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Resistance to Pseudomonas solanacearum in Lycopersicon esculentum

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Title:
Resistance to Pseudomonas solanacearum in Lycopersicon esculentum
Creator:
Ferrer Z., Alejandro, 1943-
Publication Date:
Language:
English
Physical Description:
ix, 55 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Dissertations, Academic -- Plant Pathology -- UF
Plant Pathology thesis Ph. D
Tomato wilts ( fast )
Tomatoes -- Disease and pest resistance ( fast )
Tomatoes ( jstor )
Plant roots ( jstor )
Ralstonia solanacearum ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida, 1974.
Bibliography:
Includes bibliographical references (leaves 47-49).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Alejandro Ferrer Z.

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Full Text










RESISTANCE TO Pseudomonas solanacearum IN

Lycopersicon esculentum











By

ALEJANDRO FERRER Z.









A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA


1974












ACKNOWLEDGMENTS


I wish to thank Dr. R. E. Stall, Chairman of the Supervisory

Committee, for his interest and dedication in helping me

pursue this investigation. His guidance and valuable sugges-

tion throughout this work have made possible the completion of

this manuscript.

I also wish to thank Drs. D. A. Roberts and S. J. Locascio,

members of my Supervisory Committee, for their contributions

to the work reported herein. The author is indebted to the

Compagfa Panamega de Alimentos S.A. for providing the necessary

funds for this research.

I extend my appreciation to Mr. R. E. Burns for his valuable

assistance in laboratory work and to Dr. L. H. Purdy, Chairman

of the Plant Pathology Department, for his cooperation through-

out my tenure.

Above all, I thank my wife and children for their constant

encouragement and understanding.
















ii










TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS..................................... ii

LIST OF TABLES...................................... v

LIST OF FIGURES..................................... vi

ABSTRACT......................................... vii

INTRODUCTION...................................... 1

MATERIALS AND METHODS. ............................ 6

Preparation of Inocula........................ 6
Inoculation Techniques................... .... 7
Root injury technique....................... 7
Transplant technique ... .................... 7
Stem inoculation technique.................. 8
Tomato Plant Culture ......................... 8
Root Diffusates................. ........... .. 9
Leachate medium............................. 9
Mineral-water-agar medium.................... 10

RESULTS ............. .............................. 12

Test of Inheritance.. .......................... 12
Selection within P.I. 126408............... 12
Crosses with susceptible plants............. 16
Progeny testing of F2 population of the
cross, Bonny Best x 126408-6-2 ........... 19
Crosses between resistant cultivars and
the P.I. 126408.......................... 27
126408 Field tests ........ ........... .. 29
Nature of Resistance........................... 30
Root Diffusates ............ ................. 30
Leachate tests............... ...... ..... 32
Mineral-water-agar test... .............. 34
Penetration of Noninjured Roots............ 35
Effect of temperature on resistance......... 38

DISCUSSION...................................... 40

SUMMARY........................... ................ 45

LITERATURE CITED................................. 47



iii











Page

APPENDICES .. .... ................. ...... .......... 50

BIOGRAPHICAL SKETCH................................. 55






















































iv
Vf










LIST OF TABLES


TABLE 1. Progeny test of 126408-6 and selections from it for
bacterial wilt resistance

TABLE 2. Progeny test of selections from 126408-6 for bacterial
wilt resistance

TABLE 3. Data on survival of plants from crosses of selections
from P.I. 126408 with Bonny Best after inoculation of
two levels of inoculum of Pseudomonas solanacearum

TABLE 4. Data on survival of plants from crosses of selections
from P.I. 126408 with Floradel after inoculation with
Pseudomonas solanacearum

TABLE 5. Survival of seedlings of 49 F lines of tomato to
Pseudomonas solanacearum and relationship to fruit
weight

TABLE 6. Data on survival of plants from crosses of selections
from P.I. 126408 with Saturn after inoculation with
Pseudomonas solanacearum

TABLE 7. Field evaluation of resistance to Pseudomonas
solanacearum of crosses between susceptible and
resistant cultivars with selections from P.I. 126408
at Ft. Pierce, Fla.

TABLE 8. Influence of substrate leachates on growth of
Pseudomonas solanacearum in vitro

TABLE 9. Bacterial wilt development in plants with noninjured
roots growing in vermiculite

TABLE 10. Penetration of tomato roots by Pseudomonas
solanacearum in a mineral-water-agar media

TABLE 11. Influence of temperature on bacterial wilt development
on resistant and susceptible tomato plants










v















LIST OF FIGURES


Figure 1. Number of F2 selections from the cross, Bonny Best
(S) x P.I. 126408 (H) categorized as to percent
survival three weeks after inoculation with
Pseudomonas solanacearum

Figure 2. Number of Fp selections from the cross, Bonny Best
(S) x P.I. 126408 (R), categorized as to percent
survival six weeks after inoculation with
Pseudomonas solanacearum
































vi









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


RESISTANCE TO Pseudomonas solanacearum IN
Lycopersicon esculentum

By

Alejandro Ferrer Z.

August, 1974

Chairman: Robert E. Stall
Major Department: Plant Pathology

Study of a source of resistance in tomato (Lycopersicon

esculentum Mill.) to Pseudomonas solanacearum E. F. Smith, the

causal agent of bacterial wilt, was undertaken to evaluate the

usefulness of the resistance in control of the plant disease.

Plants with a high degree of resistance to P. solanacearum were

selected from Plant Introduction 126408. These plants, however,

never produced progenies with 100% survival in inoculation tests,

and selections after a second generation of selfed plants were

not significantly different from further selections in resist-

ance. Resistance was expressed in progenies subjected to three

different inoculation techniques in the greenhouse and was also

expressed to natural populations of the pathogen in field tests

in Florida, Georgia, and Panama.

Inheritance of resistance to P. solanacearum of resistant

selections of P.I. 126408 was characterized by crossing them to

the susceptible cultivars, Bonny Best and Floradel. Segregation

at the F2 generation indicated that resistance is polygenically


vii










inherited. Inoculation results with progenies from reciprocal

crosses proved that extrachromosomal inheritance was not in-

volved. The genes were additive with no dominance, but there

was a tendency toward susceptibility in the progenies of F2

plants. Small-fruited plants were not correlated with resist-

ance.

At high temperatures, the resistance to P. solanacearum

was still evident and was equal to that of Saturn, a cultivar

with polygenic resistance. In environmentally controlled

chambers, at air temperatures of 30 or 32 C, stem-inoculated

plants from resistant lines did not wilt in 15 days, even

though some yellowing and adventitious roots were evident. At

the same temperatures, susceptible lines wilted and died within

15 days.

Tomato root exudates did not stimulate growth of P.

solanacearum. Numbers of the bacterium increased in water from

leachates from steamed soil or sand sterilized by filtration or

in an autoclave. Living roots of resistant or susceptible

plants growing in soil or sand did not increase the growth rate

of bacteria in the leachates compared to the check with no

plants. P. solanacearum cells did not multiply near tomato

roots cultured in a mineral-water-agar medium. However, the

bacterium did increase near some fungal contaminants. Such

microorganisms in the soil may produce growth factors that

stimulate multiplication of the bacterium.

Tomato seedlings of susceptible varieties growing asepti-


viii










cally in a mineral-water-agar media containing P. solanacearum

developed wilt symptoms. Penetration of the bacteria into the

roots may have occurred at the tip of the roots as well as where

secondary roots emerged. Bacteria penetrated roots of both

resistant and susceptible plants.

Resistance in P.I. 126408 is protoplasmic. No differences

could be detected in the development of the pathogen around re-

sistant and susceptible roots, and invasion seemed to occur

with equal frequency in each kind of plant. After entry, dif-

ferences in colonization of the plant types were noted. Some

development of the pathogen occurred in resistant plants after

invasion, but the pathogen often did not successfully colonized

the vascular tissues. Colonization of susceptible plants, how-

ever, continued until the plants were killed.

The polygenic protoplasmic resistance of P.I. 126408 may

be useful in breeding programs designed to develop tomatoes re-

sistant to bacterial wilt.




















ix











INTRODUCTION


Bacterial wilt of tomato, caused by Pseudomonas solanacearum

E. F. Smith, is one of the most economically important plant

diseases incited by bacteria (15). A comprehensive review of

the literature on the disease before 1953 was made by Kelman (15);

Buddenhagen and Kelman (4) later extended the review up to 1964.

The pathogen has a wide host range and the disease occurs in

almost every warm temperature, semitropical, and tropical zone

of the world (15). Host plants to P. solanacearum include

economic plants of the Solanaceae, such as tobacco, potato,

pepper, eggplant, and tomato. Non-solanaceous plants, such as

peanut and banana, are also economically damaged by strains of

this organism. A total of 33 plant families contain hosts of

P. solanacearum (15).

On tomato, Lycopersicon esculentum Mill., the disease has

been reported predominantly from the Southeastern states of the

United States. Outside the United states, losses ranging from

insignificant to severe have been reported in Mexico, all

vuuntries in Central and South America, France, Holland, Italy,

Switzerland, Algeria, Mozambique, Union of South Africa, Ceylon,

China, Formosa, India, The Philippines, Java, Sumatra and Hawaii

(15).
Detection of the organism in the field can be made by

visual observation of the symptoms of the disease on host plants.

Serological methods (13), and the use of a selective medium (14),

1






2



have also been employed to determine the presence of the

bacteria in the field. The pathogen is soil-borne and can be

found in recently cleared land as well as in cultivated land

(15). Survival of the pathogen in the soil varies from place
to place and from a few months to many years (15). Also,

since P. solanacearum invades a wide range of plants, many

of which remain symptomless (2, 3), the organism can survive

on many weed hosts.

Bacterial wilt of tomato is most serious under relative

high soil temperature and moisture. Vaughan (27), found that

the rate of disease development increased with increased soil

temperature up to 110 F. However, disease symptoms do not

develop at soil temperatures below 70 F. Wilt symptoms developed

more rapidly in stem-inoculated plants growing in wet rather

than in relatively dry soil (27). This observation was

confirmed by Gallegly and Walker (8).

The subject of root invasion by the pathogen is a contro-

versial one. In general, wounds are thought necessary for

penetration of the bacterium into the roots. These wounds may

be made during transplanting, cultivation, or nematode and in-

sect feeding on roots (15). But Van der Meer, as reviewed by

Kelman (15), found that uninjured roots can be invaded by the

bacterium, at least under high moisture levels. Kelman and

Sequeira (17), suggested that nonwounded roots may be invaded

at the points of emergence of secondary roots.

The fact that the bacterial wilt organism is soil-borne,

and that it survives for many years in the soil in the absence






3

of crop host plants (15), makes control by crop rotation imprac-

tical. Once the organism is established in a field chemical

sprays, or drenches, are of little value as a means of control
of the disease. Disease-resistant crop plants seem to be the

best hope for control.

There are various sources of resistance to bacterial wilt

in tomato, one of which seems to be determined by a single gene

or a few genes. Incorporation of these resistance genes into

commercially acceptable cultivars has been difficult. The as-

sociation of resistance to bacterial wilt with small size of

fruits (1) and the difficulties associated with transferring

polygenically inherited characteristics are the mayor problems
encountered in combining genetic resistance to P. solanacearum

with production of fruits at the commercial level.

A source of resistance selected in North Carolina was

found to be polygenically inherited (1). Venus and Saturn are

two cultivars that have the North Carolina source of resistance

to P. solanacearum (12). Even though resistance of these cul-

tivars holds up under most field conditions (20), up to more

than 90% of flowers drop under the hot field conditions of

Panama (G. Ocana, personal communication). This observations

indicates that the use of these varieties will not solve the

disease problem of bacterial wilt of tomato in the tropics.

OcaHa (21), working with crosses made with resistant breeding

material from North Carolina, found that the smaller-fruited

selections were more resistant than the larger-fruited ones.
Resistant selections did not yield well.






4

A second source of resistance was selected in Hawaii (1).

Plant introduction (P.I. 127805 (L. pimpinellifolium (Jusl.)

Mill.) was highly resistant under most field condition, but re-

sistance decreased during the warm summer weather. Fruit size

of some of the lines developed in Hawaii is acceptable for

commercial purposes. A more recent report from Hawaii in their

resistant lines indicates that "some serious breeding problems

not found in the other programs were encountered with bacterial

wilt" (9). Even though the reasons are not explained, the re-

port indicates that "resistance is more effective when fields

temperatures are below 85 F". This may explain the poor per-

formance of these lines reported by some investigators.

Ferrer (5), found the Hawaii lines susceptible to bacterial

wilt under greenhouse conditions in Florida. Ocana (21) and

McCarter (20) also found the Hawaii lines to be susceptible

under field conditions.

A selection of L. pimpinellifolium from the French Antilles,

CRA-66, is also resistant under field conditions in Panama.

The line produces very small fruit and was found by Ferrer (5)

to be susceptible under laboratory conditions. The source of

resistance of these lines was not reported.

A line, P.I. 126408 (L. esculentum Mill.), was found by
Ferrer (5) to have a higher level of resistance, in the seedling

stage, than all other lines tested. However, fruit which

average less than 1 oz. are produced on plants of P.I. 126408,

but fruit were set under relatively high temperature conditions.

Bacterial wilt resistance in tomato is highly desirable






5

for production of tomatoes in areas where soils are infested

with the agent of the disease. Research was planned to further

characterize the resistance in plants of P.I. 126408. The

mode of inheritance of the resistance and some characteristics
of the nature of the resistance are reported herein.











MATERIALS AND METHODS


A strain of Pseudomonas solanacearum, designated A-21,

was isolated from a diseased tomato plant in Florida in 1971,

and was used in all inoculation tests. It corresponds to

physiological race 1 of Hayward (11). It is a highly aggres-

sive isolate in tomato, causes wilt of tobacco when introduced

in the stem or roots, and when introduced in tobacco leaves at

concentrations greater than 1 x 10 cells/ml it incites a

hypersensitive reaction in tobacco leaves.

Preparation of Inocula

Inoculum of P. solanacearum was prepared by streaking

isolates, maintained in tubes with sterile distilled water at

approx. 27 + C in the dark, onto a tretazolium medium (16).

After 48-60 hr of incubation at 32 C, and based on colony

appearance on this medium, isolates were transferred to nutri-

ent broth and shaken for 20-24 hr at approx. 27 + 2 C. Cells

were centrifuged from the broth and resuspended in sterile tap

water, which proved in laboratory experiences to maintain the

number of living cells longer than a physiological buffered

saline solution (25). The concentration of the inoculum was

standardized photometrically using a "Spectronic 20" (Bausch &

Lomb), spectrophotometer at a wavelenght of 600 nm to either

50, 70, or 84% transmittance. Other concentrations of inocula
were derived by dilution from such standardization. A dilution

6






7


plate technique (25) was used to determine actual concentrations
of bacteria in the suspensions. Fifty percent transmittance was

equivalent to 2 x 108 cells/ml; 70%, 1 x 108 cells/ml; and 84%,
5 x 107 cells/ml.

Inoculation Techniques

Root-injury Technique

Tomato seeds were sown in 48-inch rows and 4 inches apart

in soil contained in raised benches in a greenhouse. The sus-

ceptible cultivar Bonny Best was used as control. Venus, Saturn,
Floradel, and selections of the P.I. 126408 were used as treat-
ments. Rows were thinned to 50 plants/row or left unthinned
according to the purpose of the test. Prior to inoculation, a

furrow was made with a knife about 1 inch from the plant stems

and about 2 inches deep so that one side only per line of
plants was inoculated. Then 50 ml of standardized inoculum

were poured along this furrow which was then closed to avoid
desiccation. Three to four-week-old seedlings were inoculated

using this technique.

Transplant Technique

Tomato seeds were sown in 6-inch clay pot saucers. Seven

days after emergence of the seedlings, 25 ml of standardized
inoculum were added to each plate. Seedlings were transplanted

5-7 days later to soil in the benches and placed in rows to
keep the different lines separated. Living plants were counted





8


15 days after inoculation.

