'EI

("11"1'”.
1-... 1 ,..44;'.==";u'

.1"....%¥.}1’$1

 
 
 
      

'_‘_‘1.:3" 15". 1'.” 1151‘}
w... ‘- 1 3" 3,1"

   

”do!“ A” il‘n.‘

  

   
   

I. km. |‘

. i: 111' 5.13%»? ‘fl
14, . 3,.»

"WW5?" "i " V;
I' 3' 7

2..

‘2,

5-4

    
  

   
    
   
    
      
  
 
 

\
1.1:?”

‘1“
fig‘f:g

 

     

1 L14:
2;""11' fl" 3 30+”

W591 .

.’::::.‘>'Q I\ v 51

(a :51.

424-23. 4mg- .-
-: fi‘QEE‘Wthu'J
‘0 {r'l#r

:

.
' ‘.,

   

M
u-‘I.r.l”% I '3‘

. '4'ié 3303‘.
. h“ ‘9?

   
  

 
    

  
 

'3'“.

l." L

\- mL- '_

's'll'hb; $.21“:
., I
4

      

,.I

1":
"I .1731“ . (hilt:
.wfi‘lr‘é-h-zné‘ "".'1'.‘1‘1"—'.'¢‘
. 1N ‘ «w I. a» -+
1 3' | lfgri .3 v'.|.~“$"“‘1'|
L ‘01133394. ”IN-g u ”IQ}::‘%}"C

43.} .

.. ' ‘1
l I'r‘\v.‘3 . ‘3 WY“: . "fig“, I [I K
"Q. ‘34? ' ‘v%(%§11§)‘ ’QI‘V. " ‘ '
I | Pk ‘ '

  
   

  
  
      
    
       
          

,
:-
i’g- ,
E5353"
Ir

     
 
       

 
  
 
 

 

     
   
 
   
  

     

‘_. '_‘ Wm.—

   
 
        

.
.'L_\ \- 1
. 111-3 - . f.h\1£'£i‘3‘§¢ H
I' I 3 4 ' ' I
3:.” 11:33‘3 u ‘4'\?&~;{‘:\1J'£' .1 “"V.“ KM: T34} 3.03233“. . 33.33
. 1.- 4' ' . . ‘ I. . . I 1'5, 1K3
. 1. ,4 11.313.11-‘c-3L4u; ~31 3-4-52 “1211453; 1" .s‘a'é'w; . 343-3 L 3??“
‘ ’ L '| ' 'I.) ‘ ‘V ‘ 'f; 1'1‘ I \J. . ‘ ‘ 1'
‘ ' <l: ‘I'fi'w'dei'f‘fll‘ I " "W I‘ .5)" §"" 0 _£§:";3'§ 'V“. LHi‘é‘éiis
. ,1; -, a... _ 3 4' 3. ~ {£5 *‘- ‘- t1 ~- 4
' ”' 1”- '0: fig- :3- "fi' ' ' “x . 3‘” "b ' "mF {53:53 “1‘ L'_ nib-131;,“
" I-II‘.“' ' ‘5 ' 0"! EM '5'
1 1' 4-1.1.1‘21/ I: H'R'x. ‘6
' 1 .' IL." "'1'13 3-r33 . I In mic—n
(‘1': " ' ' 'Al'." ' " I'n:'.'L ”f "11*," t 115'." U‘nvg'fl'
' NH” I . " . “31"3‘1:H
' 2:4 ' “5’? ‘1" ‘ '1 ”34' "as 15‘3"" INN
1 1‘ 3. . . 3 "L '3 "I I I" 'I I I .
l 3 M. 3A..“ " 1.9.:«A' ‘3- ’1‘ I :3”: ':'\N"
I'IL'. .'_I x l -.‘u. 13.133113“ 0 I ' I
'11. ' .4

""‘ 1‘. r'W.‘ 1121:1444! ":51. Vita/11' *4." - -

. 112’?“ '

' .11,”er u u‘

iota-a F5 {.443'135335 094'— - ~"r'5- ;-_-&.£‘-9L

A.“

 

 

_ -1
': . ' ."-
"i1 . . 3 . .. _31. . .' : . 1 z 31' 3113, ' I131. .11.. .
—- 1 -‘I ~ . 2.113 L 3' 13.3. . ”3313" ”I
3 § 133.3 3 , 33' '13' 1 ‘ 1m .13“ 1.1.3 w 1'1. ‘fl . . 'l)“
1 3.. 3 1113 13333....” 1 . 3‘.u1.3 511 3 1' 3. 3 3.333113 «1.31
L ‘ I 1 1 ‘I "

v-a

on;

1 : 1" -’1'-’...
3113 3| 1. 1 11;“ ”11114,," 1333 ”.01 .11333 ”.4":
“9.1111152 41111.22“ 442:;- .4_m44xm:.

TH E518

_.; -.-—.J-
.

t’dalt..fig'g-_‘Lx--—mm --
. 3

L I 13 RA 1-- Y
5‘4": sm Stan:

‘mversity I _.

..7—‘..-"-; .r

  
 

' '.
i
5
I
Em

' 3

 

This is to certify that the

thesis entitled

rfi‘v

In Cucumber

presented by

James Michael Dessert

has been accepted towards fulfillment
of the requirements for

Ph.D . degree in Horticulture

Date 513/? / /
/ /

0-7 639

     
    

OVERDUE FINES:

25¢ per day per i ten
RETURNING LIBRARY MATERIALS
\:

Place in book return rate remove
charge frau circulation records

‘J All/z‘ii‘?‘ A
~' \~ ~.II‘II ’l,

    
   
   

 

PSEUDOMONAS LACHRYMANS RESISTANCE IN CUCUMBER

 

By

James Michae] Dessert

A DISSERTATION

Submitted to
Michigan State University
in partiai fulfiilment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1981

ABSTRACT
PSEUDOMONAS LACHRYMANS RESISTANCE IN CUCUMBER

 

By

James Michael Dessert

Cucumber (Cucumis sativus L.) lines resistant to angular leafspot

 

caused by the bacterium Pseudomonas lachrymans react to infection by
developing necrotic lesions (NH lesion type) which lack the chlorotic
halo characteristic of the susceptible reaction (CH lesion type). This
research was conducted to determine the inheritance of the NH lesion
reaction in cucumber and its importance as a component of resistance to

E, lachrymans. Resistant pickling cucumber lines MSU 9402 and By 14A

 

were crossed with the susceptible lines SMR 18 and National Pickling and
genetic analysis of the F1, F2, BC, and E3 populations of these crosses
indicated the NH lesion type is controlled by a single recessive gene.

The role of the NH lesion type in E, lachrymans resistance was evaluated

 

based upon field disease severity ratings and E, lachrymans population
levels on the foliage of F3 lines from the cross between National Pick-
ling and MSU 9402. The F3 lines were genetically homogeneous for either
the NH or CH lesion type. Lesion type was determined to be a major com-
ponent of resistance to E, lachrymans. Disease severity on CHF3 lines
was significantly greater than on NH F3 lines; and, 87 percent of total
genotypic variance of F3 lines was due to differences in lesion type.

Broad sense heritability (Hz) for disease severity among F3 lines within

James Michael Dissert

each lesion type was medium to low (.1-.3). Bacterial populations on
CH lines were 100 times greater than populations on NH lines and were
closely associated with lesions. The development of bacterial papula-
tions and lesions over time on parental and F1 lines was monitored in
greenhouse experiments. Bacterial populations on resistant MSU 9402 and
susceptible National Pickling increased logarithmically at similar

rates after inoculation, but the peak population was lower on MSU 9402.
The NH and CH lesions developed similarly over time and were first dis-

tinguishable four to five days after inoculation.

ACKNOWLEDGEMENTS

My appreciation and thanks to Dr. L. R. Baker, my major advisor
throughout most of my Ph.D. program who taught me much about vegetable
breeding and advised me throughout this study. I also appreciate the
help and especially the encouragement of Dr. J. F. Fobes, my current
major advisor.

Thanks also to other guidance committee members: Dr. M. L.

Lacy whose lab facilities I used throughout my research, Dr. D. N.
Fulbright, especially for the help with the bacteriological aspects of
my research, Dr. A. H. Ellingboe for suggestions and interest in my
work and Dr. K. C. Sink and Dr. H. C. Price, especially for their
critique and conversation.

So many others must also be thanked: Mr. Dick Crum, Nancy Glan-
don, Horticulture and Botony field staff and fellow students.

Warmest thanks to my family: especially to my wife, Krista, for
her help with the computer analysis and editing, and to my parents who

encouraged and supported me for so many years throughout my education.

ii

Guidance Committee:

The journal-article format was adopted for this dissertation in
accordance with departmental and university requirements. Chapter I
was prepared for submission to Euphytica. Chapter II was prepared for

submission to Phytgpatholggy.

 

TABLE OF CONTENTS

 

Page
LIST OF TABLES ................. ' .......... v
LIST OF FIGURES .......................... vii
INTRODUCTION ............................ 1
CHAPTER I
INHERITANCE 0F LESION TYPE REACTION T0 PSEUDOMONAS LACHRYMANS IN
CUCUMBER .............................. 3
Introduction ......................... 3
Materials and Methods ..................... 5
Results and Discussion .................... 7
Literature Cited ....................... 19

CHAPTER II

CUCUMBER LESION TYPE IN RESPONSE TO PSEUDOMONAS LACHRYMANS INFECTION
AND ITS INFLUENCE 0N DISEASE SEVERITY AND BACTERIAL POPULATIONS . . 21

Introduction ......................... 22
Materials and Methods ..................... 25
Results ............................ 32
Discussion .......................... 57
Literature Cited ....................... 64

iv

LIST OF TABLES

Table Page
CHAPTER I

1. Lesion type frequencies of parental cucumber lines and F1
papulations in response to P, lachrymans inoculation as deter-
mined by greenhouse seedling teSts .............. 9

2. Lesion type frequencies of F2 and backcross populations of
cucumber in response to P. lachrymans inoculation as deter-
mined by greenhouse seedTing tests, with Chi squares and pro-
babilities for goodness of fit to apparent ratios ...... 11

3. Frequencies of F3 families (MSU 9402 x National Pickling)
homogeneous and segregating for lesion type in response to P,
lachrymans inoculation as determined by greenhouse seedling
tests, with Chi squares and probabilities for goodness of fit
to a 1:2:1 ratio ....................... 12

4. Lesion type frequencies in 22 segregating F3 families of the
cross MSU 9402 x National Pickling as determined by greenhouse
seedling tests, with Chi squaresw and probabilities for good-
ness of fit to a 1:3 ratio .................. 13

5. Lesion type frequencies of resistant x resistant and suscepti-
ble x susceptible cucumber crosses in response to P. lachry-
mans inoculation as determined by greenhouse seedling tests . 14

6. Lesion type frequencies of parental cucumber lines and F1, F2,
and testcross papulations in response to P. lachrymans inocula-
tion in field plantings,with Chi squares and perabiTities for
goodness of fit to apparent ratios .............. 16

CHAPTER II

1. Mean squares from analysis of variance for disease severity
ratings on F3 lines homogeneous for CH cu: NH lesion types in
field planting 1. At evaluation, plants had 12-16 nodes on
the main axis ............. . .......... 33

Table Page

2. Mean disease severity ratings on parental, F and F3 lines in
field planting 1. Individual plants were ra ed at the 12-16
node stage for disease severity. Disease rated on a 1-10
scale where 1 = no symptoms and 10 = lesions covering 50 per-
cent or more of leaf tissue ................. 35

3. Mean squares from analysis of variance for disease severity
ratings on F3 lines homogeneous for CH or NH lesion types and
over three growth stagesx in field planting 2 ........ 36

4. Mean ratings of F3 lines at specific growth stages in field
planting 2 for disease severity, lesions per leaf and loglo
CFU/cm2 leaf tissue. Disease severity ratings based on a
scale of 1-10 where 1 = no symptoms and 10 = 50 percent or
more necrotic leaf area ................... 37

5. Simple correlation coefficients between disease severity rat-
ings and total necrotic lesion number as rated on individual
plants at the 10 node stage in field planting 2 ....... 39

6. Simple correlation coefficients of mean number CFU/cm2 with
mean disease severity ratings and mean number of lesions per
leaf on F3 lines in field planting 2 ............. 41

7. Mean ratings of parental and F1 lines at specific growth
stages in field planging 2 for disease severity, lesions per
leaf and loglo CFU/c leaf tissue .............. 42

8. Genotypic variances of F3 lines for disease severity parti-
tioned into variance due to lesion type and due to F3 lines
nested within type. VT + vL(T) = total genotypic variance . . 43

9. Genotypic and environmental (error) variance components and
broad sense heritability estimates of F3 lines within CH and
NH lesion type classes for disease severity in field plantings
1 and 2 ........................... 45

