, and reactions
of any particular isolate varied from one host to another.
Effect of humidity on symptom

development in beans inoculated
with isolates of common blight

 

 

 

It is common practice, in pathogenicity tests, to
subject test plants to a high humidity environment before
and immediately following inoculation. In field studies,
Coyne et_a1. (12) furrow—irrigated plants immediately after
inoculation with common blight and wilt bacteria in order
to create a favorable microclimate for disease development.
They reported excellent infection and spread of the disease
under repeated irrigations. Zaumeyer (68) in a series of
experiments confined plants in a moist chamber for 24 hours
before and 24 hours after inoculation with Bacterium

(XanthomOnas) phaseoli. Infection was obtained without

 

premoisture treatment when plants were held in an infection
chamber for 24 hours after inoculation. He suggested that
post-inoculation moisture treatment was a factor in disease
development.

Seeds were planted in sterile soil in the green—
house. Four weeks after planting, plants were inoculated
by the water'soaking method. One set of plants (2 plants/
pot and 3 replicates per treatment) was immediately placed
in mist chamber for 48 hours; another set was maintained

at the same temperature (27 i 2 C) but without moisture

31

treatment. Disease reactions were noted three weeks after
inoculation on all plants (Table 9) but time required for
expression of first observable symptoms was scored for
Xle, Xp23, Xp24, XpU-2 and a mixture of the 4 Xps on

four varieties (Table 8). The experiment was repeated
under identical conditions.

The effect of humidity on time of symptom expres—
sion is shown in Table 8. Infection generally began either
as small discrete chlorotic and necrotic spots or as
water-soaked spots all of which eventually developed into
the typical blight lesions with necrosis and chlorosis as
observed in a susceptible-isolate—host combination. The
water soaking reaction was the most virulent reaction (most
compatible pathological combination) which always resulted
in large lesions and was observed only on susceptible—
isolate-host systems. Necrotic flecks and chlorotic spots
were observed on both susceptible and tolerant hosts. The
flecking response is a hypersensitive response characteris-
tic of a highly incompatible host-parasite relationship.
The flecks covered most of the leaf surface particularly on
primary leaves of susceptible varieties and usually led to
a general chlorosis of the entire leaf.

Moisture seemed important during the latent or
incubation period between inoculation and symptom expres-
sion. Specifically, moisture appeared to reduce the incu—
bation period in several isolate/host combinations (Xp23——

and XpU—2—-MCC systems) and gave rise to a more uniform

32

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HO HO HO HO HO HO O a a O HO HO HO HO HO HO NONOO~.H.O
0m 0m O02 O02 mm 0m O02 O02 HO HO HO O02 O02 O02 O02 O02 002
mm 0m mm mm mm mm U02 602 002 MVOZ U02 U02 mm mm 0m 0m SOHHGME
m 4 m 4 m 4 m 4 m 4 m 4 m 4 om n4 mumHum>

«sax mmmx 4402 Ommx Ommx Hmmx mme «me

 

mmumHomH nuH3 mcoHuommH mmmmmHo

 

.mx mo mmumHomH nqu pmumHsoosH mammn 2H pcmemon>m© Eoumahm so wquHfiss mo pommmmnn.m mqmde

34

reaction time in each variety regardless of the challenged
isolate. The latent periods were more variable when plants
were not given post-moisture treatment following inocula-
tion. In general, the latent period of each isolate varied
with the bean variety. There was no direct relationship
between the reaction time and the reaction type.

Moisture treatment following water—soaking
inoculation was not necessary for disease development
(Table 8). Also, symptom type and severity of disease were
not influenced by moisture as long as all plants were main—
tained at the same temperature. Moisture also had no
apparent effect on the virulence of the isolates. It was
apparent that once bacteria were placed in the intercellular
spaces of well—watered plants, the infection process pro-
ceeded independently of external moisture treatment. There—
fore, moisture treatment was omitted in all subsequent
experiments.
Effect of inoculum concentration on
infection efficiency of X. phaseoli

and X3*§haseoli var. fuscans in a
suscep 1 e ean

 

 

 

Klement et_al. (32) observed that the time of
symptom development with phytopathogenic bacteria in bean
pod infection Was independent of inoculum level and was
regulated by the rate of bacterial multiplication. On the
other hand, the intensity of the reaction of host tissues

depended on the inoculum level. In the present study,

35

Manitou bean leaves (particularly young trifoliolate leaves)
were uniformly susceptible to all 15 isolates of Xp and pr
when the inoculum contained 2.8 x 10.7 cells/ml. With such
a high inoculum density, it was difficult to detect any
pathogenic variation among isolates of both pathogens. In
order to use Manitou seedlings as a functional differential
host, an infectivity experiment was conducted to study the
comparative virulence of isolates based on the general
problem of the relationship of inoculum density to success-
ful development of disease reaction.

Three serial dilutions (2.8 x 107, 2.8 x 105 and
2.8 x 103 cells/ml) of inocula were prepared from 48-hour
cultures. Eighteen-day-old Manitou seedlings (2 plants/pot)
maintained in the greenhouse (28 i 2 C and 14 hours daylight)
were inoculated with each suspension using the modified
water-soaking procedure. Inoculated plants (3 replicates
per treatment) were returned to greenhouse conditions.
Disease reactions and percent leaf infection were recorded
14 days after inoculation (Table 10).

A direct relationship was evident between the necro—
tic potential (virulence) and inoculum density for all iso-
lates except pr28 and pr29. The slight virulence asso-
ciated with Xp2l and Xp23 at 2.8 x 107 cells/ml was completely

3

lost at 2.8 x 10 and 2.8 x 105 cells/ml. The dose—response

relationship was quite different for pr28 and pr29. In this

case, a higher pathogenic capability was expressed at 2.8 x

3

10 cell/ml than at 2.8 x 105 or 2.8 x 107 cells/ml. The slight

.ON H mm um
sOHHmHsoosH Hmpmm mmmp OH pmmmmmHU mmmHHOH UmpmHsooaH mo mmmusmoumm smmE n soHuommsH O0

.psmHmHou n a

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36

 

 

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0.00 0O 0.00 O02 0.0 HO OH 02

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w OmMOmHD w OmmmeD w Ommmmfifl

 

 

 

HE\OHHO0 HOHxO.m H2\OHH00 OOHxO.m Ha\mHH00 OOHxO.O

GOHHmesmocoo EsHsoosH

 

m.smmn sopHsmE
pHOlmmplmH 2H ucmfimon>m© mmmmmHU so sOHumHucmoaoo EDHSUOGH mo Hommmmll.OH mqm¢e

37

disease reaction observed in pr29/tissue combination at 2.8 x

3

10 cells/m1 was completely lost at inoculum density of 2.8 x

107

cells/ml.

The isolates differed in terms of disease reaction
and percent infection. Sometimes isolates incited identical
disease reactions but differed quantitatively in terms of
total necrosis. The existence of pathogenic variation
among the tested isolates was more evident at low inoculum
than at high inoculum densities. The data strongly suggest
the existence of pathogenic heterogeneity among isolates of
X. phaseoli and X, phaseoli var. fuscans. Based on the
quantitative response, three virulent classes were arbitrarily
recognized; the most virulent Group I comprised Xp15,

XpU-2, Xp24, pr16, pr844, and pr Ciat A. Group II with
moderate virulence comprised Xp12 and prl9, while the
least virulent Group III contained Xp21, Xp23, Xp25, pr28
and pr29.

Effect of leaf and plant age on the

development of common and fuscuous
blights of bean

 

 

 

In the preliminary studies, it was commonly observed
that leaves on the same inoculated plant developed different
sizes and numbers of lesions. This indicated that leaf age
and plant age should be examined in a study of pathogenic
variation in Xp and pr. The assumption was that if
isolates were homogeneous with respect to disease reaction—

factors, then the disease reactions should be similar or

38

identical in comparable tissues of the same age; however,
if isolates were heterogeneous, then they should differ
with respect to the degree of virulence and symptom type.
The virulence of pr isolates was studied using
plants of the same age (28 days at inoculation); each
plant, however, possessed leaves of different maturities.
At this stage the plants were in a 5—1eaf stage; the
primary leaf and first trifoliolate leaves were fully
expanded, the second trifoliolate leaves were still expand—
ing, the third trifoliolate leaves were just unfolding,
while the fourth trifoliolate leaves were completely
unfolded. Disease reactions were compared on all leaves

present at the time of waterfisoaking inoculation.

 

For the comparison of isolates of Xanthomonas
phaseoli, plants of different ages were grown (staggered
planting) and inoculated simultaneously using water—soaking
procedure. The three age groups (all vegetative) included
10 days, 20 days, and 30 days. Ten—day—old plants were in
the primary leaf stage; plants 20 days at inoculation
possessed fully open first and second trifoliolate leaves
and partially unfolded third trifoliolate leaves; 30—day-old
plants were in their fourth trifoliolate—leaf stage with the
'youngest leaf partially unfurled. In this case, disease
reactions were studied on the primary, first and second
trifoliolate leaves.

Seedlings were grown from diseasevfree seeds of MCC

and Manitou (for isolates of pr) and Manitou (for isolates

39

of Xp) beans. Seedlings were grown at the rate of 2 plants
per pot of compost soil at 27 :32 C and 14 hour photoperiod.
Selection for uniformity in leaf stage and leaf size and
freedom from malformation was done at the time of inocula—
tion. Leaves were numbered from the base of the plant
upward. Inoculation was made with a 48~hour culture of
isolates scraped into sterile distilled water. All leaves
were individually sprayed with the bacterial suspension on
the lower surface to cause visible water soaking. Water
soaking was progressively more difficult to effect as leaf
age increased. Longer exposure times and a reduction in
distance between the spray nozzle and tissues ensured
adequate water-soaking of older tissues. Plants were
placed at 27 2 2C and observed daily for symptom development.
Final disease rating was done 14 days after inoculation.
Time of first appearance of disease reaction in
infection with isolates of Xp is shown in Table 11. Suc-
cessful infection was evidence by the occurrence of water!
soaked lesions associated with bacterial ooze. With all
isolate/tissue combinations, the reaction time increased
‘with leaf age. A minumum reaction time of three days was
found for all isolates in lO-day—old primary leaves.
Typical disease reactions were not observed in 30—day—old
primary leaves regardless of isolate; in 20—dayaold primary
leaves, disease reactions were produced only by Xp24 and
XpU—Z at 5 days. The reaction time in first and second

trifoliolate leaves varied from 3-6 days depending on the

40

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.mSOOHH UmpmHsoosH so mHmonomc
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u H «EOHQEMO on u o umpmH0mH\mpsmHm O HO somm mummEHHmmxm N m0 mmmum>m n Hmavm
H.m m.H m.m O.o v.0 o.H o.m o.N mm 2mm:
m O O m m m m N m H m m m m m O
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4l

leaf-isolate combination. Isolates Xp22, and Xp23 failed
to incite typical disease reactions even at 14 days after
inoculation on comparable leaves of 30-day-old plants.