Stem Inoculation Technique

Tomato seedlings in the cotyledonary stage were trans-

planted into 4-inch pots containing steam sterilized soil.

About 3 weeks after transplanting, when they were about 6

inches tall, seedlings were inoculated by piercing the hypo-

cotyl about 0.5 cm above the soil level. The inoculum con-

tained about 2 x 10 cells/ml. Plants were then placed in

Sherer CEL 4-4, environmental chambers, at various tempera-

tures, which were kept within 2 C of the desired temperatures

throughout the experiment. A 16-hr light period/day from

fluorescent and incandescent lamps giving approx. 680 lux 12

inches from the lights was used in this test. Living plants

were counted 15 days after inoculation.

Tomato Plant Culture

All plants raised to maturity were planted in soil in

raised benches. All secondary buds were kept pruned and the

plants were trellised on strings attached to wires overhead.

The plants were watered twice daily, fertilized with a 6-6-6

commercial fertilizer every 2-3 weeks, and sprayed with pesti-

cides as needed. Steamed soil was always used in the green-

house benches.

Crosses were made by transferring pollen to emasculated

flowers. Selfing was allowed to occur naturally,.since very










few flower-visiting insects or wind existed in the greenhouse.

All inoculation tests to determine levels of resistance

were made in a greenhouse. Temperatures fluctuated greatly

during most tests and from one test to another. During some

tests, soil temperatures fluctuated between 20 and 30 C. In

others, the temperatures fluctuated between 28 and 32 C.


Root Diffusates


Leachate Medium


Three tests were undertaken to assess the role of root

diffusates on the growth of P. solanacearum. In the first and

second tests, steamed soil was placed in 115 ml "Nalgene" dis-

posable filter units with an 0.20p membrane filter under the

soil. Tomato plants in the first true-leaf stage were trans-

planted to the soil and the units were kept in a Sherer CEL 4-4

growth chamber at 28-30 C and with 12 hr of light daily. During

the two weeks of plant growth, water percolated through the soil

and through the sterilizing membrane. This water was collected

and used as a medium for P. solanacearum.

In a third test, washed white sand was placed in steam-

sterilized 4-inch pots. Tomato seeds were planted in the sand,

which was watered daily with a 4-salt mineral solution with

minor elements (18). Three weeks after germination of the

seedlings, the sand was flooded with water and left 20 min for

excess water to drain. The pots were then placed on a "Nalgene"

filter unit with an 0.20. membrane filter. Ten ml of water






10



were added to each pot and a vacuum of 25 inches of mercury

was applied to the filter unit. The excess water from the pots

was drawn through the filter and was used as a medium for

P. solanacearum.

All media obtained from the pots were again filter-steri-

lized by forcing them through a "Swinny" filter containing an

0.20h- membrane filter. In the first two tests, half of each

batch of medium was also autoclaved.

Cells of P. solanacearum, isolate A-21, were added to the

soil-water medium. A 20-hr broth culture of the bacterium was

centrifuged at 2,000 g to remove the bacteria. The pellet was

resuspended in sterile tap water and standardized to 50% trans-

mittance in a "Spectronic 20". Using this standard suspension,

equivalent to 2 x 10 cells/ml, a suspension was obtained by

dilution, containing about 1 x 103 cells in a 0.05-ml drop.

The latter amount was added to each ml of soil-water medium.

The tubes of media were placed on a reciprocal shaker at 30 C.

The growth of bacteria in the medium was determined by

periodically diluting the medium 10-fold and planting measured

volumes of the diluted medium on nutrient agar plates (25).

Colonies were counted and numbers were converted to bacterial

cells concentration in the original medium. Two or three dilu-

tions were used to obtain average readings.


Mineral-Water-Agar Medium

A mineral-water-agar medium (MWA) was used to culture






11


tomato plants in the presence of a dilute suspension of P.

solanacearum. This medium was prepared by adding Bacto-Agar

(Difco) at 1.3% w/v to a modified 4-salt nutrient solution with

minor elements (18). Cells of P. solanacearum, isolate A-21,

from a 20-hr culture were added before solidification of the

medium at 47 C. Twenty ml of this medium were placed in ster-

ile 50-ml tubes with a cotton stopper. Tomato seeds were sur-

face-sterilized by dipping them in 95% alcohol and then trans-

ferring them to a 10% "Clorox" solution for 2 minutes. A ster-

ile seed was placed in each tube with the medium and the tubes

were placed in a Sherer CEL 4-4 growth chamber, at a tempera-

ture of 30 + 2 C. Plants were illuminated for 12 hr daily with

fluorescent and incandescent lamps giving approx. 680 lux 12

inches from the lamps. Plants were placed about 12 inches from

the lights to obtain a fair growth of the seedlings. Seedlings

were observed periodically and tubes containing no seeds were

used to compare growth of bacterial colonies where tomato plants

were absent.










RESULTS


Tests of Inheritance


Inheritance of the resistance to bacterial wilt of plants

of P.I. 126408 was investigated by screening progeny of resist-

ant selections from this line crossed with plants of the sus-

ceptible cultivars, Bonny Best and Floradel. Crosses were also

made with plants of the cultivars, Venus and Saturn, which have

polygenic resistance to bacterial wilt, to observe if such a

cross would increase the resistance among the progeny over

progeny of either parent.


Selection within P.I. 126408


Preliminary experiments demonstrated the resistance of

plants of P.I. 126408 to bacterial wilt, but since P.I. acces-

sions are usually heterozygous, seed were obtained from resist-

ant plants through successive generations in attempts to in-

crease homozygosity. Progeny of these selections were screened

for resistance.

Preliminary screening tests indicated that seedlings from

the sixth selection from P.I. 126408 were the most resistant

of 10 selections made and this selection was labeled 126408-6.

Eight plants from 126408-6 were selected after screening progeny

for resistance. Selfed seed were obtained from the 8 plants

and the resulting seedlings were not significantly different

from the progeny of parent selections in survival after inocu-


12






13



lation if data from all selections were lumped together. How-

ever, the progeny of selections 126408-6-2, -6-4, -6-7, and

-6-8 (Table 1) were more resistant than the parent selection,

and this criterion was used for further selection within those

lines.

In the third generation from P.I. 126408 selections from

resistant progeny of 126408-6-2 and 126408-6-8 were made after

inoculations of the lines. The progeny of these selections

were screened for resistance and compared with populations

representing each previous generation (Table 2). Three inoculum

levels of the pathogen were used.

As the inoculum level increased survival of plants de-

creased. At the lower level of inoculum, not all susceptible

control plants, Bonny Best, were diseased. Even with the low

level of inoculum resistance of selections was evident, however,

by a higher percentage of living plants 2 weeks after inocula-

tion. The data from the three levels of inoculum were lumped

to evaluate the resistance of the selections.

After an analysis of variance and Duncan's multiple range

test were performed on the data (Appendix 1,2), wide differences

in survival of selections in inoculation tests were noted among

the representatives of the generations. Resistance was increased

by selection through the three generations. However, no sig-

nifficant differences occurred between the second and third gen-

eration. This indicated that selection in future generations

would probably not significantly increase resistance in the

lines.





14






Table 1. Progeny test of 126408-6 and selections from it
for bacterial wilt resistance


Selection Total No. Plants Livinga) Living

126408-6 24 8 33.3
126408-6-1 24 9 37.5
126408-6-2 24 16 75.0
126408-6-3 24 5 20.8
126408-6-4 24 16 75.0
126408-6-5 24 13 54.2
126408-6-6 24 9 37.5
126408-6-7 24 16 75.0
126408-6-8 24. 17 79.2


a) living plants 2 weeks after root inoculation with
2 x 108 cells/ml of Pseudomonas solanacearum.






Table 2. Progeny test of selections from 126408-6 for bacterial wilt resistance

50%a) 70%a) 84_ a) Totals
126408 No. b) No. b) No. b) No. a %
Selection plants living plants living plants living plants livingb) livin
1st gen.
6 9 0 16 4 7 4 32 8 25.0
2nd gen.
6-2 18 8 26 13 32 23 76 44 57.9
6-8 22 4 33 26 32 28 87 58 66.7
6-9 31 14 33 23 17 15 81 52 64.2
6-10 16 6 20 -9 21 20 57 35 61. 4
3rd gen.
6-2-1 22 10 24 10 36 21 82 41 50.0
6-2-2 8 0 12 7 15 12 35 19 54.3
6-2-3 17 7 26 22 38 32 81 61 75.3
6-2-4 25 7 28 25 37 34 90 66 73.3
6-8-1 25 11 25 25 29 18 79 54 68.3
6-8-2 35 23 32 22 32 13 99 58 57.6
6-8-3 31 9 29 22 36 32 96 63 65.6
6-8-4 14 6 26 12 25 22 65 40 61.6
Bonny Best
(Ck) 27 7 18 9 24 14 69 30 43.4
a) % transmittance in Spectronic 20 of bacterial suspensions used in inoculum. 50, 70, and
84% transmittance refers to 2 x 10, 1 x 108, and 5 x 10l cells/ml
b) living plants 2 weeks after root inoculation.






16



Crosses with susceptible plants

The cultivar, Bonny Best, was used as a susceptible

parent and crossed with a plant from 126408-6-4. A few F1

plants were raised to maturity and these were used to obtain

an F2 population and to backcross to the resistant and suscep-

tible parents. The data after root inoculation of the popula-

tions at two different levels of inoculum are in Table 3.

Again, survival increased as inoculum level decreased. Progeny

of 126408-6-2-3 had a high percentage of survival compared to

the plants of the susceptible cultivar, Bonny Best. The F1

population survival figures were intermediate between those of

both parents. The F2 population survival numbers were also

intermediate between those of the parents. A backcross to the

susceptible parent resulted in a population with a survival

percentage below of that of the F1.

With Floradel as the susceptible parent and 126408-6-8 as

resistant parent populations were also obtained of Fl, F2 and

backcrosses to susceptible and resistant parents. Results

similar to the ones with Bonny Best were obtained except the

backcross to a resistant parent resulted in a higher percentage

survival than that of the F1 population, as expected (Table 4).

The latter did not occur when Bonny Best was used as the sus-

ceptible parent.

Reciprocal crosses were made between Floradel and 126408-6-8

to establish if cytoplasmic inheritance was involved in the re-

sistance. All populations from reciprocal crosses behave alike,






17






Table 3. Data on survival of plants from crosses of
selections from P.I. 126408 with Bonny Best after inoculation
of two levels of inoculum of Pseudomonas solanacearum


50% b) 70% c)
No. a) No.
Generation plants living living plants living living


Bonny Best (BB) 80 2 2.5 92 13 14.1
6-2-3* x self 63 20 31.7 67 38 56.7

F1 (BB x 6-4*) 94 18 19.1 74 22 29.7
F2 (BB x 6-4*) x
self 83 9 10.8 77 21 27.3
Bc (6-4* x BB)
6-2-3 59 9 15.2 40 8 20.0
Bc (BB x 6-4*) x
BB 73 6 8.2 67 10 14.9


* the prefix 126408 has been omitted.
a) living plants 2 weeks after root inoculation.
b) inoculum concentration: 2 x 10 cells/ml.
c) inoculum concentration: 2 x 10 cells/ml.






18






Table 4. Data on survival of plants from crosses of

selections from P.I. 126408 with Floradel after inoculation
with Pseudomonas solanacearum

No. a) %
Line or cultivar plants livinga living

Bonny Best (Control) 92 20 21.7
Floradel 79 14 17.7

126408-6-2-3 x self 78 56 71.8

F1 (Floradel x 126408-6-8) 69 14 20.3

F2 (Floradel x 126408-6-8) x self 58 8 13.8

Be (126408-6-2 x Floradel) x
126408-6-2-3 66 19 28.8
Be (Floradel x 126408-6-2) x
Floradel 41 4 9.7



a) living plants 2 weeks after root inoculation with 2 x 108

cells/ml of Pseudomonas solanacearum.






19


which means that the resistance is probably not cytoplasmically

inherited.


Progeny testing of F2 population of the cross, Bonny Best x

126408-6-2

Variation due to increased homozygosity of genes contribu-

ting to resistance was investigated in a progeny test of an F2

population of the cross, Bonny Best x 126408-6-2. Seeds were

obtained from 49 self pollinated F2 plants and approximately

100 seedlings from each were inoculated with P. solanacearum.

The percentage survival of the F3 plants (Table 5) was used as

an indication of the degree of resistance of the parent. Read-

ings for plant survival were taken 3 and 6 weeks after inocula-

tion. The selfed plants had a variation as expected for a

normally distributed curve (Fig. 1 and 2). Data used to calcu-

late-expected and actual.curves are in Appendix 3-4. Both

curves obtained were similar and both agreed with a normally

distributed population curve at the 1% level using the X2 test.

The characteristic of resistance, measured as survival percentage

and which is normally distributed, indicates that this character

is polygenic in nature with no dominant genes. The resistance

genes have an additive effect.

Average weight per fruit of F plants from the cross, Bony

Best x 126408-6-4 were recorded (Table 5) and the survival per-

centage of progeny to bacterial wilt was correlated with fruit

weight. The correlation coefficient between weight/fruit and

survival percentage was found to be -0.2124 for the reading






20


Table 5. Survival of seedlings of 49 F3 lines of tomato to
Pseudomonas solanacearum and relationship to fruit weight


% F % F
F plaAts plaAts
slection No. Ave. No. living living
No. fruits oz./fruit plants 3 weeks 6 weeks

2 8 1.69 100 28.0 23.0
3 3 2.00 100 32.0 28.0
5 1 2.00 82 25.6 17.1
6 13 1.15 100 30.0 20.0

7 5 1.60 95 28.4 22.1
8 9 1.39 100 54.0 48.0
11 5 1.20 100 34.0 28.0
12 5 1.80 96 24.0 20.0
14 7 1.00 100 50.0 44.0
15 2 2.00 100 34.0 28.0
16 4 1.75 100 42.0 35.0
17 2 2.00 87 19.1 6.9
18 6 1.58 100 36.0 32.0
19 9 0.78 89 47.2 38.2
20 5 1.10 98 39.8 37.8
21 7 1.57 100 32.0 27.0
22 5 2.60 100 31.0 25.0
23 4 1.50 100 24.0 16.0
24 6 1.17 100 41.0 36.0
25 3 2.67 100 30.0 20.0
28 2 2.75 99 34.3 31.3






21


Table 5. Continued



F F3 % F3
2 plants plants
selection No. Ave. No. living living
No. fruits oz./fruit plants 3 weeks 6 weeks

29 4 2.50 100 21.0 12.0
30 4 1.88 100 55.0 47.0
31 3 2.50 100 22.0 16.0
33 9 1.22 100 42.0 36.0
34 5 1.20 98 17.3 14.3
35 4 1.88 99 18.2 13.1
37 3 1.50 100 14.0 13.0
38 5 1.90 100 24.0 22.0
40 5 1.60 100 27.0 20.0
41 8 1.13 100 47.0 44.0
43 4 2.13 100 54.0 46.0
44 5 1.30 100 31.0 23.0
45 8 1.38 100 27.0 19.0
46 4 1.63 90 13.1 11.1
47 3 2.00 100 36.0 30.0
48 4 1.63 60 36.2 21.7
49 7 1.21 100 51.0 50.0

50 3 1.50 97 27.8 18.6
51 10 1.45 100 47.0 42.0
52 5 1.20 100 14.0 10.0
53 4 1.75 98 28.6 21.4






22


Table 5. Continued


F% F3 % F3
2 plants plants
selection No. Ave. No. living living
No. fruits oz./fruit plants 3 weeks 6 weeks

54 3 1.67 98 19.4 11.2
55 4 2.13 100 31.0 25.0
56 5 1.20 99 41.4 37.4
57 10 1.00 97 36.1 29.9
58 5 1.60 100 40.0 32.0
59 5 1.20 99 29.3 28.3
60 2 2.25 100 45.0 38.0
126408-6-2-3 100 63.0 56.0
Bonny Best 95 15.0 8.4






















Figure 1. Number of F2 selections from the cross, Bonny Best (S) x P.I. 126408 (R),
categorized as to percent survival three weeks after inoculation with
Pseudomonas solanacearum. (--o- ) theoretical normal curve, (--A--)
actual observed frequency.
S= 32.9, s = 10.9, p = 0.75-0.50








I0
I

9 I
I


C/
c7
0






2-
U) /


1-

S 3- /
z



O 2


0 10.5 20.5 30.5 40.5 50.5 60.5
Percent Survival






















Figure 2. Number of F2 selections from the cross, Bonny Best (S) x P.I. 126408 (R),
categorized as to percent survival six weeks after inoculation with
Pseudomonas solanacearum. (-ao--- ) theoretical normal curve, (--A--)
actual observed frequency.
y = 26.86, s = 11.5, p = 0.50-0.25







I0

9- A

8

//



- I \ /
S4- / \/





E / /

2 3-
2--



|4-








O 111
2-




o 1 1 t I I
0 10.5 20.5 30.5 40.5 50.5 60.5
Percent Survival






27


taken 3 weeks after inoculation. The correlation coefficient

calculated 6 weeks after inoculation was -0.2566. Thus, fruit

size was not correlated with resistance in the tests reported

here.-

Crosses between resistant cultivars and the P.I. 126408

Saturn and Venus, two resistant cultivars were used as

parents in crosses with a selection from P.I. 126408 with the

purpose of determining if the resistance of the two parents

would be additive in the progeny. A cross between Saturn and

126408-6-4 produced an F1 population with as many resistant

plants as that of the parents (Table 6). Similar results were

obtained with a cross, Venus x 126408-6-4. The F2 population

of the Saturn cross had more resistant plants than those of

either parent, suggesting that genes from both parents were

additive in the cross, Saturn x 126408-6-4. This increased

resistance was not observed in the cross with Venus, in which

the F2 population had about as many resistant plants as the

parents. A backcross to Saturn produced a population with as

many resistant plants as that of the parents. These results

indicate that when a selection from P.I. 126408 is crossed with

Venus or Saturn, the resulting population has a resistance at

least as great as that of their parents. In the cross with

Saturn, resistance to bacterial wilt may have been increased

through increasing homozygosity in this population.





