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1962 Evaluation of Several Bactericides as Seed Treatments for the Control of Black Rot of Crucifers and Studies on an Antibacterial Substance From Cauliflower Seed. (Parts I and II). Fereydoon Malekzadeh Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Malekzadeh, Fereydoon, "Evaluation of Several Bactericides as Seed Treatments for the Control of Black Rot of Crucifers and Studies on an Antibacterial Substance From Cauliflower Seed. (Parts I and II)." (1962). LSU Historical Dissertations and Theses. 787. https://digitalcommons.lsu.edu/gradschool_disstheses/787

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MALEKZADEH, Fereydoon, 1933- EVALUATION OF SEVERAL BACTERICIDES AS SEED TREATMENTS FOR THE CONTROL OF BLACK ROT OF CRUCIFERS AND STUDIES ON AN ANTIBACTERIAL SUBSTANCE FROM CAULI­ FLOWER SEED. (PARTS I AND II).

Louisiana State University, Ph.D.,1962 Agriculture, plant pathology University Microfilms, Inc., Ann Arbor, Michigan EVALUATION OF SEVERAL BACTERICIDES AS SEED TREATMENTS FOR THE CONTROL OF BLACK ROT OF CRUCIFERS AND STUDIES ON AN ANTIBACTERIAL SUBSTANCE FROM CAULIFLOWER SEED

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Botany and Plant Pathology

by Fereydoon Malekzadeh B.Sc., University of Teheran, 1956 M .Sc., University of Teheran, 1958 August, 1962 ACKNOWLEDGMENT

The writer wishes to express his sincere appreciation and gratitude to Dr. T. P. Pirone for his assistance, constructive criti cism, and stimulating suggestions, during the course of this work and in the preparation of the manuscript. He wishes to express his appreciation to Dr. S. J. P. Chilton, Dr. H. E. Wheeler, and

Dr. J. B. Baker for making facilities available. He also wishes to thank Dr. I. L. Forbes and Dr. A. R. Colmer for their helpful criticisms in the final preparation of this manuscript and Dr. E. C

Tims for his assistance in photography. TABLE OF CONTENTS

Page

ACKNOWLEDGMENT . . ii

LIST OF TABLES...... v

LUST OF FIGURES...... vi

ABSTRACT...... vii

PART I

INTRODUCTION...... 1

REVIEW OF LITERATURE...... 4

MATERIALS AND METHODS...... 10

EXPERIMENTAL RESULTS . . . . . „ ...... 14

Levels of Chemicals Inhibitory to the Pathogen ...... 14 Chemical Seed Treatments of Artificially Inoculated Cabbage Seed , ...... 16 Assay of Treated Seeds for Chemical Absorption...... 18 Stability of Chemicals on Treated Seed...... 20 Effect of Chemical Treatments on Germination of Cauliflower Seed...... 22 Effect of Chemical Seed Treatment on Root and Stem Growth of Cauliflower Seedlings ...... 27 Effect of Chemical Solutions on Cabbage Seedlings Grown from Treated Seed...... 27

DISCUSSION...... 32

SUMMARY...... 37

LITERATURE CITED...... 40

PART II

INTRODUCTION...... 43

iii Page

REVIEW OF LITERATURE...... 44

MATERIALS AND METHODS . . 57

EXPERIMENTAL RESULTS...... «... 65

Antibacterial Effect of Cauliflower Seed ...... 65 Agglutination T ests ...... 70 Antibacterial Spectrum ...... 71 Antibacterial Activity of the Extract at Various Dilutions...... 75 Inhibitory Effect on Seed Germination...... 77 Antibacterial Activity of Seeds of Various Cruciferous Plants...... 78 Physical and Chemical C haracteristics ...... 81