10. L0910 mean number CFU/cm2 at lesion or non-lesion leaf sites
on parental and F1 lines ................... 46

11. Simple correlation cgefficients of the number CFU/cm2 with the
number of lesions/cm in individual leaf samples of greenhouse
grown plant§ 11 days after leaf infiltration with approximate-
ly 4.5 x 10 CFU/ml inoculum ................. 56

vi

LIST OF FIGURES

Figure Page
CHAPTER II

1. Multiplication of Pseudomonas lachrymans in leaves of resis-
tant MSU 9402 (9402), susceptible National Pickling (NP) and
9402 x NP. The second leaf of three-leaf plants
was infiltrated with 1. 45 x 105 CFU/ml using a Devilbis ato-
mizer at time = 0. Values are averages of two experiments,
three replications per experiment .............. 48

2. Multiplication of P. lachrymans in leaves of resistant MSU
9402 (9402), susceptible National Pickling (NP) and
9402 x NP. The second leaf of three-leaf plants was infil-
trated with 1.16 x 103 CFU/ml using a Devilbis atomizer at
time = 0. Values are means of three replications, two plants
per replication ....................... 51

3. Necrotic and total lesion number/cm2 and 10910 CFU/cm2 on MSU
9402 (9), National Pickling (N) and on MSU 9402 x
National Pickling (9 x N) at day 11 after bacterial infiltra-
tion. Second leaf 8f three-leaf Slants was infiltrated with
1.8 x 103, 5.0 x 10 and 7.1 x 10 CFU/ml for experiments 1-3
respectively. Values are means of four replications. Means
of each character within same experiment with same letter are
not significantly different at p = .05 by Duncan‘s Multiple
Range Test .......................... 54

vii

INTRODUCTION

Pickling cucumbers are a major vegetable crop in the United
States. Approximately 125,000 acres of pickling cucumbers are grown in
the U.S. Michigan is the leading production state producing 25 percent
.of the total crop. The average yield in Michigan is 150-220 bu/acre,
and 95 percent of the cucumber acreage in Michigan is mechanically har-
vested. Cucumber culture is often very intensive, including high plant
density and overhead irrigation. Under such intensive growing condi-
tions, yields of 400 bushels per acre are common.

Disease management is essential to insure the vigorous uniform
growth necessary for high yields. Angular leafspot caused by Pseudomo-
nas lachrymansa is the current major disease of Michigan cucumbers.

Cucumber scab, caused by the fungus Cladosporium cucumerinum and cucum-

 

ber mosaic, a viral disease, were major diseases of cucumbers in Michi-
gan until controlled by incorporation of high levels of resistance to
these diseases into Michigan adapted cucumber cultivars.

Angular leafspot infects cucumber foliage, causing limited, angu-
lar lesions. Cucumber fruit may also become infected. The disease may
cause serious yield and quality losses, especially during warm, wet

weather under intensive growing conditions. Angular leafspot is

 

aPseudomonas syringae pathovar lachrymans is the current correct
name of this bacterium according to the International Journal of Syste-
matic Bacteriology, 1980. However, the more familiar term, PSeudomonas
lachrymans,will be used throughout this dissertation.

 

1

partially controlled by copper containing chemical sprays; however,
genetic resistance would provide more efficient and complete control.

Some P, lachrymans resistant cultivars have been developed, but

 

transfer of resistance into adapted varieties has been difficult and
inefficient because the mode of inheritance and nature of resistance is
unclear. Chand and Walker (1964) reported that resistance in cucumber
to P, lachrymans is multigenic. Nhen resistant lines are infected with
IE, lachrymans they develop fewer and smaller lesions than susceptible
. lines. In addition, the appearance of lesions on resistant lines is
different than on susceptible lines. Susceptible lines typically deve-
lap orange-brown necrotic lesions surrounded by chlorotic halos (CH
lesions). Lesions on resistant lines are typically white or tan with no
or very slight chlorotic halo (NH lesion). This difference in lesion
type is easily distinguished on inoculated seedlings. Furthermore, pre-
liminary studies indicate the NH lesion type appears to be associated
with resistance to P, lachrymans. If NH lesion type is found to be
simply inherited and closely associated with E, lachrymans resistance,
then selection for the NH lesion type in a seedling population would
increase the efficiency of developing adapted cultivars resistant to
P, lachrymans.

Therefore, the objectives of this research were to: 1) determine
the genetic control of lesion type in response to E, lachrymans inocu-
lation of the pickling cucumber, and 2) evaluate lesion type as a compo-

nent of resistance to E, lachrymans.

CHAPTER I
INHERITANCE 0F LESION TYPE REACTION
TO PSEUDOMONAS LACHRYMANS IN CUCUMBER

INHERITANCE 0F LESION TYPE REACTION
TO PSEUDOMONAS LACHRYMANS IN CUCUMBER

Abstract

Cucumber (Cucumis sativus) lines resistant to angular leafspot

 

caused by Pseudomonas lachrymans react to an infection by developing
necrotic lesions that lack the chlorotic halo characteristic of the sus-
ceptible reaction. The inheritance of the non-halo lesion reaction was
studied in crosses between resistant lines MSU 9402 and Gy 14A, and
susceptable cultivars Wisc. SMR 18 and National Pickling. Genetic
analysis of the F1, F2, backcross, and F3 populations revealed that the
non-halo lesion type, associated with resistance, is controlled by a
single recessive gene. This character appears to be an important com-

ponent of resistance to P, lachrymans.

 

Introduction

Angular leafspot caused by Pseudomonas lachrymans (Smith and Bryan)
Carsner causes serious yield losses to pickling cucumber (Cucumis
sativus L.) crops in northern U.S. and Canadian production areas
(Kennedy and Alcorn, 1980). Early symptoms include both water-soaked,
vein-limited angular lesions on the foliage and circular, water-soaked
lesions on the fruit. As the disease progresses, the necrotic centers
of the foliar lesions become dry and may fall out. Lesions on the
fruit often crack open and exude'amber-colored bacterial ooze (Wiles

and Walker, 1951). Chand et al. (1963) reported that many fruit

infected with P, lachrymans were misshapen, developing into "crooks,"
thus reducing the quality of the harvested crap.

W. C. Barnes (1966) reported that South Carolina breeding lines
had a high degree of resistance to E, lachrymans. He noted that P.I.
197087 from India was undoubtedly the source of resistance. Since then,
cultivars with varying levels of resistance have been developed.
Pohronezny et al. (1977) found that P, lachrymans caused significantly
lower yield losses in resistant cultivars than in susceptible ones when
tested under artificially inoculated field conditions.

Because of difficulty in reproducing disease screening results and
the unclear genetic control of resistance, the development of new
resistant cultivars has been difficult. Breeding programs have used
seedling screening programs to increase the ease, efficiency, and relia-
bility of selection programs (de Zeeuw et al., 1971; de Ponti, 1975;
Haley et al., 1977). Chand and Walker (1964) reported that resistance
to E. lachrymans in the accession P.I. 169400 from Turkey was multigenic
in crosses with two susceptible Wisconsin cuItivars, SMR 15 and SMR 18.

Resistant cultivars develop fewer and smaller lesions when
infected with P, lachrymans. In addition, lesions on susceptible culti-
vars become surrounded by conspicuous chlorotic halos, whereas lesions
on resistant cultivars have little or no chlorotic halo development.
Haley and Palmer (1977) found high positive correlations between cucum-
ber cultivars that produced halos surrounding lesions and severe foliar
symptoms. In response to P, lachrymans inoculation, the non-halo
lesion type appears to be an important component of resistance in cucum-
ber. This character is also easily identified for selection in cucumber

breeding programs. The objective of the present study was to

determine the genetic control of lesion type in response to P. lachry-

mans in the pickling cucumber.

Materials and Methods

 

Parents and Crosses

TWO pickling cucumber lines resistant to E, lachrymans. MSU 9402
(9402) and By 14A, were crossed with two susceptible cultivars National
Pickling (NP) and Wisconsin SMR 18. These parental lines were selected
on the basis of disease severity ratings in replicated field trials.
Lines Gy 14A, and especially 9402, repeatedly exhibit low dis’ease sever-
ity under the epiphytotic conditions of MSU angular leafspot field
trials. Gy 14A, a gynoecious line (Clemson University), is commonly a
parent in F1 hybrid cultivars. The line, MSU 9402 is of value chiefly
as a source of resistance to P, lachrymgns. National Pickling and SMR
18, older cultivars (Michigan State University and the University of
Wisconsin, respectively) are highly susceptible to P, lachrymans. All
lines were inbred and screened for their reaction to P, lachrymans
prior to this study. Reciprocal Fl's and F2, and backcross ponulations
were produced. In addition, a random group of 9402 x NP F2 plants were

selfed to produce F3 families.

Pathogen
A single colony isolate (MSU #5) of E, lachrymgns was used

throughout this study. This isolate was originally supplied by C. A.
John, J.lL Heinz Co., Bowling Green, Ohio. A total of six isolates from
Michigan, Wisconsin, and Ohio were compared for pathogenicity and viru-
lence on several pickling cucumber lines. No differential pathogenicity

was observed, but small differences in virulence were noted. Likewise,

Van Gundy and Walker (1957) reported six other isolates of £3 lachrymans

 

uniform in pathogenicity. Isolate #5 was selected because of its rela-
tively high virulence. As previously observed by Keen et al. (1967), a
series of inoculations and re-isolations from cucumber did not noticea-
bly increase the virulence of these isolates.

Isolates were stored either on yeast dextrose carbonate agar
slants or in sterile distilled water at 4-5°C. The isolates were trans-
ferred on a regular basis to maintain viability. Prior to inoculation,
the bacteria were increased for 48 hours in the HK broth of Husain and
Kelman (1958), as modified by Keen et al. (1967). Immediately before
inoculation, the cultures were diluted with deionized water and adjusted
turbidimetrically using a Baush and Lamb Spectronic 20 calorimeter to
an optical density of .30 absorbance units at 620 nm. This approximated

8

2 x 10 colony forming units (CFU)/ml.

Greenhouse inoculation technique

Seeds were sown in wooden flats containing a greenhouse soil mix
and fertilized once with a water soluble (20-20-20) fertilizer to pro-
vide 300 ppm N. Flats were placed on a bottom-heated bench (30°C soil
temperature) to insure uniform rapid germination. Parental lines were
included in each flat to standardize reaction types. Plants were ino-
culated at the 'candle' stage (first true leaf just emerging) 8-10 days
after sowing. Inoculum was atomized onto the underside of the cotyle-
dons with a Devilbis atomizer held at a distance of 10-15 cm.. Inocula-
tions were made between 8:00 a.m. and 12:00 noon and flats were imme-
diately placed in an unlighted incubator at 21-24°C with 95-100 percent
relative humidity. After a 48 hour incubation period, flats were

removed to a greenhouse maintained at approximately 18°C night and 24°C

day temperature. Each seedling was visually classified for its lesion

type reaction to P, lachrymans 7-9 days after inoculation.

Field studies

Field plots of parental, F1, F2 and backcross generations were
sown in rows spaced 1.3 meters. Plants were thinned to a within row
spacing of approximately 10 cm. Standard Michigan cultural practices
were used. Inocula were increased and prepared as specified for the
greenhouse studies. At the five-leaf stage, plants were lightly
sprayed with a suspension of P. lachrymans (107 CFU/ml.) using a back-
pack sprayer. A second inoculation was made 1 week later. Plots were
frequently irrigated lightly to ensure optimum conditions for disease
development. Typical angular lesions developed approximately 5 days
after the first inoculation. Lesion type was classified 10 days after

the second inoculation.

Results and Discussion

Disease symptomology

In greenhouse studies, cotyledons of susceptible lines NP and SMR
18 developed rounded or irregularly shaped, orange-brown necrotic
lesions surrounded by chlorotic halos. Lesion diameter varied from 1-
10 mm, and the surrounding chlorotic halo ranged from 1-5 mm in width.
Moreover, diffuse chlorosis often developed between lesions. This
symptom will be termed the "chlorotic halo" (CH) lesion type reaction.