28






Taole 6. Data on survival of plants from crosses of
selections from P.I. 126408 with Saturn after inoculation
with Pseudomonas solanacearum

No. %
Line or cultivar plants livinga) living

Bonny Best (Control) 60 9 15.0
Saturn 60 40 66.7
126408-6 x self 60 41 68.3
F1 (Saturn x 126408-6-4) 60 38 63.3
F2 (Saturn x 126408-6-4) x self 60 51 85.0
Bc (Saturn x 126408-6-4) x
Saturn 60 37 61.7


a) living plants 2 weeks after inoculation by the trans-
planting technique using 25 ml of 2 x 10 cells/ml of
Pseudomonas solanacearum per seeded plate.





29


126408 Field Tests

Tests under laboratory conditions (5) indicated that

certain selections of the P.I. 126408 were highly resistant to

bacterial wilt caused by one isolate, A-21, of P. solanacearum.

Field trials were necessary to confirm this resistance under

conditions where natural populations of the pathogen exist.

Three investigators cooperated in planting selections of P.I.

126408 in field trials.

S. M. McCarter, University of Georgia, Athens, planted

populations of progeny of the selection 126408-7 in infested

plots at Midville and Athens, Georgia (20). Only 3 out of more

than 300 plants had evidence of bacterial wilt at the completion

of the experiment. Plants of the susceptible check, Marion,

had 81.7% mortality and plants of the selection 126408-7 had an

0.0% mortality in the test at Midville, whereas 67.9% and 2.3%

mortality, respectively occurred at Athens.

G. Ocana, University of Panama, Panama City, Panama,

planted 45 seedlings of a selection of the P.I. 126408-6 in a

field naturally infested with P. solanacearum. All the resist-

ant plants survived whereas all plants of the variety Floradel
died.

R. Sonoda, IFAS, Agriculture Research Center, Ft. Pierce,

Florida, planted individuals from 126408-6, from crosses between

selections of 126408-6, and the resistant cultivars, Venus and

Saturn, and individuals of the susceptible cultivars, Bonny Best

and Floradel. The plots were heavily infested with P. solanacearum






30



since previous tomato trials in the same plots were devastated

by the disease. This test was in late Spring 1973 and tempera-

tures were above that normally occurring during tomato produc-

tion. Thus, conditions were extremely favorable for disease

development.

The crosses between the 126408-6-4 and the resistant

cultivars had survival percentages equal to those of the resist-

ant cultivars and a little higher than those of the selfed

126408-6 selection (Table 7). Crosses between the 126408-6

and the susceptible cultivars Bonny Best and Floradel produced

F1 populations just as susceptible as the susceptible parents.

Results of the Ft. Pierce test were consistent with the

seedling inoculation tests. Crosses of 126408 with the resist-

ant Venus and Saturn do not decrease resistance in the progeny,

but crosses with the susceptible Bonny Best and Floradel do

decrease resistance in progeny.


Nature of Resistance


Studies of the nature of the resistance in plants from

P.I. 126408 were undertaken to gather information on the nature

of the resistance of these plants for future use of this re-

sistance in field control of bacterial wilt.

Root Diffusates


Experiments were undertaken on the role of root diffusates

on multiplication of P. solanacearum around the roots of plants

resistant and susceptible to bacterial wilt. Root diffusates






31







Table 7. Field evaluation of resistance to Pseudomonas

solanacearum of crosses between susceptible and resistant
cultivars with selections from P.I. 126408 at Ft. Pierce, Fla.


Cultivar, line No. plants No. plantsa)
or cross transplanted living living

Bonny Best 56 11 19.6
Venus 56 34 60.7

Saturn 56 32 57.1

Floradel 56 18 32.1
126408-6 x
Bonny Best 56 6 10.7
Venus x
126408-6-4 56 32 57.1
Saturn x
126408-6-4 56 31 55.6
Floradel x
126408-6-2 56 12 21.4
126408-6 x self 56 25 44.6


a) living plants 4 weeks after transplanting in naturally
infested soil.






32


have been found to be important in development of some other

diseases involving root pathogens. Root exudates play an

important role in the germination of spores or sclerotia of

many fungi, chemotaxic responses such as in the case of

Phytophthora spp., and maintaining growth of pathogens in the

rhizosphere (23).


Leachate test


Root diffusates were collected in water from pots contain-

ing plants resistant to bacterial wilt, Saturn and 126408-6-2-3,

and plants susceptible to bacterial wilt, Bony Best. Controls

consisted of steamed soil or white sand without plants.

In three tests, P. solanacearum grew in the control medium,

except in one treatment (Table 8). Since only mineral nutrients

were added to the medium and no source of carbon was added, it

was felt that the growth factors released in the soil probable

were of microorganism origin. Since P. solanacearum grew in

leachates from washed white sand, probably algae or other auto-

trophic organisms were involved in the stimulation of growth

of the bacteria.

The growth of P. solanacearum in the leachate media was

relatively slow, compared to growth of the bacteria in nutrient

broth. Populations of this organism reach 109 cells/ml in 48 hr

in nutrient broth (5). Therefore, if growth factors were re-

leased from the tomato roots, growth of the bacteria in the

media from pots with plants should have increased over the






33


Table 8. Influence of substrate leachates on growth of
Pseudomonas solanacearum in vitro

Water extracted Bacteria/ml of substrate leachates
from substrate Hr
+ or plant 0 24 48

Test 1
Bonny Best F (a) 5.0 x 102 3.0 x 106 2.9 x 10
FA (b) 1.0 x 103 2.7 x 10 1.8 x 107
126408-6-8 F 2.4 x 102 2.5 x 10 1.7 x 107

FA 2.9 x 102 3.1 x 105 1.9 x 107
Soil only (Ck) F 5.0 x 102 0 0
FA 2.2 x 102 2.1 x 105 2.6 x 106
Test 2
Bonny Best F 1.8 x 103 4.8 x 106 3.0 x 107

FA 1.5 x 103 8.2 x 106 3.7 x 10
126408-6-8 F 1.6 x 103 5.2 x 106 1.1 x 107

FA 1.6 x 103 8.7 x 106 1.5 x 107
Soil only (Ck) F 1.1 x 103 6.0 x 105 6.5 x 106

FA 1.3 x 103 3.1 x 105 5.7 x 106
Test 3
Saturn F 1.1 x 103 2.8 x 106 1.8 x 107
Bonny Best F 9.0 x 102 4.0 x 10 1.8 x 107
126408-6-2-3 F 1.1 x 103 2.7 x 10 1.8 x 107

Sand only (Ck) F 1.2 x 103 3.4 x 106 1.4 x 107


(a) filter-sterilized
(b) filter-sterilized and autoclaved






34


control pots. In no instance, however, did increased growth

occur in media from pots containing tomato plants (Table 8).

Therefore, it can be concluded that diffusates from the tomato

plants had no effect on the observed growth of P. solanacearum

in the aqueous medium.


Mineral-water-agar test


The above experiments had a limitation of possible dilut-

ion of released growth factors from roots to the point of mak-

ing detection impossible. A different approach was used in an

attempt to determine whether factors that might influence growth

of P. solanacearum are released in minute amounts from roots.

Bacterial cells were added to a mineral-water-agar (MWA) medium

at a concentration of approximately 1 x 103 cells/ml. After

4-5 days, minute colonies, 0.1 mm in diam, were visible with

a binocular microscope at 100 X and also with the unaided eye

when the tube containing the medium was placed against a source

of light, such as a fluorescent lamp. The colonies did not

increase in size over the next two months, when the temperature

remained at approximately 30 C.

Seeds of two cultivars, Saturn (resistant), and Bonny Best

(susceptible), and the line 126408-6-2-3-Bk were placed on the

agar surface after colonies of P. solanacearum had become

visible. Germination of seed and growth of the plants occurred

on this medium. Colonies of P. solanacearum, near or far from

the roots never enlarged beyond the size of colonies in tubes

without plants. However, in about 5% of the tubes, fungal






35


contaminants occurred, and the zise of P. solanacearum colonies

near these fungal colonies did increase in size. The contami-

nant fungi were not identified. This was considered as further

evidence that tomato root diffusates do not affect the growth

of P. solanacearum, but possibly microorganisms in the soil do

produce growth factors that effect multiplication of the

bacterium.


Penetration of Noninjured Roots


Preliminary experiments in which inoculum of P.

solanacearum was poured over roots of tomato plants growing in

vermiculite indicated that the pathogen could penetrate non-

injured roots (Table 9). This observation was confirmed in

the NWA medium.

Penetration of noninjured roots of resistant and susceptible

tomato seedlings occurred when 100 or 1,000 cells/ml of

P. solanacearum were added to the medium (Table 10). At the

higher density of bacterial colonies, both resistant and sus-

ceptible plants had browned roots. Browning symptoms were

observed at the root tips and areas where secondary roots

emerged in both resistant and susceptible plants. Constriction

of stems and wilting of seedlings were also observed, as well

as development of adventitious roots in some cases in both

types of plants.

In the absence of mechanical injuries, and assuming that

opening in the roots are necessary for ingress, the infection

court could theoretically be the places where secondary roots






36


Table 9. Bacterial wilt development in plants with

noninjured roots growing in vermiculitea)


Cultivar or line No. plants livingb) % living

Venus 10 10 100.0

Bonny Best 12 4 33.3

126408-6-9 12 10 84.0

126408-6-2-1 12 11 91.7

126408-6-8-1 12 11 91.7

08
a) roots were drenched with suspensions of 2 x 10 cells/ml
of P. solanacearum.

b) living plants after 2 weeks after addition of bacterial

suspension to the substrate.



Table 10. Penetration of tomato roots by Pseudomonas

solanacearum in a mineral-water-agar media

total % % with
Line or cultivar plants livinga) brown roots

Test 1 *

Bonny Best 16 37.5 100.0
Test 2 **

126408-6-2-3-Bk 7 57.1 100.0

Bonny Best 6 33.3 100.0

a) living plants 6 weeks after germination in MWA.

* inoculum concentration = 100 cells/ml.
** inoculum concentration = 1,000 cells/ml.






37



emerge. Movement of bacteria in this environment is restrict-

ed, therefore, they had to be present at the site of injury

for penetration. The probability of a bacterium being exactly

at the site where a secondary root emerged was calculated. At

the density of 1,000 bacteria per ml in the medium, 3 out of

109 times a bacterium would be in the area where a secondary

root emerged (Appendix 5). Observed results utilizing the MWA

technique did not fit the probability, since invasion occurred

far more frequently than the probability dictated.

To account for such a high rate of invasion of plants

without artificial injures, it can only be speculated that the

pathogen might have multiplied on the surface of the root and

that populations of the pathogen near the secondary root

emergence sites might have been much'greater than could be

determined by the techniques used in these experiments.

Evidence was found that the bacterium might multiply ex-

tensively near the root tip. In several instances, turbidity

was noticed around root tips, in the case of the susceptible

cultivar Bonny Best, which then became brown and ceased to grow.

In isolations made after carefully removing root tips from the

agar medium, only colonies of P. solanacearum could be recov-

ered. That the bacterium multiplies on root tips and enters

them directly can only be suggested at this time. More refined

techniques must be developed before multiplication and entry

of P. solanacearum into root tips can be established with

certainty.






38


Effect of Temperature on Resistance

Resistance to bacterial wilt of some tomato lines is

affected by temperature (9). For this reason plants of

126408-6-2-3-Bk were tested for resistance at various tempera-

tures. Plants of Saturn and Bonny Best were included, respec-

tively, as resistant and susceptible controls. Plants were

inoculated by the stem-inoculation technique and placed at 25,

28, 30, and 32 C after inoculation. Almost all of the three

types of plants developed some symptoms of the disease, such

as adventitious root formation on the stem and some degree of

dwarfing or vascular discoloration, but very little wilting

occurred in any of them at 25 and 28 C, even among plants of

the susceptible Bonny Best (Table 11). Plants of Saturn and

126408-6-2-3-Bk remained resistant at 30 and 32 C whereas those

of Bonny Best were almost totally susceptible. Since Saturn

remains resistant to bacterial wilt even under some field

conditions where all susceptible plants die, plants of

126408-6-2-3-Bk will probably hold their resistance under the

same field conditions.

This experiment also demonstrated that resistance of the

P.I. 126408 plants operates even after the pathogen had been

introduced into the stems.






39






Table 11. Influence of temperature on bacterial wilt
development on resistant and susceptible tomato plants


Line or cultivar Temperature C
25 28 30 32

Bonny Best 5/8 6/8 1/8 0/8
126408-6-2-3-Bk 7/8 7/8 8/8 8/8
Saturn 8/8 8/8 8/8 7/8



* healthy plants/total inoculated using the stem injury
technique with 2 x 107 cells/ml of Pseudomonas solanacearum
15 days after inoculation.












DISCUSSION



Inheritance of the resistance to bacterial wilt of plants

of the tomato line P.I. 126408 was observed in crosses with sus-

ceptible cultivars. Under greenhouse conditions, where the

observations were made, soil temperature and inoculum levels

greately influenced the survival of tested lines. But in a

given experiment, where different lines were exposed to the

same environmental conditions, the higher survival rate occurred

in lines with the higher level of resistance. From the cross,

Bonny Best (susceptible) x 126408 (resistant), the survival

percentage of the F1 progeny was between that of the parents.

Probably no dominance occurred but there was a slight tendency

toward the susceptible side in the population observed. The

levels of resistance in the F2 population followed a curve

close to a normal curve. The significance of these data is

that it confirms that the resistance behaves as expected for

a polygenically inherited character with an additive gene.

effect.

The possibility of extrachromosomal inheritance was discarded

by making reciprocal crosses with the susceptible cultivar

Floradel and the P.I. 126408. The resukts were that when either

parent was used as female, the F1 progeny behaved the same.

In both cases, the resulting survival percentage was between

that of parents, but again a little toward the susceptible

parent.