The disease reactions were scored using disease
index classes ranging from 0-5 as follows: 0 = no visible
disease symptoms; 1 = necrosis on less than 20% of inoculated
tissue; 2 = necrosis on 20—40% of tissue; 3 = necrosis
covering 40—60% of tissue; 4 = necrosis covering 60—80% of
tissue; and 5 = necrosis on more than 80% of tissue, often
accompanied by wilting and defoliation. The youngest leaf
on any plant of any age was most susceptible to both Xp and
pr blight isolates (Tables 11 and 12, respectively. Com—
parable leaves in different age groups reacted differently
to isolates of Xp; leaves on 30~day~old plants were the
least susceptible while those on lOOdayeold plants were the
most susceptible (Figure 4). The isolates were quite
heterogenous in their reaction phenotypes; they markedly
differed on the basis of their reaction in comparable tissues
of the same age group and in the same tissues of varying ages.
Based on the mean disease reading for all plant ages (Table
11), four virulence groups were easily identified for Xp
isolates. Virulence Group I comprised Xp15, Xp24, and
XpU2 with very similar and sometimes identical disease
reactions. These isolates were the most virulent on all
three plant—age groups and were associated with mean disease
ratings of 3.0, 3.3, and 3.1, respectively. Xp12 with a

mean disease rating of 2.0 was less virulent than members

42

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n

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002

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U

 

m

.mammn SOHHsmS cam 00: GH uanHQ msoomsm mo pcmfimon>mU so mmm mmmH mo HommmmII.NH mamma

43

 

 

 

 

Figure 4.——Effect of leaf age (A) and plant age (B) on
symptom development in Manitou bean inoculated
at 10, 20, and 30 days after planting.

44

of virulence Group I, but more virulent than the remaining
isolates; Xp12 constituted a single member of virulence
Group II. Xle and Xp25 with disease ratings of 1.0 and
1.3, respectively, were similar in their reaction and were
put in virulence GrOUp III, while Xp22 and Xp23 fell into
Class IV, the least virulent group with disease scores of
0.7 and 0.4, respectively.

On the basis of disease ratings obtained on
Manitou and MCC beans, three virulent groups or strains
were recognized among isolates of pr (Table 12). The
most virulent Group I consisted of pr844 and pr Ciat A,
each with a mean disease score of 3.4 and 3.0 in MCC and
4.0 and 3.8 in Manitou, respectively. Group II comprised
pr l6, prl8, and pr 19, each with disease scores of
2.6, 2.4, and 2.0 in MCC and 3.2, 3.4, and 3.2 in Manitou,
respectively. The third and least virulent group comprised
pr28 and pr29, each with a disease rating of 0.6 and 1.0
in MCC and 2.6 and 1.6 in Manitou, reSpectively.

In all cases, the most virulent isolates were able
to incite disease reactions in both old and young plant
tissues; infection with less virulent isolates was restricted
primarily to the young, more succulent tissues of the plant.
Separation of isolates into virulence groups was easier on
older tissues than on younger tissues where disease reac-

tions tended to be uniform for all tested isolates.

45

Comparative virulence of Xp and pr
blight isolates in Phaseolggspp. ‘
inoculated at vegetative and
reproductive stages of development

 

 

 

 

Reports relating to the effect of plant age on
susceptibility to infection by blight bacteria are incon-
sistent. Goss (22) found that older leaves were more
susceptible than younger leaves while Patel and Walker (46)
reported that the younger leaves were more susceptible. In
both cases, the workers failed to consider the physiological
stage of the plant as related to disease reaction. In
recent studies, Coyne et_al, (14) reported that suscepti—
bility or tolerance of beans to common blight depends
greatly upon the developmental stage of the plant; plants
are more susceptible when in reproductive stage than when
in the vegetative stage. They suggest that, in a breeding
program, it is important to select plants which exhibit high
tolerance during pod development, when the plants are most
susceptible. Their suggestion was based upon disease reac—
tion ratings of two bean lines, G.N. Nebraska #1, Sel. 27
(late) and the nearly—isogenic G.N. Nebraska #1, Sel. 27
(early), which were in vegetative and reproductive stages
of growth at inoculation. In essence, the major source of
variation in disease reaction may be plant age, but one of
the previous workers actually compared disease reactions in
the vegetative and reproductive stages of the same bean line
in a standardized environment. In order to test the claim

that bean plants are more susceptible in the reproductive

46

stage than in the vegetative stage of growth, a sequential
inoculation of plants (seeded at the same time) and/or
simultaneous inoculation of plants (employing staggered
planting) at different physiological stages under identical
experimental conditions is necessary.

In the present study, staggered planting technique
was employed. The reaction of different bean varieties to
Xp and pr isolates in vegetative and reproductive stages
of development was studied with seven varieties: G.N. Tara,
MCC, Manitou, Tepary (only vegetative in greenhouse),

G.N. Jules, P.I. 207262, and G.N. #1 Sel. 27. Plants in
the vegetative stage (21 days after seeding) and plants in
the reproductive stage (35 days after planting) were inocu»

7 cells/ml) using

lated with standard inoculum (2.8 x 10
leaf-water—soaking technique. Inoculated plants were placed
on greenhouse benches at 27 i 2 C and 14 hours photoperiod
and watered alternately with tap water and fertilizer solu—
tion (sequestrene, Fe as metallic 1.8 ppm and Rapid gro,
1 teaspoon per 2 liters of water). Symptoms were scored on
comparable inoculated leaves three weeks after inoculation.
There were differences in the reaction pattern of
host varieties to infection by isolates; some isolates
incited similar or identical disease reactions regardless
of the developmental stage of plant at inoculation, whereas
other isolates sometimes became slightly more virulent on

plants in the reproductive stage (Tables 13 and 14). These

observations suggest the existence of pathotypes with_varying

47

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48

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49

virulence which.may be affected by the stage of plant
development. The change in susceptibility or tolerance
with plant maturity depended on the specific host/isolate
combination. For example G.N. Jules and P.I. 207262 were
uniform in their reaction to individual isolates of Xp
(Table 13) both in vegetative and reproductive stages of
growth. However, these varieties were more susceptible

to most isolates of pr (Table 14) in the reproductive than
in the vegetative stages of growth. This general pattern
did not hold for a variety such as Manitou. For example,
the greater virulence of Xp22, Xp21, and pr 28, and pr29
associated with the vegetative growth was absent at the
reproductive stage of plant growth. It was difficult to
explain the decrease in virulence during the reproductive
stage of the Manitou variety. In general, the results in
Tables 13 and 14 showed that depending on the host/isolate
combination, some isolates which appeared slightly patho—
genic during the vegetative stage of plant growth became
more pathogenic during the reproductive stage. The data
confirm the report by Coyne et_al. (14) that many lines
may have a moderate level of tolerance in the vegetative
stage but become susceptible during the reproductive stage
of development. A comparison of disease reactions on all
seven differential hosts indicates the existence of patho—
genic variation among the tested isolates of Xp and pr

(Tables 13 and 14).

50

Effect of mixed inoculum of prand pr
on disease development in bean plants
assayed by leafuinciSion and waterO
soaking techniques *—

 

 

 

 

In a survey of internally—borne bacterial diseases
in Michigan Navy (pea) beans, Saettler and Perry (51)
reported that both Xp and pr were found together in about
50% of the seed lots. However, there is no report regarding
the co—existence of both bacteria in leaf tissues artifia
cially inoculated with a mixture of both.pathogens. Previous
workers have always studied infection using individual
isolates of either X, phaseoli or X. phaseoli var. fuscans.
In so doing, they have overlooked the possible aspect of
multiple infection with several isolates of each bacterial
type, as well as mixed infection with a combination of
common and fuscous blight isolates. Pompeu and Crowder
(48) reported that resistance of dry bean lines to Xp is
conditioned by a few genes; resistant and susceptible lines
have different genes for reaction. Coyne ep_al. (l4) and
Pompeu ep_al. (48) in isolated studies reported that reaction
to Xp is inherited quantitatively and that lines can be
developed with greater resistance than the parents. Arti—
ficial inoculations using several isolates of the same
species or a mixture of Xp and pr, have a direct bearing
in resistance-breeding programs since infection involving
different pathogen genotypes can very readily reveal which
plant entries have the desirable horizontal (quantitative)

resistance. The present study examined interactions

51

resulting from the co—existence of many pathogen cell types
in the same tissue in relation to disease development. Two
inoculation procedures were used:

(a) Leaf-Incision technique: Bean varieties G.N.

 

Jules, G.N. Tara, Manitou, MCC and Tepary, were grown in
vermiculite—peat mixture (3 parts vermiculite: 1 part
peat) in the growth chamber at 26 C and 14 hour photo—
period. SuSpensions of seven Xp and seven pr isolates
(Table 15) were prepared from 48 hour YCA—cultures. Mixed
inocula were prepared by combining appropriate suspensions
in equal volumes so that each composite inoculum contained
seven cell types. Xp composite inoculum was combined with
pr composite inoculum in a 1:1 (vol/vol) ratio and mixed
thoroughly to obtain an Xp—pr inoculum containing 14 cell
types. Prepared in this way, each inoculum type contained
approximately 107 cells/ml. Inoculation was performed on
lZ—day-old plants (2 seedlings/carton) using the half—leaf
method on uniform primary leaves. Inoculations with Xp12,
pr18 and distilled water were included as controls. Plants
were placed in the growth chamber at posteinoculation
temperature of 26 C, watered with nutrient solution, and
examined for type and size of lesion 10 days after inocula—
tion.

(b) Water-Soaking inoculation: Bean varieties were

 

grown in compost soil in the greenhouse. Plants were
thinned to two plants per pot and kept adequately watered

with nutrient solution. Inocula were prepared as described

 

 

52

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53

above and plants were inoculated 31 days after planting
using the water—soaking procedure. At the time of inocula—
tion, all plants except Tepary were in the reproductive
stage. Inoculations with Xp12, prl8 and distilled water
were included as controls. The experiment was run in
triplicate and disease reactions were rated three weeks
after inoculation.