Cotyledons of resistant MSU 9402 and Gy 14A developed white or
tan necrotic lesions of similar or smaller diameter, with very slight
or no chlorotic halo development. This resistant symptomology will be

termed the "non-halo" (NH) lesion type reaction. Both lesion types

developed in 5-7 days after removal from the incubation chamber. Within
72 hours, they were seen as water-soaked, sunken areas of 1-2 mm dia-
meter on the lower side of the cotyledons. Small chlorotic spots formed
on the upper surface opposite the water-soaked area. These water-soaked
areas then expanded and developed into the CH or NH lesion type, depend-
ing on the cucumber genotype. The NH reaction resembled hypersensiti-
vity, but was distinguished from it in its development. Typical
hypersensitive lesions develop within 24-48 hours after inoculation,
preceding development of lesions on susceptible plants by 1-2 days
(Mfiller, 1959; Klement et al., 1964). Furthermore, hypersensitive
reactions are typically not preceded by water soaking of the leaf tissue
(Klement and Goodman, 1967). NH lesions, on the other hand, developed
over 3-5 days, along the same time course as CH lesions. In some cases,
water soaking preceded the necrosis of the NH reaction. Resistant and
susceptible plants developed similar numbers of lesions on inoculated
cotyledons. Upon inoculation of true leaves in the field, the two
lesion types developed as in seedling tests, except they often became
vein limited and angular in shape. In contrast to the seedling tests,
NH genotypes in the field developed fewer observed lesions than CH

genotypes.

Inheritance studies

In greenhouse studies most F1 seedlings of resistant and suscep-
tible crosses developed CH lesions, indicating that the CH was dominant
to the NH reaction (Table 1). Some misclassification was evident in the
F1 as in the parental generation. Progeny tests indicated that off-

types were genetically similar to the expected. The intensity of the

Table 1. Lesion type frequencies .of parental cucumber lines and F1 popu-
lations in response to P, lachrymans inoculation as determined
by greenhouse seedling tests.

 

 

No. Total no. Observed Expected
individual Pedigree of plants ratio ratio
tests ‘ * tested w NHx CHy NH CH
PARENTS
5 9402 76 h 76 0 1 o
6 Gy 14A 90 85 5 1 o
8 SMR 18 119 1 118 o 1
7 NP 97 1 96 o 1
51
10 9402 x NP 201 1 200 o 1
2 NP x 9402 62 2 60 o 1
2 9402 x SMR 18 36 2 34 o 1
1 SMR 18 x 9402 12 1 11 o 1
4 SMR 18 x Gy 14A 86 8 78 o 1
1 Gy 14A x SMR 18 50 1 49 o 1
1 NP x Gy 14A 18 o 18 o 1
1 Gy 14A x NP 62 o 62 o 1

 

xNumber of plants with non-halo lesion type.
yNumber of plants with chlorotic halo lesion type.

10

CH reaction, though variable, was not always as great in the F1 genera-
tion as the susceptible parent. This was possibly due to incomplete
dominance of genes controlling the CH reaction and/or due to the inter-
action with the genetic background.

All F2 populations, except Gy 14A x NP fit the ratio of 1NH:3CH
(Table 2). Chi square analysis of the F2 plants from the cross Gy 14A
x NP gave a P value.which exceeded .05 due to an excess of NH types.
This low P value is most likely due to misclassification, especially
since the reciprocal F2 population (NP x Gy 14A) had an excess of CH
reaction individuals. All progenies from testcrosses to resistant
parents segregated 1NH:1CH (Table 2) whereas backcrosses to susceptible
parents resulted in only CH progeny (Table 2 ).

The F3 families from the cross 9402 x NP could be classified into
three distinct groups, as follows: CH, segregating, and NH. The pheno-
types of the F3 families gave an acceptable fit to the ratio of 1CH:2
segregating:1NH; although, there was some excess of true breeding CH
families (Table 3). Within family segregation ratios were determined
fur the 22 segregating F3 families. Most families gave a good fit to
the expected ratio of 1NH:3CH. Pooling the data from the 22 segregating
F3 populations yielded a total of 97 NH and 315 CH plants, P = .4-.5 for
a 1:3 ratio (Table 4). Observed segregation ratios indicated single
gene control of lesion type with the NH reaction recessive to the CH
reaction.

The F1 and F2 p0pulations from crosses between the two reSistant

parents, 9402 and Gy 14A, were all resistant. In addition, there were

2
two susceptible parents NP and SMR 18 (Table 5). Lack of segregation

no resistant segregates in thel= populations<rfthe cross between the

11

Table 2. Lesion type frequencies of F2 and backcross populations of
cucumber in response to P. lachrymans inoculation as deter-
mined by greenhouse seedTing tests, with Chi squares and
probabilities for goodness of fit to apparent ratios.

 

 

 

 

 

 

No. Total no. Observed EXpected 2
individual Pedigree plants ratio ratio x P value
tests tested NHY CH2 NH CH
2 (9402 x NP)-1 110 24 86 1 3 .594 .4-.5
10 (NP x 9402)-1 287 63 224 1 3 1 423 .2-.3
3 (SMR 18 x 9402)-1 172 46 126 1 3 316 .5-.6
4 (SMR 18 x 14)-1 165 45 120 1 3 455 .5
1 GY 14A x NP)-1 52 20 32 1 3 5 026 025
1 (NP x GY 14A)-1 67 15 52 1 3' .244 .1-.2
Pooled" values 853 213 640 1 3 .0004 .98
3 (NP x 9402) 9402 115 56 59 1 1 .078 .7-.8
3 (SMR 18 x 9402) 9402 156 79 77 1 I .026 .8-.9
1 Gy14A(SMR 18 x Gy14A) 32 17 15 1 1 .125 .7- .8
2 (SMR 18 x Gy14A)Gy14A 113 61 52 1 1 .717 .3-.4
1 (NP x Gy 14A)Gy 14A 60 30 30 I 1 0 1
Pooledx values 476 243 233 1 1 .210 .6-.7
2 NP (NP x 9402) 86 0 86 0 1 - -
2 SMR 18(SMR 18 x 9402) 36 4 32 0 1 - -
1 (SMR 18 x 9402)SMR 18 20 2 18 0 1 - -
1 (SMR 18 x Gy 14A)SMR18 13 0 13 0 1 - -
1 (NP x Gy 14A) NP 17 0 17 0 1 - -
Pooled values 172 6 166 0 1 - -
w 2 . _
x heterogeneity - 8.057 (p - .1-.2)
xxzheterogeneity = 0.736 (p = .95)

yNumber of plants with non-halo lesion type.

2Number of plants with chlorotic-halo lesion type.

12

Table 3. Frequencies of F3 families (MSU 9402 x National Pickling)
homogeneous and segregating for lesion type in response to
P, lachrymans inoculation as determined by greenhouse seedling
tests, with Chi squares and probabilities for goodness of fit
to a 1:2:1 ratio.

 

 

 

Class 2
NHx segy.. CHz x P value
NP x 9402 8 22 14 1.636 .4-.5

 

xNumber of F3 families homogeneous for non-halo lesion type.
yNumber of F3 families segregating for lesion type.

2Number of F3 families homogeneous for chlorotic-halo lesion type.

13

Table 4. Lesion type frequencies in 22 segregating F3 families of the
cross MSU 9402 x National Pickling as determined by greenhouse
seedling tests, with Chi squaresw and probabilities for good-
ness of fit to a 1:3 ratio.

 

 

 

Number of plants per F3 family, 2w

NHy CHZ Total X P‘Va‘ue
5 20 25 .12 .7-.8
3 13 16 .08 .7-.8
4 7 11 .27 .6-.7
3 13' 16 .08 .7-.8
2 22 24 4.08 .025-.05
8 15 23 .71 .3-.4
5 21 26 .21 .6-.7
5 13 18 .00 1
8 19 27 .11 .7-.8
3 9 12 .00 1
1 6 7 .05 .8-.9
1 4 5 .00 1
5 16 21 .00 1
7 12 19 .86 .3-.4
6 14 20 .07 .7-.8
2 13 15 .56 .4-.5
3 13 16 .08 .7-.8
5 14 19 00 1
5 14 19 .00 1
6 23 29 .10 .7-.8
6 16 22 .00 1
4 18 22 .24 .6-.7

Pooledx values
97 315 412 .47 .4-.5

 

wYates correction for continuity (Yates, 1934).
xxzheterogeneity = 11.41 (p = .95).
yNumber of plants with non-halo lesion type.

2Number of plants with chlorotic-halo lesion type.

14

Table 5. Lesion type frequencies of resistant x resistant and suscep-
tible x susceptible cucumber crosses in response to E, lachry-
mans inoculation as determined by greenhouse seedling tests.

 

Total no. Observed Expected

Pedigree Generation plants xratio ratio
tested NH CHy NH CH

Gy 14A x 9402 F1 11 11 0 1 0
(Gy 14A x 9402)-I F2 49 48 1 1 0
SMR 18 x NP ' F1 13 0 13 '0 1
(SMR 18 x NP)-1 F2 39 0 39 0 1

 

xNumber of plants with non-halo lesion type.

yNumber of plants with chlorotic-halo lesion type.

15

indicated that lesion type is conditioned by the same allele in all
parental lines of this study. This was also suggested by the resistant
x susceptible crosses where a single gene controlling lesion type was

indicated by data from all the crosses.

Field studies

Cucumber breeding programs have screened seedlings under con-
trolled conditions for resistance to E, lachrymans. The relationship of
these seedling tests to the lesion reaction on mature plants, especially
in the field, in unclear. To elucidate this relationship, segregating
generations and their parents were inoculated and evaluated for reaction

to P, lachrymans in the field. These field studies resulted in similar

 

segregation patterns for the F2 and testcross generations as did the
greenhouse seedling tests (Table 6). Therefore these data confer that
foliar lesion type in response to P, lachrymans inoculation is monogenic
with the NH lesion type recessive t0 the CH lesion type.

Variation of lesion size on both cotyledons and true leaves was
evident within parental and segregating CH and NH classes. Because of
this variation, even within homogeneous parental lines, lesion size was
not considered in the classification of lesion type. Halo intensity
and size also varied in the CH class, but in this case variation was
much more evident in segregating generations. These slight differences
in chlorotic halo intensity and size might be classified with a more
refined system than the visual rating used in this study. Classifica-
tion for lesion type was usually distinct in segregating populations of
reciprocal crosses between 9402 and NP. However, classification for

lesion type in other crosses, especially involving Gy 14A, required

16

 

 

 

Table 6. Lesion type frequencies of parental cucumber lines and F} F2,
and testcross p0pulati0ns in response to P. lachrymans inocu ation
in field plantings, with Chi squares and probabilities for
goodness of fit to apparent ratios.

. Total no. Observed EXPECPBd 2

Ped1gree plants ratio ratio x P value

tested NHx CHy NH CH

Parents

9402 48 48 0 1 0 - -

Gy 14A 17 17 0 1 0 - -

NP 54 0 54 0 1 - -

SMR 18 40 0 40 0 1 - -

_F.

NP x 9402 41 0 41 0 1 - -

9402 x NP 33 0 33 0 1 - -

Gy 14A x SMR 18 53 0 53 1 - -

£2

(NP x 9402)-1 116 27 89 1 3 .184 .5-.75

(9402 x NP)-1 45 10 35 1 3 .185 .5-.75

(SMR 18 x 9402)-1 95 20 75 1 3 .789 .3-.4

(SMR 18 x Gy 14A)-1 123 29 94 1 3 .133 .7-.8

Testcross

(NP x 9402) 9402 150 74 76 1 1 .027 .8-.9

(SMR 18 x 9402) 9402 133 55 78 1 1 3.977 .025-.050

(SMR 18 x Gy 14A) Gy 14A 59 26 33 1 1 .830 .3-.4

Gy 14A (SMR 18 x Gy 14A) 47 24 23 1 1 .021 .8-.9

 

xNumber of plants with non-halo lesion type.

yNumber of plants with chlorotic-halo lesion type.

17

meticulous observation of the lesion reaction for proper classification.
This might be explained by the observation that 9402, and especially

Gy 14A, infrequently expressed a slight chlorotic halo of less than 1 mm
width surrounding an otherwise typical NH lesion.

Variability in intensity of the chlorotic halo could probably be
reduced by closer control of environmental factors, especially tempera-
ture. Van Gundy and Walker (1957) found severity of angular leafspot in
cucumber to be dependent on both host nutrition and temperature. Patel
and Walker (1963), working with halo blight of bean caused by Pseudomo-
nas phaseolicola, found that the size of the chlorotic halo around
lesions increased as temperatures decreased from 28° to 16°C.

Cucumber foliage color sometimes interfered with lesion type
classification in both seedlings and mature plants. Susceptible parents
SMR 18 and NP, had a lighter green foliage color which is representative
of northern U.S. pickling cucumber genetic backgrounds. The resistant
parents, MSU 9402, and Gy 14A, are lines derived from southern U.S.
breeding programs which are characterized by darker green foliage. In
segregating populations, foliage color displayed an apparent continuous
segregation pattern which tended to either mask or enhance chlorotic
halo development depending on the intensity of green foliage color.