40






41


The back crosses to both parents did not always behave as

expected for a polygenically inherited character, i.e., the

survival percentage did not fall between that of the F1 popula-

tion and their respective recurrent parent. A p"osible expla-

nation is that the resistant parents were not selfed with the

specific purpose of achieving complete homozygosity. It was

assumed that they were nearly homozygous for the character of

resistance from the results of inoculation tests. Variation

among resistant lines used as parents were detected, but this

variation was difficult to measure accurately and it was

disregarded as not significant.

Robinson (22) found that measuring resistance of potato

varieties to P. solanacearum was a difficult task. Some

potato lines were resistant and later became susceptible and

these were considered to have vertical resistance (22).

According to Robinson, a high mutability of the pathogen

accounted for the poor performance of potato lines thought to

have had vertical resistance. Because of this Robinson

suggested that horizontal resistance, in this case of resistance

to P. solanacearum, has much greater agricultural value.

In all crops in which resistance to P. solanacearum,

has been found, stable resistance has been determined to be

inherited in a polygenic manner (15). Transfer of this

resistance to economically accepted cultivars has been slow.

The reason for this is because in each cross to susceptible

plants with commercial quality, approximately half of the

genes governing resistance are replaced in the F1 generation.






42

Some of them can be recovered by careful selection for

resistance in the F2 population. In practice, however certain

levels of resistance are usually lost with each cross.

Polygenic resistance is affected by environmental factors

to a greater degree than vertical resistance (26). In the

work reported here, resistance was still present at 32 C, but

one would expect both temperature and moisture levels to

greatly affect the degree of resistance. Also, such factors

as solar radiation (22), fertility (7), and certain nematode

damage (19) may alter the level of resistance.

French (6), and Gonzales et al. (10), working on potato
with a polygenic resistance, found that selection of clones

from a cross between resistant and susceptible plants can be

done best by planting inoculated potato plants under lower

temperature conditions in the field where resistance is best

expressed. Work reported here suggests that selections can

best be made of tomato segregating lines where polygenic

resistance is involved at about 30 or 32 C.

The level of resistance of plants of P.I. 126408 to

bacterial wilt does not appear superior to the North Carolina

resistance that occurs in the cultivars Saturn and Venus.

However, the resistance appears equally as effective. There-

fore, one wonders about the usefulness of the resistance in

future commercial application.

The resistance might be useful in crosses with Venus and

Saturn to obtain relatively large-fruited lines that will set
fruits in tropical climates. P.I. 126408 was originally






43

collected in Panama (24), and seems to set many fruits under
relatively high temperatures. Crosses of Saturn or Venus with

susceptible lines to obtain good fruit-setting properties under

high temperatures results in decreased bacterial wilt resist-

ance (21). But crosses of these cultivars with P.I. 126408

would not decrease resistance to bacterial wilt and selection

for fruit setting ability could be made in the progeny without

fear of losing the level of resistance to bacterial wilt

Small-fruit size has often been associated with bacterial

wilt resistance using the North Carolina resistance (1, 21).
In the F2 population from the cross, Bonny Best x 126408, no

correlation of fruit size and bacterial wilt resistance was

noted. This is encouraging, but not necessarily conclusive

evidence that fruit-size and bacterial wilt resistance are

not linked in the P.I. 126408 plants. However, if this is cor-

rect, the bacterial wilt resistance of P.I. 126408 may be

incorporated into large-fruited lines with much less effort
than was the case with the North Carolina resistance.

The nature of bacterial wilt resistance in the P.I. 126408
is protoplasmic. Experiments designed in this work have

failed to show any differences in the effect of the resistant

plants on growth of the bacterium in the medium around the
roots. The bacterium also seemed to invade resistant plants

as effectively as susceptible plants. Invasion occured in
both types of plants without artificial injury. Resistance

seems to involve mechanisms that reduce colonization of the
host after invasion. After invasion, the pathogen seems to






44


spread slowly, and then gradually declines in colonization of

the host tissues. In susceptible plants, however, the pathogen

does not decline in colonization, but continues to grow within

infected plants until wilting and death of the plants occurs.

The mechanism of resistance in plants of P.I. 126408 seems to

be similar to that of the North Carolina resistance.

Breeding for resistance to bacterial wilt of tomato is

possibly the best way to control the disease under field

conditions, but problems of inheritance of all sources of re-

sistance available make it clear that development of commer-

cially acceptable cultivars will be difficult. There is still

need for a source of resistance to minimize problems in

breeding for disease resistance to bacterial wilt in tomato

so that resistant varieties with a high degree of resistance

can become available for commercial use. Finding a source of

resistance to bacterial wilt in tomatoes which could be easily

transferred to commercial varieties is still of foremost

importance.










SUMMARY

Selections from P.I. 126408 were found that had a high

level of resistance to bacterial wilt of tomato. The resist-

ance level was increased by selection, but not significantly

so after the second generation of selfing. The resistance

was evident in experiments involving three inoculations

techniques and was present to natural pooulations of the

pathogen in field tests in Florida, Georgia, and Panama.

Progeny of crosses of the susceptible cultivars Bonny

Best and Floradel, with selections from P.I. 126408,

established that the resistance was inherited in a polygenic

manner. Reciprocal crosses with Floradel produced progenies

similar in their resistance to P. solanacearum, suggesting

that the resistance was not extrachromosomal. Progenies of

crosses with resistant cultivars Venus and Saturn, with se-

lections from P.I. 126408, were as resistant as either parent.

The bacterium multiplied in leachates from steamed soil

and washed-white sand. Increased multiplication was not

noted in leachates from soil or white sand that contained

living roots of resistant of susceptible tomato plants.

Diffusates from tomato roots growing in a mineral-water-agar

medium containing cells of P. solanacearum did not influence

growth of the bacterium. Some fungal contaminants, however,

did result in increased growth of the bacterium in the medium.

The pathogen entered roots that were not artificially

injured. Entry may have occurred where secondary roots emerged


45






46


but the probability calculated for such entry was much less than

that actyally observed.

Observations made suggest that the bacterium multiplies

near the root tips and that entry occurs on living nonwoumded.

No differences were found between invasion of resistant or

susceptible plants.

The resistant plants from P.I. 126408 were as resistant

as Saturn at 30 and 32 C. Very little wilting occurred at 25

or 28 C, even in susceptible plants.

Observations suggest that resistance of the P.I. 126408

plants was protolasmic in nature and was expressed during

colonization of the host. The resistant plants are not

colonized at the same rate as susceptible plants.











LITERATURE CITED

1. Acosta, J. C., J. C. Gilbert, and V. L. Quinon. 1964.
Heritability of bacterial wilt resistance in tomato.
Amer. Soc. Hort. Sci. Proc. 84:455-462.

2. Belalcazar, C. S., G. Uribe M., and H. D. Thurston. 1968.
Reconocimiento de hospedantes a Pseudomonas solanacearum
(E. F. Sm.) en Colombia. Revista ICA Vol. III. p. 37-46.

3. Buddenhagen, I. W. 1960. Strains of Pseudomonas
solanacearum in indigenous hosts in banana plantations
of Costa Rica, and their relationship to bacterial wilt
of bananas. Phytopathology 50:660-664.

4. Buddenhagen, I. W., and A. Kelman. 1964. Biological and
physiological aspects of bacterial wilt caused by
Pseudomonas solanacearum. Ann. Rev. Phytopathol. 2:203-
230.

5. Ferrer, A. 1971. Techniques to identify the tomato race
of Pseudomonas solanacearum and to screen for resistance
in tomato. M.S. Thesis, Univ. of Florida, Gainesville.
47 p.
6. French, E. R. 1973. Evaluaci6n de la resistencia de la
papa a Pseudomonas solanacearum en un fitotr6n. Phyto-
pathology 63:1435 (Abstr.).

7. Gallegly, M. E., and J. C. Walker. 1949. Plant nutrition
in relation to disease development. V. Bacterial wilt of
tomato. Amer. J. Bot. 36:613-623.

8. Gallegly, M. E., and J. C. Walker. 1949. Relation of
environmental factors to bacterial wilt of tomato.
Phytopathology 39:936-946.

9. Gilbert, J. C., J. L. Brewbaker, J. S. Tanaka, J. T. Chinn,
R. W. Hartman, J. A. Crozier, Jr., and P. J. Ito. 1969.
Vegetable improvement at the Hawaii Agricultural Experi-
ment Station. Research Report 175. Hawaii Agr. Expt.
Sta. Coll. Trop. Agri., Univ. of Hawaii. p. 16.
10. Gonzales, L. C., L. Sequeira, P. R. Rowe, and R. Bianchini.
1972. Field resistance to bacterial wilt in hybrid
potato progenies. Phytopathology 62:760 (Abstr.).
11. Hayward, A. C. 1964. Characteristics of Pseudomonas
solanacearum. J. Appl. Bacteriol. 27:265-277.


47






48



12. Henderson, W. R., and S. F. Jenkins, Jr. 1972. Venus and
Saturn two new tomato varieties combining desirable
horticultural features with southern bacterial wilt re-
sistance. North Carolina Agr. Expt. Sta. Bull. 444. 13 p.

13. Jenkins, S. F., Jr., D. J. Morton, and P. D. Dukes. 1967.
Comparison of techniques for detection of Pseudomonas
solanacearum in artificially infested soils. Phyto-
pathology 57:25-27.

14. Karganilla, A. D., and I. W. Buddenhagen. 1972. Develop-
ment of a selective medium for Pseudomonas solanacearum.
Phytopathology 62:1373-1376.

15. Kelman, A. 1953. The bacterial wilt caused by Pseudomonas
solanacearum. North Carolina Agr. Expt. Sta. Tech. Bull.
99. 194 p.
16. Kelman, A. 1954. The relationship of pathogenicity in
Pseudomonas solanacearum to colony appearance on a tetra-
zolium medium. Phytopathology 44:693-695.

17. Kelman, A., and L. Sequeira. 1965. Root-to-root spread of
Pseudomonas solanacearum. Phytopathology 55:304-309.

18. Klein, R. M., and D. T. Klein. 1970. Research methods in
plant science. The Natural History Press. Garden City,
N. Y. 756 p.

19. Libman, G., J. G. Leach, and R. E. Adams. 1964. Role of
certain plant-parasitic nematodes in infection of tomatoes
by Pseudomonas solanacearum. Phytopatholdgy 54:151-153.

20. McCarter, S. M. 1973. A procedure for infesting field
soils with Pseudomonas solanacearum. Phytopathology 63:
799-800.
21. Ocaia, G., and G. Silvera. 1970. Determinaci6n de la
factibilidad del cultivo del tomate a nivel del mar
durante la estaci6n lluviosa. Progreso de labores de
investigaciones agropecuarias, 1969. Facultad de Agro-
nomfa de la Universidad de Panama. p. 89-121.

22. Robinson, R. A. 1968. The concept of vertical and hori-
zontal resistance as illustrated by bacterial wilt of
potatoes. Phytopathol. Papers, No. 10. Commonwealth
Mycol. Inst., Kew, Surrey, England. 37 p.

23. Schroth, M. N., and D. C. Hildebrand. 1964. Influence of
plant exudates on root infecting fungi. Ann. Rev. Phyto-
pathol. 2:101-132.






49


24. Skrdla, W. H., L. J. Alexander, G. Oakes, and A. F. Dodge.
1968. Horticultural characters and reaction to two
diseases of the world collection of the genus Lycopersicon.
Ohio Agr. Res. and Development Cent. Res. Bull. 1009.

25. Stall, R. E., and A. A. Cook. 1966. Multiplication of
Xanthomonas vesicatoria and lesion development in resist-
ant and susceptible pepper. Phytopathology 56:1152-1154.

26. Van der Plank, J. E. 1963. Plant Diseases: Epidemics and
control. Academic Press. N. Y. and London. 349 p.

27. Vaughan, E. K. 1944. Bacterial wilt of tomato caused by
Phytomonas solanacearum. Phytopathology 34:443-458.


































APPENDICES






51

APPENDIX 1. Analysis of variance of data on resistance of

plants in three generations from P.I. 126408


Source DF SS MS F Prob.> F
Selection 2 0.4775 '0.2387 3.456 0.0434
Residual 30 2.0725 0.0691
Inoculum 2 2.1232 1.0616 15.367 0.0001
Residual 30 2.0725 0.0691
Selection- 4 0.0031 0.0508 0.735 0.5775
Inoculum
Residual 30 2.0725 0.0691











APPENDIX 2. Duncan's multiple range test of significance
of differences in resistance among generations for
P.I. 126408

Duncan's Test


Generation of Mean Mean
Selection Transformed Original
1)
3 0.7042 0.6062 a
2 0.6979 0.6097 a

1 0.2870 0.2738 b


1) Figures with same letter are not significantly different.






52










APPENDIX 3. Observed frequency of selections compared to a
a)
normal curve


1) 2) 3) 4) 5) 6) 2
Range Sd. Z values Area exp. no. obs. X
0-10.5 2.51 .4940 .0197 0.97 0 0.97
10.5-15.5 2.06 .4803 .0351 1.72 3 0.95
15.5-20.5 1.60 .4452 .0723 3.54 4 0.06
20.5-25.5 1.14 .3729 .1212 5.94 5 0.15
25.5-30.5 0.68 .2517 .1646 8.07 10 0.46
30.5-35.5 0.22 .0871 .1819 8.91 9 0.00
35.5-40.5 0.24 .0948 .1632 8.00 5 1.13
40.5-45.5 0.70 .2580 .1190 5.83 5 0.12
45.5-50.5 1.16 .3770 .0693 3.40 4 0.11
50.5-55.5 1.16 .4463 .0345 1.69 4 3.16
55.5-60.5 2.07 .4808 .0192 0.94 0 0.94

a) 3 weeks after inoculation with 2 x 10 cells/ml of
Pseudomonas solanacearum.
1) Range corresponding to percent living.
2) Standard deviation from mean.

3) Z values corresponding to 5% range.
4) Area of normal curve.

5) Expected frequency of selections per area.
6) Observed frequency of selections per area.






53






APPENDIX 4. Observed frequency of selections compared to a
b)
normal curve



Area1) Sd) Z values3) Area) Expected5) Actual6) X2
0-5.5 1.91 .4719 .0281 1.38 0 1.38
5.6-10.5 1.46 .4279 .0440 2.16 2 .12
10.6-15.5 1.02 .3461 .0818 4.01 6 .99
15.6-20.5 0.57 .2157 .1304 6.39 8 .41
20.6-25.5 0.12 .0478 .1679 8.23 9 .07
25.6-30.5 0.32 .1255 .1733 8.49 7 .26
30.6-35.5 0.77 .2794 .1539 7.54 4 1.66
35.6-40.5 1.22 .3888 .1094 5.36 6 .76
40.6-45.5 1.67 .4525 .0631 3.12 3 .46
45.6-50.5 2.12 .4830 .0305 1.49 4 4.23
50.5-55.5 2.56 .4948 .0118 0.58 0 .58
55.6-60.5 3.01 .4087 .0039 .19 0 .19


b) 6 weeks after inoculation with 2 x 108 cells/ml of
Pseudomonas solanacearum.
1) Range corresponding to percent living.
2) Stndard deviation from mean.
3) Z values corresponding to 5% range.
4) Area of normal curve.

5) Expected frequency of selections per area.
6) Observed frequency of selections per area.





54




APPENDIX 5. Calculations used to figure probability of a

bacterium being at injury caused by emergence of secondary

root.



Bacterial size = 2,0 x 0.5


Root diameter = 0.25 mm


Bacterial density in agar = 1 cell/mm3

1 1 1
Formula = --x-.- x-- = probability


a = No. of bacterium diam in an mm = 2000

2
b = No. of root diam in an mm = 16


c = probability of a secondary root touching ane bacterium
at the point of emergence
= No. of cells in area occupied by root
total cells in mm3


1 1 1 1 x 9
2~0UX-Tt- xyg- = 3.1 x 10













I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.





R. E. Stall, Chairman
Professor of Plant Pathology




I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully-adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.