The effect of mixed infection on disease development
using leaf—incision technique is presented in Table 15.
Infection was more severe with.multiple or mixed isolates
of Xp and pr than with Xp12 and pr18 alone. Composite
inocula of seven Xps consistently produced larger lesions
on all susceptible varieties than did Xp12 alone. The
same observation held for the seven prs inocula as com—
pared to isolate pr18. In most cases, a mixture of seven
Xps and seven prs resulted in slightly larger lesions.
Co-existence of multiple isolates did not result in any
apparent change in symptom type. All inoculum/host combi—
nations (except XplZ/Jules) produced necrotic lesions
associated with spreading chlorotic halo > 2 mm from the
lesion edge. Xp12/G.N. Jules system resulted in small
necrotic areas associated with restricted halos. Tepary
was tolerant not only to Xp12 and pr18 but also to all
composite inocula; symptoms consisted of a general yellowing
of tissue.

Disease reactions obtained with the watervsoaking

technique are shown in Table 16. The results were quite

54

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55

similar to those obtained with leaf—incision. There was no
evidence of antagonism among isolates in the composite
inocula since disease intensity was not reduced. With this
inoculation procedure, Tepary, G.N. Jules and G.N. #1,

Sel. 27 were tolerant to Xp12; Tepary and G.N. Jules were
slightly susceptible while G.N. #1, Sel. 27 was moderately
susceptible to composite inocula of seven Xps. Tepary was
tolerant to pr18 but slightly susceptible to a mixture of
seven prs. In general, mixed infection with composite

inocula was more severe than with either Xp12 or pr18.

Comparative virulence ofX, phaseoli

and X Ihaseoli var. fuscans isolates
in bean pods

Pod infection was studied in seven varieties of

 

 

Phaseolus spp. including G.N. Jules, G.N. Tara, G.N. #1,

 

Sel. 27, Tepary, Scarlet Runner (3. coccineus), MCC, and

 

P.I. 207262. The plants were grown at the Michigan State
University Farm using commercial production practices.

Pods were harvested when still green and about half—filled.
They were washed in running tap water to remove sand and
debris and any chemical residue from the surface, and rinsed
in sterile distilled water. After drying on sterile paper
towel, pods were carefully needle—inoculated with appro—
priate suspensions of blight isolates. Bacterial suspension
was introduced into the pod tissue by wounding the tissue
with a needle. Control pods were inoculated with sterile

distilled water. Following inoculation, the bacterial

56

suspension diffused in the mesocarp covering an area
ranging from 0.5-1.0 mm depending on the bean variety. In
most cases, the water soaked spots disappeared after 5—10
minutes and the affected tissue regained its original
color following absorption of the inoculum suspension.
All samples were incubated in 14 cm diam.-petri dish
moist-chambers at room temperature (25 i l C). Pods were
examined six days after inoculation and disease reactions
were compared both qualitatively and quantitatively on the
basis of lesion type (Figure 3) and lesion size, respec—
tively. Lesion size was determined by measuring two
diamEters at right angles to each other. Measurements were
performed only on green looking pods; yellow pods in the
senescent stage were discarded at the time of measurement
to eliminate a potential source of variability among
isolates.

Disease reactions were evident in most isolate/pod
combinations 2—3 days after inoculation but final readings
were obtainedsix days after inoculation. The reactions

were categorized into five symptom classes as follows:

'D' = dry needle hole showing no necrosis;

'WC' = dry needle hole with white chlorotic halo,
tissue not macerated;

'W' = water-soaked light to dark green necrotic
lesion with or without bacterial ooze,
tissue macerated;

'N' = necrotic, dry—looking brown lesion, tissue
not macerated;

'B' = wet looking dark brown necrotic lesion,

tissue macerated.

57

The 'W' class of lesions was indicative of the
virulence potential of the isolates in a susceptible
isolate-pod-combination; class 'B' was regarded as a more
advanced state of 'W'. It was difficult to determine the
nature of 'WC' and 'N' lesion types but their occurrence
indicated further variability in disease reaction. The
isolate/pod combination resulting in 'D' symptom class
indicated a tolerant pathological relationship similar to
water-inoculation controls. Based on the total number of
inoculations, the distribution of the percent lesion types
(Table 17) showed a wide spectrum of variation depending
on the host/isolate combination. Among isolates of X,
phaseoli, Xp12, Xp15, Xp24 and Xp25 consistently caused
a higher percentage of the 'W' lesion type than Xle and
Xp22. The 'WC‘ lesion type was uncommon and was incited
mainly by Xp2l. Among isolates of pr, infection with
pr16, pr18 and pr19 consistently resulted in higher per—
centages of the 'W' reaction type than did infection with
pr28 and pr29. None of the compared pr isolates incited
the 'WC' lesion type.

Table 18 gives the quantitative estimate of
necrosis incited by isolates of Xp in the tested bean
varieties. The data show that isolates markedly varied
in their invasive ability on the same and different hosts.
The quantitative virulence of Xp15 and Xp24 was significantly
greater than that of Xp12, Xp21, Xp22 and Xp25 at the 5%

level. Variation in virulence was also observed in

58

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60

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61

isolates of pr (Table 19). Two virulence groups were
recognized; Group I with greater virulence contained prl6,
pr18 and pr19 and the less virulent Group II contained
pr28 and pr29.

Pathogenic variability in_§p_pgg§gplit

and X, phaseoli var. fuscans in
field-grown bean plants

 

Previous greenhouse studies based on leaf~incision
and water-soaking inoculation procedures have consistently
shown considerable variation in the virulence of Xp and pr
isolates. Laboratory infection of green pods also indi—
cated qualitative as well as quantitative differences among
isolates of these bacteria. To establish and confirm exis—
tence of pathogenic variation among isolates of these
bacteria, it was desirable to compare, in an appropriately
designed field experiment, representative isolates of
common and fuscous blight bacteria originating from dif—
ferent geographical zones.

Four isolates each of Xp and pr, representing
different major bean—growing regions were employed in this
study. The isolates included XpU2 from Uganda, Xp21 and
pr Ciat A from Colombia, Xp23 and pr29 from Nebraska,
Xp15 and pr16 from Michigan, and pr844 from Guatemala.
Isolates were established from diseased seed and were main—
tained on YCA for a period of three years. Pathogenic
comparisons were performed on eight commercial bean

varieties. Susceptible varieties included Sanilac,

62

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63

Seafarer, MCC, Manitou, Ouray and Red Mexican U.I.#3,
tolerant varieties were G.N. #1, Sel. 27 and G.N. Tara.
Seedlings were grown from disease-free seed. A randomized
split-plot design, consisting of three replications was
used with main plots consisting of isolates and sub«plots
bean varieties. Each sub-plot contained two single rows,
10 feet long and spaced 28 inches apart. Each sub-plot
was isolated from the other by a compact row of corn
planted to buffer and check spread of bacteria from one
plot to another. Bacterial inocula were prepared from

48 hour-old YCA cultures in tap water and diluted to 106
cells/ml. Primary infection was established in one row
(spreader row) in each sub-plot by individually water-
soaking leaves with appropriate bacterial suspension using

a hand operated atomizer. Control rows were sprayed with
tap water only. At the time of inoculation (38 days after
seeding), Sanilac, Seafarer, MCC, Red Mexican U.I.#3, Tara
and Ouray were in the blossom stage of development while
G.N. #1, Sel. 27 and Manitou were in the vegetative stage.
Initial disease readings were taken 21 days after inocula-
tion and final disease reactions were taken 35 days after
inoculation. The virulence of isolates was compared using
the following criteria: (a) primary infection on inoculated
rows (Figure 5) described as severely susceptible (Se),

moderately susceptible (Mod), slightly susceptible ($1),

64

 

Figure 5.-—Disease reaction classes in field-grown beans
inoculated with blight bacteria; T = tolerant,
81 = slightly susceptible; Mod = moderately
susceptible, Se = severely susceptible.

65

 

Figure 5.——continued.

66

or tolerant (T); (b) secondary spread to adjacent non-
inoculated rows, based on the number of lesions/plant;

(c) pod infection on spreader (inoculated) row; (d) seed
infection/100 gm wt of seed from spreader row; and (e)
yield reduction relative to control. Quantitative measure-
ments were analyzed statistically to establish significant
differences resulting from differential virulence of
isolates.

Table 20 summarizes disease reactions on spreader
rows and shows the intensity of secondary spread of the
disease to adjacent tester rows in infections with isolates
of Xp. The isolates differed not only in the host reaction
types (amount of necrosis) but also in the amount of
secondary spread to adjacent rows 28 inches away. Based
on the amount of necrosis, XpU2 was the most virulent
isolate followed by Xp15. Xp21 and Xp23 were the least
virulent on the same varieties giving, in most cases, a
tolerant reaction. XpU2 and Xp15 could not be differen—
tiated in their reaction in Sanilac, Seafarer, MCC and
G.N. Tara. However, they differed in their reaction in
Manitou, Red Mexican U.II#3 and Ouray where XpU2 was con—
sistently more virulent than Xp15. Secondary infection
was dependent upon primary infection and varied among
isolates. In all compatible combinations, secondary spread
was more effective with Xp15 and XpU2 than with Xp23 and
Xp21. Infection with Xp23 usually resulted in no secondary

spread since primary infection was very slight.

67

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68

prl6, pr Ciat A and pr 844 were quite virulent
and tended to be identical in their disease reactions in
the same bean varieties (Table 21). The slight variation
in disease reaction observed in MCC, G.N. #1, Sel. 27 and
Manitou was not distinct enough to differentiate these
isolates into virulent classes. pr29 was less virulent
than prl6, pr844 and pr Ciat A. Primary infection with
prl6, pr844 and pr Ciat A resulted in more secondary
spread than infection with pr29. As noted for Xp,
secondary spread in infection with isolates of pr was
dependent upon the level of primary infection.

Virulence of Xp and pr isolates were distinct
(Tables 20 and 21). Of interest was the susceptibility of
G.N. #1, Sel. 27 and G.N. Tara to isolates of pr and
their tolerance to isolates of Xp. In general, isolates
of pr resulted in higher disease reactions and more
secondary spread than did isolates of Xp in the same bean
varieties.