Chand and Walker (1964) concluded that P, lachrymans resistance
in P.I. 169400 appeared to behave as a multigenic factor. Segregation
patterns of the F2 and testcross p0pulations would not fit any simple
genetic ratio. Our study has determined that the lesion type reaction

in response to E, lachrymans infection is controlled by a single gene,

 

where the NH lesion type associated with resistance, is recessive to

the CH lesion type reaction. This apparent discrepancy can be simply

18

resolved: Chand and Walker (1964) based their inheritance study on a
disease index incorporating lesion number and size, whereas out study
focused on lesion type. Lesion number and size in response to E, lggh;_
rymggg infection may indeed be controlled by two or more genes as their
study indicated. Their disease index approximated the total disease
reaction, where lesion type is more likely a single component of resis-
tance. From our observations and the work of Haley and Palmer(1977),
the NH reaction is directly associated with low disease severity. It is
easily selected for by using the seedling screening procedure empha-
sized in this study. We suggest that this character confers a very

useful degree of resistance to P, lachrymans; other loci interact to

 

effect varying levels of resistance. We propose nh_as the symbol for
this recessive gene conditioning the non-halo lesion tYPe reaction to P-

lachrymans in cucumber.

10.

11.

12.

13.

19

Barnes, W. C. 1966. Development of multiple disease resistant
hybrid cucumbers.’ Proc. Amer. Soc. Hort. Sci. 89:390-393.

Chand, J. N., E. K. Wade, and J. C. Walker. 1963. Sprays for
control of angular leafspot or cucumber in Wisconsin. 1. Plant
Dis.43eptr. 47:94-95.

 

Chand, J. N. and J. C. Walker. 1964. Inheritance of resistance to
angular leafspot of cucumber. Phytopathology.. 54:51-53.

 

dePonti, 0. M. 8., A. C. van der Giessen, H. Inggamer. 1975.
Inpasse in het Toetsen op resistentie tegen bakterievlekkenziek-
te Pseudomonas lachr ans in augurk doorbroken! Overdruk uit
Zaadbelangen. 123-12;.

de Zeeuw, D. J., R. Crum, J. E. Wilson, L. R. Baker. 1971.
Screening cucumber seedlings for angular leafspot resistance.
HortScience. 6:276 (Abstr.).

 

Haley, A. B. and M. J. Palmer. 1977. Resistance of cucumber to
angular leafspot disease--correlation of greenhouse and field
reactions. Pickling Crops Research, Univ. of Wisc., Madison.

Husain, A., and A. Kelman. 1958. Relation of slime production to
mechanism of wilting and pathogenicity of Pseudomonas solana-
cearum. Phytgpathology. 48:155-165.

Keen, N. T., P. H. Williams, and J. C. Walker. 1967. Protease of
Pseudomonas lachrymans in relation to cucumber angular leafspot.
Phytopathology. 57:263-271.

Kennedy, 8. W. and S. M. Alcorn. 1980. Estimates of U.S. crop
losses to procaryote plant pathogens. Plant Disease. 64:674-
676.

 

Klement, Z., G. L. Farkas and L. Loviekovich. 1964. Hypersensi-
tive reaction induced by phytopathogenic bacteria in the
tobacco leaf. Phytopathology. 54:474-477.

Klement, Z., and R. N. Goodman. 1967. The hypersensitive reaction
by bacterial plant pathogens. Ann. Rev. Phytopath. 5:17-44.

Mfiller, K. 0. 1959. Hypersensitivity. p. 469-519. 12 J. G.
Horsefall and A. E. Dimond (ed.). Plant pathology, an advanced
treatise, Vol. 1. Academic Press, New York.

Patel, P. N. and J. C. Walker. 1963. Relation of air temperature
and age and nutrition of the host to the development of halo
and common bacterial blights of bean. Phytopathology. 53:407-
411.

14.

15.

16.

17.

20

Pohronezny, K., P. 0. Larsen, 0. A. Emmatty, and J. D. Farley.
1977. Field studies of yield losses in pickling cucumber due
to angular leafspot.’ Plant Dis. Reptr. 61:386-390.

Van Gundy, S. D. and J. C. Walker. 1957. Relation of temperature
and host nutrition to angular leafspot of cucumber. Phytopatho-
logy. 47:615-619.

Wiles, A. B. and J. C. Walker. 1951. The relation of Pseudomonas
lachr ans to cucumber fruits and seeds. Phytopathology. 41:
1059-I064.

Yates, F. 1934. Contingency tables involving small numbers and
the x2 test. J. Roy. Stat. Soc. Suppl. 1 217-235.

CHAPTER II
CUCUMBER LESION TYPE IN RESPONSE TO PSEUDOMONAS LACHRYMANS INFECTION
AND ITS INFLUENCE ON DISEASE SEVERITY AND BACTERIAL POPULATIONS

CUCUMBER LESION TYPE IN RESPONSE TO PSEUDOMONAS LACHRYMANS INFECTION
AND ITS INFLUENCE ON DISEASE SEVERITY AND BACTERIAL POPULATIONS

Abstract

Resistance to Pseudomonas lachrymans in cucumber (Cucumis sativus

 

L.) is a multigenic character. Lesion type on cucumber foliage in
response to E, lachrymans infection is controlled by a single gene.
Susceptible lines develop orange-brown necrotic lesions surrounded by
chlorotic halos (CH lesion type). Lesions also develop on resistant
lines and are typically white or tan with very slight or no chlorotic
halo (NH lesion type). The purpose of this study was to evaluate lesion

type as a component of resistance to E, lachrymans. The role of lesion

 

type in E, lachrymans resistance was evaluated based upon field disease
severity ratings and E, lachrymans p0pulations on the foliage of F3
lines. These lines were genetically homogeneous for either the NH or CH
lesion type. The F3 lines were selected from a cross between resistant
line MSU 9402, and the susceptible cultivar, National Pickling. In
addition, the development of bacterial populations and lesions over time
on parental and F1 lines was monitored in greenhouse experiments. The
disease severity ratings for CH lesion type F3 lines were generally
twice that for NH F3 lines. The degree of resistance on NH lines
approached that of the resistant parent. Eighty-seven percent of the
total genotypic variance was due to lesion type. Broad sense heritabi-
lity estimates in disease severity among F3 lines within each lesion
type were moderately low (.1-.3). In field experiments, the average

21

22

bacterial population on CH lines was 100 times that on NH lines. Bac-
terial populations on cucumber foliage was closely associated with
specific lesion sites. Bacterial populations on the resistant parent
MSU 9402 increased logarithmically at similar rates to those on the
susceptible parent National Pickling, but peak populations were lower.
Lesions on both MSU 9402 and National Pickling first appeared 2-3 days
after inoculation. “Differences in lesion type were distinguishable 5
days after inoculation. Based upon the results of this research, the
NH lesion type in response to E, lachrymans infection is a major compo-

nent of resistance to E, lachrymans.

Introduction

Angular leafspot of cucumber (Cucumis sativus L.), caused by
Pseudomonas lachrymans (Smith and Bryan) Carsner, is a serious problem
of pickling cucumbers grown in humid production areas. Initial E,
lachrymans infestations of cucumber crops usually are from seed borne
bacteria or from overwintered bacteria on plant debris (6, 24, 33, 37).
Secondary dispersal throughout the crop is predominantly by rain splash
or mechanical means (3, 7, 19, 24, 33). Infection takes place through
the stomata, wounds, or other openings in the epidermis. The disease
initially appears as small, water-soaked spots which later evolve into
characteristic vein-limited angular lesions. .

Sources of resistance to E, lachrymans have been identified (2,
9). Some resistant cultivars have been developed and are in common use.
Use of resistant cultivars results in significantly reduced yield losses
under epidemic conditions (30). Chand and Walker (9) reported that

disease resistance in cucumber, based on lesion number and size, was

23

quantitatively inherited. Resistant cultivars developed fewer and
smaller lesions when infected with P, lachrymans.

The type of lesions on resistant lines differs from that on sus-
ceptible lines. Susceptible lines typically develop orange-brown necro-
tic lesions surrouhded by chlorotic halos (“CH“ chlorotic-halo lesion
type). Lesions on resistant lines are typically white or tan with very
slight or no chlorotic halo ("NH" non-halo lesion type). The lesion

type in reaction to E, lachrymans infection has been found to be con-

 

trolled by a single gene (15). The NH lesion type reaction associated
with resistance, is recessive to the CH lesion type reaction. The NH
reaction has been correlated with low disease severity (20) and appears
to be an important component of resistance to E, lachrymans.

A similar lesion type is also associated with resistance to Eggp-

domonas phaseolicola in common bean (Phaseolus vulgaris). Jensen and

 

Boss (23) noted that small, inconspicuous brown circular spots, lacking
the typical chlorotic halo, occurred on inoculated leaves of some bean
cultivars found to be resistant in field tests. Several studies on the
inheritance of resistance to P, phaseolicola followed (13, 32, 35).
Schuster (32) and Walker and Patel (35) defined resistance to race 1 of
.3. phaseolicola on the basis of lesion type and the absence of systemic
chlorosis. After a second race of E. phaseolicola was discovered, Patel
and Walker described a tolerant reaction (also based on lesion type) to
both races and reported that the tolerant reaction was under indepen-
dent genetic control (28, 29). Coyne, Schuster, and Gallegos (11)
first reported that the foliar lesion reaction and systemic chlorosis
reaction were separately controlled by two linked genes. Later, two

other components of resistance to E, phaseolicola, the pod reaction and

24

a wilting reaction of primary leaves, were found to be controlled by
independent major genes (10, 21).

Relatively low bacterial populations in host tissue have been
associated with resistance to bacterial diseases (1, 14, 18, 26, 27,
31, 34). Homologous bacteria typically multiply logarithmically in

susceptible host tissue to population levels of 10 colony forming
units (CFU)/cm2. Bacteria multiply in resistant tissue at similar or
slightly slower rates, but reach a lower maximum population. The bac-
terial population maxima in resistant tissue are typically 10-100 times
less than in susceptible tissue. Chand and Walker (8) studied the mul-

tiplication of E, lachrymans in resistant cucumber lines PI 169400,

 

susceptible cultivar SMR 18, and their F1 progeny. Large differ-
ences were found in bacterial population levels among these lines at all
sampling times after inoculation. At maximum, the bacterial population
in susceptible lines SMR 18 was 300 times that in resistant PI 169400.
The bacterial population in F1 plants was intermediate but close to
the resistant parent. The number and size of lesions in the resistant
parent and F1 plants were also correspondingly lower.

The purpose of the present study was to evaluate lesion type as a
component of resistance to P, lachrymans. The specific objectives were
to:

1. Examine the interrelationship between lesion type (CH or NH) and
disease severity in the field.

2. Define the relationship between P, lachrymans populations-and foliar

 

lesions on cucumber.

25

Materials and Methods

 

Bacterial Isolates

An isolate of P, lachrymans, 01, originally supplied by 0.M.B.

 

dePonti of Instituut voor de Veredeling van Tuinbouwgewassen (IVT),
Wageningen was used for lesion type classification and greenhouse con-
ducted experiments. A spontaneous mutant (NR 22) resistant to 500 ug/ml
nalidixic acid (NA) and 50 ug/ml rifampin was used for field studies.
Use of NR 22 allowed the selective isolation of this isolate on antibio-
tic-containing media, greatly reducing bacterial contamination.

The E, lachrymans isolate, NR 22, was derived from isolate 01 us-

 

ing selective plating methods. Isolates were initially selected for
resistance to NA using the gradient plate technique of W. Szybalski (4).
Petri plates were poured with two layers of Kings 8 agar, adjusted to

pH 7.4 with 1N Na0H to prevent precipitation of nalidixic acid. The
bottom layer, supplemented with 1 mg/ml NA was allowed to harden with
the plate in a slanted position. A second layer of unsupplemented Kings
8 medium was poured with the Petri plate in the normal horizontal posi-
tion to just cover the highest part of the slanted layer. This tech-
nique established a uniform concentration gradient of the antibiotic.
Resistant mutants were obtained by plating a 24 hour culture of approx-

imately 109

bacteria over the freshly poured bilayer gradient plates.
As colonies appeared at the lower concentration of NA, they were con-
tinually streaked towards the highest concentration. Only NA resistant
cells were able to form colonies at the higher antibiotic concentra-
tions. Colonies growing at the higher concentrations were subcultured

onto Petri plates of Kings 8 agar (pH 7.4) containing 500 ug/ml NA.

26

 

Approximately 109 cells of £3 lachrymans previously selected for
NA resistance were plated on Kings 8 medium (pH 7.2) supplemented with
50 ug/ml rifampin (Weller and Saettler, 1978). The gradient plate tech-
nique was not necessary in order to obtain resistant isolates. Several
colonies per plate appeared, and these rifampin resistant colonies were
subcultured.