D. A. Roberts
Professor of Plant Pathology




I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.





S. J.f/ocascio
Professor of Vegetable Crops










This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate Council, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.

August, 1974





;n, College o Agriculture





Dean, Graduate School





















V




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mods:subject SUBJ650_1 lcsh
mods:topic Tomatoes
Disease and pest resistance
SUBJ650_2
Tomato wilts
SUBJ650_3 fast
Tomato wilts
SUBJ650_4
Tomatoes
Disease and pest resistance
SUBJ690_1
Plant Pathology thesis Ph. D
SUBJ690_2
Dissertations, Academic
Plant Pathology
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PAGE 1

.BESISTANCE TO Pseudomonas solanacearum IN Lycopersicon esculentum By ALEJANDRO PERHER Z. A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OP THE UNIVERSITY OP FLORIDA IN fftHTIAL PULPILLMEST OP THE REQUIREMENTS FOR TM DEGREE OP DOCTOR OP PHILOSC^HIC UNIVERSITY OP PLORIDA 197^

PAGE 2

ACKNOWLEDGMENTS I wish to thank Dr. R. E. Stall, Chairman, of the Supervisory Committee, for his interest and dedication in helping me pursue this investigation. His guidance and valuable suggestion throughout this work have made possible the completion of this manuscript. I also wish to thank Drs. D. A. Roberts and S, J. Locascio, members 'Of y Supervisory Committee, for their contrilmtioiis to the work reported herein. The author is indebted to the Compania Fanamena de Aliment os S.A. for providing the n#PfSi^iT funds for this research. I extend my appreciation to Mr. R. E, Bums for his valuable #sistanoe in laboratory work and to Dr. L. H Purdy, QhmtlN^m of the Plant Pathology Department, for his cooperatlozi throughout my tenure. all, I thank my wife and children f0r their is^stant encouragemit and under staiiding. ii

PAGE 3

TABLE OF CONTENTS Page ACKHOWL^eilWS ii LIST OF TABLES. him OF FIGURES. vi ABSTRACT, i vii INTRODUCTION 1 mfOTALS iUfD METHODS 6 Preparation of Inocula 6 Inoculation Techniques 7 Root injury technique. 7 Transplant technique 7 Stem inoculation technique. 8 Tomato Plant Culture 8 Root Diffusates 9 Leachate medium 9 Mineral-water-agar medium 10 RESULTS 12 Test of Inheritance.. 12 Selection within P.I. 126^08 12 Crosses with susceptible plants ........ 16 Progeny testing of Fp population of the cross, Bonny Best x 126^^-08-6-2 19 Crosses between resistant cultivars and the P.I. 126^+08... 27 12640& Field tests i 29 Nature of Resistance,, ^ 30 Root Diffusates.,.. 30 Leachate tests 32 Mineral-vjater-agar test... 3^ Penetration of Noninjured Roots....... 35 Effect of teraperature on resistance 2^ DISCUSSION 40 SUGARY.. 45 LITERATURE CITED 47 iii

PAGE 4

Page APPIMDICES.,. 50 BIOGRAPHICAL SKETCH '. 55 IV

PAGE 5

LIST OF TABLES TABIS 1. Progeny test of 126408-6 and selections from it. for bacterial wilt resistance TAilE 2. Progeny test of selections from 126408-6 f or baer:ter4al wilt resistance TAI^jE 3 Data on survival of plants from crosses of selections froim P.I. 126408 with Bonny Best after inoculation of two levels of •inoculum of Pseudomonas solana ce arum TABLE 4. Data on survival of plants from crosses of selections from P.I. 126408 with Ploradel after inoculation with Pseudomonas solanacearum TABLE 5. Survival of seedlings of 49 lines of tomato to Pseudomonas solanacearum and relationship to ftrait weight 7ABIi£ 6. Data on survival of plants from crosses of selections from P.I. 126408 with Saturn after inoculation with Pseudomonas solanacearum .!0ABLE 7. Field evaluation of resistance to Pseudomonas solanacearum of crosses between susceptible and resistant cultivars with seleciii^mS from P. I. I^$k08 at Ft. Pierce, Fla. TABIiB 8. Influence of substrate leachates on growth of Pseudomonas solanacearum in vitro fAfil^ 9. Bacterial wilt development in j^atSr|rith noninJu?d roots growing" in vermlculite : ; TABIiE 10. pjenetriatibn of tomato roots by Pseudomonas solanaeearum in a mineralwateragar madia TABliB 11. Influence of temperature ori "bacterial wilt development on resistant and susceptible; tomato plants V

PAGE 6

LIST OP FIGURES Figure 1, Number of Fo selections from the cross, Bonny Best (S) X P.I. 126408 (R) categorized as to percent survival three weeks after inoculation with Pseudomonas solanacearum Figure 2, Number of Fo selections from the cross. Bonny Best (S) X P.I. 126408 (R), categcFPlzed am to pwttsm^' survival six weeks after inoculation with Pseudomonas solanacearum J vi

PAGE 7

Abstract of Dissertation Presented to the Graduate Cdimeil of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BE3ISTANCE TO Pseudomonas solanacearum IN Lycoperslcon esculentum By Alejandro Ferrer Z. August, 197^ Chairman: Robert E. Stall Major Department: Plant Pathology Study of a source of resistance in tomato ( Lycopersicon egoulenttun Mill.) to Pseudomonas solanaoearta^ 1. P. Smitll, ti^e oausal agent of bacterial wilt, was undertadcen to evaluate %lh6 usefulness of the resistance in control of the plant disease. Plants with a high degree of resistance to P. solanaceafSiai pil^^ selected from Plant Introduction 126408. These plants, however, never produced progenies with 100^ survival in inoculation .tes,ts and selections after a second generation of selfed plants ifi^ not significantly different from further selections in resistance. Resistance was expressed in progenies, subjected to thre different inoculation techniques in the greenhouse and was t^i& expressed to natural populations of the pathogen in field tests in Florida, Georgia, and Panam^. Inheritance of resistance to P. solemacearum of resistant selections of P.I. 126408 was characterized by crossing them to the susceptible cultivars, Bonny Best and Floradel* Segregation at the Fg generation indicated that resistance is polyg^cally

PAGE 8

inherited. Inoculation results with progenies from reciprocal crosses proved that extrachromosoraal inheritance was not involved. The genes were additive with no dominance, hut there was a tendency toward susceptibility in the progmim of plsmts. Snailfruited plants were not correlat€s4 witli reaftjB^ ance. At high temperatures, the resistance to P." mXsmmmrm was still evident and was equal to that of Saturn, a cultiTikr with polygenic resistance. In environmentally controlled dhambers, at air temperatures of 30 or 32 st€W<#ilk0c^atKd plants from resistant lines did not wilt in 15 days, even though some yellowing and adventitious roots were evident. At the am^ temperatures, susceptible lines wilted and difd witMii 15 days. Tomato root exudates did not stimulate growth of ^, ^ianacear^itm Numbers of the bacterium increcMied in wate# f fcte leachates from steamed soil or sand sterilized by filtration or in an autoclave. Living roots of resistant or sasceptible plaats growing in soil or sand did not increase the growth rate of bacteria in the leachates compared to the check with no plants, P. SQlanaoearum cells did not multiply near tomato roots cultured in a mineral -water-agar fliedluA, However, the bacterium did increase near some fungal contamingoits. Such microorganisms in the soil may produce growth factors that stimulate multiplication of the bacterium. Tomato seedlings of susceptible varieties growing aseptiviii

PAGE 9

cally in a mineral -wateragar media containing P. solanacearum developed wilt symptoms. Penetration of the bacteria into titf roots way Wave occurred at the tip of the roots as #sli as tiiire secondary roots emerged. Bacteria penetrated roots of both iNislSt^ait and susceptible plants. Resistance in P.I. 126^K)8 is protoplasmic. No diffsrences could be detected in the development of the pathogen around resistant and susceptible roots ^ and invasion seeni^ to occur with equal frequency in each kind of plant. After entry, diffarences In colonization of the plant types were noted. Soma devaiopment of the pathogen occurred In resistant piasts afte^ invasion, but the pathogen often did not successfully colonized the vascular tissues. Colonization of susceptible plants, iiom-^' ever, continued until the plants were killed. The polygenic protoplasmic resistance of P.I, 126^08 may be useful in breeding programs designed to develop to^toes resistant to bacterial wilt.

PAGE 10

INTRODUCTION Bacterial wilt of tomato, caused by Pseudoa^ttas fol^a^^anffi E. P. Smith, is one of the most economically important plant diseases incited by bacteria (IS)A comprehensive review of the literature on the disease before 1953 was mi4fe by Kelman (15|^ Buddenhagen and Kelman (4) later extended the review up to 196^ The pathogen has a wide host range and the disease occurs in almost every warm temperature, semi tropical, and tropical stmt of the world (15). Host plants to P. solanacearum include economic plants of the Splanaceae such as tobacco, potato, pepper, eggplant, and tomato. Non-solanaceous plants, such as peanut and banana, are also economically damaged by strains of this organism. A total of 33 plant families contain hosts of P. 6olanacearum (IS)*, On tomato, Lycopersicon esculentum Mill., the disease has been reported predominantly from the Southeastern states of tli UnitiBd States. Outside the United states, losses ranging fwmi insignificant to severe have been reported in Mexico, all (toittwntries in Central and South America, France, Holland, Italy, Switzerland, Algeria, Mozambique, Union of South Africa, Ceylon, China, Formosa, India, The Philippines, Java, Sumatra and Ha)5fai.i (15). Detection of the organism in the field can be made by visual observation of the symptoms of the disease on host plaints. S^KftSi@gical methods (13), and the use of a selective mediiMi Cl^)i 1

PAGE 11

2 have also been employed to determine the presence of the bacteria in the field. The pathogen is eoil-bome and cffisi W found in recently cleared land as well as in cultivated land (15). Survival of the pathogen in the soil varies from place to place and from a few months to many years (15). Also, since P. solanacearum invades a wide range of plants, many of which remain symptomless (2, 3), the organism can survive, on mmy weed hosts. Bacterial wilt of tomato is most serious under relative high soil temperature and moisture. Vaughan (27), found that the rate of disease developnent increased with lj[ef^aeil sdl^ teagHSFature up to 110 F. However, disease symptoms do not develop at soil temperatures below 70 P. Wilt aymptma 4ip|ftoped mort rapidly in stem-inoculated plants growing in wet lather than in relatively dry soil (27), This observation was confirmed by Gallegly and Walker (8). ^ The subject of root invasion by the pathogen is a ecttttroversial one. In general, wounds are thought necessary for peni^ration of the bacterium into the roots. These tfouiiBis i&ay be made during transplanting, cultivation, or nematode and insect feeding on roots (15). But Van der Meer, as revieipjed by Kelman (15), found that uninjured roots can 'berfij!ided % bacterium, at least under high moisture levels. Kelman and Sequelra (17), suggested that nonwounded roots may be invaded at the points of emergence of secondary roots. The fact that the bacterial wilt organism is soil-borne, and that it survives for many years in the soil in the absaiiiii^

PAGE 12

3 of erop host plants (15), makes control by crop rotation impractical. Once the organism is established in a field chemical sprays, or drenches, are of little value as a means of control of the dlmme' Disease-resistant crop plants seem to be the best hope for control. There are various sources of resistance to bacterial wilt in t^^to, one of which seems to be determined by a single feja# > or a few genes. Incorporation of these resistance gries in'to commercially acceptable cultivars has been difficult. The association of resistance to bacterial Wilt with small size of fruits (1) and the difficulties associated with transferring polygenically inherited characteristics are the mayor problems encountered in combining genetic resistance to jP. solanaceafigft with production of fruits at the commercial level. A source of resistance selected in North Carolina was fowid to be polygenically inherited (1). Venus and Saturn asw two cultivars that have the North Carolina source of resistance to P. solanacearum (12). Even though resistance of these cultivars holds up under most field conditions (20), up to mom than 90fo of flowers drop under the hot field conditions of ^Qia8ia.(G. Ocana, personal communication). This observations ihdicates that the use of these varieties will not solve thu disease problem of bacterial wilt of tomato in the tropics. Ocana (21), working with crosses made with resistant breeding material from North Carolina, found that the smaller-fruited selections were more resistant than the largerfruited ones. Resistant selections did not yield well.

PAGE 13

A second source of resistance was selected in Hawaii (1). Plant introduction (P.I. 127805 (L* pimpinellifoliuy (Jusl) Mill.) was highly resistant under most field coMltlon, but Te- sistance decreased during the, warm summer weather. Fruit size of some of the lines developed in Hawaii is acceptable for eommercial purposes. A more recent report from Hawaii in their resistant lines indicates that "some serious breeding problems not found in the other programs were encoiintei'ed with bacteipial wilt" (9)' Even though the reasons are not explained, the report indicates that "resistance is more effective when fields taperatures are below 85 F". This may explain the poor p&t" formance of these lines reported by some investigators. Ferrer (5)t found the Hawaii lines susceptible to bactBr|.|ll wilt under greenhouse conditions in Florida. Ocana (21) and McCarter (20) also found the Hawaii lines to be susceptible under field conditions. A selection of L. pimpinellifolium frort the PireftCh Antilles, CRA-66, is also resistant under field conditions in Panama. The line produces very small fruit and was f otind by f&rreT (5) to be susceptible imder laboratory conditions. The source of resistance of these lines was not reported. A line, P. I. 126408 (L. esculentum Mill.), was f otmd % Ferrer (5) to have a higher level of resistance, in the seedUlik^., Stage, than all other lines tested. However, fruit which average less than 1 oz. are produced on plants of P.I. 126438, but fruit were set under relatively high temperature conditions. Bacterial wilt resistance in tomato is highly desiirabl^

PAGE 14

for production of tomatoes in areas where soils are infested, with the agent of the disease. Research was planned to further characterize the resistance in plants of P.I, 126408. The mode of inheritance of the resistance and some characieristicil of the nature of the resistance are reported herein.