DISCUSSION...... 86

SUMMARY...... 91

LITERATURE CITED...... 94

VITA...... 100

iv LIST OF TABLES

TABLE Page

PART I

1. Inhibition of_X. cam pestris by various chem icals as determined by filter paper disc-agar diffusion method ...... 15

2. Stability of chemicals on treated seeds ...... 21

3. Effect of chemical seed treatments on root and stem growth of cauliflower seedlings...... 28

4. Phytotoxic effect of various chemical seed treatments to emergent cabbage seedlings ...... 30

PART II

1. Presence of antibacterial substances in relation to viability of seed and seed parts ...... 68

2. Activity spectrum of cauliflower seed extract against different bacteria ...... 72

3. Effect of cauliflower seed extract on seed germination...... 79

4. Inhibitory effect of seed extracts of different species and varieties of cruciferous plants on X. cam p estris ...... 80

5. Antibacterial activity of cauliflower seed extract as influenced by temperature and pH ...... 83

v LIST OF FIGURES

FIGURE Page PART I

1. Inhibition of Xanthomonas campestris by (counter­ clockwise from disc showing the least inhibition): 25, 500, 1800, 2500 and 4000 ppm of EP-166, using the filter paper disc-agar diffusion method . . 12

2. Method of assaying of chemicals on artificially inoculated seed ...... 17

3. Regardation of germination of cauliflower seed treated with chemicals at a concentration of 250 ppm ...... 24

4. Retardation of germination of cauliflower seed treated with chemicals at a concentration of 5000 ppm...... 25

5. Retardation of germination of cauliflower seed treated with chemicals at a concentration of 1000 ppm...... 26

PART II

1. Inhibitory effect of the cauliflower seed extract on growth of campestris , as tested by filter paper disc method ...... 60

2. Inhibitory effect of cauliflower seed on X. cam pestris ...... 66

3. Comparison of inhibitory activity of seeds of low viability (right), and high viability (left). .... 69

4. Reaction of various bacteria in suspension to the cauliflower seed extract...... 73

5. Inhibitory effect of several dilutions of aqueous cauliflower seed extract on growth of X. campestris...... 76

vi ABSTRACT

PART I

Aqueous solutions of EP-166 (9-(p-n-hexyloxyphenyl)-10- methyl-acridinium chloride), Morven (sodium 2,4,5-trichlorophenate),

Nurelle (2,4,5-trichlorophenol), and Septigard (Alkyl tolyl methyl - trimethyl ammonium chloride) were tested in the laboratory and greenhouse as seed treatments for control of the black rot disease of crucifers caused by the bacterium Xanthomonas campestris.

Seeds artificially inoculated with X. campestris were com­ pletely disinfested when treated with EP-166, Nurelle, and Septigard at concentrations of 500 and 1000 ppm, whereas complete disinfes­ tation of seeds treated with Morven at the same concentrations was not obtained.

Assays for absorption of chemicals by seeds treated with these chemicals did not reveal the presence of the chemicals on the internal seed parts, hence complete disinfection of naturally infected seed apparently is not possible, using the techniques em­ ployed in this study.

The effect of chemical seed treatments on the germination and root and stem growth of cauliflower seedlings was studied. Of the chemicals used, EP-166 was nonphytotoxic at concentrations up to

vii 1000 ppm, and remained stable on treated seed for 10 months. These properties indicate that it may be of some use as a seed treatment.

The phytotoxicity of Nurelle and Septigard at concentrations required to inactivate the bacteria, and the failure of Morven to completely disinfest the seeds make these chemicals impractical for use as seed treatment.

PART II

The presence of an antibacterial substance in cauliflower seed and certain of its properties are reported.

The antibacterial effect of cauliflower seed extract on repre­ sentative genera and species of phytopathogenic and other bacteria was studied/ and the extract was found to exhibit the greatest activity against species of the genus Xanthomonas. When the extract was added to water suspensions of species of Xanthomonas. immediate agglutination occurred. Species of Erwinia. Pseudomonas.