Selected isolates were checked for resistance to both antibiotics
. by plating on Kings 8 agar pH 7.4 + 50 ug/ml NA + 50 ug/ml rifampin
(KBNR media). The observed colony type and growth rate on Kings 8 was
not distinguishable from the parent isolate, 01. In addition, colonies
were checked for oxidase reaction and flourescence under ultraviolet
light prior to testing for virulence on cucumber.

Virulence of resistant isolates was tested using the following two
inoculation techniques. Cucumber cotyledons were inoculated with a bac-
terial suspension of 107 colony forming units (CFU)/ml using the "multi-
ple toothpick" inoculation procedure described below; additionally,
first and second-true leaves were injected with 105 CFU/ml using a
hypodermic syringe. In both cases, virulence was rated according to
lesion size and severity of chlorotic halo on the cucumber cultivar,
National Pickling. Antibiotic resistant isolates were compared to D1
and other wild type isolates. Only slight differences in virulence were
observed among resistant isolates. The NR 22 isolate was selected for
use in this study because it appeared identical to the parental isolate,
01, in all aspects other than antibiotic resistance. Single colonies of ‘
NR 22 were sequentially transferred three times on KBNR media prior to

field inoculations, insuring inoculum uniformity. Bacterial isolates

27

were maintained for long term storage in sterile distilled water at 4°C
or on Kings 8 Petri plates at 4°C for up to 2 weeks. Prior to all ino-
culations, bacteria were increased for 24 hours in the modified liquid
medium of Husain and Kelman (22), as modified by Keen et al. (25).
Immediately before inoculations, bacterial cultures were diluted with
phosphate buffer (0.01M, pH 7.2) and adjusted turbidimetrically using a
Bausch and Lomb Spectronic 20 calorimeter, and by serial dilution as
needed to obtain the desired inoculum concentration. Inoculum concen-
tration was checked with dilution plate counts taken immediately prior

to inoculation.

Lesion Type Classification

Prior to the field and/or greenhouse experiments, parental, F1,
and F3 generations were each classified according to lesion type in
response to P. lachrymans infection. A multiple puncture inoculation
technique was used. Inoculations were made with eight round, wooden
toothpicks fastened with a rubber band. Advantages of this inoculation
technique included the ability to clearly distinguish between lesion
types and a lack of escapes. The disposability of the toothpicks was
especially advantageous when testing various isolates of the pathogen
for virulence on cucumber. The underside of cotyledons of 10-day-old
cucumber seedlings were punctured with toothpicks dipped in_a suspension
of 108 CFU/ml. Cotyledons were supported with the index finger during
inoculation. For inoculation, seed was sown in flats of greenhouse
soil mix and fertilized once with 20-20-20 water soluble fertilizer to
provide 300 ppm N. Seeded flats were placed on a greenhouse bench

supplemented with bottom heat (30°C soil temperature) to ensure rapid,

uniform germination.‘ The plants were inoculated 10 days after sowing,

28

when the first true leaf was just emerging. Seedling flats were moved
to a greenhouse bench after inoculation. Each seedling was classified
for lesion type 7 days after inoculation. All inoculated plants of
National Pickling and reciprocal F1 plants developed typical CH lesions.
All inoculated plants of MSU 9402 developed typical NH lesions.

Selection of Fegtines

When studying the effect of a single character, such as lesion
type, it is necessary to differentiate between the specific character
effects and effects due to other traits. To permit differentiation, two
groups of F3 lines were chosen for field studies. These lines were
genetically homogeneous for either the NH or CH lesion type. The F3
lines were selected from a cross between the E. lachrymans resistant
line, MSU 9402 (9402), and the susceptible cultivar National Pickling
(NP). Both parental lines were inbred several generations prior to this
study. Since lesion type is controlled by a single gene, three types
of F3 lines appear in a ratio of 1:2:1, respectively: (1) homogeneous
f0r the NH lesion type, (2) segregating, or (3) homogeneous for the CH
lesion type. Due to the random segregation of other genes, selected
groups of F3 lihes only have lesion type as a common distinguishing

character.

Field Studies

Disease severity and bacterial populations were monitored in
parental, F1, and F3 populations in two field plantings (in late summer,
1980) at the Botany and Plant Pathology research farm, Michigan State
University, East Lansing, Michigan. Seed for two field plantings were

germinated in the greenhouse in 6 cm peat pots and transplanted two

29

weeks later into the field with 45 cm square Spacing. Field planting I
(planted July 17, 1980) including 24 lines: 8 NH lines, 12 CH lines,
reciprocal Fl's and parents. Field planting 2 (planted August 18),
included 14 lines: five randomly selected NH and CH lines, reciprocal
Fl's and parental lines. A randomized complete block design with

12 and 6 single plant replications was used for field plantings 1 and 2,
respectively. Two direct seeded border rows were planted around each
field plot 2 weeks prior to transplanting. Standard Michigan cultural
practices were used.

8 CFU/ml.

Each field planting was inoculated with approximately 10
Inoculum was misted on the foliage to run off with a hand pump back
pack sprayer. Care was taken not to infiltrate any leaf tissue with the
inoculum. Inoculations were made in the a.m. while foliage was wet with
dew or after a light irrigation. After inoculations, field plantings
were frequently lightly irrigated to ensure good angular leafspot devel-
0pment.

Field planting 1 was inoculated August 11 and 15 at the 10-14 leaf
stage. Typical angular lesions appeared August 14. Field planting 2
was inoculated August 23 and September 6 at the 2-3 leaf stage and first
symptoms appeared August 28.

Field plantings were rated for foliar disease severity on a rela-
tive scale of 1-10, the score of 1 denoting no visible lesidns, and 10
denoting severe infection including coalescing of lesions and tearing of
leaf tissue on 50% or more of the foliage. Both lesion types, with or
without chlorotic halos, were treated equally in rating disease sever-

ity. In field planting 2, the actual number of lesions per plant was

also counted and the number of lesions per leaf was calculated.

30

The disease severity scores for F3 lines in field planting 1 and
the mean severity scores (over three growth stages) for F3 lines in
field planting 2 were partitioned into variance components due to lesion
type (VT)’ and line within type (VL(T))' The VT + VL(T) is equivalent
to the total genetic variance for disease severity within these popula-
tions. Variance components were claculated by equating mean square
values from the analysis of variance for type, and for line nested with-
in type to their expected mean squares and solving for the specific
variance component.

Genotypic and error variances f0r disease severity were calculated
for each class of F3 lines. These variance components were calculated
by equating the appropriate expected mean squares to the error mean
square and the mean square for F3 lines. Heritability of disease sever-
ity within each lesion class was computed using the formula:

Heritability = VG/(VG + VE)
where VG and VE are the genotypic and error variances, respectively.
This formula estimates heritability both in the broad sense and on a
per plot basis.

Bacterial populations in CH and NH classes were estimated in both
field plantings using conventional serial dilution plate count methods.
All plants were sampled at least one week after inoculation in order to
allow bacterial p0pulations to develop and equilibrate. SyStematically
selected leaf discs were removed from individual plants and cooled with
ice until grinding. Each sample of tissue was ground in 0.01 M phos-
phate buffer, pH 7.2. Ten-fold serial dilutions of ground suspensions
were made in buffer, and 0.1 ml aliquots were pipetted onto Petri plates

of Kings 8 medium supplemented with 50 ug/ml rifampin, 400 ug/ml

31

nalidixic acid and 50 ug/ml cycloheximide. Colonies were counted after

three days incubation at approximately 22°C.

Greenhouse Studies

Lesion number and bacterial population were monitored on parental
and F1 populations grown under greenhouse conditions. Cucumber seeds
were sown in an artificial peat lite medium in 6 cm peat pots and trans-
planted 14 days later into 11 cm clay pots. Seedlings were fertilized
with 20-20-20 at 300 ppm N after germination and transplanting. Plants
were grown under fluorescent lighting at approximately 18°C night and
24°C day temperatures. All experiments were conducted in the winter and
early spring. Since minimal contamination occurred in the greenhouse,
antibiotic resistance and selective plating was not necessary. There-
fore, P, lachrymans isolate 01 was used. In all experiments, the second
leaf of plants of uniform leaf size and development was inoculated.
Plants were inoculated by thoroughly infiltrating the lower leaf surface
with a bacterial suspension. A Devilbis atomizer held at 2 cm from the
lower leaf surface was used for infiltration. Infiltration was facili-
tated by covering the plants with a plastic tent the evening prior to
inoculation. Leaf tissue appeared completely watersoaked after infil-
tration. Inoculated leaves were washed with a stream of distilled water
and the plants were placed on greenhouse benches. Bacterial popula-
tions were monitored in time course experiments at 24-48 hour intervals
after inoculation by removing 1.15 cm diameter leaf discs (#8 corkborer)
in a systematic pattern. Leaf samples were taken immediately after
watersoaking disappeared and up to 6 days after inoculation. The sam-

ples were ground in buffer and the suspensions serially diluted and

32

plated on unsupplemented Kings 8 medium. Colonies were counted after 3
days incubation at approximately 22°C to estimate CFU/cm2 of leaf tis-
sue.

With low inoculum concentrations (103 cells/ml), specific lesions
devel0ped on inoculated leaves. These were especially visible on the

2 disc (1%" cookie cutter) was removed

upper leaf surface. An 11.34 cm
from the center of the leaf 11 days after inoculation and the number of
lesions on the disc were counted. The disc was then ground in buffer

2

and CFU/cm was estimated as above.

Results

Field Studies

No angular leafspot symptoms were present prior to inoculation.
All cucumber lines in field plantings 1 and 2 developed typical angular
lesions after inoculation. Both the resistant line.9402,.and F3 lines
within the NH lesion class developed typical NH lesions without a sur-
rounding chlorotic halo. NP, reciprocal F1 lines and CH class F3
lines produced CH lesions, easily identified by the presence of a chlor-
otic halo surrounding the lesion. Chlorotic halo intensity on the F1
lines was sometimes less than that on NP.

Disease severity in field planting 1 was evaluated (measured on
an entire plant basis) on August 21, 10 days after inoculation when
plants were at the 11-16 node stage. Analysis of variance for disease
severity scores revealed highly significant differences both between the
NH and CH lesion type classes and among lines within each lesion class
(Table 1). The mean severity score of the NH class (4.02) fell between
the mid-parent value (4.79) and the resistant parent 9402 value (2.50).

33

Table 1. Mean squares from analysis of variance for disease severity
ratings on F3 lines homogeneous for CH or NH lesion types in
field planting 1. At evaluation, plants had 12-16 nodes on

 

 

main axis.

Source Degrees of Freedom Mean Square
Replication 11 4.30
Lesion type 1 324.90**
Line within type 18 9.58**
Error 207 1.99

 

**Significant at .01 level.

34

The mean severity score for the CH class (6.40) fell between the mid-
parent value and the susceptible parent, NP (7.08). There was some
overlap of disease severity ratings between classes. 9402 had signifi-
cantly lower disease severity ratings than either reciprocal F1 plants
or NP (Table 2). F1 scores fell above the mid-parent value indicating
a degree of dominance for susceptibility.

Experimental lines in field planting 2 were evaluated for disease
severity at three time periods. The initial disease severity evaluation,
at the 3 node stage, was made on the first and second leaves August 31,
eight days after inoculation. Leaves above the second node were evalu-
ated at the 6 node stage; and leaves above node five and on laterals
were evaluated at the 10 node stage.

As in field planting 1, differences between lesion type classes
were significant (Table 3). There were also differences among lines
within each lesion class at all growth stages. At the 3 and 10 node
stage no differences among lines within either type were found. Lesion
type x time interaction was significant. The mean disease severity of
CH lines increased slightly over time; whereas, severity ratings on NH
lines decreased with maturity (Table 4).

The mean disease severity ranking of individual F3 lines within
the NH class in field planting 2 corresponded closely to the ranking of
these same lines in field planting 1. F3 line N3 in both field planting
1 and 2 had the most severe symptoms of all the NH lines. In contrast
to the NH lines, interplot rankings did not correspond closely for the
CH lines.

The total number of lesions per leaf and lesions 3 5mm diameter

were counted at the 10 and 14 node stage, respectively (Table 4).