PAGE 15

MATERIALS AND METHODS A strain of Pseudomonas solanacearuin designated A-21, was isolated from a diseased tomato plant in Florida in 1971$ and was used in all inoculation tests. It corresponds to physiological race 1 of Hayward (11). It is a highly aggressive isolate in tomato, causes wilt of tobacco when introduced in the stem or roots, and when introduced in tobacco leaves at concentrations greater than 1 x 10^ cells/ml It incites a hypersensitive reaction in tobacco leaves. Preparation of Inocula Inoculiim of P. solanacearum was prepared by streaking isolates, maintained in tubes with sterile distilled water at approx. 27 + C in the dark, onto a tretazolium medium (16). After 48-60 hr of incubation at 32 C, and based on colony appearance on this medium, isolates were transferred to nulaFi* ent broth and shaken for 20-24 hr at approx. 2? 2 C. Cells were entrifuged from the broth and resuspended in sterile tap tisiter:^' which proved in laboratory experiences to maintain iim nunber of living cells longer than a physiological buffered saline solution (25X. The concentration of the inocu2:iia was standardized photometrically using a "Spectronic 20" (Bausch ft Lomb), spectrophotometer at a wavelenght of 600 nm to either 50, 70, or 84^ transmittarice Other concentrations of inooiila ifre derived by dilution from such standardization. A dilutii^

PAGE 16

plate technique (25) was used to determine actual concentrations of bacteria in the suspensions. Fifty percent transmlttsric^ mi equivalent to 2 x 10^ cells/ml; 70?5, 1 x 10^ cells/ml; and 84f. 5 X 10 cells/ml. Inoculation Techniques Root^in.lurv Technique Tomato seeds were sown in 4^8inch rows and > inches apart in soil contained in raised benches in a greer^isuse. The susceptible cultivar Bonny Best was used as control. Venus, Saturn, Ploradel, and selections of the P.I. IZSkOB were used as trejst* ments. Rows were thinned to 50 plants/row or left unthlittied according to the purpose of the test. Prior to inoculaticin, a furrow was made with a knife about 1 inch from the i4at st^s and about 2 inches deep so that one side only per line of plants was inoculated. Then 50 ml of standardized inoculum were poured along this furrow which was then closed to avoid desiccation. Three to four-week-old seedlings were inoculated using this technique. Transplant Technique Tomato seeds were sown in 6-inch clay pot saucers. Seveii days after emergence of the seedlings, 25 ml of standardiised inoculum were added to each plate. Seedlings were transplanted 5-7 days later to soil in the benches and placed In rows to keep the different lines separated. Living plants were counted

PAGE 17

8 15 days after inoculation. Stem Inoculation Technique Tomato seedlings in the cotyledonary stage were transplanted into /j-inch pots containing steam sterilized soil. About 3 weeks after transplanting, when they were about 6 inches tall, seedlings were inoculated by piercing the hypocotyl about 0.5 cm above the soil level. The inoculum contained about 2 X 10 cells/ml. Plants were then i^aca in Sherer GEL ^-4, environmental chambers, at various temperatures., which were kept within 2 C of the desired temperatures throughout the experiment. A l6-hr light period/day feora fluorescent and incandescent lamps giving approx. 680 lux 12 inches from the li^ts was used in this test. Living plsaits were counted 15 days after inoculation. Tomato' Plant Culture All plants raised to maturity were planted in soil in raised benches. All secondary buds were kept pruned and the plants were trellised on strings attached to wires OTerlli!ad* The plants were watered twice daily, fertilized with a 6-6-6 coBsnercial fertilizer every 2-3 weeks, and sprayed with pesti cideS as needed. Steamed soil was always used in the greenhouse benches. Crosses were made by transferring pollen to emasculated. flowejTS. Selfing was allowed to occur naturally since very

PAGE 18

few flower-visiting insects or wind existed in the greenhouse. All inoculation tests to determine levels of resi8taxie were made in a greenhouse. Temperatures fluctuated greatly during most tests and from one test to another. During some tests, soil temperatures fluctuated between 20 and 30 C. In others, the temperatures fluctuated between 28 and 32 C. Root Diffusates Leachate Medium Three tests were undertaken to assess the role of root diffusates on the growth of P. solanacearum In the first and second tests, steamed soil was placed In 115 ml "Nalgefie* disposable filter units with an 0 20|x membrane filter under the soil. Tomato plants in the first true-leaf stsipe ifer transplanted to the soil and the units were kept in 6 Sherer CEL growth chamber at 28-30 C and with 12 hr of light daily. During the two weeks of plant growth, water percolated through the soil and through the sterilizing membrane. This water was collected and used as a medium for P. solanacearum In a third test, washed white sand was piaeed In steaft. sterilized ^-inch pots. Tomato seeds were planted in the sand, which was watered daily with a ^-salt mineral solution with minor elements (18). Three weeks after germination of th seedlings, the sand was flooded with water and left 20 min for excess water to drain. The pots were then placed on a "NalgiawS* iRllter unit with an 0.20|x membrane filter. Ten ml of waier

PAGE 19

10 were added to each pot and a vacuum of 25 inches of mercury ,:m9B allied to the filter unit. The excess water from ppts wftS drawn through the filter and was used as a mediiUB for P. solanacearuai All isedia Obtained from the pots w4m 8gi^k flltr-f3teriliteA hy fbrcing them through a "Swinny* filter containing an 0,2^|4. membrane filter. In the first two tests, half of each batch of medium was also autoclaved. Cells of P. solanacearum isolate A-21, were added to the soil-water medium, A 20-hr broth culture of the bacterium w^ centJPifnged at 21,000 g to remove the bacte^ria. |0S resuspended in sterile tap water and standardized to ^0% transmi ttance in a "Spectronic 20". Using this standaz*d suspension, 8 equlTi^ent to 2 x 10 cells/ml, a suspension was obtained by dilution, containing about 1 x 10-^ cells in a 0.05-ml drop. The latter amount was added to each ml of soiX<^water iDOi^tp* thB tubes of media were placed on a reciprocal shs^er at JO ii The growth of bacteria in the medium was determined by pei*lodlea3,ly diluting the medium 10-fold aail jgHmatins mmmred olUBies of the diluted medium on nutrient agar plartes (25). Colonies were counted and numbers were converted to bacterial cellf concentration in the original medium. Two or three dilutions were used to obtain average readings. Mineral -Water-Agar Medium A mineral -water-agar medium (MWA) was used to culture

PAGE 20

11 tomato plants in the presence of a dilute suspension of P. SQlimeeattnim This medium was prepared by adMag Sd^t^^^i! (Difco) at 1.3% w/v to a modified ^-salt nutrient solution with minoi* elements (18). Cells of P. solanacearum isolate A^21. from a 20-hr culture were added before solldiflatioii of the medium at ^7 C. Twenty ml of this medium were placed in sterile 50-ml tubes with a cotton stopper. Tomato soe^lt nepi^ ptfw face-sterilized by dipping them in 95^ alcohol and then treUis^* ferring them to a 10% "Clorox" solution for 2 minutes. A sterlie seed was placed In eaoh tube with the me4iijm mA. tHe tubes were placed in a Sherer CEL k-ii growth chamber, at a temperature of 30 + 2 C. Plants were illuminated for 12 hr daily with flttorescent and incandescent lamps giving appjsdx. 61^0 liix iJg inches from the lamps, flants were placed about 12 inches from the lights to obtain a fair growth of the seedlings. Seedlings were oliierved periodically and tubes contaiiaittg bo se^ used to compare growth of bacterial colonies where tomato plants were, absent.

PAGE 21

RESULTS Tests of Inheritance Inheritance of the resistance to bacterial wilt of plants of P.I. 126408 was investigated by screening progeny of resist*' ant selections from this line crossed with plants of the susdfptible cultivars. Bonny Best and Floradel. Cros8e9 were i^i; made with plants of the cultivars, Venus and Saturn, which hVt polygenic resistance to bacterial wilt, to observe if such a crolis would increase the resistance among the progeny over ja^ogeny of either parent. Selection within P.I. 126^08 Preiimihary experiments demonstrated the resistance of plants of P.I. 126408 to bacterial wilt, but since P.I. aboes* sions are usually heterozygous, seed were obtained from resit
PAGE 22

13 lation if data from all selections were lumped together. Howwr, the progeny of selections 126408-'6-2, -6-4, -6-7 an(I -6-8 (Table 1) were more resistant than the parent selection, and this criterion was used for further selection within tJipse lines. In the third generation from P.I. 1264-08 selections from resistgtnt progeny of 126408-6-2 and 126408-6-8 w#re made after inoeiiiiations of the lines. The progeny of these selections were screened for resistance and compared with populations representing each previous generation (Table 2). Three inofii^pm levels of the pathogen were used. As the inoculum level increased survival of plants decreased. At the lower level of inoculum, not iiil .su^^efffetlSCe control plants. Bonny Best, were diseased. Even with the low level of inoculum resistance of selections was evidentr however, by a hi^er percentage of living plants 2 weeks after inomiia^ tion. The data from the three levels of inoculum were lumped to evaluate the resistance of the selections* After an analysis of variance and Duncan's laultiple range test were performed on the data (Appendix 1,2), wide differences in survival of selections in inoculation tests were noted mm^ the representatives of the generations. Resistance was increased by selection through the three generations. However, no signiffiCi^t differences occurred between the second and third generatien. This indicated that selection in future generations would probably not significantly increase resistance in the lines*

PAGE 23

1^ Tafble 1. Progeny test of 126iK)8-6 and selections fr^ it fpr bacterial, wilt resistance, Selection Total No. Plants Livins^^ 2if 8 33.3 126^^-08-6-1 9 37.5 126^8-6-2 2i^ 16 75.0 126JI08-6-3 2i^ 5 20.8 126-4-08-6-^ 24 16 75.0 126ifrO0-6-5 24 13 5^.2 126**08i6-6 24 9 37.5 126408-6-7 24 16 75.0 X26#08*6-8 24 17 79.2 6) living plants 2 weeks after root inoculation with 8 2 X 10 cells/ml of Pseudomonas solanaoeatrum

PAGE 24

''"'W;v2H!<;!?T!"' J5 n rH Oj -P O C •H c •H > -P O CO •H CO o 03 o 03 O VP CO O C 2 C3 iH P c •H > to O C CM OS CM -4* • • • VO H \rv vo NO vo 00 CM ^ 00 CM V> ^ VA VTv MD l>H fv. CO 00 VTi O 00 >A O CM CM H CM CM CM 1>H cnMDvo\o v\ Es. (N-MD xrvNO vO. HONHVO-:tOOC^O -:t H VO AO vr\ ir\ M3 ^ CMVpjHOC3sO\vovrv 00 C^OO CJN On On M3 HCMCM^OOc^CMCM CMHC^O^rHiHC^CM VO u^ 00 £N. 0\ CM VO iTv c^H cnc^cM C^C^CM O Cv. CM.U^V\CM CM l H CM CM CM CM -l>-r-lr^0\V0 H H CM CM 00 ^-w^v^\r^H^ CM H CM CM C^ H en o en rH •siCM 00 CM o • • •H C C 00 -P 0), o o o VO rH p CM CD CO H CO H VO CM CM 00 Ov rH I I I I VO VO VO VO a HCMf^-^rHcMm^ I r I I I I I I CMCMCMCMCOOOOOOO I I t I I I I I vovovovovovovovo o o pq w O vr\ B rH o o •H rH h Q) rH o 9 £v C O O H •H W $^ Q) ft CO TJ H Cd 00 •H O o Jh rH •rj 0) -P -P tcJ o H 3 O o ^00 a o o •H o CM o •H o +> O Q) ft CO +> J"( o o CM U u CO H C •H (U o C cd + -P •H S CO cd p u Q) M (U 0) O cd -p -p •H E CO Cd -p 00 Cd CO 0) <1> CM CO +> c Cd H ft •H > •H rH

PAGE 25

16 Crossea with suscep tible plants The cultivar, Bonny Best, was used as a susceptible parent and ci?ossed with a plant from 126408-6-*4. A few plants were raised to maturity and these were used to obtain an population and to backcross to the resistant and sussf^ tible parents. The data after root inoculation of the populations at two different levels of inoculum are in Table 3* Again, survival increased as inoculum level ^?rased* i^ogffir^ of 126i08-^6-2-3 had a high percentage of survi"^! compared td the plants of the susceptible cultivar, Bonny Best. The F^^ population survival figures were intermediate between these tf. both parents. The population survival numbers were also intermediate between those of the parents. A backcross to the susceptible parent resulted in a population with a sanrifml percentage below of that' of the F^^. With Floradel as the susceptible parent and 126408-6-8 as resis-^t parent populations were also obtained of J^2 ^2 backcrosses to susceptible and resistant parents. Results slftilax* to the ones with Bonny Best were obtained eixcept 1^ backcross to a resistant parent resulted in a Higher percentage survival than that of the F^^ population, as expected (Table 4). The latter did not occur when Bonny Best was used as the siis-, oeptible parent. Reciprocal crosses were made between Floradel and 1264Q8-6-8 to establish if cytoplasmic inheritance was involved in the resistance. All populations from reciprocal crosses behave alike,

PAGE 26

17 Table 3. Data on survival of plants from crosses of selections from P.I. 126408 with Bonny Best after inoculation of two levels of inoculum of Pseudoroonas sol^ac^earuiB 50% b) 70^ c) Generation Noa) plants livinK livlnp No. plants Bonny Best (BB) 80 2 2.5 92 13 14.1 6-2-3* x self 63 20 31.7 67 38 56.7 ?! (BB X 6-4* ) 94 18 19.1 74 22 Pp (BB X 6-4*) X self 83 9 10.8 77 21 27;^ Be (6-4* X BB) 6-2-3 59 9 15.2 40 8 20.0 Be (BB X 6-4*) X BB 73 6 8.2 67 10 1^0 the prefix 126408 has been omitted. a) living plants 2 weeks after root inoculation. o b) inoculum concentration: 2 x 10 cells/ml. o) inoculum concentration: 2 x 10^ cells/ml.

PAGE 27

18 Table ''l'. Data on survival of plants from crosses of selections from P.I. 126408 with Floradel after inoculation with Pseudomonas solgmacearum Line or cultivar No. plants living^^ % living Bonny Best (Control) 92 20 21.7, Floradel 79 14 17.7 126^*08-6-2-3 X self 78 56 71.8 F-^ (Floradel x 126^08-6-8) 69 14 20.3 Pg (Plpradel x 126408-6-8) x self 58 8 13. i Be (126408-6-2 x Floradel) x 126408-6-2-3 66 19 28.8 Bo (Floradel x 126408-6-2) x Floradel 41 4 9.7 a) living plants 2 weeks after root inoculation with 2 x 10 cells/ml of Pseudomonas solsmacearum

PAGE 28

which means that the resistance is proba}}iy not cytoplasmicalX;)^ iah^pited., Prop:eny testing of F q population of the Uroea, Boiwar Best x 126it^o8-6-2 Variation due to increased homozygosity of genes contributing resistance was investigated in a progeny test of an F2 population of the cross, Bonny Best x 126^M)8-6-2. Seeds were obtained, from ^9 self pollinated plants and approximately 100 saedlings from each were inoculated witfe SQXtistearum The percentage survival of the Fj plants (Table 5) was used as an indication of the degJee of resistance of the par^t*. JispdIngs for platt survival were taken 3 and 6 weeks after ii^oMIItt^ tion. The selfed plants had a variation as expected for a nonaally distributed curve (Fig. 1 and 2). Data used to calculate' expected and actual, curves are in Appaidix 3-^* B0th curves obtained were similar and both agreed with a normally distrllnitied population curve at the 1% level ufH^ the test, •fhe eharacteristlc of resistance, measured as survival percentage and which is normally distributed, indicates that this character is poX^^'genlc in nature with no dominant genes. The realtisi[l^ genes have an additive effect Average weight per fruit of P plants from the cross, Bjpny Best X 12648-6-if were recorded (Table 5) and the surviiral percentage of progeny to bacterial wilt was correlated with fruit weight. The correlation coefficient between weight/frulit and survival percentage was found to be -0.212^ for the reiy|i%

PAGE 29

Table 5. Survival of seedlings of 49 lines of tomato to. Pseudomonas solanacearum aM relationsl^ip ta fS?ttit irei^t F2 selection No. No. fruits Ave oz. /fruit No. plants ^ F^ plants living 3 weeks '.--iJ.. % F^ plants living 6 weeks 2 8 1.69 100 28.0 23.0 3 3 2.00 100 32.0 28.0 5 1 2.00 82 25*6 17.1 6 13 1.15 100 30.0 20.0 7 5 1.60 95 28.4 22.1 8 9 1.39 100 48.0 11 5 1.20 100 34.0 28.0 12 5 1.80 96 24.0 20.0 14 7 1.00 100 50.0 44.0 15 2 "2.00 100 34.0 28 .0 16 4 1.75 100 17 2 2.00 87 19. 1 6.9 18 6 1.58 100 36.0 32.0 19 9 0.78 89 47.2 20 5 1.10 98 39.8 37. 8 n f J.UU Z/* w 5 2.60 100 31.0 25.0 23 4 '1.50 100 24.0 16. .0 24 6 1.17 100 41.0 36.0 25 3 2.67 100 30.0 20.0 28 2 2.75 99 34.3 31.3

PAGE 30

21 Table .5. Continued 2 selection No. No. fruits Ave. OZ. /fruit No. plants 9S F3 plants living 3 weeks 5^ f 3 plants living 6 week£ 29 k 2.50 100 21.0 12.0 30 k 1.88 100 .55.0 47.0 Jl 3 2.50 100 22.0 16.0 33 9 1.22 100 42.0 • 36 '9 34 5 1.20 98 17.3 14^3 35 1.88 99 18.2 13.1 37 3 1.50 100 14.0 13.0 38 5 1.90 100 24.0 5 1.60 100 27.0 20.0 /fl 8 "1.13 100 47.0 44,0 43 2.13 100 54.0 46.0 5 1.30 100 31.0 23-0 8 1.38 100 27.0 19.0 46 k 1.63 90 13.1 11.1 47 3 2.00 100 36.0 30.0 48 1.63 60 36.2 21.7 49 7 1.21 100 51.0 50.0 50 3 1.50 97 27.8 18.6 51 10 1.45 100 47.0 42;x) 52 5 1.20 100 14.0 10.0 53 4 1.75 98 28.6 21.4