Corynebacterium. Bacillus. Escherichia, and Staphylococcus were also inhibited by the extract but to a lesser degree. Some turbidity occurred when the extract was added to water suspensions of these bacteria/ but the characteristic agglutination obtained with species of Xanthomonas did not occur.

Cauliflower seed extract was also capable of inhibiting seed germination. Of the various seeds tested, germination of rice and

viii oat seeds was completely inhibited, whereas radish seed was not affected greatly.

The antibacterial substance, as well as the germination in­ hibitor, was associated with seed centers. Seed coats were devoid of either kind of activity. In addition, the presence of the anti­ bacterial agent was correlated with the viability of the seed. Seeds with low viability ( ^5 per cent) possessed more activity than did

seeds with high germinability.

Of the seeds of various varieties and species of Cruciferae tested, cauliflower seed (vars. Master Original and Early Snowball) with low viability exhibited the greatest activity, followed by viable

seeds of the same varieties. Cabbage (var. Globe) and broccoli

(var. Waltman) showed the least activity against X. campestris.

Radish (var. Champion) did not give agglutination with bacterial sus­

pensions but the diameter of inhibition zone produced on agar plates was between that produced by cabbage or broccoli, and cauliflower

seeds with high viability.

The active principle is water soluble, stable under slightly acid

conditions for more than 2 1/2 months at 8-10°C, and dialyzable.

The results of qualitative tests indicated the presence of protein,

phenols and sulfur and lack of the aldehyde or carbohydrates in a

partially purified extract. When chromatographed, however, the area

ix of the paper showing antibacterial activity gave negative results when tested for the presence of phenols and amino acids.

x PART I

INTRODUCTION

Black rot of crucifers, caused by the bacterium Xanthomonas campestris (Pam.) Dowson, is a serious disease of cruciferous plants. It is more prevalent wherever rainfall or heavy dews are plentiful and average temperatures are between 16° and 22°C. In areas of low rainfall the development of the disease is limited or practically absent.

The disease was first reported by Gorman in Kentucky in 1891

(13) and by Pammel in Iowa in 1892 (24). Later the disease was recognized as being destructive in numerous cabbage growing areas in the United States and was studied intensively by Russell and by

Smith (27, 29).

Symptoms of the disease vary depending upon the part of the plant being attacked, the stage of disease, and environmental con­ ditions. Usually on cabbage leaves it first makes its appearance on the edge of the leaves as yellow lesions which progress toward the midrib in a "V11-shaped manner. In the chlorotic tissue the veins and veinlets become dark in color and when the lesion is held before a source of light a network of black venation stands out prominently.

The black rot organism may be seed-borne (23). Seed may become infected either by systemic invasion of the seed stalks, the bacteria presumably invading the seed coat through the vascular system of the funiculus (10), or by local infection of the seed podD

Clayton (7) reported that the organism remains viable in naturally infected seed for 3 years. There are also numerous re­ ports indicating that the pathogen remains alive in soil at least for one year (27, 29). Thus seed and soil become important means of dissemination of the pathogen. Mildly infected or infested seeds are capable of germination the following year and from their seed coats bacteria gain access to the cotyledon. Infested cotyledons become yellow with darkened veinlets, in the affected tissue. Affected leaves and cotyledons drop off prematurely (24). Later the bacteria progress systematically throughout the plant.

Control of black rot has been a difficult problem. The first scientific studies of the disease made by Smith (28) and Russell (27) were most interesting and productive of many suggestions for control.

The use of disease-free seed, clean seed beds, crop rotation with plants not related to crucifers, insect control, livestock control, pulling and destroying of diseased plants as soon as they appear, and seed treatment, were their recommendation for control of the d ise a se .

The hot water treatment is recognized as being the most effec­ tive treatment for disinfecting black rot contaminated seed. However, 3 danger of impairing seed germination exists if the treatment is im­ properly applied and if seed vitality is low.

Control of various bacterial diseases of plants with chemicals has been reported during the past decades (6). However, control of numerous other plant diseases caused by bacteria or fungi has not been completely successful, providing only partial control and in some cases no control.