35

.A—m>m_ mo. V amok mmcmm mpapupzz m .cmuczo
an noepssmumu mm acmemwmpu xpucwoee_cmpm no: men emuuop osmm use me cmzoppom memos .30; some :Pguwz
.ummp mmcmm mpawupaz m.:mucao an _m>m— mo. um mcopueemamm cemzx

.oo=e_ee> eo mwmapece
>3 em:PELmume we momma—o cowmmp cmmzumn new mmcw_ macaw Apm>mp Ho.v mmucmcmmmwe ucmopewcawm x—gmwzxx

 

o¢.m mm~.m muom.n comm.“ muuoc.m wcomm.m uunm¢.c unwo.m unmo.o unwo.m amom.m nm~¢.m mmn.¢ xpwgw>mm x

 

M. mu e0 ofiu go no no Nag mu Nu Lao 08 No ttmee__ 1m
No.4 eeefi.m unms.e eemfi.e eemo.e demo.e deco.e ammo.m emm.m xapetesmm.m «x
M. m2 “2 wz Nz mz Hz oz ez 44meeep :2
33
ewe.“ eme.e amm.e eom.N sewte>em.w
az woes x az az x woes woes xemee__ fie ._eeeetea

 

.mammwu Loop mo ages so “amoeba om mcwew>oo meowmo— u o“ new meoaaeam o: u a meme: mpmom
oHtH e :0 count me: manomvo .xupcm>mm mmeomvu toe woman one: o~-- as“ an vogue mew: mucapa
pcauw>_ucm .H mcwucmpa u—owe cw mwcwp me new .Hu .Pmucmemq :o mmewuee xuwem>mm mmeow_u cam: .N m_aeh

36

Table 3. Mean squares from analysis of variance for disease severity
lines homogeneous for CH or NH lesion types and

ratings on F3
over three growth stagesx in field planting 2.

 

 

Source Degrees of Freedom Mean square
Replication 5 2.52
Lesion Type 1 206.94**
Line (Type) 8 4.98**
Error (a) 45 1.50
Time 2 5.22*
Type x Time 2 9.44**
Time x Line (Type) 16 1.75 n.s.
Error (b) 97 1.35

 

*, ** Significant at the 0.05 and 0.01 probability levels, respectively.

x3, 6, and 10 node stage: approximate number of nodes on main axis.

37

.umoh m oo=a_ea>
mo «_m»_ue< we po>op ocean—m—em.m “o. mew an mocpp :u sate acmeoum_u xpucuu.mw=m.m mo:__ :2 no mucoum~
.umo» m ou:a_go> uo mwmzpmc< xa a.m.:v

ueoufie.capm we: age: to Aaav _o>m— He. no acmeoumvu xpueau_mv:m_m moax» :z to :u =_:u_3 mmcvp uo museum»
.A_o>o— mc.v emu» magma mpapu—az couczo an umcvscouou ma

ucocoempo »_u:eu?w—ea.m no: men eouuop usem use xn nozoppom m:eoE.cE:—ou none can mmupo :owmm— gone evguwzx

.mpxa spas co move: we cones: mumswxoeaaettomoum cuzocw:

.AmcovueUV—ame omega *o mceoev u\mu_:= newscou ace—oo-1~2u\=mu>

.m:o_»auwpaoe agape opmzvm x.m mo memos men came e 95:: copmo_ was xuveo>um mmeom_o=

 

 

 

 

 

waxh
as «a «a. .13 «i .3 «a ~m3—0> u
mc—a noummz
.m.: as .m.: as .m.: «e .m.= mospm> a
me.o o~.m o~.- mm.e o~.e um.¢ om.¢ asapoo.w
om~.o n~w.e omm.- a-.m mom.m acoo.m eoo.m mu
em~.o oom.~ a-.oH ammm.v oom.¢ amm.¢ o~m.v cu
av~.o one.~ umm.o~ moo.e oso.e amm.m eso.v mu
amm.c em~.m om~.m~ eooé moo.¢ m~m.m umm.e Nu
u-.o em~.m www.mg a-.m emm.e noo.o o~o.c Ho
mmcvb :u
m¢.e mm.o om.~ e¢.~ ON.” um.~ so.m can—ou.x
amc.m a~¢.o uo~.m am¢m.~ ecod nooo.m oom.~ mz
afim.c emm.o a-.~ no-.~ emm.~ use.“ com.m oz
oom.m am~._ om~.m a-.m omm.~ aoo.¢ omo.m mz
u~m.c a-.~ mmm.m anem.~ coo.~ mom.~ ooo.m Nz
amm.m oom.o use." now.“ emm.~ xemo.~ axo.~ Hz
mmuo: - womb: «H mono: o~ phi. move: o“ mono: o .3mmuo: m mm:_4 :z
mammpu emm_ Ned 55m “w pouch ammcpumc xw_co>mm oma6m_a mwcwa mm
>=uu mop ammo—\mcovmop o'uoeomz

 

.ooca Coop upuocum: ocoe co acouema on u o” use mEouaexm o: u _ meme: oH-~
mo opeom a no names mac_umc xuweo>mm mmmomvo .osmm.a Loop EU\=uu ogmop new ‘66— eon meowmap
.»u_eo>om «momm'u to» N m=_u=u~a u_ovm :' mwmoum guzocm oww_uoam no mocwp me we mac—ace coo: .e «page

38

Lesions on leaves one and two were n0t evaluated. Again, there was a
large difference in both the total number of lesions and the number of
lesions 3_5mm diameter between lesion classes. There was not a differ-
ence in total lesion number among lines within each lesion type. Only
within the CH type was there a significant difference in number of
lesions 3 5mm diameter.

Total lesions were counted primarily to validate the subjective
disease severity ratings. The number of lesions_: 5mm were also evalu-
ated to determine if there was a difference in number of large lesions,
in addition to total lesions, between lesion classes. Thirty-three
and twenty-six percent of total lesions were 3 5mm diameter in NH and
CH F3 lines, respectively. This difference was not significant (T
test).

Correlation coefficients were calculated for individual plant
disease severity ratings and the number of lesions per leaf at the 10
node stage. Correlation coefficients were .67 and .58 for NH and CH
populations, respectively. The pooled correlation coefficient for the
two populations was .81 (Table 5).
2

of leaf tissue in F3 lines were

estimated from 3.8 cm diameter leaf discs. A single sample was taken

Colony forming units per cm

from leaf five or six on three plants per line (blocks 1-3) and
repeated each day for 3 days. As with disease severity estimations and
lesion counts, there was a large difference in bacterial populations
between lesion classes. The mean bacterial population of CH F3 lines
was 100-fold greater than the mean bacterial population in NH F3 lines.
There were not significant differences in bacterial populations among

lines within each type. Bacterial p0pulations were highly correlated

39

Table 5. Simple correlation coefficients between disease severity rat-
ings and total necrotic lesion number as rated on individual
plants at the 10 node stage in field planting 2.

 

NHx F3 lines CHy F3 lines Pooled F3 lines
.565** .575** .806**

 

**Significant at .01 level.
xNon-halo lesion type.

yChlorotic-halo lesion type.

40

with mean severity and lesion number among CH lines, but poorly corre-
lated among NH lines (Table 6).

In addition to the F3 lines, parental and reciprocal F1 lines were
evaluated in field planting 2 for disease severity, lesion number and
bacterial population (Table 7). All ratings of 9402 were less than half
those of NP. The F1 ratings, relative to those in field planting
1, were lower and fell very close to mid-parent values. In no case were
there significant differences in disease severity, lesion number, or
bacterial populations between reciprocal F1 lines. Unlike NH class F3
lines, the interaction between growth stage and the resistant parent
9402 was not significant. However, the same trend was reflected:
disease severity ratings on 9402 decreased with increasing maturity.

In field planting 2, mean ratings of NH class F3 lines for sever-

ity, lesion number, and CPU/cm2

were very close to those of the resis-
tant parent 9402. Mean rating for CH class F3 lines ranged from
mid-parent values to values for National Pickling. In field planting 2,
there were large differences between the highest NH class values and
lowest CH class values for disease severity, lesion number, and bacter-
ial populations (Table 4). Some overlap may have occurred if all CH
lines of field planting 1 had been included in field planting 2. None
of the three lowest ranked CH lines in field planting 1 were included
among the randomly selected CH lines in field planting 2.

The disease severity scores for F3 lines in field planting 1 and
the mean severity scores (over three growth stages) for F3 lines in
field planting 2 were partitioned into variance components due to lesion

type (VT), and line within type (VL(T)) (Table 8). Based upon the
equation VT / (VT + VL(T))’ 81 and 93% of the genetic variance for

41

Table 6. Simple correlation coefficients of mean number CFU/cm2 with
mean disease severity ratings and mean number of lesions per

leaf on F3 lines in field planting 2.

 

Necrotic
Disease severity lesions/leaf
3:10 node stage 10 node stage TotEl'» 3_5mm dia.

 

CH Class F3 lines .878* .794+ .797+ .858*
NH Class F3 lines .808+ .102 n.s. .281 n.s. .512 n.s.
All F3 lines .890** .861** .892** .928**

 

+, *. **, n.s.: Significant at the .10, .05, .01 level and not signi-
ficant, respectively.

42

Table 7. Mean ratings of parental and F1 lines at specific growth
stages in field planting 2 for disease severity, lesions per
leaf and loglo CFU/cm2 leaf tissue.

 

Line 3nodesw 6’06385 10 nodes 17 10 nodes 14 nodes 11'nod€§

Necrotic
. u Lesions/leafu . log
Disease severity rating; Total 25 mm CFU/cm2v

 

 

 

9XN 4.00a 4.17b 5.00b 4.39b 14.82bc 2.523 6.07

9402 4.17ax 2.50a 1.67a 2.78a 3.283 1.30a 4.39

 

NX9 4.83a 3.83b 3.83b 4.17b 9.03ab 2.07a 6.70 H
5.50a 5.67b 5.33b 5.50c 18.92c 4.68b 6.33 i
F valuey -- -- -- ** x ** n.s.

 

Disease severity and lesion number data are means of six single plant
replications. Disease severity rates on scale of 1-10 where 1 = no
symptoms and 10 = 50 percent or more necrotic leaf area.

CFU/cm2--means of three replications. Estimated from three 3.8 cm
diameter leaf discs per replication. Samples were from leaf five or
six on three plants per line (blocks 1-3). Sampling was repeated
each day f0r 3 days.

Growth stage: approximate number of nodes on main axis.

Within each column, means followed by the same letter are not signi-
ficantly different as determined by Duncan's Multiple Range Test (.05
level .

Mean scores among lines within each column are significantly differ-
ent at the .05 level (*), .01 level (**) or not significantly differ-
ent (n.s.) by F test. Growth stage or line x growth stage not
significant for disease severity ratings as determined by F test.

43

Table 8. Genotypic variances of F3 lines for disease severity parti-
tioned into variance due to lesion type and due to F3 lines
nested within type. VT + VL(T) = total genotypic variance.

 

 

VType vLine(Type) VT/(VTI'VL(T))
Planting 1u 2.74 .63 .81
Planting 2v 2.16 .17 .93

 

uField planting 1: Variance calculations from disease severity rated
once at approximately the 11-16 node stage.

vField planting 2: Variance calculations from mean of three ratings at
the 3, 6, and 10 node stage.

44

disease severity was attributed to differences between lesion types in
field planting 1 and 2, respectively.

Heritability values for disease severity within each F3 lesion
type class ranged from .10 to .30 (Table 9). Within each field plant-
ing, heritability was higher among CH lines than among NH lines. The
heritability estimates for field planting 1 were possibly more accurate
than field planting 2 due to the larger sample size.'

Leaf tissue was sampled a second time from 9402, 9402 x NP, and NP
to determine the relationship between bacterial populations and lesions.
A split-plot design was used with cultivar as the main plot, and sample
types (with or without lesions) as the sub-plots. Each experimental
unit homogenized for bacterial population estimations consisted of nine
0.9 cm diameter discs--three from each of three randomly selected plants
per line in this study. Each sub-plot experimental unit, with or
without lesions, was taken from the same individual leaves. At isola-
tion, plants were 12 nodes in length. All samples were taken from
leaves five, six, or seven. Samples without lesions were taken from
green tissue without visible necrosis. For samples with lesions, each
leaf disc included a single lesion of approximately 3 mm in diameter.
The average bacterial p0pulation at lesions was 1000 times that of non-
lesion areas (Table 10). The bacterial population at lesion sites on NP
and 9402 x NP was 10 times as great as the bacterial p0pulation at
lesion sites on 9402. No significant difference in bacterial popula-

tions was found among lines in non-lesioned sites.

Greenhouse Studies
Infiltration of cucumber leaves with 1.5 x 105 CFU/ml resulted in

an initial population of about 2.8 x 102 CFU/cm2 (Figure 1). Bacteria

 

45

Table 9. Genotypic and environmental (error) variance components and
broad sense heritability estimates of F3 lines within CH and
NH lesion type classes for disease severity in field plant-
ings 1 and 2.

 

 

. 2
F3 lines Exp. No. VG VE H
N 1-8 1x .52 2.56 .17
c 1.12 1 .70 1.66 .30
N 1-5 2y .12 1.13 .10
c 1-5 2 .23 1.53 .13

 

xField planting 1: variance calculations from disease severity rated
once at approximately the 11-16 node stage, 12 replications.

yField planting 2: variance calculations from mean of three ratings at
the 3, 6, and 10 node stage, 6 replications.