Table 22 shows pod infection data. There was slight
variation among isolates but in general, number of lesions
per pod was very low. The data on seed infection of
Sanilac bean is shown in Table 23. The number of diseased
seeds was lower in infection with isolates of Xp than with
pr isolates. Statistical analysis of data showed no
difference among isolates of Xp; among isolates of pr,
pr16 was similar to pr844 and pr Ciat A but differed

significantly from pr29.

69

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71

TABLE 23.—-Seed infection in field—grown Sanilac bean
inoculated with isolates of Xp and pr.X '

 

 

Isolate Seed infectionY
XpU2 0. 4 bZ

Xp15 0.4 b

Xp21 1.4 b

Xp23 0.0 b

pr16 12.1 a

pr844 5.2 ab

pr Ciat A 6.4 ab

pr29 0.0 b
Control(H20) 0.0 b

 

XPlants were in blossom stage at inoculation.

yNumber of diseased seeds/100 gm. wt. (samples ranged
from 150—200 gm. wt.)

ZMean separation by Duncan's multiple range test, 5%
level.

 

 

72

The relationship between disease reactions and yield
(seed weight) was studied in two varieties: Sanilac and Red
Mexican U.I.#3 (Table 24). Variation in yield due to the
differential virulence of isolates was significant at 5%
level. The difference between the 'check' mean and each
isolate mean was significant in both bean varieties. On
Sanilac, Xp21 and Xp15 were similar in virulence but
differed from both XpU2 and Xp23; prl6 and pr Ciat A were
similar in virulence and differed only slightly from pr844
but significantly from Xp29. The following schematic

diagram illustrates the virulent groups observed on Sanilac:

prl6 XpU2 Xp15

n H > Xp21 :> Xp23

Ilv

pr Ciat A pr844 pr29

 

 

I II III

A comparison of isolate means on Red Mexican showed
that Xp15, Xp21 and Xp23 were similar in virulence but they
differed significantly from XpU2. Among isolates of pr,
pr844 was similar to pr Ciat A but differed significantly
from pr16 and pr29. Schematically, the following
virulence scale was proposed for isolates of Xp and pr on

Red Mexican:

 

73

TABLE 24.-—Effect of Xp and pr isolates on yield (seed
weight) of fieldegrown Sanilac and Red
Mexican U.IZ#3 beans.X

 

Seed weight (gm)y

 

 

Isolate Sanilac Red Mexican U.I.#3
XpU2 96.7 cdeZ 99.4 de

Xp15 112.8 bc 123.5 bc

Xp21 117.1 bc 141.7 b

Xp23 134.4 b 143.5 b

pr16 78.6 de 117.3 ed
pr844 88.0 cde 92.3 e

pr Ciat A 72.1 e 105.3 cde
pr29 103.3 bcd 126.6 bc
Control(H20) 184.9 a 187.5 a

 

XPlants were inoculated in the blossom stage.
yAverage of 3 replicates with 10 plants each.

zMean separation by Duncan's multiple range test,
5% level.

74

 

 

 

pr844
" pr29 Xp21
pr Ciat A Z pr16 > u > u
n
XpU2 Xp15 Xp23
I II III

In general, disease was more severe with most
isolates of pr than with isolates of Xp on both Sanilac
and Red Mexican U.I.#3.

Population trends of Xp and pr isolates

in tolerant and susceptible bean
cultivars as related to disease reaction

 

 

 

The ability of a phytopathogen to grow and multiply
in the host is often considered as a compatible pathogen/
host relationship which involves the recognition of the
host by the pathogen. Pr0ponents of the so—called Fgene-
for-gene' theory argue that an incompatible relationship
results from the lack of recognition of the host by the
pathogen. Studies by previous workers (56) have demonstrated
the existence of pathotypes among isolates of Xp. However,
we do not know whether the ability of the pathogen to
recognize and concomitantly multiply in one host, and its
inability to recognize and multiply in another host, is
what accounts for the compatible and incompatible relation-
ship with the hosts. Leben (35) has reported the recovery

of Xanthomonas vesicatoria from non-diseased tomato seed-

 

lings; the lack of internal multiplication and invasion of

 

75

the tissue by the pathogen may explain lack of disease
development. Stall and Cook (52) related hypersensitivity
and susceptibility in pepper to bacterial concentration;

the concentration of §.vesicatoria associated with necrosis

 

in hypersensitive pepper tissues was lower than that in
susceptible tissues. Their observation suggests that the
ability of bacteria to grow and multiply is a factor in
the pathogenicity or virulence of the bacteria in the
susceptible pepper. The present investigation concerns

the population trends of g, phaseoli and x3 phaseoli var.

 

 

fuscans isolates relative to symptom development in several
commercial bean varieties.

Populations were determined in G.N. Jules, Manitou
and G.N. Tara. The first part of the experiment involved
infection with individual isolates of Xp: Xp21, Xp23,

Xp24 and XpU2. These isolates were selected because of

their reported pathogenic variation in Rhaseolus spp. (56).

 

The second study concerned mixed infection with: (a) com0
posite inocula containing Xp21, Xp23, Xp24 and XpU2, and
(b) a mixture of seven isolates of Xp and seven isolates of
pr. Manitou was selected as a suitable host because it is
reported to be susceptible to both Xp and pr (5) while
G.N. Tara and G.N. Jules were chosen because of their
previously reported tolerance to both Xp and pr (5, 10)
and Xp (11), respectively.

Bacterial cells washed from plates of two—day-old

YCA cultures were suspended in distilled water at a standard

76

concentration of 2.8 x 107 cells/m1, or at a lower concen—
tration of 2.8 x 104 cells/m1. In mixed inoculations,
individual isolate suspensions were prepared and combined
in equal volumes (10 ml/isolate) togive a multincomponent
or composite inoculum. The suspension was vigorously
shaken to mix the cells and produce a uniform inoculum
which, in the case of 7 Xps + 7 prs, yielded a l Xp:

lpr cell ratio on YCA—dilution«plate counts in repeated
trials.

Disease-free seeds were planted (2 seedlings/
carton) in steamed compost soil contained in 32 oz. wax-
lined cardboard cartons in the greenhouse at 28 i 2 C and
14 hours photoperiod. All leaves were inoculated by the
water-soaking method within 1—2 hours after inoculum
preparation two weeks after planting (vegetative period).
Multiplication in mixed infection with four isolates of Xp
was studied in first trifoliolate leaves of G.N. Tara,
while population trends in infection with individual
isolates were examined in first trifoliolate and primary
leaves of G.N. Tara and.Manitou, respectively. Multiple
infection with pooled inoculum of Xp and pr isolates was
studied in first trifoliolate leaves of Manitou and G.N.
Jules.

Populations of viable bacterial cells were deter-
mined from ten 9 mm—diameter leaf discs from three
inoculated leaves selected at random. Discs were surface

sterilized in 2.6% NaOCl for 10—15 seconds followed by

 

77

rinsing in three changes of sterile distilled water. The
discs were placed in 5 ml of sterile distilled water and
thoroughly comminuted in a glass tissue grinder. Tenfold
serial dilutions of ground suspensions were made in sterile
distilled water, and 0.1 ml aliquots pipetted into empty
sterile Petri plates. YCA previously cooled to about 45 C
was poured into each plate (about 15 ml/plate) and mixed
with the suspension by gently swirling the plate in two
directions. Plates were incubated at 25 C and colonies
were generally counted after 305 days of incubation in
individual infections and after 5—7 days in mixed infection
with Xp + pr.

Population after inoculation (Figure 6) resembled
that of a typical bacterial growthrcurve. Four phases of
growth were recognized as follows: (a) a short lag phase
(0-24 hr.); (b) the logarithmic or exponential phase;

(0) the stationary phase; and (d) the decline or death
phase. No host/isolate specificity was apparent although
1.5—2 times as many viable XpU2 cells were sometimes
recovered as compared to Xp21, Xp23 and Xp24. The isolates
multiplied at different rates particularly during exponen-
tial and decline phases but the growth patterns were quite
similar at the stationary growth phase. The growth curve
generated by the composite inoculum of the four isolates
in multiple infection closely resembled those of the
individual isolates, thus showing no apparent existence of
antagonism or complementarity among the isolates in growth

requirements.

Figure 6.——Population trends of Xp isolates in trifoliolate
leaves of G.N. Tara inoculated with.2.8x10
cells/ml.

Figure 7.—-Di1uti0n plate used for isolating bacteria from
G.N. Tara leaves 12 days after inoculation with
a mixture of Xp21, Xp23, Xp24, and XpU2; (A) =
large colonies of Xp; (B) = small colonies of
orange~yellow new phenotype (XpE); and (C) =
grayish white colonies of resident bacteria.

78

 

0

01AM DISCS G MCIED TISSUE
-

u.S/TEN9MM

0'

L

 

 

 

LOCBMVIABLE CE

 

9 12 15 18 2
DAYS A"!!! INOCULATION

Figure 6

 

Figure 7

 

 

79

Symptoms on G.N. Tara differed with each isolate.
No symptoms developed up to four days after inoculation:
the first disease reactions were chlorotic spots develOped
with Xp24 and XpU2, five days after inoculation. Necrotic
lesions developed at six days with Xp24 and XpU2 and
after 21 days, XpU2 lesions developed into large necrotic
lesions (> 5 mm diam) to give a 'severely susceptible'
(Se) reaction-type. Lesions with Xp24 remained restricted
in size (< 3 mm diam) and gave rise to a 'slightly—
susceptible' (Sl) reaction type. Xp21 initially produced
a general chlorosis 7—8 days after inoculation; at 21 days,
a 'Sl' reaction type was associated with Xp21 while a
'tolerant' (T) reaction associated with neither chlorosis
nor necrosis, was observed in Xp23/Taracombination.
Symptoms were delayed with composite inoculum (9 days)
but the final disease reaction was ‘Se'.

A new orange yellow phenotype (designated XpE,
Figure 13) was recovered from G.N. Tara leaves inoculated
with the composite inoculum. The occurrence of the 'new'
type was first observed nine days after inoculation; the
frequency of isolation generally ranged for 005 colonies
per plate. XpE appeared to differ from the parental types
(Xp21, Xp23, Xp24, XpU2) in its growth requirements; it
grew very slowly on YCA but very actively in yeast extract
liquid medium. In initial tests, it was highly pathogenic
in susceptible Manitou but it seemed to belong to a

serotype different from that of the parental types

 

80

(Figure 13). Periodic inoculation of Manitou seedlings
with transferred cultures of XpE maintained on YCA indi—
cated apparent decrease in pathogenic potential with a
concomitant increase in ability to grow in YCA.