This investigation was undertaken with the objective of con­ trolling black rot by using the seed treatment method and employing the fairly new chemicals: EP-166 (9- (p-n-hexyloxyphenyl)-lO- methyl-acridinium chloride), Morven (sodium 2,4,5-trichlorophenate),

Nurelle (2,4,5-trichlorophenol), and Septigard 25 (Alkyl tolyl methyl- trimethyl ammonium chloride). All of these chemicals had previously been found to be effective bactericides and had been used with relative safety on certain plants. REVIEW OF LITERATURE

The organisms causing many diseases of plants are present in

or on the seed. Disinfection of the seed is an important method of

reducing losses from seed-borne diseases, and treatment of seeds with chemicals for the control of plant diseases is a commonly ac­

cepted practice.

Seed treatment is used for protecting seeds and seedlings from

plant pathogens in the soil and to prevent infection by pathogens

borne in or on the seed. Chemicals in some cases decontaminate

seed surfaces, resulting in a lower incidence of disease, and in

other cases act internally to eradicate systemic seed infections.

One of the most important methods in seed treatment is the

heat method. Since its recommendation in 1882 by Jensen (as cited

by Martin, 22) hot water seed treatment has been, in spite of the

danger of impairing seed germination, widely used against fungal

and bacterial diseases in which the pathogen is carried within the

seed. Hot water treatment of seeds has been recommended for con­

trolling such seed-borne diseases of Cruciferae as black rot, black

leg, and Alternaria blight. Haskell and Doolittle (15) summarized

the data on temperature and duration of hot water treatments, as

presented by various investigators, and recommended hot water at

a temperature of 50°C for 25 minutes for cabbage, broccoli and brussels sprouts, and 15 minutes for cauliflower seeds„

The powerful bactericidal action of mercuric chloride naturally

led to its trial as a seed disinfectant. Kellerman and Swingle (18)

used mercuric chloride for the treatment of cereal grains. Hiltner

(as cited by Martin, 22) used it for control of Fusarium disease of

rye. Hiltner discovered that Calonectria graminicola (Bark & Br.)

Wr, is not carried over as spores on the exterior of the grain but

as dormant mycelium within the seed. The fungicide must remain

on the seed to prevent infection of the coleoptile. Hiltner*s obser­

vation thus indicated the possibility of utilizing a protective fungi­

cide for the control of diseases carried over within the seed.

Cook (9) and Gilbert (12), applied Semesan, Vasco 4 and

Ceresan to prevent decay of cabbage and spinach seeds, and seed

treatment with mercuric chloride to control cucumber angular leaf

spot. The most common concentration of mercuric chloride used

for treatment of vegetable seeds is 1:1000 for 20 minutes (9).

Mercuric chloride, because of its poisonous properties, has

largely been replaced by organic mercury compounds which are

less toxic (25). Kreitlow (20) used several chemicals, including

mercuric chloride in diethyl ether, 1:5 00, mercury bichloride 1:500

in 70 per cent ethyl alcohol plus 3 per cent acetic acid, and

1:20,000 gentian violet in 5 0 per cent ethyl alcohol plus 3 per

cent acetic acid, as seed treatments for control of bacterial bean

blight. He reported positive results not only in controlling the disease but also in increased yield and better quality.

In the last 30 years the number of chemicals used for the purpose of plant disease control has increased considerably. Some of the Dithiocarbamates and thiuram derivatives are used for seed treatment. For example thiram gives satisfactory control of

Colletotrichum lini (Westerd) Toch. on flax seed (22). Chloranil was selected in the trials at the Crop Protection Institute (11) for the protection of lima beans from damping-off and has since been used for the treatment of the seed of many other vegetables (3 ,4 ,

21).

The application of antibiotic substances in seed treatment for the control of seed-borne diseases has been investigated by numerous workers. In some cases they decontaminate seed surfaces, resulting in a lower incidence of disease, and in other cases act systemically to eradicate internal seed infections.