46

Table 10. Loglo mean number of CFU/cm2 at lesion or non-lesion leaf
sitesZ on parental and F1 lines.

 

 

 

 

 

Main Plots* _4__ §ub-plots**
9402 9402 x NP NP X All Lines
+ Lesion 5.903y 6.98b 7.04b 6.64
- Lesion 3.33a 2.90a 3.74a 3.32
Y 4.51a 4.94ab 5.39b

 

*Significant difference between lines by F test; lines x sample type
(+ or - lesion) not significant.

**Highly significant difference between samples with or without lesions.

y Cultivar means followed by the same letter are not significantly
different as determined by Duncan‘s Multiple Range Test.

2 Leaf tissue was sampled with .9 cm diameter discs containing either a
single lesion or no lesion. Leaves 5, 6 or 7 were sampled at the 12
node growth stage. Corresponding sub-plot samples (+ or - lesions)
were from the same leaf in all cases.

 

Figure 1.

47

Multiplication of Pseudomonas lachrymans in leaves of resis-
tant MSU 9402 (9402), susceptible National Pickling (NP) and
9402 x NP. The second leaf of three-leaf
plants was infiltrated with 1.45 x 105 CFU/ml using a Devil-
bis atomizer at time = 0. Values are averages of two exper-
iments, three replications per experiment.

10

acute

LOG CPU/0M2
. GI

48

 

 

 

 

 

1 r r 1 I r I
and
.1
d
‘
0 NP
6 9402 XI? ..
A 9402
L l J L L

 

24 48 72 96 120 I44 168
‘ HOURS AFTER NOCULATION

49

in NP and 9402 x NP increased in a typical log phase manner with no ap-
parent lag time. The log phase ended 48-72 hours after inoculation and
the bacterial population leveled out at approximately 4.26 x 106 CFU/cm2
until 144 hours after inoculation. Bacteria on 9402 multiplied at a

8 CFU/cmzz a peak popu-

slightly lower rate and leveled off at 2.3 x 10
lation two-fold less than NP and 9402 x NP. Similarly, when both paren-
tal and F1 lines were infiltrated with a lower inoculum concentration
(1.2 x 103 CFU/ml), bacteria in 9402 multiplied at the same rate as NP
and 9402 x NP (Figure 2). Bacterial populations in all lines increased
for 48 hours at a rate almost identical to that observed with the higher
inoculum concentration. After 48 hours, bacterial populations leveled
off to approximately 6 x 106 CFU/cmz. Regardless of inoculum concen-
tration, peak bacterial p0pulations (at 96 hours) were greater than
the initial population by a factor of 2 x 106 (Figures 1 and 2).

When plants were infiltrated with the higher inoculum concentra-
tion, pinpoint water-soaked spots first appeared on NP and 9402 x NP 48-
72 hours after inoculation. Similar pinpoint spots appeared on 9402 72
hours after inoculation. By 96 hours, water-soaked areas expanded,
coalesced, and collapsed into irregular necrotic areas between major
veins. This occurred on all lines, but less necrosis was observed on
9402. A slight, general chlorosis was observed on NP and 9402 x NP at
96 hours post-inoculation, which intensified by 120 hours. »At approxi-
mately 120-144 hours, necrotic tissue began to dehydrate on all lines.

Lesions developed over a similar time course for both high and
low inoculum concentrations, however, lesions did not coalesce with the

latter. At the lower inoculum concentration, lesions in all three

lines first appeared at 48 hours as water-soaked, pinpoint spots. By

 

50

Figure 2. Multiplication of P, lachrymans in leaves of resistant MSU
9402 (9402), susceptible National Pickling (NP) and
9402 x NP. The second leaf of three-leaf plants was infil-
trated with 1.16 x 103 CFU/ml using a Devilbis atomizer at

time = 0. Values are means of three replications, two plants
per replication.

LOG cru I 6M2
-5 U! 0 V O

(a)

51

 

 

 

7
. \

 

 

 

‘fyi

u NP '
o 9402qu
A 9402 _.

 

l l l l l l

24 48 72 96 120 I44 168
HOURS AFTER NOCULATION

 

52

96 hours all lesions were light green round spots, 1mm in diameter with
a 0.2mm pinpoint necrotic center. From 120 to 168 hours after inocula-
tion, lesions on NP and 9402 x NP increased to approximately 2-3mm dia-
meter with the periphery of the necrosis turning an orange-brown color.
Lesions on 9402 rarely enlarged beyond 1mm in diameter, and remained a
light green color. The white, necrotic centers on 9402 enlarged
slightly or not at all. In the greenhouse environment, lesions of both
classes were not vein-limited as is typical in the field.

2 of leaf tissue was

3

The number of necrotic lesions per 20 cm

counted at 96 hours after inoculation with 1.2 x 10 CFU/ml. The mean

number of ncrotic lesions/20 cm2 was: NP = 42.3, 9402 x NP = 28.3, and
9402 = 3.8.

At 192 hours after inoculation, three .48 cm diameter discs con-
taining a single lesion or none were taken from each plant. The isola-

tions were replicated twice with three plants per replications. With
2 7

one lesion per disc, the mean number of CFU/cm was: NP = 2.5 x 10 ,
9402 x NP = 1.8 x 107, and 9402 = 4.1 x 105. 411 samples without
lesions had less than 104 CFU/cmz.

2

Colony forming units, necrotic lesions and total lesions per cm
of leaf tissue 11 days after infiltration for three experiments were
determined (Figure 3). Inoculum concentrations for experiments one,
two and three were 1.8 x 103, 5.0 x 103, and 7.1 x 103 CFU/ml, respec-
tively. Individual necrotic lesions developed after inoculation in
these experiments similar to the inoculations with 1.2 x 103 CFU/ml
described above. Lesions on 9402 x NP ranged in intensity both among

experiments and among plants within an experiment. In experiment one,

lesions on the hybrid were as intense as those on National Pickling.

Figure 3.

53

Necrotic and total lesion number/cm2 and loglo CFU/cm2 on MSU
9402 (9), National Pickling (N) and on MSU 9402 x

National Pickling (9 x N) at day 11 after bacterial infiltra-
tion. Second leaf of three-leaf glants was infiltrated with
1.8 x 103, 5.0 x 103 and 7.1 x 10 CFU/ml for experiments

1-3 respectively. Values are means of four replications.
Means of each character within same experiment with same
letter are not significantly different at p = .05 by Duncan's
Multiple Range Test.

LOGCFU/CM2
NOD-hUIQthoO
l

54

 

r
U

 

 

 

 

 

 

 

   

1 r
9 9XN N
EXP. 1
El LOG BAC.

TOTAL LESIONS (EXP. 2 , 3 ONLY)
I NECROTIC LESIONS

LESION No.7 0M2

 

55

In experiments two and three, there was more variability in F1 lesion
intensity. F1 lesions ranged from identical to NP to only slightly more
conspicuous than 9402. Necrotic lesions on MSU 9402 were uniformly
light green with pinpoint white, necrotic centers.

Bacterial populations were significantly different among lines in
experiments one and two (p = .01) and in experiment three (p = .05).
Average bacterial populations over all three experiments were: ,NP = 3.4

9 8. and 9402 = 8.5 x 107. In these experi-

x 10 , 9402 x NP = 8.9 x 10
ments, the mean bacterial populations in NP were 40 fold greater than
the population in 9402 eleven days after inoculation. This difference
among lines was much greater than the 2-10 fold difference seen at fOur
days after inoculation (Figures 1 and 2). There were also large differ-
ences in the number of lesions among lines, with the F1 lines usually
intermediate (Figure 3). Lesions counted as necrotic had typical necro-
tic centers. Total lesions were counted by holding leaf discs up to a
background light. This technique permitted non-necrotic, immature
lesions to be easily identified. The percentage of the total lesions
that was necrotic was calculated from experiments two and three. Only
36% of the total lesions on 9402 were the larger necrotic lesions;
whereas 69 and 88% were necrotic on 9402 x NP and NP, respectively.
Correlation coefficients between the number of bacteria and number of
necrotic and total lesions across all three lines, were highly signifi-
cant at .71 and .85, respectively (Table 11). Within lines 9402 and

9402 x NP there was little if any correlation between number of bacteria

and lesion number.

56

Table 11. Simple correlation coeffgcients of the number CFU/cm2 with
the number of lesions/cm in individual leaf samples of
greenhouse grown plants 11 days after leaf infiltration with
approximately 4.5 x 103 CFU/ml inoculum.

 

 

Line Necrotic Lesions Total Lesions
MSU 9402 -.240 n.s. -.221 n.s.
9402 x NP -.435 n.s. .534 n.s.
National Pickling .613 * .885 **
All Lines .711 ** .852 **

 

*, **, n.s.: Significant at the .05, .01 level and not significant,
respectively.

57

DiScUssion

 

Compared to National Pickling (CH lesion type) MSU 9402 (NH lesion
type) showed high levels of resistance to Pseudomonas lachrymans in both
field and greenhouse environments. The F1 plants were intermediate in
disease severity, but biased towards the susceptible parent, National
Pickling. There was not a significant difference in disease severity
between reciprocal F1 lines which indicated no maternal effects for
resistance to E, lachrymans.

Based upon a previous inheritance study, a single gene controls
the expression of lesion type (15). In this study, the importance of
this single gene as a component of resistance to E, lachrymans was
determined by a comparative study of F3 lines homogeneous for either the
NH or CH lesion type. The F3 lines were used to ascertain the effect of
lesion type on disease resistance since the parental lines had different
genetic backgrounds. The F3 lines were selected at the seedling stage
only on the basis of lesion type, and unlinked genes were assumed to
segregate randomly between the two lesion classes. Differences in
disease severity between the two groups of F3 lines were due to lesion
type.

The disease severity of NH lines remained consistenlty lower than
CH lines throughout several growth stages. Other studies have shown
that resistance to bacterial diseases varies with maturity (8).

Disease ratings of CH F3 lines, NP, and F1 lines were relatively stable
over three growth stages; in contrast, disease ratings of NH F3 lines

and MSU 9402 decreased with increasing maturity.

58

In order to quantify the importance of the lesion type as a compo-

nent of resistance to P, lachrymans, genotypic variance of disease
severity for both field experiments was partitioned into variance compo-
nents due to lesion type and due to F3 lines nested within type.
Eighty one and ninety-three percent of the total genotypic variance in
field plantings 1 and 2 were accounted for respectively by lesion type,
indicating that NH lesion type is a major component of resistance to P.
lachrymans.

Heritability estimates among F3 lines within each lesion class
were medium to low (.1-.3). Even so, heritability estimates on a per
plot basis of this magnitude indicate there is sufficient genetic
variance within each lesion class to successfully select fOr higher
levels of resistance within each lesion class. The importance of other
genes controlling resistance to g. lachrymans is also indicated by the
overlap of disease severity ratings between the two classes.

Cucumber lines, homozygous for the CH lesion type, with a genetic
background conferring high levels of resistance may have more resis-
tance to P, lachrymans than a homozygous NH line with a background con-
ferring low level resistance. In field plantings 1 and 2, several NH
and CH F3 lines had disease severity ratings close to the parental
extremes indicating that only a few (less than four) additional genes
condition the reaction to E, lachrymans.

Some caution should be used in extrapolating these results beyond
this F3 population. These quantitative estimations illustrate that
lesion type, controlled by a single gene, is a major component of resis-

tance to E, lachrymans and that the interaction of minor genes can

 

modify the level of resistance. An analogous situation has been

59

reported for resistance to bacterial blight in cotton, caused by lgnthg-
monas malvacearum, where minor genes influence the expression of major
genes for resistance (17).

Differences in disease severity between CH and NH lesion types
were also reflected in foliar bacterial populations. Bacterial popula-
tions on F3 lines of the CH lesion class were, on the average, IOO-fold
those on NH F3 lines. These differences between lesion type classes
indicated that the gene coding for the NH lesion type limits bacterial
multiplication in addition to reducing disease severity under epiphyto-
tic conditions.

In field studies, cucumber lines were monitored for bacterial
population levels 16 days or more after inoculation; when they were
assumed to be beyond the logarithmic phase of population increase and
at a steady state. Greenhouse experiments were initiated to compare
growth of bacteria and lesion development among parental and F1 lines
and to determine the association of bacterial pOpulations with lesions.

In greenhouse experiments, bacterial populations in resistant MSU
9402 increased logarithmically after inoculation at a similar or slight-
ly lower rate relative to bacteria in National Pickling and F1 plants.
Beyond the logarithmic stage, and up to 8 days after inoculation,
there were only small differences in bacterial population levels, even
when leaves were inoculated with a low bacterial concentration of 1.2 x
103 CFU/ml. In three other greenhouse experiments, 11 days after inocu-

lation with an average of 4.6 x 103

CFU/ml, bacterial populations on
NP averaged 40 fold those on 9402, and four fold those on 9402 x NP.