Multiplication pattern in Manitou inoculated with
low inoculum concentration (104 cells/ml) is shown in
Figure 8. The growth pattern also resembled the general
pattern observed for these isolates in G.N. Tara. The
more virulent XpU2 and Xp24 showed an enhanced growth rate
over the less virulent Xp21 and Xp23 during the early
stages of infection; more advanced stages of infection
sometimes yielded more viable cells of the less virulent
Xp21. The least number of viable cells was recovered from
Xp23—infected leaves. In all cases, no symptoms were
observed up to 14 days after inoculation; the first indi—
cation of disease (a general chlorosis associated with a
few necrotic areas) was associated with XpU2 and Xp24, 16
_days after inoculation. At the low inoculum density, plants
inoculated with Xp21 and Xp23 were not different from water
controls. However, in all cases, the population of viable
cells rose rapidly from a level of about 102 cells one hour
after inoculation, to a high population level of about 107
cells/ten 9 mm-diameter leaf discs, 16 days after inocu—
lation.

The populations of Xp and pr in mixed infection of
Manitou and G.N. Jules (Figure 9 and 10, respectively) were

similar over 21 days, increasing rapidly one hour after

81

 

O
—
‘
fl
-
-

l

o
I

on
I

‘
T

 

lOG‘oNO mu CELLS/TEN 9m DIAM oascs or mam I’ISSUE

 

 

21 ‘. 1 l l 1 1
o 1 4 1 1o 13 10

DAYS AFTER INOCULAIION

Figure 8.——P0pulation trends of Xp isolates in primary
leaves of Manitou bean inoculated with
2.8x104 cells/ml.

 

 

82

 

 

 

 

 

8 I I I I I I
g n——-—I
2 .///°”““\
7 —- /
Is "7
§
3
3.
i 0
9
:39
l l 1 J _MJ
9 12 15 18 21

 

DAYS AFTER INOCULATION

Figure 9.—-Effect of mixed infection on the number of viable
Xp and pr cells in diseased Manitou leaves.

 

on
—1
—
-(
-
—(
d

N

0’

 

 

 

LOG” NO VIABLE CELLS/TEN9MM DIAM DISCSOF IWECTED TISSUE
m

00
O '0;
w L—

 

J J l l
6 9 12 15 13 21

DAYS AFTER NOCULATION

 

Figure lO.--Effect of mixed infection on the number of
viable Xp and pr cells in diseased G.N.
Jules leaves.

 

83

inoculation to a peak 15 days later followed by a decline
through 21-days. The growth curves indicated differential
growth rates between Xp and pr in mixed infection.
Between 0-9 days (in G.N. Jules, Figure 10), and 0-6 days
(in Manitou, Figure 9), the total population of viable
cells was composed of twice as many Xp as pr; at nine
days, the total population of viable cells consisted of
about equal numbers of Xp and pr in Manitou; between 12
and 15 days, Xp and pr were recovered at a frequency of
l Xp: 4 pr in Manitou and l Xp: 3 pr in G.N. Jules; but
at 18 days, the population of viable cells comprised Xp
and pr in a 1:1 ratio in G.N. Jules and twice as many pr
as Xp in Manitou. pr exhibited a typical stationary
phase of growth in both bean varieties; a gradual decline
phase immediately followed the exponential growth phase in
Xp/host combinations. In most cases, 1.5—2 times as many
total viable cells were recovered from the less susceptible
or 'tolerant' G.N. Jules as from the susceptible Manitou
bean. The disease reaction in Manitou was 'Se' (more than
80% of inoculated leaves necrotic) and 'Mod' (30-40% of
tissue affected) in G.N. Jules, at 21 days, although G.N.
Jules contained higher populations of viable cells than did
Manitou throughout the period of determination.

There was a gradual build—up of a new orange-
yellow phenotype among mixed p0pulations of Xp and pr
nine days after inoculation in both Manitou and G.N. Jules;

the frequency of occurrence increased in later stages of

84

infection to a maximum 18 days after inoculation. The
sudden appearance of the new cell type was concomitant
with a rapid decline in recovery of both Xp and pr; the
new phenotype was not observed in tissues inoculated with
sterile distilled water. Growth of the new phenotype was
very limited on YCA. The small discrete colonies remained
hard and depressed at the center. Subsequent transfers on
YCA failed to generate measurable growth and growth in
yeast extract liquid medium was negative. The pathogeni—
city of the 'new' cell type could not be verified because
of insufficient inoculum supply; the phenotype was subse—
quently lost in culture.

Pathogenicity of isolates of X. haseoli

and z, phaseoli var. fuscang‘in cowpeas
(Vigna unguigulata) I

 

It has hitherto been observed that g. phaseoli is

 

pathogenic on Phaseolus vulgaris but non-pathogenic on

 

Vigna spp. (personal communications with Dr. N. Vakili,
U.S.D.A. Research Plant Pathologist, Puerto Rico) whereas

g. vignicola is pathogenic on both hosts. The deve10pment

 

of yellow halo in association with lesions distinguishes

x. phaseoli from x, vignicola (Vakili unpublished). We

 

 

consistently observed chlorotic halos around lesions incited

by all isolates of Xp and pr in P. vulgaris. Pathogenicity

 

of Xp and pr originating from different ecological zones

was determined on Vigna unguiculata.

 

85

Cultivars Purple Hull Southern Pea and Mississippi
Silver Pea, were grown in steam—sterilized compost soil in
the greenhouse at 27 i 2 C. Inoculum consisting of
individual isolates of Xp and pr (Table 25) was prepared
from 48-hour old YCA cultures and adjusted to standard
concentration. Seedlings were inoculated at primary leaf
nodes 14 days after planting using a seedling injection
procedure (50) and incubated on greenhouse benches.

Xp23 was highly pathogenic on Purple Hull Southern
Pea (Table 25). Infected plants were stunted and wilted
and extensive necrotic lesions on the stem were associated
with brownish—yellow bacterial ooze indicative of active
multiplication and tissue invasion. All other isolates were
non—pathogenic on this host; the needle—injection punctures
were dry-looking and showed no observable evidence of
browning or ooze production. Except for Xp23, the isolates
were not readily differentiated based on their reaction in
Southern Pea. Host reaction types were more evident on
Mississippi Silver Pea; this host was Slightly susceptible
to Xp15 and pr18; moderately susceptible to Xp21, prl6
and pr Ciat A; severely susceptible to Xp23 and pr844;
and resistant to Xp12, Xp22, Xp24, Xp25, XpU2, pr19,
pr28 and pr29.

Serological studies of 5p and
pr insolates

 

 

Xp and pr comprise a large number of isolates that

cannot be distinguished from one another by symptomatology

86

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87

in the field. Significant variation, however, exists among
isolates within the same group and between isolates of Xp
and pr in their necrotic or disease potential. Phage
typing and pigment production are additional methods used

to identify and differentiate the two closely—related groups

of Xanthomonas species. Both Xp and pr produce a non—

 

diffusable yellow pigment on culture but pr also produces
a brown diffusable pigment on certain high carbohydrate
media. Each group belongs to a different lysotype (27, 31).
Phage typing and pigment production are useful only for
separating Xp from pr and not for separating isolates
within the same group. Prior to the report by Schuster and
Coyne (55), a pathological homogeneity was supposed for
isolates of Xp; they first reported the existence of patho—
genic variability among Xp isolates. Our broad collection
of Xp and pr isolates from wide geographical locations not
only confirms their findings but further shows that there
exist pathotypes among isolates of pr as well. Knowledge
on the serology of these groups of bacteria is still incom-
plete; previous investigators (18) have suggested that Xp
and pr may belong to the same serotype but no definitive
studies have been carried out to justify such claims.
Pathogenic variation in the studied isolates strongly
suggests the probable existence of serological heterogeneity
in the two groups of blight bacteria. The purpose of this
study was to provide further information on the serological

identities and relationships among phenotypically—identical

88

isolates within a group and between isolates of the two
groups, and in addition, an attempt was made to correlate
the findings to the differential pathogenicity on commer-
cial bean varieties.

Eight isolates of Xp and seven isolates of pr
were included in the study. Stock cultures were grown on
YCA and antigens consisting of living cells ("0" antigen)
were prepared from polysaccharideafree cultures; polysaccharide
production is reported to interfere with agglutination tests
when cultures are grown on high carbohydrate medium (18).
The elimination of most of these interfering poly—
saccharidic antigens was achieved by growing cells in
buffered yeast extract (10 gm yeast extract/l PO4 buffer,
pH 6.9). The phosphate buffer used was .OlM Na HPO4 -

2
KH PO . Antigen samples were harvested in the logarithmic

2 4

phase of growth, usually 12-18 hours after incubation with
continuous shaking. The cells were sedimented by centri—
fuging at room temperature for 20 min. at 10,000 g to free
them from growth medium and pigments. The sediment was
resuspended and washed at least two times with physiolo—
gical buffered (PO4 buffer pH6.9) saline (0.85% NaCl w/v)
by centrifuging. The final titer was adjusted turbide-
mitrically to 109 - 1010 cells/ml.

The antisera were prepared in rabbits and provided
through the courtesy of Dr. A. W. Saettler (Department of
Botany and Plant Pathology, Michigan State University).

They included antisera prepared against Xp15, Xp24, prl6

 

 

89

and pr28. The rabbits were immunized by five intravenous
injections with.live cells and 4—5 blood samples were
obtained by ear bleeding at 5+7 day intervals following
the last injection. The last sample with the highest
antibody titer was the test serum and was preserved in
sodium azide (500 ppm NaN3), stored in the freezer, and
used as needed.

Serological relationships were tested by cross—
agglutination, agglutinin—absorption and immunodiffusion
techniques.

For cross—agglutination, bacterial suspensions were
compared at antisera dilutions up to 1:2560; one drop of
antigen was added to a drop of anti-serum in a spot depres-
sion slide. The antigen and antiserum were mixed and
allowed to react undisturbed for 10—20 minutes at 25 i l C;
antiserum plus buffered saline and antigen plus buffered
saline were included as controls. Slides were examined
microscopically using obliquely—transmitted light and
reaction end points were recorded (Table 26).