The first report of the use of an antibiotic as a seed treatment material was that of Brian and Hemming (5), who showed that gliotoxin had fungitoxic activity in tests with 3 small grain diseases.

Gliotoxin was applied as a dust treatment at a rate of 2 ounces of a

5 to 10 per cent mixture per bushel of seed. The higher concentra­ tions of gliotoxin caused a marked disease reduction of leaf spot

(Helminthosporium avenae) of oats and covered smut (Ustilago hordei) of barley, and bunt of wheat. Henery et_ al (16) used Acti-dione and streptomycin as a seed treatment for oat seed infected with Ustilago killeri Wille, and demonstrated that Acti-dione at a concentration of 10 ppm gave almost complete control of covered smut of oats without injuring the seed significantly, whereas streptomycin at 1000 ppm failed to control this disease. They concluded that the control of an important seed-borne fungus is possible by Acti-dione seed treat­ ment.

The possibility of using streptomycin against seed-borne phytopathogenic bacteria was suggested by Ark (l). Streptomycin was found to be toxic to 14 species of both gram-positive and gram- negative pathogenic bacteria. Cucumber seeds artificially inocu­ lated with Pseudomonas lachrymans and treated for 20 minutes with streptomycin solution at a concentration of 100 ppm were freed from the pathogen. In greenhouse studies (2) cucumber seeds which were field contaminated with Pseudomonas lachrymans and treated prior to planting with streptomycin at 100 ppm for 20 minutes produced plants which were free of the disease.

Katznelson and Sutton (17) compared the activity of six com­ mercially available antibiotics against a large number of species of plant pathogenic bacteria in vitro and found aureomycin to be the most potent. Aureomycin completely inhibited the growth of most xanthomonads, including Xanthomonas campestris. These investigators concluded that aureomycin not only can disinfest but also can dis­ infect the seeds which are internally infected with Xanthomonas campestris,. and that it might replace the hot water treatment.

They gave no evidence to support their speculation that the seeds had been disinfected, however.

Sutton and Bell (30) reported in 1954 that aureomycin used in water solution as a seed treatment was effective in controlling the black rot organism (X. campestris) of Cruciferae. Artificially in­ oculated rutabaga seed was treated with aureomycin at different concentrations, ranging from 1:1000 to 1:10,000 for a period of 30 minutes, and later dusted with Arasan or Spergon. Complete control was provided with aureomycin at 1:1000 in laboratory plate tests, greenhouse soil tests, and field trials with no impairment of seed germination. At a concentration of 1:500 germination was slightly retarded and temporary purple discoloration of young rutabaga seedlings occurred.

Recently Klisiewicz and Pound (19) investigated the possibility of controlling black rot of crucifers by treating seeds with antibiotics.

Streptomycin sulfate, Agri-mycin 100, Terramycin hydrochloride,

Aureomycin hydrachloride, and Achromycin hydrochloride were used in this work. Artificially inoculated seeds, when treated with anti­ biotic solutions for 30 minutes and plated on nutrient agar, developed no bacterial colonies except those treated with Streptomycin sulfate and Terramycin. Treated seed assayed for antibiotic absorption failed to show the penetration of the antibiotics through the seed coat of cabbage and broccoli seeds. The results of this work indi­ cated that complete disinfection of naturally infected cabbage and broccoli seed was not obtained with concentrations as high as

3,000 ppm, although the percentage of seedling infection was re­ duced appreciably. MATERIALS AND METHODS

Cabbage (Brassica oleracea var. capitata L.) and cauliflower

(Brassica oleracea var. botrytis) seeds were obtained from the Reuter

Seed Company, New Orleans, Louisiana.

The black rot organism (Xanthomonas campestris (Pammel)

Dowson, isolated from field infected cauliflower leaves by the dilution plate technique, was used for all inoculations.