It appears that under these greenhouse conditions, 3, lachrymans popu-

 

lations continued to increase slowly beyond the log phase. Using a

60

similar inoculation procedure, Chand and Walker (8) found that E.
lachrymans multiplied slower in leaf tissue of the resistant cucumber
lines PI 169400 than in the susceptible line Wisconsin SMR 18. Peak
bacterial populations were reached in both lines 4 days after inocula-
tion. In contrast to this research, Chand and Walker (8) found large
differences in bacterial populations. At 4 days after inoculation,
there were about 300 times as many bacteria in SMR 18 as in P1 169400.
Bacterial populations in the F1 lines were only twice the population
of'the resistant line. In this study, bacterial populations in the F1
were closer to those in the susceptible parent.

Other bacterial plant pathogens have been shown to multiply in
resistant tissue at the same (1; 16, 31, 34) or at slower rates (14, 18,
27) relative to susceptible tissue. With most bacterial/host systems
studied, maximum pOpulation levels in susceptible tissue are typically
100-1000 fold those in resistant tissue (14, 27, 31, 34). In several
cases where large differences in bacterial populations were reported,
resistance was described as hypersensitive (27, 34). The NH lesion
resistance is not hypersensitive. NH lesions develop over the same
time course as the CH lesion type and often is preceded by water soak--
ing (15). Small differences in bacterial population levels have been
reported for "tolerant" or moderately resistant lines (5, 12, 31).

These studies with parental and F1 lines also illustrated that
under greenhouse conditions CH and NH lesions develOped over the same
time-course. Differences between lesion types gradually became evident
once they became necrotic (120 hours after inoculation). Necrotic
lesions on 9402 were typically light colored and restricted in size

while those on NP were larger, orange-brown in color and surrounded by

61

a chlorotic halo. Lesions on plantsunder greenhouse environmental
conditions never enlarged to become vein-limited. Differences in lesion
type were also indicated by the percentage of lesions that became necro-
tic. At 11 days after inoculation of greenhouse plants, 88, 69, and

36% of the total lesions were necrotic on NP, 9402 x NP, and 9402,
respectively.

Bacterial populations were found to be closely associated with
lesion sites. Means of bacterial populations on F3 lines in field
plantings were highly correlated (.86 to .93) with mean disease severity
and lesion number. Examining each lesion class separately, bacterial
population levels and disease severity or lesion number were highly
correlated within the CH lesion class, but poorly correlated within the
NH lesion class. Correlations of bacterial populations with lesion
number on individual samples under greenhouse conditions also reflected
this pattern. There were high correlations between bacterial popula-
tions and lesion numbers on National Pickling, but these were poorly
correlated within samples from 9402 or 9402 x NP. Bacterial populations
at specific lesions on plants in the field were 1000 times greater than
the populations at non-lesion sites of the same leaves. The line 9402
had significantly fewer bacteria than 9402 x NP and NP at lesions of the
same size. At non-lesion areas on the same leaves, there was little
difference in bacterial populations among lines. A similar association
of bacteria with lesions was also observed in the greenhouse where

bacterial populations at lesions on all lines were greater than 4 x 106

2

CFU/cm and populations at non-lesion sites on the same leaves were

4

less than 10 CFU/cmz. Differences in bacterial population levels

among these experimental lines were primarily due to differences in

62

bacterial number at specific lesions and differences in lesion number.
There may have been bacterial population differences at non-lesion
sites, but population levels at these sites were very low relative to
those at lesions. The poor correlation of bacterial populations and
lesion numbers on foliage of 9402 and NH F3 lesion classes might have
been due to the small number of lesions on these lines which would
increase the relatively importance of bacterial populations at non-
lesion areas.

Even though bacterial multiplication in MSU 9402 is limited rela-

tive to National Pickling, P, lachrymans still mutliplied to high

 

levels in this resistant line. This is also true for other bacterial-
host systems (5, 31, 34). Haas and Rotem (19) suggested that high num-
bers of bacteria are not of particular importance epidemiologically.
They noted that once the initial inoculum is present in a crop, the epi-
phytic population of the pathogen is not a limiting factor in epidemic
development.

The multiplication of the bacterium in the host has another impli-

cation. Coyne et al. (12) found large pOpulations of Xanthomonas pha-

 

§gglj_developed on tolerant lines of Phaseolus vulgaris. Wellhausen
(36) noted that virulence of some bacterial pathogens has been shown to
increase during passage through a tolerant host and suggested that seed
of tolerant cultivars should be saved only from plants that are free of
bacteria. As with the Xanthomonads, there are varying degrees of viru-
lence among strains of Pseudomonads. Cultivars resistant to Specific
strains were later found susceptible to more virulent strains (35).

We have, however, observed only slight differences in virulence among

tested 3, lachrymans isolates. Since first introduced in 1966, cucumber

63

cultivars resistant to P. lachrymanshave remained resistant to all P,
lachrymans isolates encountered, indicating that the bacterium has not
had a tendency to mutate towards higher levels of virulence. However,
this fact does not preclude the possibility of a new highly virulent

strain appearing in the future. Because 3, lachrymans is often seed-

 

borne, we suggest that special attention should be given to the control
of P. lachrymans in cucumber seed production fields.

The NH lesion type on cucumber in response to E. lachrymans infec-

 

tion is associated with relatively low levels of bacterial multiplica-
tion. Lesion size and number on NH lines is also limited and disease
severity in the field is significantly reduced. Minor genes interact
to affect varying levels of resistance to E, lachrymans. In summary,
lesion type in response to P, lachrymans infection is a major component

of resistance to this pathogen.

10.

11.

12.

13.

64

Literature Cited

 

Allington, W. C., and D. W. Chamberlain. 1949. Trends in the
population of pathogenic bacteria within leaf tissue of suscep-
tible and immune plant species. Phytopathology. 39:656-660.

Barnes, W. C. 1966. Development of multiple disease resistant
hybrid cucumbers. Proc. Amer. Soc. Hort. Sci. 89:390-393.

Beecher, F. S., and S. P. Doolittle. 1950. Dissemination of
angular leafspot of cucumber. Plant Dis. Reptr. 34:382-383.

Bryson, V., and W. Szybalski. 1952. Microbial selection.
Science. 116:45-51.

Cafati, C. R., and A. W. Saettler. 1980. Effect of host on multi-
plication and distribution of bean common blight bacteria.
Phytopathology. 70:675-679.

Carsner, E. 1917. Do the bacteria of angular leafspot of cucumber
overwinter on the seed? Phytopathology. 7:61-62.

Carsner, E. 1918. Angular leafspot of cucumber: dissemination,
overwintering, and control. J. Agric. Res. 15:201-220.

Chand, J. N. and J. C. Walker. 1964. Relation of age of leaf and
varietal resistance to bacterial multiplication in cucumbers
inoculated with Pseudomonas lachrymans. Phytopathology. 54:
49-50.

Chand, J. N. and J. C. Walker. 1964. Inheritance of resistance to
angular leafspot of cucumber. Phytopathology. 54:51-53.

Coyne, D. P. and M. L. Schuster. 1974. A morphological and phy-
siological component genetic approach to breeding for tolerance
to the bacterial pathogens, Pseudomonas phaseolicola and Xantho-
monas phaseoli. Ann. Rep. Bean Imp. Coop. 17:23-24.

Coyne, D. P., M. L. Schuster and C. C. Gallegos B. 1971. Inheri-
tance and linkage of the halo blight systemic chlorosis and
leaf watersoaked reaction hiPhaseolus vulgaris variety crosses.
Plant Dis. Reptr. 55:203-207.

Coyne, D. P., M. L. Schuster, and K. Hill. 1973. Genetic control
of reaction to common blight bacterium in bean (Phaseolus
vulgaris) as influenced by plant age and bacterial multiplica-
tion. J. Amer. Soc. Hort. Sci. 98:94-99. '

Coyne, D. P., M. L. Schuster, and L. Shaughnessy. 1966. Inheri-
tance of reaction to halo blight and common blight bacteria in
a Phaseolus vulgaris variety cross. Plant Dis. Reptr. 50:
29-32.

 

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

65

Daub, M. E. and D. J. Hagedorn. 1979. Resistance of Phaseolus
line WBR 133 to Pseudomonas syringae. Phytopathology. 69:
946-951.

Dessert, J. M., L. R. Baker, and J. F. Fobes. 1981. Inheritance
of reaction to Pseudomonas lachrymans in cucumber. In Review.

Diachum, S. and J. Troutman. 1954. Multiplication of Pseudomonas
tabaci in leaves of burley tobacco, Nicotiana longifTOra, and
Hybrids. Phytopathoquy. 44:186-187.

 

El-Zik, E. M. and L. S. Bird. 1970. Effectiveness of Specific
genes and gene combinations in conferring resistance to race of

Xanthomonas malvacearum in upland cotton. Phytopathology, 60:
441-447?

 

Ercolani, G. L. and J. E. Crosse. 1966. The growth of Pseudomonas
phaseolicola and related plant pathogens jg VIVO. J. Gen.
Microbiol. 45:429-439.

Haas, J. H. and J. Rotem. 1976. Pseudomonas lachrymans inoculum
on infected cucumber leaves subjected to dew- and rain-type
wetting. PhytOpathology. 66:1219-1223.

Haley, A. B. and M. J. Palmer. 1977. Resistance of cucumber to
angular leafspot disease--correlation of greenhouse and field
reactions. Pickling Crops Research, Univ. of Wisconsin,
Madison. -

ffill, K., D.P. Coyne, and M. L. Schuster. 1972. Leaf, pod, and
systemic chlorosis reactions in Phaseolus vulgaris to halo
blight controlled by different genes. J. Amer. Soc. Hort. Sci.
97:494-498. '7

Husain, A. and A. Kelman. 1958. Relation of slime production to
mechanism of wilting and pathogenicity of Pseudomonas solana-
cearum. Phytopathology. 48:155-165.

Jensen, J. H. and R. W. Goss. 1942. Physiological resistance to
halo blight in beans. Phytopathology. 32:246-253.

Jones, L. R., S. P. Doolittle, and W. W. Gilbert. 1921. Field
tests with cucumber leafspot and anthracnose. Phytopathology.
11:298-299.

Keen, N. T., P. H. Williams, and J. C. Walker. 1967. Protease of
Pseudomonas lachr mans in relation to cucumber angular leafspot.
PhytopatholOgy. 57:263-271.

Lyon, F. and R. K. S. Wood. 1976. The hypersensitive reaction and
other responses of bean leaves to bacteria. Ann. Bot. 40:
479-491.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

66

Omer, M. E. H. and R. K. S. Wood. 1969. Growth of Pseudomonas
phaseolicola in susceptible and in resistant bean pTants. Ann.
Appl. Biol. 63:103-116.

Patel, P. N. and J. C. Walker. 1965. Resistance in Phaseolus to
halo blight. Phytopathology. 55:889-894.

Patel, P. N. and J. C. Walker. 1966. Inheritance of tolerance to
halo blight in bean. PhytopatholOgy. 56:681-682.

Pohronezny, K., P. O. Larsen, 0. A. Emmatty, and J. D. Farley.
1977. Field studies of yield losses in pickling cucumber due to
angular leafspot. Plant Dis. Reptr. 61:386-390.

Scharen, A. L. 1959. Comparative population trends of Xanthomonas
phaseoli in susceptible, field tolerant, and resistant hOsts.
Phytqpathology. 49:425-428.

Schuster, M. L. 1950. A genetic study of halo blight reaction in
Phaseolus vulgaris. Phytopathology. 40:604-612.

Smith, E. F. and M. K. Bryan. 1915. Angular leafspot of cucumber.
J. Agr. Res. 5:465-476.

Stall, R. E. and A. A. Cook. 1966. Multiplication of Xanthomonas
vesicatoria and lesion development in resistant and susceptiblE
pepper. Phytopathology. 56:1152-1154.

 

Walker, J. C. and P. N. Patel. 1964. InheritanCe of resistance
to halo blight of bean. Phytopathology. 54:952-954.

Wellhausen, E. J. 1937. Effect of the genetic constitution of the
host on the virulence of Phytomonas stewarti. Phytopathology.
27:1070-1089.

 

Wiles, A. B. and J. C. Walker. 1952. Epidemiology and control of
angular leafspot in cucumber. Phytopathology. 42:105-108.

   

IIIIIIIIIIIIIIIIIIIIIIII

iiiiiiiNiIsiiiialiiiiill

)
3 1293 10