PAGE 31

Tattle 5Continued p i J Z : plants plants stitetin No. Ave. No. living living No. ^ M^uits ,oz. /fruit plants 3 weeks 6 yM^^s 5^ 3 1.67 98 19.4 il^ 55 4 2.13 100 31.0 25.0 56 5 1.20 99 >1.4 37.4 57 10 1.00 97 36.1 29.9 58 5 1.60 100 40.0 32.0 59 5 1-20 99 29.3 28.3 60 2 2.25 100 k5,0 3.d 126408-6-2-3 100 63.0 56.0 Bonny Best 95 15,0 8.4 ;

PAGE 32

(X) -P 1 CM C'*-' H O •H • +> QJ M Cj > • rH S-i o o X o C H ^ • -H TO ^ ^^ 4) O -P +• c to Ih O rH • o Q) > Y H Pi O CO U n § § O c o 0) ,o u rt 3 • •H a> 0) o V r-t O Rt h o> o c II Q) pH rJ 0) CO m o > CM fx, t3 cn Q) CO O -H O O f:^ E p o rH CM 0) Mtj P
PAGE 34

03 a CM CM 0) I •H 1^

PAGE 35

26 suoiP9|es ^0 J8qujn|\|

PAGE 36

2? taken 3 weeks after inoculation. The correlation coefficient cttlciirlated 6 weeks after inoculation was -0,2566. Thus, fni*i size was not correiatfid Irlth resistance in the tellts reported here.Crosses between res istant cultivara and the P.I. 126408 Saturn and Venus, two resistant cultivars. wei^ uiped as parents in crosses with a selection from P.I. 126408 with the purpose of determining if the resistance of the two parents wotild t>9 additive in the progeny. A cross h^lm^tn Saturn and 126408-6-4 produced an population with ad many i*e#ist#it plants as that of the parents (Table 6). Similar results were Obtained with a cross, Venus x 126408-6-4. The populatimk 61* the Saturn cross had more resistant plants than those of either parent, suggesting that genes from both parents were additive in the cross, Saturn x 126408-6-4. This incre8flrd resistance was not observed in the cross with' Venus, in which the Fg population had about as many resistant plants as the parftiitsv A backcross to Saturn produced a population with ma many resistant plants as that of the parents. These results indicate that when a selection from P.I. 126408 is cz^0ssed .with 'Venus or Saturn, the resulting population has a resist&te least as great as that of their parents. In the cross with Saturn, resistance to bacterial wilt may have been increasad through increasing homozygosity in this population.

PAGE 37

28 Tade 6. Data on survival of plants from crosses of selections from P.I. 126^8 with Saturn aftfr Inoeulaticm with Pseudomonas solanacearum Line Qte fliAlti^ No. \ /o Bonny Best (Control) 60 9 15.0 Saturn 60 40 126408-6 X self 60 41 68.3 (Saturn x 126408-6-4) 60 38 63.3 (Satum X 126408-6-4) x self 60 5X 85.0 Be (Saturn x 126408-6-4) x Saturn 60 37 61.7 a) living plants 2 weeks after inoculation by the transplanting technique using 25 ml of 2 x 10 cells/ml of Pseudomonas solanacearum per seeded plate.

PAGE 38

29 126408 Field Tests Tests under laboratory conditions (5) indicated that certain selections of the P.. I. 126408 were highly Resistant to teioterial wilt caused by one isolate, A-21, of P. selanacearum Field trials were necessary to confirm this resistance under conditions where natural populations of the pathogen exist* ^ree investigators coopi^rated in pl^tii^ selections of P.I* 126408 in field trials. S* M. McCarter, University of Georgia* Athens t |>3jtn1>6d populai^ons of progeny of the selection 12640S*'? in infested plots ai; Midville and Athens, Georgia (20). Only 3 out of more than plants had evidence of bacterial wilt at the eei^etion of the experiment. Plants of the susceptible check, Marion, had 81.7^ mortality and plants of the selection 126408-7 had an 0*Ofl lEWjrtaiity in the test at Midville, whereas 67r0 aa^ 2. 3J5 mortality, respectively occurred at Athens. G. pcana. University of Panama, Panama City, Panami, plan^ 45 seedlings of a selection of the P.I. 126408-6 in & field naturally infested with P. solanacearuBl All the resistant plants survived whereas all plants of the variety Floradel "iS'led. R. Sonoda, IFAS, Agriculture Research Center, Ft. Pierce, Ploridat planted individuals from 126408-6, from crosses betwemi selections of 126408-6, and the resistant cultivars, Venus and Saturn, and individuals of the susceptible cultivars, Bonny Best iaa4vf^(lpadel. The plots were heavily infested with £. 'H^ l fgn f^-WM

PAGE 39

30 iince previous tomato trials In the same plots were devastsiteA by the disease. This test was in late Spring 1973 and ^^i^eratures were above that normally occurring during tomato production. Thus, conditions were extremely favorable fpr disease, development. The crosses between the 126408-6-4 and the resistant cultlvars had survival percentages equal to those of the pe#4stnt cultlvars aiid a little higher than those of the selfed 126408-6 selection (Table 7). Crosses between the 126408-6 > and the susceptible cultlvars Bonny Best and Ploradel prodttcNnl Fi populations Just as susceptible as the susceptible parents. Results of the Ft. Pierce test were consistent with the seedling Inoculation tests. Crosses of 126408 with the resistant Venus and Saturn do not decrease resistance in the progeny, but crosses with the susceptible Bonny Best and Ploreuiel do decrease resistance in progeny. Nature of Resistance Studies of the nature of the resistance In plants from P.I. 126408 were undertaken to gather information on the nature of the resistance of these plants for future use of tlds reslstanee In field control of bacterial wilt. Hoot Dlffusates Experiments were undertaken on the role of root dlffusates on multiplication of P. solanacearum around the roots of plants resistant and susceptible to bacterial wilt. Root dlffuat

PAGE 40

31 Table ?• Field evaluation of resistance to Pgeudoyioinas solanaCggrum of crosses between susceptible and resistant cultivars with selections from P.I. 126408 at Ft. Pierce, Fla. or crosli 1??:?^nspl^nte4 No nlpniti*^^ living 1^ 1 livine Bonny Best 56 11 19.6 56 3^ 60 7 Saturn 56 32 57.1 Floradel 56 18 32.1 126/108-6 X Bonny Best 56 6 10.7 Venus X 126408-6-4 56 32 57.1 : Saturn x 126408-6-4 56 31 55.6 Floradel x 126408-6-2 56 12 21.4 126408-6 X self 56 25 a) living plants 4 weeks after transplanting in natmrally infested soil.

PAGE 41

have been found to be important in development of some other diseases involving root pathogens. Root exudates play an impoHant role in the germination of spores op sclerotia of many fungi, chemotaxic responses such as in the case of Phytophthora spp, and maintaining growth of jjathpgens in tj| phizosphere (23). X€^w?|iate teat Root diffusates were collected in water froA pote ooittMk* ing plants resistant to bacterial wilt, Saturn and 126408-6-2-p, and plaxtts suiiceptible to bacterial wilt, Box^ Bet. Conl^F^ljti consisted of steamed soil or white sand without plants. In three tests, P. solanacearum grew in the control medium,, except in one treatment (Table 8). Since only mineral nutrie^# were added to the medium and no source of carbon was added, it was felt that the growth factors released in the soil probabla were of microorganism origin. Since P; solanacearum lerelf la leachates from washed white sand, probably algae or other autotrophic organisms were involved in the stimulation of growth of the bacteria. The growth of P, solanacearum in the leachate media was relatively slow, compared to growth of the bacteria in ati*i^t broth. Populations of this organism reach 10^ cells/ml in 48 hr in nutrient broth (5). Therefore, if growth factors were released from the tomato roots, growth of the bacteria in tJW; media frcHDi pots with plants should have increased over the

PAGE 42

33 Table 8. Influence of substrate leachates on growth, of gsettdOMftonas solan^ce^tyun) in vitro Water extracted Bacteria/ml of substrate leachates from substrate Hr + or plant 0 Test 1 Bonny Best F (a) 5.0 X 10^ 3.0 X 10^ 2.9 X FA (b) 1.0 X io3 2.7 X 10^ 1.8 X 126408-6-8 P z.h X 10^ 2.5 X io5 1.7 X lol FA 2.9 X 10^ 3.1 X io5 1.9 X Soil only (Ck) F 5.0 X 10^ 0 0 FA 2.2 X 10^ 2.1 X 10^ 2.6 X Test 2 Bonny Best F 1.8 X 103 4.8 X 10^ 3.0 X PA 1.5 X io3 8.2 X 10^ 3.7 X lo' 126408-6-8 P 1.6 X 103 5.2 X 10^ 1.1 X FA 1.6 X 103 8.7 X 10^ X 10'' Soil only (Ck) F 1.1 X lo3 6.0 X 10^ 6.5 X 10 PA 1.3 X 103 3.1 X 10^ 5.7 X Test 3 Saturn F 1.1 X io3 2.8 X 10^ 1.8 X io7 Bonny Best F 9.0 X 2 lO'^ 4,0 X 10^ 1.8 X lO^ 126408-6-2-3 P 1.1 X 103 2.7 X 10^ 1.8 X 10? Sand only (Ck) F 1.2 X 103 3.4 X 10^ 1.4 X io7 (a) filtersterilized flj)^; ^^ter-sterilized and autoclaved

PAGE 43

3^ contz*ol pots. In no Instance, however, did increased growth occur in media from pots containing tomato plants (Table 8), Therefore, it can be concluded that diffusates from the tomato plants had no effect on the observed growth of P. solanacearum in the aqueous medium. Nineral-water-agar test The above experimen,ts had a limitation of issible dilution of released growth factors from roots to the point of mak-ing detection impossible. A different approach was used in an attempt to determine whether factors that might influence growth of P. solanacearum are released in minute amounts from roots. Bacterial cells were added to a mineral-water-agar (MWA) medium at a concentration of approximately 1 x 10^ cells/ml. After 4-5 days, minute colonies, 0.1 mm in diam, were visible with a binocular microscope at 100 X and also with the unaided eye when the tube containing the medium was placed against a soopM of light, such as a fluorescent lamp. The colonies did not increase in size over the next two months, when the temperature remained at approximately 30 C. Seeds of two cultivars, Saturn (resistant), and Bonny. Best (susceptible), and the line 126^08-6-2-3-.Bk were placed on tl^ agar surface after colonies of Pi solanacearum had become visible. Germination of seed and growth of the plants occurred on this medium. Colonies of P. solanacearum near or far tr
PAGE 44

35 contfiUninarits occurred, and the zise of P. solanacearum colonies near these fungal colonies did increase in size. The contamisaiil^ fungi were not identified. This was considered as twe^>imi evidence that tomato root diffusates do not affect the growth of P. solanacearum but possibly microorganisms in the soil dp produce growth factors that effect multlplloatian Of the bacterium. Penetration of Noninjured Roots Preliminary experiments in which inoculum of P. golmsMNjearum was poured over roots of tomato pla&t growlng tft vermleulite indicated that the pathogen could penetrate noninjured roots (Table 9). This observation was confirmed in the NWA medium. Penetration of noninjured roots of resistant and susceptible tomato seedlings occurred when 100 or 1,000 cells/ml of P. mtimamAVom were added to the medium (Table 10). At the higher density of bacterial colonies, both resistant and susceptible plants had browned roots. Browning symptoms were observed at the root tips and areas where sec
PAGE 45

36 Table 9. Bacterial wilt development in plants with a) nonin jiired roots growing in vermiculite GialtlyaE or line No. plants living ^ ^ Iprixi^ fenus 10 10 100.0 Bonny Best 12 h 126#08-6-9 12 10 ^ 8*^.0 126408-6-2-1 12 11 91.7 126408-6-8-1 12 11 91*7 a) roots were drenched with suspensions of 2 x 10 cells/ml of p. solanacearum b) living plants after 2 weeks after addition of bacterialsuspension to the substrate. Table 10. Penetration of tomato roots by PseudcMPonas aolanaeearum in a mineral-water-agar media Line or cultivar total plants livin^^' with ....... .)mm roptf Test 1 Bonny Best 16 37.5 100.0 feat 2 ** 126408-623-Bk 7 57.1 100.0 Bonny Best 6 33.3 100.0 a) living plants 6 weeks after germination in MWA. inpculvun concentration = 100 cells/ml. ** inoculum concentration = 1,000 cells/ml.

PAGE 46

37 emerge. Movement of bacteria in this environment is restricted, therefore, they had to be present at the site of injury for penetration. The probability of a bacterium being axacfeiy at the site where a secondary root emerged was calculated. At the density of 1,000 bacteria per ml in the medium, 3 out of 10^ times a bacterium would be in the area Where a secondary root emerged (Appendix 5). Observed results utilizing the MWA techttique did not fit the probability, since invasion occurred far more frequently than the probability dictated. To account for such a high rate of invasion of plants without artificial injures, it can only be speculated that th pathogen might have multiplied on the surface of the root and that populations of the pathogen near the secondax^ root emergence sites might have been much "greater than could be determined by the techniques used in these experiments. Evidence was found that the bacterium might maltipXf exr tensively near the root tip. In several instances, tui^bldity was noticed around root tips, in the case of the susceptible oultivar Bonny Best, which then became brown and ceased to gs*0il; In isolations made after carefully removing root tips from the agar medium, only colonies of P. solanacearum could be reoov-. ered. That the bacterium multiplies on root tips amd enters them directly can only be suggested at this time. More refined techniques must be developed before multiplication and entry of P. aolanaoearum into root tips can be established with certainty.

PAGE 47

38 Effect of Temperature on Hesistamee Resistance to bacterial wilt of some tomato Ilne3 is affected by temperature (9). For this reason plants of 126^8-6>2-3~Bk were tested for resistance at various t^qpera* tures. Plants of Saturn and Bonny Best were included, respectively, as resistant and susceptible controls. Pleuats were inoculated by the stem-inoculatipn technique and placed at 25, 28, 30, and 32 C after inoculation. Almost all of the three types of plants developed some symptoms of the disease, such as adventitious root formation on the stem ahft some degree of dwarfing or vascular discoloration, but very little wilting occurred in any of them at 25 and 28 C, even among plants of the susceptible Bonny Best (Table 11). Plants of Saturn and 126408-6-2-3-Bk remained resistant at 30 and 32 C whereas those of Bonny Best were almost totally susceptible. Since Sltt^m remains resistant to bacterial wilt even under some field conditions where all susceptible plants die, plants of 126408-6-2-.3-Bk will probably hold their resistance under the same field conditions. This experiment also demonsti*ated that reslstanoe of the P.I. 126408 plants operates even after the pathogen had been introduced into the stems.