The experimental samples of EP-166 were supplied by the Morton

Chemical Company, Septigard by the Thompson-Hayward Chemical

Company, and Morven and Nurelle by the Dow Chemical Company.

Preparation of chemical solutions throughout the experiments con­

sisted of mixing the chemicals with distilled water. All concentra­ tions tested are listed as ppm of active material of the chemicals.

Nutrient dextrose broth used for culturing the organism con­ tained the following ingredients: dextrose 10 g, peptone 10 g, beef

extract 3 g, and one liter of distilled water. For the agar medium

17 g of agar were added.

Commercial cabbage seed of high germinability was used for

germination tests of seed treated with various chemicals and for

tests of phytotoxicity of the chemical treatments to cabbage seedlings.

Cauliflower seed was used to determine chemical absorption by seed. 11

Artificially inoculated seed was used for seed treatment studies in the laboratory. Commercial cauliflower seed was artificially in­ oculated by soaking the seed in a heavy suspension of virulent X. campestris grown in nutrient broth. Seed was placed in an Erlenmeyer flask on a shaker and soaked for 5 to 6 hours. A period not less than

3 days was allowed for the seed to dry on filter paper in petri dishes at room temperature, prior to use in experimental work.

In treating seeds with chemicals, seed samples were placed in small bags made of cheesecloth and fastened with a rubber band.

The bags were immersed in the solutions for a determined period of time. A 30 minute seed treatment was generally used.

Chemical assay studies were made to determine the levels of the chemicals inhibitory to X. campestris. The filter paper disc-agar diffusion method was used as described by Grove and Randall (14).

Pyrex plates 6 inches in diameter were flooded with 3 0 ml of nutrient dextrose agar and 2 ml of a suspension of X. campestris in sterile distilled water with an optical density of 0.20 at 625 mu. Filter paper discs (Schleicher and Schull Analytical Filter Paper) with a diameter of 12.7 mm were dipped in chemical solutions at various concentrations ranging from 25 to 5 000 ppm, placed in a sterile glass plate and after drying for 24 hours were placed on the solidified agar

(Figure 1). Measurements of inhibition zones were made after a 24 hour incubation period at room temperature by measuring the diameter 12

Figure 1. Inhibition of Xanthomonas campestris by (counter­ clockwise from disc showing the least inhibition): 25, 500, 1800, 2500 and 4000 ppm of EP-166, using the filter paper disc-agar diffusion method. A similar arrangement was used in testing the other chemicals. 13 of the circle of inhibition . For each concentration of a chemical

10 paper disc replicates were used.

Seed assays similar to the paper disc assays were made to observe chemical absorption by treated cauliflower seeds. Whole seeds, seed coats, and seeds from which the seed coats were re­ moved were plated on nutrient dextrose agar seeded with bacteria.

Three of each of the seed parts were plated for each of the 6 chemical concentrations assayed on a single Pyrex plate. Zone measurements were made after a 24 hour incubation period at room temperature.

Treated seeds of each sample were stored at room temperature for 10 months for retesting to determine the stability of chemicals on treated seeds.

In comparative laboratory tests of various chemicals, a study was made to determine the effectiveness of the chemicals in dis­ infestation of seeds superficially contaminated with X. campestris.

Phytotoxic effects of the chemicals were studied in the green­ house. Seeds were treated with various chemical solutions. After a

24 hour drying period, they were planted in greenhouse flats. First readings were taken 7 days after planting. EXPERIMENTAL RESULTS

Levels of Chemicals Inhibitory to the Pathogen

Levels of the four chemicals inhibitory to _X. campestris were

determined by the filter paper disc-agar diffusion method, at con­

centrations ranging from 25 to 5 000 ppm. After a 24-hour incubation

period at room temperature, a uniform bacterial growth was present

in the inoculated agar plates and inhibition zones were clearly de­ veloped around the discs. The data in Table 1 show the inhibition of the organism by these chemicals at various concentrations. Al­ though 25 ppm was the lowest concentration assayed, activity of

EP-166 at that concentration indicated that this agent might inhibit the organism at less than 25 ppm.