In general, titers (Table 26) were highest with
Xp antisera and Xp isolates than with pr isolates. With
pr antisera the reverse was obtained. The wide range of
reaction end points suggest heterogeneity in antigenic
action of the tested isolates. Reactions between homo-
logous antigen/antisera combinations gave higher titer
end points than did certain reaction between heterologous

pairs. Certain heterologous combinations equalled

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91

homologous combinations. Cross agglutination of Xp12,
Xp15, Xp24 and XpU2 against antisera for Xp15 and Xp24
indicated that similarities exist; the antisera agglutinated
these isolates to a titer of 1:1280. Isolates Xp21, Xp22
and Xp25 were intermediate. The lowest dilution end points
of 1:10 (with antiserum for Xp15) and 1:160 (with anti—
serum for Xp24) was obtained with Xp23. End points with
antisera for pr16 and pr28 were markedly lower than those
obtained with antisera for Xp15 and Xp24; they varied from
1:640 for XpU2 to 1:120 for Xp24. Again the reaction of
Xp23 was quite different fromthose of the other isolates

of Xp; no agglutination was observed with antiserum for
pr28 while a titer as low as 1:60 was obtained with anti-
serum against pr16.

The cross-agglutination reactions of pr isolates
except pr19 and possibly pr844 were quite similar with
all antisera; the titer was 1:640 to 1:1280 for pr16 and
pr28 antisera and 1:640 for Xp15 and Xp24 antisera. pr
isolates except pr19 were not differentiated by cross
agglutination test. The reaction of pr19 was quite dif—
ferent; the antisera against pr16 and pr28 agglutinated
this isolate to a titer of 1:640 while antisera against
Xp15 and Xp24 gave titer end points of 1:160 and 1:320,
respectively.

The lack of adequate differentiation of isolates
by cross agglutination reaction made it desirable to further

compare them by agglutinin—absorption procedure (Table 27).

€92

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93

Antisera were absorbed overnight at 4 C with excess antigen
in test tubes. The supernatant was transferred to sterile
plastic tubes and centrifuged to remove all agglutination
products. The supernatant was then cross—reacted with
freshly prepared antigens (109 cells/ml) in depression
slides.

There was greater similarity among isolates of the
same group and a greater serological difference between
isolates of Xp and pr. Among Xp isolates, Xp15, Xp22
and XpU2 were quite similar in their agglutinin—absorption
reaction; Xp21 and Xp23 were very similar; and Xp12, Xp24
and Xp25 were each different from the others. Among
isolates of pr, pr844 and pr Ciat A were very Similar
in their reaction, pr18 and pr29 were similar; and pr16,
pr19 and pr28 were quite different from the others. The
results thus suggested the existence of serotypes among
isolates of Xp and pr. The observed serological relation—
ships among isolates of the same group and between isolates
of Xp and pr was further tested on immunodiffusion plates.

The immunodiffusion agar medium contained 8.5 gm
purified agar, 10 ml 1% Orange G (dye) and 500 ppm NaN3 per
liter of buffered saline (0.85% w/v NaCl). The medium was
steamed to dissolve the agar and then dispensed into
unscratched 9 cm diam. plastic Petri plates at the rate of
15 ml/plate. In each plate, six antigenic wells equidistant
from the central antiserum well were dug out using previously

0

prepared patterns. Antigens (2.8 x 101 cells/ml) consisting

94

of lS-hour—old cultures ('0' antigens) were used. When
antisera were previously absorbed, the absorption was done
with excess homologous antigens in test tubes at 25 i l C
overnight. The supernatants were separated from the
pellets and further purified by centrifugation. The
supernatant-antiserum or unabsorbed antiserum was placed'
in the central well and reacted with antigens contained in
outside wells.

The difference between the number of bands asso—
ciated with unabsorbed antisera and antisera previously
absorbed with homologous antigens (Table 28) indicate the
number of antigenic constituents common to each isolate
pair. The complete elimination of precipitin bands (except
for pr29) associated with isolates of pr and the reduc—
tion in the total number of bands obtained with isolates
of Xp following previous homologous absorption of pr28
antiserum, showed the serological affinity of pr28 to the
other pr isolates and the more distant relatedness of
pr28 to Xp isolates (Figure 11). pr16 was less related
to Xp isolates than was pr28; pr16 shared only one
specific antigen with Xp12 and XpU2, two antigens with
Xp22, Xp24 and Xp25, and no specific antigens with Xp15,
Xp21 and Xp23 (Figure 12). Absorption of Xp15 antiserum
with Xp15 was even more interesting; the bands associated
with unabsorbed antiserum were completely eliminated by
previous absorption with the homologous antigen (Figure 13).

Complete absorption of Xp24 antiserum with Xp24 and pr28

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Figure ll.——Immunodiffusion patterns obtained from the
reaction of pr28 antiserum (central well)
with pr isolates before (A), and after (B)
homologous absorption of antiserum and,
reaction of pr28 antiserum (central well)
with Xp isolates before (C), and after (D)
absorption with pr28.

97

 

Figure 12.-—Immunodiffusion patterns obtained from the
reaction of pr16 antiserum (central well)
with Xp isolates before (A) and after (B)
homologous absorption of antiserum with prl6.

 

Figure 13.——Immunodiffusion patterns obtained from the
reaction of Xp15 antiserum (central well)
with Xp isolates before (C) and after (D)
homologous absorption of antiserum with Xp15.

 

 

 

 

 

98

antiserum with pr28 in repeated trials could not be
effected but the high number of percipitin bands obtained
with heterologous antigens were either completely elimi—
nated or effectively reduced to l or 2 in most cases after
previous absorption with homologous antigen. It is possible
that Xp24 and pr28 isolates used in this study are not
identical with isolates used for antiserum preparation.

Xp15 and Xp24 antisera produced arc—shaped bands very close
to antigen wells of isolates Xp12, Xp15, Xp22, Xp25 and
XpU2. In general, there were marked differences in the
number of bands associated with individual isolates; in

most cases, the highest number of bands were obtained with
homologous antigen/antiserum pairs but sometimes heterolo—
gous pairs gave rise to more bands than did homologous

pairs (e.g., pr28 antiserum/pr28 antigen = 3 bands whereas

pr28 antiserum/pr29 antigen = 4 bands).

99

 

 

Figure l4.——Disease reaction (A) and reaction categories
(B) of P.I.207262 inoculated with pr isolates.

 

DISCUSSION

This study has examined in detail the existence of

pathogenic variation among isolates of Xanthomonas phaseoli

 

first observed by Schuster et al. (55). Their studies,
however, concerned four isolates of common blight assayed
by water-soaking procedure only, but the present study was

broadened to include isolates of both g, phaseoli and g.

 

phaseoli var. fuscans originating from distinct ecological

 

bean producing areas represented by Nebraska, Michigan and
Idaho in the U.S.A.; Colombia in South America; Uganda in

Africa; and Guatemala in Central America. By comparing the
pathogenicity of these representative isolates on selected

commercial varieties of Phaseolus spp. and Vigna anguiculata

 

 

using different inoculation techniques detailed in Material
and Methods, I have marshalled an evidence for the exis—

tence of pathogenic variation in both Xanthomonas phaseoli

 

and §. phaseoli var. fuscans.

 

Attempts to more closely simulate natural conditions
of infection were made by gently spraying the inoculum to
run-off onto leaf surfaces; this treatment neither caused
wounding of leaf surfaces nor internal soaking of tissues

so that cell penetration of the tissue would be through

100

101

stomata. This inoculation procedure, although desirable,
proved unsuitable since uniform infection was not obtained
consistently. Results were consistent when inoculum was
physically placed in the cellular environment; this was
achieved by pin-prick inoculation of submerged pods, leaf—
incision procedure, and water-soaking of leaves. The
placement of cells inside the tissue has the advantage of
ensuring each cell type an equal opportunity for infection.
The method directly compared the physiological ability of
each isolate to invade the cellular environment following
intromission into cellular spaces; such ability is a
reflection of the genetic potential which involves patho—
gen nutrition, induction of gene products or enzymes, and
the elaboration of metabolites for cell growth and multi«
plication. Theoretically, the reaction phenotypes in
inoculated tissues under identical conditions would be
expected to be uniform or identical if reaction factors

in the isolates were homogeneous; on the contrary, however,
a heterogeneous population of isolates should separate into
distinct phenotypes on the basis of their reaction with a
physiologically—dynamic intracellular environment. Pheno-
typic reaction types were identified in this study. In
addition to adequately standardizing disease reaction con-
ditions, these inoculation procedures have some practical
utility in screening for resistance, since active biochemical
resistance can readily be distinguished from a conditional
rather passive morphological resistance exhibited by some

plants.

 

102

Leaf clipping has been used for study of other
bacterial diseases. Winstead and Kelman (67) used clip—

pings technique to evaluate resistance to Pseudomonas

 

solanacearum and McCarter et a1. (42) in field studies

 

reported spread of g. solanacearum and tobacco mosaic

 

virus (TMV) from diseased to healthy tomato plants by
clipping with rotary mower. Recently, Kauffman et a1. (28)
have reported the use of clipping procedure for inoculating

rice, Oryza sativa with Xanthomonas oryzae. However, until

 

 

the present study, bean pathologists have not explored the
utility of leaf—clipping and leaf-incision techniques in
screening for varietal resistance and pathogenic varia—
bility among isolates of bean blight bacteria. In the
present study, leaf—incision inoculation allowed a reliable
qualitative and quantitative evaluation of infection and
the separation of isolates into virulent groups or strains;
disease indices from leaf to leaf and from plant to plant
showed little or no variation and the large number of
replications gave valid disease readings.

Pod infection was not only useful in demonstrating
the existence of pathogenic variation but also provided
additional information on varietal susceptibility or
resistance of commercial hosts to infection by blight
isolates. Resistances associated with vegetative tissues
of some bean varieties were not necessarily expressed during
pod infection. For example, leaves of P.I.207262 were

resistant to infection by Xp21, Xp22 and Xp23 but these

 

 

 

 

103

isolates incited measurable lesions in inoculated pods.
This observation indicates that the infection process in
the leaf may be quite different from that in the pod and
mandates the use of leaf as well as pod inoculations in
studies designed to screen for resistance in commercial
bean varieties. A disease may be overt in one part of a

plant and latent in another part; for example, Xanthomonas

 

vesicatoria, the cause of bacterial spot of tomato and

 

pepper, has been isolated from the interior of symptomless
fruits on plants that showed evidence of infection on
leaves (35). To be acceptable, a bean variety should be
resistant both in its vegetative and reproductive stages
of growth.