PAGE 48

39 Table 11. Influence of temperature on bacterial Wilt de'r^Iofffient on resistant and susceptible tomato plants Line or cultlvar Temperature C 2^ 28 i 30 32 Bonny Best 5/8 6/8 1/8 0/8 126^08-6-2-3-Bk 7/8 7/8 8/8 8/8 Saturn 8/8 8/8 [ 8/8 7/8 healthy plants/total inoculated using the stem injury t
PAGE 49

DISCUSSION Inheritetnce of the resistance to bacterial Hilt of pl^aJt*.-,, of the tomato line P.I. 126408 was observed la et^sds^iith susceptible cultivars. Under greenhouse conditions, where the observations were made, soil temperature and Inai^i^ |t,ei|ii,# greately Influenced the survival of tested linds. But in a given experiment, where different lines were exposed to the B9xm environmental conditions, the hlgh^z^jpe^JHFi^ l^t# #lMiurred In liiies with the higher level f resistance. Prom the cross, Bonny Best (susceptible) x 126408 ( resistant ), the survival per^atage of the P]^ progeny was between that of the parents. Probably no dominance occurred but there was a slight tflldency toward the susceptible side in the population dfejierir#^ $he levels of resistance in the population followed a curve close to a normal curve. The significance of these data is that it ecmflriBS that the resistance behaves as mepm&t^ f&t. a pol3Pgenlcally inherited character with an additive gene, effect. The possibility of extrachromosomal inheritance discarded by making reciprocal crosses with the susceptible cultivar Floradel and the P.I. 126408. The resukts were that when |jther paipeat nas used as female, the Pj progeny behaved the same. In both cases, the resulting survival percentage was between that of parents, but again a little toward the susceptibla : parent 40

PAGE 50

The l>ack crosses to both parents did not always behaTt wm', expected for a polygenically inherited character, i.e. the surviv?tl percentage did not fall between that of the population and their respective recurrent pareht. A plitible e-xpla^ nation is that the resistant parents were not selfed with thse specific purpose of achieving complete homozygosity. It was* assumed that they were nearly homozygous for "thie character of resistance from the results of inoculation tests. Variation SMong resistant lines used as parents were d^^eted* but Idilji variation was difficult to measure aceurttt^^Utnd it \fms disregarded as not significant. Robinson (22) found that measuring resonance of patate varieties to P. solanacearum was a difficult task. Some potato lines were resistant and later becaate jpusceptible and these were considered to have vertical resistance (22). According to Robinson, a high mutability of the pathogen accounted for the poor performance of potato lines thou^t t; have had vertical resistance. Because of l^is Robinson suggested that horizontal resistance, in this case of resistanc to £. solanacearum, has much greater agricultural value. In all crops in which resistance to f. solanacearum has been found, stable resistance has been determined to be inherited in a polygenic manner (15). Transfer of this resistance to economically accepted cultivars has been slow. The reason for this is because in each cross to susceptible plants with commercial
PAGE 51

42 S^ne of them can be recovered by careful selection for resistance in the population. In practice, however certain levels of resistsuice are usually lost with each cross. Polygenic resistance is affected by environmental, faeters to a greater degree than vertical resistance (26). In the work reported here, resistance was still present at 32 G, but €ie would expect both temperature and moisture l#els to greatly affect the degree of resistance. Also, such factors aa solar, radiation (22), fertility (7), and obtain negstfkoda Mismge (19) may alter the level of resistance. French (6), and Gonzales et al. (10), working on potato with a polygenic resistance, found that selection of clones from a cross between resistantand susceptible plants can be done best by planting inoculated potato plants under lower temperature conditions in the field \i^ere resistaaeit is best expressed. Work reported here suggests that selections can best be made of tomato segregating lines where polygenic resistance is involved at about 30 or 32 C. The level of resistance of plants of P.I. 126^08 to bacterial wilt does not appear superior to the North Caapolina resistance that occurs in the cultivars Satum and Ventis. However, the resistance appears equally as effective. Therefore, one wonders about the usefulness of the resistancrin future commercial application. The resistance might be useful in crosses with Venus and Saturn to obtain relatively largefruited lines that will set illt In -ta^opical climates. P.I, i26408 was originally

PAGE 52

k3 collected in PanamS (2^), and seems to set many fruits under relatively high temperatures. Crosses of Saturn or YemHSt with susceptible lines to obtain good fruit-setting properties under high temperatures results in decreased bacteria!^ wilt resistance (21). But crosses of these cultivars with P. I. lZ0tGB would not decrease resistance to bacterial wilt and selection for fruit setting ability could be made in -Uie frogeny iri-^cmt fear of losing the level of resistance to bacterial wilt Smallfruit size has often been associated with bacterial wilt x#9istance using the North Carolina resistfyiM^e (1, 21). In the population from the cross, Bonny Best x 126408, no correlation of ffuit size and bacterial wilt resistance waus noted. This is encoursLgihg, but not tt€^essarily oonelusive evidence that fruit-size and bacterial wilt resistance are not linked in the P.I. 126408 plants. Hwirfi(|?p^ if this, is correct, the bacterial wilt resistance of P.I. 126408 may be incorporated into large-fruited lines with much less effort than was the case with the North Carolina reaijii^iRee. The nature of bacterial wilt resistance in the P.I. 126408 is protoplasmic. Experiments designed in this work have. fai|.ed to show any differences in the effefct of the resistazii plffiita on growth of the bacterium in the medium around the roots. The bacterium also seemed to invade resistant plants me effectively as susceptible plants. Invasion occured in both types of plants without artificial injury. Resistance seems to involve mechanisms that reduce colonization of the te@e%.i^itr invasion. After invasion, the pathogen seems to

PAGE 53

spread slowly, and then gradually declines in oolonizatli^ #f the host tissues. In susceptible plants, however, the paiiii|||Q does not decline in colonization, but continues to grow wi'ljsi^ inf^eted plants until wilting and death of the plaatS oomtrsv The mechanism of resistance in plants of P.I, 126^8 eeB8 to b slnilar to that of the North Carolina resistance. Breeding for resistance to bacterial wilt of tomato is possibly the best way to control the disease under field conditions, but problems of inheritance of al% Wmmea of r sistance available make it clear that development of commercially acceptable cultivars will be difficult. There is still ne0d a source of refiistanoe to roininize probl^s In breeding for disease resistance to bacterial wilt in tomato so that resistant varieties with, a high degree cli" rrnXutma^ oan lid^one available for commercial use. Finding a souroe of resistance to bacterial wilt in tomatoes which could be easily transferred to commercial varieties is still of forenost >

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SUMMARY Selections from P.I. 126408 were found "ttiat had a high level of resistance to bacterial wilt of tomato. The resistance level was increased by selection, but not signifiosaitlj^ so after the second generation of selfing. The resistance was evident in experiments involving three inoculations techniques and was present to natural pooulations of "tehe pathogen in field tests in Florida, Georgia, and Panama. Progeny of crosses of the susceptible cultivars Boimy Best and Ploradel, with selections from P.I. lZ6ti<^B, established that the resistance was inherited in a polygenic manner. Reciprocal crosses with Ploradel pro^uc^edi progenien similar in their resistance to P. solanacearum suggesting that the resistance was not extrachromosomsil. Progenies of crosses with resistant cultivars Venus and S&tuzfit with ae' lections from P.I. 126408, were as resistant as either parent. The bacterium multiplied in leachates from steamed soil and washed-white sand. Increased multiplication was not noted in leachates from soil or white sand that contained living roots of resistant of susceptible tomato plants. Diffusates from tomato roots growing in a mineral -water-a|5ar medium containing cells of P. solanacearum did not influence growth of the bacterium. Some fungal contaminants, however, di4 ^sult in increased growth of the bacterium in the medium. The pathogen entered roots that were not artificially injured. Entry may have occurred where secondary roots iMI|||pd

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but the probability calculated for such entry was much less than that ^tyally observed. Observations made suggest that the bacterium multiplies near the root tips and that entry occurs on living nonwouraded. No differehces were found between invasion of resistant or susceptible plsdnts. The resistant plants from P.I. 126408 were as resistant as Saturn at 30 and 32 0. Very little wilting occurred at 2$ or 28 C, even in susceptible plants. Observations suggest that resistance of the P.I. 126400:' "plants was protolasmic in nature and was expressed during colonization of the host. The. resistant plants are not colonized at the same rate as susceptible plants*

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LITERATURE CITED 1. Acosta, J. C, J. C. Gilbert, and V. L. Quinon. 196'!|. Heritability of bacterial wilt resistance in tomato. Amer. Soc. Hort. Sci. Proc. 84:^55-462. 2. Belalcazar, C, S,, G. Uribe M., and H. D. Thurston. 1968. Hecotiooimlento de hospedantes a Pseudomonas solanacearum (E. P. Sm.) en Colombia. Revista ICA III. p. 37-46, 3. Buddenhagen, I. W. I96O. Strains of Pseudomonas solanacearum In indigenous hosts in banana plantations of Costa Rica, and their relationship to bacterial wilt of bananas. Phytopathology 50:660-664. 4. Buddenhagen, I. W., and A. Kelman. 1964. Biological and physiological aspects of bacterial wilt caused by Pseudomonas solanacearum Ann. Riev. Phytopathol. 2:203235: 5. Ferrer, A. 1971. Techniques to identify the tomato race of Pseudomonas solanacearum and to screen for resistance in tomato. mTS. Thesis, Univ. of Florida, Gainesville. 47 p. 6. French, E. R. 1973. Evaluacion de la resistencia de la papa a Pseudomonas solanacearum on fitotr6n. Phytopathology 63:1435 (Abstr.K 7. Gallegly, M. E., and J. C. Walker. 1949. Plant nutrition in relation to disease development. V. Bacterial wilt of tomato. Amer. J. Bot. 36:613-623. 8. Gallegly, M. E. and J. C. Walker. 1949. Relation of environmental factors to bacterial wilt of tomato. Phytopathology 39:936-946. 9. Gilbert, J. C., J. L. Brewbaker, J. S. Tanaka, J. Chtltft^' R. W, Hartman, J. A. Crozier, Jr., and P. J. Ito. 1.^^ Vegetable improvement at the Hawaii Agrloultux^ Sjcpsri-* ment Station. Research Report 175. Hawaii Agr. Expt. Sta. Coll. Trop. Agri., Univ. of Hawaii, p. 16. 10. Gonzales, L. C., L. Sequeira, P. R. Rowe, and R. Bianchlsi; 1972. Field resistance to bacterial wilt in hybrid potato progenies. Phytopathology 62:760 (Abstr. ) 11. Hayward, A. C. 1964. Characteristics of Pseudomonas solanacearum j. Appl. Bacterid. 27:2631^777 47

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12. Henderson, W. R., and S. F. Jenkins, Jr. 1972. Venus and Satum two new tomato varieties combining desirable horticultural features with southern bacterial wilt resistance. North Carolina Agr. Expt. Sta. Bull. 4^4, 13 p 13. Jenkins, S. P., Jr., D. J. Norton, and P. D. Dukes. 1967. Comparison of techniques for detection of Pseudomonas / solanacearum in artificially infested soils. Phytopathclogy 57:25-27. Ik, Karganilla, A. D., and I. W. Buddenhagen, 1972. Development of a selective medium for Paeudiatsilftg seitnacearum Phytopathology 62:1373-1376. 15. Kelman, A. 1953. The bacterial wilt caused by Pseudomonas solanacearum North Carolina Agr. Expt, Sta, Tech, xJBiUJL. W. W p. 16. Kelman, A. 195^. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazollum medium! Phytopathology 44:693-*695, 17. Kelman, A., and L. Sequeira. 1965. Root-to-root spread of Pseudomonas solanacearum Phytopathology 55i 304-369 18. Klein, R. M., and D, T, Klein. 1970. Besea^Qh methods in plant science. The Natural History Pj^isi/'. ? 21. Ocana, G., and G. Silvera, 1970. Determinacion de la factibilidad del cultivo del tomate a nivel del mar durante la estacldn lluviosa. Progreso de labores de investigaciones agropecuarias, I969. Facultad de Agro<*. nomia de la Universidad de Panamll. p. 89-121. 22. Robinson, R. A. 1968. The concept of vertical and horizontal resistance as illustrated by bacterial wilt of potatoes. Phytopathol. Papers, No. 10. C(Bmonwei^tii Mycol. Inst., Kew, Surrey, England. 37 p. 23. Schroth, M. N., and D. C. Hildebrand. 1964. Influeciee o^f plant exudates on root infecting fungi. Ann. fiev. Phytopathol. 2:101-132.

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^9 24. Skrdla, W. H,, L. J. Alexander, G. Oakes, and A, P. Dodge. 1968, Horticultural characters and reaction to two diseases of the world collection of the genus Lycoperalip i ^ Ohio Agr. Res. and Development Cent, Res. Bull. IOO9. f 25. Stall, R. E., and A, A. Cook, I966. Multiplication of Xanthomonas vesicatoria and lesion development in resistant and sudeeptlble pepper. Phytopathologjr 56:1152*115^ 26. Van der Plank, J. E. I963. Plant Diseases: Epidemics and. control. Academic Press. N. Y. and London. 3^ P* 27. Vaughan, E. K. 1944. Bacterial wilt of tomato caused by Fhytomonas solanacearum Phytopathology 3^tW$J-'^$$,

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APPENDICES

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51 APPENDIX 1. Analysis of variance of data oh resistance of plants in three generations from P.I. 126408 Source DP SS MS F Prob.> F Selection 2 0.4775 0.2387 3.456 0.0434 Residual 30 2.0725 0.0691 Inoculum 2 2.1232 I,06l6 15.367 0.0001 Residual 30 2.0725 0.0691 SelectionInoculum 4 0.0031 0.0508 0.735 0.5775 Residual 30 2.0725 0.0691 APPENDIX 2. Duncan's multiple range test of sigaifiesiio^ of differences in resistance among generations for P.I. 126^8 Dundan*s Test Generation of Mean Mean Selection Transformed Orip;inal 3 0.7042 1) 0.6062 a 2 0.6979 0.6097 a 1 0.2870 0.2738 b 1) Piguj[(9s with same letter are not significantly different. i' •

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52 APPENDIX 3. Observed frequency of selections compared to a a) normal curve 1) 2) 3) 4) 5) 6) 2 Ranp;e Sd. Z values Area exp. no. obs. X 0-10.5 2.51 49^0 .0197 0.97 0 0.97 10.5-15.5 2.06 .^803 .0351 1.72 3 0.95 15.5-20.5 1.60 .0723 3.54 0.06 20.5-25.5 1.1^ .3729 .1212 5.94 5 0.15 25.5-30.5 0.68 .2517 8.07 10 0.46 3
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53 APPENDIX ^. Observed frequency of selections compared to a non.al curve"* Sd^^ 3) Z values-'^ Area Expected^ ^ Actual^ ^ x2 0-5.5 1.91 .0281 1.38 0 1.38 5.6-10.5 1.46 .4279 .0440 2.16 2 .12 10.6-15.5 1.02 .3^61 .0818 4.01 6 .99 15.6-20.5 0,57 .2157 .1304 6.39 8 .41 20.6-25.5 0.12 .0478 .1679 8.23 9 .07 25.6-30.5 0.32 .1255 .1733 8.49 7 .26 30.6-35.5 0.77 .2794 .1539 7.54 4 1.66 35.6-/^0.5 1.22 .3888 .1094 5.36 6 .76 ^0.6-45.5 1.67 .4525 .0631 3.12 3 .46 ^5.6-50.5 2.12 .4830 .0305 1.49 4 4.23 50.5-55.5 2.56 4948 .0118 0.58 0 .58 55.6-60.5 3.01 .4087 .0039 .19 0 .19 b) 6 weeks after inoculation with 2 X 10^ cells/ml of Pseudomonas solanacearum. 1) Bsmge corresponding to percent livi|jg, 2) Stndard deviation from mean. 3) Z values corresponding to 5% range. 4) Area of normal curve. 5) Expected frequency of select l^s per 8rea< 6) Observed frequency of selections per area.

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5^ APPENDIX 5. Calculations used to figure probability of a bacterium being at injury caused by emergence of secondary root Bacterial size =2,0 x 0.5 Root diameter = 0.25 mm 3 Bacterial density in agar = 1 cell/mm Formula = -^x-^" probability 2 a = No. of bacterium diam in an mm = 2000 b = No. of root diam in an rom^ = 16 : ^ c = probability of a secondary root tmiehing ane bacterium at the point of emergence ; ; ^ : No. of cells in area occup ied by root total cells m ram-^ ^^-TT^TTJ^ = 3.1 X 10^

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. R. E. Stall, Chairman Professor of Plmit Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree df Doctor of Philosophy. D. A. Roberts Professor of Plant Pathology I certify that I have read this study and that in my. opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope soid^ iquality, as a dissertation for the degree of Doctor of Philosophy. S. J.(4jOcascio Professor Of Vegetable Crops

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This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 197^ Dean, Graduate School


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