Inhibition zones developing around the discs treated with

EP-166 at concentrations ranging from 25 to 1800 ppm were greater

than those of Morven and Nurelle at this range, while at concentra­ tions ranging from 1800 to 5 000 ppm, the diameter of inhibition zones

around the Morven and Nurelle treated discs exceeded those of EP-166.

The inhibitory level of Morven and Nurelle increased as the

concentration of the solutions increased. Septigard showed a low

level of activity at all concentrations tested.

With EP-166 and Septigard a point was reached, at a concentra­ tion of 4000 to 5 000 ppm, where no significant further increase in

14 15

Table 1. Inhibition of _X. campestris by various chemicals as deter­ mined by filter paper disc-agar diffusion method.

Chemical concentration Diameter** of inhibition zones (mm) ppm EP-166 Morven Nurelle Septigard

25 25.5 21.2 14.5 15.0

50 31.5 24.0 14.5 15.5

200 34.1 24.2 15.7 . 16.0

500 35.4 30.1 18.4 16.1

800 36.4 30.5 24.8 16.7

1000 39.0 30.8 26.7 17.0

1800 39.1 37.1 35.5 17.5

2500 39.5 42.0 47.0 18.6

3200 40.6 51.1 47.2 18.6

4000 44.5 58.0 52 o 0 19.6

5000 44.5 60.1 55 „ 2 19.8

*Diameter of filter paper disc is 12.7 mm.

**Average diameter of 10 paper disc replicates. 16 inhibition occurred. Morven and Nurelle showed an increase in inhibition between the two concentrations.

Chemical Seed Treatments of Artificially Inoculated Cabbage Seed

To determine whether chemicals were bactericidal or bacterio­ static, seeds artificially inoculated with X. campestris were treated at concentrations of 500, 1000, 1800, and 2500 ppm and then plated on nutrient agar (Figure 2). The plates were incubated at room temperature. Untreated seeds soaked in sterile distilled water were used as controls. Three replications of 100 seeds each were made for each chemical treatment. Twenty seeds were placed in each petri plate. All seeds were dusted lightly with Arasan prior to plating in order to reduce contamination by fungi. No bacterial colonies developed from seeds treated with EP-166, Nurelle and

Septigard at these concentrations, while colonies developing on agar plates from the untreated control seeds reached 96 per cent 3 to 4 days after plating. Seeds treated with the chemicals at 1000 ppm were plated and examined daily for a period of 16 days for the ap­ pearance of bacterial colonies. Again no bacterial colonies de­ veloped around EP-166, Nurelle, and Septigard treated seeds. From seeds treated with Morven, a few colonies began to appear 8 days after plating (Figure 2). Bacterial colonies which were suspected as

X. campestris were positively identified by inoculating cabbage plants 17

Figure 2. Method of assaying of chemicals on artificially inoculated seed. Plate at the lower right is an untreated check, the other three plates contain seeds treated with Morven at 1000 ppm. Disinfestation is measured by the reduced number contaminated seeds. 18 in the greenhouse and observing development of characteristic black rot symptoms. The colonies developed from Morven treated seeds were less yellow than those appearing on the control plates.

However, when cabbage plants were inoculated with these bacteria, characteristic symptoms of black rot were produced. It is possible that the whitish colonies resulted from a combination of bacterial pigment and the chemical with which the seeds were treated.

Assay of Treated Seeds for Chemical Absorption

To effectively control the black rot bacteria which invade the seed internally it is essential that the chemicals penetrate into the internal seed parts through the seed coat in sufficient amounts to kill the bacteria present. Assays of cabbage and cauliflower seeds treated separately with EP-166, Morven, Nurelle, and Septigard for

30 minutes at concentrations ranging from 5 00 to 5 000 ppm failed to show absorption of the chemicals into the seed.