Water-soaking procedure was more difficult to effect
than either the pineprick inoculation of pods or the leaf—
incision procedure; physical characteristics of the leaf
such as number of stomata and degree of internal cellular
barriers, affected the rate of penetration, but in most
leaves the flooding took place readily. At 17 p.s.i., water—
soaking was easily effected without causing evident physical
damage to the inoculated tissue; severe disease reactions
on susceptible bean varieties were produced both in field
and greenhouse experiments and varietal reactions induced
were highly reproducible.

Qualitative and quantitative characterization of
isolates based on their pathogenic reactions in commercial

bean varieties separated the isolates arbitrarily into

 

104

virulence—phenotypic groups. Among isolates of‘E: phaseoli,
XpU2, Xp15 and Xp24 were the most virulent isolates con—
stituting Group I; Xp12 and Xp25 expressed intermediate
virulence and were placed in Group II; and Xp21, Xp22 and
Xp23, with the least virulence were put in Group III.
Similarly, three virulence groups were constructed for
isolates of x, phaseoli var. fuscans; the pooled data on
infectivity placed pr844 and pr Ciat A in the most

virulent Group I; pr16, pr18 and pr19 belonged to the

 

intermediate virulence——Group II; while pr28 and pr29
constituted the least virulent Group III.

In considering the reasons for the observed
differential virulence, a number of alternative hypotheses
were entertained. Firstly, the differential pathogenicity
might result from either 'long association‘ by some isolates
or 'strange contact' by some isolates with the tested com—
mercial bean varieties. If this hypothesis is true, then
high virulence may be a consequence of long exposure and
adaptability to a particular host while avirulence may
result from short—term contact with the host tissue.

Whether virulence preceeded avirulence or vice versa needs
genetic verification but under this theory it is reasonable
to speculate that one pathogenic level evolved from the
other in a stepwise manner.

The second hypothesis assumes that what accounts for
the observed pathogenic variation may involve interactions

between the isolates and the normal resident microflora;

 

 

105

complementary or antagonistic interactions may specify the
type and level of disease. During in;yiyg multiplication
studies, four resident phenotypes ranging in colour from
red, brown and purple to chocolate white were often iso-
lated from field—grown, healthy—looking beans (David Weller,
personal communications); their interaction in mixed infec-
tion with individual isolates was not examined but they
were apparently non—pathogenic when individually tested on
susceptible hosts.

The third hypothesis relates virulence to the ability
of the isolate to recognize the tissue microenvironment. In
genetic terms, this hypothesis requires that for an isolate
to be virulent, it must possess an ‘early gene‘ whose
product or function can recognize the cellular environment
and cause the production of specific degradative enzymes
which act on host metabolites and result in successful infec—
tion. In contradistinction, an avirulent phenotype may
result from non—recognition of the micro-environment, a
probable consequence of loss or change of function of
pathogen gene products. Under this hypothesis, it is rea—
sonable to speculate that different host systems require
different pathogen gene products for the establishment of
compatibility relationships. A large number of early genes
would convey an extensive virulence whereas a small number
of such genes would specify a limited virulence to the
isolate. The 'wild' phenotype (exemplified by pr844,

pr Ciat A, Xp15 and XpU2) may belong to the former

 

106

genotype whereas a 'mutant' phenotype (typified by Xp21,
Xp23, Xp22, pr28 and pr29) might represent the latter
genotype. Under this model, virulence is a function of
early and late gene products; early gene product in the
absence of late gene product (degradative product) results
in avirulent phenotype or latent infection. The isolation
of the gene loci specifying the production of functional
pathogenic factors requires a detailed knowledge of the

genetic map of a standard blight isolate and is a subject

 

for further investigation.

The final hypothesis holds that the ability of an
isolate to actively multiply in a host is of secondary
rather than primary importance in infection bionomics;
in the in_yiy9 multiplication studies, such ability did
not necessarily determine the ability to cause visible
disease symptoms. Multiplication may involve a symbiotic
relationship in which case the pathogen actively synthe—
sizes the precursors of its progeny without adversely
upsetting the physiology or gross anatomy of the host.
This type of multiplication results in a tolerant reaction
phenotype as in the case of Xp23/Tara combination. If,
however, multiplication is accompanied by the production
of infection factors, then infection ensues; in this case,
a high rate of multiplication will effectively enhance
disease severity since the amount of degradative factors
would be directly proportional to bacterial cell concen«

tration. The different levels of disease incited in the

107

universal suscept, Manitou, may be related to the tissue.
The development of necrosis or pathogenic relationship in
a susceptible isolate—host combination is seen as a morbid
bite—the-hand-that—feeds existence of isolate while no
symptom expression associated with active multiplication
in tolerant varieties is a symbiotic or commensal relation—
ship. If pathogenicity in the latter parasitic system is
based on the so-called geneafor—gene relationship, then
some genetically-controlled biochemical deficiencies
emanating from some functional gene mutation may account
for the observed variation in virulence among Xp and pr
isolates toward the host.

The delayed symptom expression in primary leaves
of Manitou inoculated with a low inoculum density (2.8 x
104 cells/ml) may be associated with a bacteriostatic
action postulated by Allington et_§1, (1); when Manitou
leaves were inoculated with 107 cells/ml, necrotic lesions
were observed in about 7-8 days after inoculation. Perhaps,
the more concentrated inoculum increased too rapidly to a
necrosis level before any bacteriostasis developed. Leben
and co-workers (37) observed that a low inoculum concentra~
tion resulted in lower population maxima and delayed symptom
expression in soybean. The ability of an isolate to
multiply to a high population level without observable
disease reaction may occur under conditions less exacting
than those required for infection. The invasion of a plant

by a pathogen not accompanied by symptom development, is in

 

108

accordance with the idea that parasitism and pathogenicity
are not necessarily related. The invasion of symptomless
carrier plants, however, has important epidemiological
consequences since such hosts, through normal cultural
operations or through the activities of ubiquitous insects
may serve as a primary source of inoculum for nearby sus-
ceptible hosts and an important overewintering source
(survival value) for the next season crOp. This poses a
major threat to control programs because, if conditions
favorable for infection and disease development prevail for
a prolonged period, a sudden outbreak of bacterial blight
may originate from previously healthynlooking plants and
spread to adjacent rows.

Active bacterial multiplication associated with no
visual infection has been reported for other phytopatho-
genic bacteria. Leben (35) reports active multiplication

of g. vesicatoria on tomato seedlings without apparent

 

effect on seedlings; English and co—workers (19) and Crosse

(15) also isolated bacterial pathogens (Pseudomonas

 

syringae) from non-diseased hosts such as almond, peach

 

and cherry trees. Kennedy (29) has shown that Pseudomonas.

 

glycinea, the cause of leaf spot of soybean, is often

 

present within host stems during the growing season, but
without profuse multiplication and without evidence of
systematic infection. Thomas and others (63) in another

study demonstrated the presence of Xanthomonas_phaseoli.

 

and Corynebacterium flacumfaciens, the cause of bean common

 

 

 

109

blight and wilt, respectively, in the stems of apparently
healthy pinto bean plants. Gaumann (21) terms this type

of infection "inapparent" or "symptomless" and Leben (36)
describes the host/pathogen combination as a "resident
phase" during the life cycle of the pathogen in which active
growth and multiplication are not necessarily followed by
signs and symptom expression. What accounts for the absence
of disease symptoms in a host which actively supports
pathogen multiplication is not known, but I submit that

such "latent infections" result from some genetically con—
trolled alteration in essential functions of parasite which
specify compatible pathogenic interactions. Surface sterili-
zation of leaves followed by serial washings in sterile
distilled water strongly support an endophytic rather than
epiphytic association of Xp23 and Xp21 with the symptomless
tolerant G.N. Tara bean.

Leaf age and plant age affected the virulence of
some of the tested isolates. The pooled data on infecti-
vity of isolates as affected by plant age and leaf age
(Tables 11, 12) separated the isolates into virulence
phenotypes. It is possible that developmental alterations
in the metabolism of older tissues may lead to the produc-
tion of toxic products which inactivate degradative enzymes
or functions of the pathogen. Dye (17) points out that
§. phaseoli and g, phaseoli var. fuscans utilize similar
metabolites in culture; however, a successful in yit£9_

utilization of metabolites does not necessarily imply that

110

metabolism of the same compounds would be effected in ziyg.
In_yiyg_metabolism may require regulatory factors in a
physiologically—dynamic host tissue. Johnson (26) points
out that the amount of polymorphic variation at the enzyme
loci of natural populations is quite high. Enzyme poly—
morphisms increase adaptiveness by providing a means of
metabolically compensating for a varying environment; thus
individuals with multiple molecular forms of an enzyme may
be capable of coping with different cellular environments.
Whether enzyme polymorphism at several gene loci is
responsible for the observed pathogenic variation among
isolates of Xp and pr awaits verification by detailed
biochemical experimentation in conjunction with a detailed
map of the genome.

An increase in tolerance with age of leaf has been
reported in cucumber against the angular leafspot pathogen,

Pseudomonas lachrymans (65) and in tomato against §.

 

vesicatori (43). In cucumber it was correlated with a

 

decrease in amino nitrogen and in tomato it was associated
with a decrease in total and soluble nitrogen. In bean
leaves, Patel and Walker (46) point out that no single
amino acid showed a pattern of change with aging that could
be associated with high susceptibility of younger leaves
and the high tolerance of the older leaves. Their studies,
however, did not involve plants in different physiological
stages. The present study showed that bean plants were
more susceptible to blight infection in the reproductive

than in the vegetative stage of growth; some isolates with

 

111

a suppressed pathogenic potential at the vegetative stage
of plant growth showed enhanced virulence when plants
entered the flowering or reproductive phase (Tables 13
and 14). Xp21, Xp22, pr28 and pr29 were, however, more
pathogenic on Manitou when in the vegetative than when in
the reproductive stage of growth.

Multiple infections with several isolates each of
Xp or pr and mixed infection with combined Xp and pr
isolates have not been reported in literature. This study
examined this aspect of infection and the results indicate
that several isolates of the same bacterium as well as a
misture of isolates of Xp and pr can compatibly co—exist
and cause disease in the same plant tissue. Mixed infec—
tion with Xp and pr isolates indicated no biocidal or
biostatic effect of one cell type upon another. Both
phenotypes were recovered from infected tissue but at
varying frequencies on culture media. Symptomvwise, viru—
lence complementation was not observed in multiple infec-

tions with isolates of the same Xanthomonas sp. or in

 

infected plants inoculated with isolates of Xp and pr.

The results, however, did not preclude the possibility that
complementary biological functions do exist among isolates;
studies on synthetic media may reveal their existence. In
mixed inoculations, proportionately fewer of the total
population of cells comprised Xp in later infection thus
indicating a less saprophytic ability of Xp or a reduced

competitive ability in mixed infection with pr. The rapid

 

112

decline in viable pOpulation of Xp might be a result of
rapid depletion in synthesis precursor pool more actively
competed for and accumulated by pr cells.

The recovery of a new phenotype in mixed infections
was interesting and noteworthy; it pointed to a possible
means for the evolution of new isolates or cell types which
may complicate blight—control programs. The sudden appear-
ance of the 'new' type was concomitant with a rapid decline
in the total population of Xp and pr thus suggesting a
higher selective saprophytic advantage for the new pheno—
type over that of the parental types. The absence of the
new type in water—inoculated control plants indicated that
the new type was not a normal resident of leaf surface or
intercellular leaf spaces.

Comparative studies in field infections with

isolates of §. phaseoli and §. phaseoli var. fuscans indi—

 

cated that the two groups may differ in moisture requirement
for infection. Coyne et al. (12) obtained excellent infec—

tion in field trials with g, phaseoli and reported rapid

 

spread from inoculated plants to the surrounding bean crOp
causing severe foliage damage and reduction in yield. In
the present study, spread occurred under natural conditions
without the introduction of irrigation water, a likely
source of injury for intromission of bacteria into succulent
bean tissues. The practice of Spraying inoculated plants
with water at 180 pounds' pressure at two-week intervals (12)

may account for the frequently reported spread of disease

 

113

from a local primary source of inoculum to healthy sur~
rounding plants. Under natural conditions of rain and
insect damage (notably from members of the Order Orthoptera,
Coleoptera and Homoptera), personal observations showed
little spread from inoculated spreader rows to adjacent
tester rows, 28 inches apart. However, such spread
appeared more efficient and common with isolates of pr

than with Xp isolates; the observed differences in the
degree of secondary spread had a functional relationship
with the amount of primary infection on the inoculated
spreader rows. The yield losses accompanying infection with
most isolates of pr were higher than those caused by iso—
lates of Xp. The unusually hot, dry weather during the
growing season could account for the reduced virulence of
Xp isolates in field tests as compared to their pathogenic
efficiency in greenhouse and growth chamber studies where
plants were adequately watered. The observation suggests
the importance of water potential or turgor pressure in
infection bionomics. Isolates of pr appeared to be indif-
ferent to moisture stress and showed no apparent attenuation
in their pathogenic potential under the same conditions in
the field.

It is a truism that weather determines whether a
compatible pathogen—host relationship will develop into
disease whereas climate is an important indicator of the
probable limits of spread and the destructive potential of

the pathogen. Humidity is one of the weather factors

 

114

important in bean blight infection. In greenhouse studies
where plants were kept well watered, moisture was not impor—
tant in disease development once the bacteria had been
placed in the cellular space. The pooled data from field
and greenhouse studies, however, indicate that disease
forecasting based exclusively on weather predictions will
yield little positive results if the population and compo“
sition of the bacterial isolates to be encountered are not
included in such studies.

The result of serology studies presented interpre—
tative difficulties but the data generally substantiate
isolate differentiation. Isolates shared some antigenic
components in common but the different titer end-points
and precipitin bands indicated the existence of serotypes
among isolates of Xp and pr; the serological relationship
was closer between isolates within a group than between
isolates of Xp and isolates of pr. We do not know the
primary significance of antigenicity in pathogenesis of
blight; the observed variability in antigenic constituents
in relation to pathogenic variation among isolates of blight
bacteria awaits further verification. Strider (59) and
Strider and Lucas (60) demonstrated no serological differ-

ence in isolates of Corynebacterium michiganense although

 

these isolates varied in virulence. In Pseudomonas

 

lachrymans Lucas and Grogan (39) reported that little or no

 

difference could be detected in the virulence of smooth and

rough isolates although serological variability was reported

115

among isolates of the same pathogen (38). Guthrie (24)

reported no serological differences in Pseudomonas

 

phaseolicola races 1 and 2.

 

The earlier reports regarding the tolerant or
resistant reactions of various bean varieties to Xp and
pr (Table 2) needs to be re—examined. The present study
has shown that some of these varieties, for example, P.I.
207262 and G.N. Tara, are susceptible to certain isolates
of both groups of bacteria (Figure 14), thus suggesting

the existence of host—specialized pathotypes in Xanthomonas

 

pathogenic on beans. This study emphasizes the importance

of using breeding material with different germplasms in
crosses aimed at evolving a variety with desirable horizontal
resistance. It is equally essential to include representa—
tive isolates of a given geOgraphical area in resistance
screening programs for effective evaluation.

At the present time, little or no success has been
achieved with chemical protectants for controlling bean
blights; immediate hope rests with use of disease-free seed
under proper cultural practices, but long term hope rests
in the development of resistant and tolerant varieties.
However, while the outlook for successful exploitation of
resistance in control of bacterial blight of beans looks
promising and fascinating, the studies required to effect
it are rather complex and require new techniques and
ingenuity. Further research in this area requires a more

thorough understanding of the behavior of blight pathogens

 

116

in nature, their existence and distribution, survival,
genetics, and population dynamics, both in the presence
and absence of the hosts and vectors. The biology and
virulence of these pathogens in nature on a world basis
constitute a challenging approach for phytopathologists
engaged in breeding bean for resistance. This study sug—
gests an urgent need for a comprehensive centralized

collection of all reported isolates of Xanthomonas phaseoli

 

and g. phaseoli var. fuscans that would be readily avail-
able to all laboratories engaged in bean crop management

and bacterial blight studies.

 

 

 

S UMMARY

This study examined the existence of pathogenic

variation in Xanthomonas phaseoli (Xp) and §, phaseoli

 

var. fuscans (pr), the incitants of common and fuscous

blights of beans, respectively. Eight isolates of Xp and

 

seven isolates of pr were selected from bean production
areas located in different geographical regions, and were
maintained at room temperature (25 i l C) on YCA throughout
the period of investigation. Pathogenic variation was
studied on 13 commercial bean varieties selected on the
basis of their reported tolerance or susceptibility to
common and fuscous blight pathogens. Different inoculation
techniques were examined; inoculation by seed infiltration
and multiple needle leaf-puncture methods were less satis-
isfactory than leaf—incision, leaf waterusoaking and excised
pod techniques. Inoculum consisted of 48 hour YCA bacterial
cultures suspended in sterile distilled water and adjusted
to a standard concentration of 2.8 x 107 cells/ml using
turbidimetric procedure.

The results of leaf-incision inoculation for patho—
genicity tests under growth chamber and greenhouse condi—

tions indicated that disease reactions associated with

117

 

 

118

individual isolates differed qualitatively and quantita-
tively. The quantitative differences were based on mean
lesion size. Postuinoculation moisture treatment was not
necessary for optimum symptom development but did shorten
the latent period between inoculation and symptom
expression.

Isolates differed in their ability to cause disease
at different inoculum levels. Using serial dilutions of
inocula, the tested isolates were easily separated into

three virulent classes based on their necrotic potential

 

on susceptible Manitou bean. Pathogenic variation among

isolates was more evident at low inoculum than at high

inoculum concentrations. The data suggest the existence

of pathogenic heterogeneity among the compared isolates.
Leaf age and plant age had a marked effect on the

virulence of isolates. The most virulent isolates were

able to incite disease reactions in both old and young

plant tissues; infection with less virulent isolates was

restricted primarily to the young, more succulent tissues

of the plant. Separation of isolates into virulent groups

or strains was more evident on older tissues than on younger

tissues where disease reactions tended to be uniform for all

tested isolates. The developmental stage of the plant was

important in disease development. Depending on the host/

isolate combination, some isolates which appeared slightly

pathogenic during the vegetative stage of plant growth

became more virulent during the reproductive stage. The

 

 

 

 

119

disease reactions suggest the existence of pathogenic
variation among isolates of Xp and pr.

Several isolates of Xp or pr and a mixture of
isolates of both bacteria co—existed in the same tissue
without any evidence of antagonism. In most cases, mixed
infection with composite inocula resulted in more severe
infection than when individual isolates were used. Mixed
infections with composite inocula generated "new“ cell
phenotypes which were not observed in control plants

similarly inoculated with.water. The occurrence of the

 

new phenotypes suggests a possible source of new isolates
of blight bacteria.

Isolates differed in their ability to cause necrotic
reactions in excised bean pods. Some isolates that were not
pathogenic on leaf tissues caused measurable lesions on
excised pods of the same variety. Statistical analysis
indicated significant differences in quantitative virulence
of the tested isolates. In the field-grown beans, marked
differences were also noted in disease reactons incited
by isolates. Statistical comparisons of yield reductions
associated with individual isolates separated the isolates
into three virulent groups or strains. In general, disease
was more severe with isolates of pr than with isolates of
Xp on both Sanilac and Red Mexican U.I.#3 in field tests.
The isolates also showed marked variation in virulence on

Vigna unguiculata; Xp23 was highly pathogenic on Purple

 

 

Hull Southern Pea, but all other isolates were non—pathogenic

 

 

120

on this host. Pathotypes were more evident on Mississippi
Silver Pea where three virulent classes were evident.

The population variation with time (colony counts)
of Xp21, Xp23, Xp24 and XpU2 in tolerant G.N. Tara bean
resembled a typical bacterial growth—curve (Figure 6) with
a lag phase, exponential growth phase, stationary, and
decline phases of growth. Xp24 and XpU2 incited disease
symptoms, but active growth of Xp21 and Xp23 was not

accompanied by symptom production. The result indicates

 

that active bacterial multiplication in cell tissue may
not necessarily result in disease development. A low
inoculum concentration of 104 cells/ml resulted in delayed
symptom expression in a susceptible Manitou tissue which
was readily diseased at 107 cells/ml level. Serological
variability was observed among isolates of Xp and pr, but
such variability could not be directly correlated to the

observed pathogenic variation among these isolates.

 

LITERATURE CITED

10.

 

LITERATURE CITED

Allington, W. R. and D. W. Chamberlain. 1949. Trends
in the population of pathogenic bacteria within
leaf tissues of susceptible and immune plant species.
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Andersen, A. L. 1951. Bacterial diseases of Michigan
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