At all concentrations of the chemicals used, clear zones of inhibition occurred around whole seeds and seed coats. Inhibition zones around the seed centers were limited in number and not con­ sistent. These inconsistent zones were probably due to tiny cracks in the seed coat through which the chemical solutions entered the seed. There may be some EP-166 absorption by the seeds, although it was not shown consistently with all seed pieces. The evidence 19 for this is that the percentage of EP-166 treated seeds showing inhibition zones around the seed centers was greater than those of Morven, Nurelle and Septigard treated seeds. This was true especially at higher concentrations.

Very few seed centers showed zones of inhibition when the seeds were treated with Morven, Nurelle and Septigard at 5 00 ppm.

In all seed assays, an increase in inhibition with increasing chemical concentrations occurred around whole seeds and seed coats. Although uniform seeds were selected for plating, some variations in the diam­ eter of inhibition zones occurred within the concentrations used. The average diameter of inhibition zones around seed coats was approxi­ mately 2 to 5 mm less than that around whole seeds.

Chemicals were adsorbed on the seed coat rather than absorbed into the seed. When seeds were subjected to longer treatments of 1 to 3 hours, the number of pieces showing inhibition zones was in­ creased as compared to the 30 minute treatment. However, this in­ crease was slight and the presence of chemicals was not detected in all seed pieces. This may have been due to rupturing of the seed coat, thereby enabling the chemicals to come in contact with the seed centers.

Seed lots were also treated in the presence of sterile, fine sand on an electric shaker for 30 minute and 1 hour periods. These treat­ ments failed to show very favorable effect on chemical absorption, however. 20

The most interesting point was that when sterile distilled water treated control seeds were plated after removing the seed coats, the center parts of some seeds showed distinct zones of inhibition, while no inhibition zones were observed around the seed coats. This ob­ servation made the detection of the chemical absorption by the treated seeds more difficult. This finding led to further research on the inhibitory effect of seeds on growth of X. campestris, which is the subject matter of the second part of this dissertation.

Stability of Chemicals on Treated Seed

Treated seeds can be stored before they are planted, even at room temperature, if the chemical with which seeds have been treated is stable and has no detrimental effect on seed germination.

In an experiment conducted to determine the stability of EP-166,

Morven, Nurelle, and Septigard, seeds were treated at 1000 ppm for

30 minutes, dried, and stored in Erlenmeyer flasks at room tempera­ ture. Mercuric chloride treated seeds were used in this experiment for comparison. Each month 100 seeds were plated on nutrient agar seeded with X._ campestris. The data in Table 2 show that the diam­ eters of the inhibition zones around the seeds treated with Morven,

Nurelle and Septigard were 20, 15, and 10 mm, respectively, 24 hours after treatment. Morven treated seeds did not show any inhibition zones after a month, and this was true for Nurelle, Septigard and 21

Table 2. Stability of chemicals on treated seeds.

Treatments Diameter* of inhibition zone 1000 ppm 2 1 2 3 4 5 6 7 8 9 10 (days) — - --(months)

Check 0 0 0 0 0 0 0 0 0 0 0

Mercuric chloride 14 12 0 0 0 0 0 0 0 0 0

EP-166 25 24 21 21 20 20 21 20 20 20 20

Morven 20 0 0 0 0 0 0 0 0 0 0

Nurelle 15 8 0 0 0 0 0 0 0 0 0

Septigard 10 5 0 0 0 0 0 0 0 0 0

*Average diameter in mm of inhibition zones around 100 seeds. 22 mercuric chloride after 2 months. EP-166 treated seeds showed distinct inhibition zones when plated on nutrient agar seeded with

X. campestris even if the treated seeds were stored for 10 months.

During this period there was no evidence of impairment of seed germination.

Comparison between the diameter of inhibition zones of treated seed after 24 hours and 10 months indicated that the EP-166 could remain active on treated seed, without impairing seed germination and thus can act as a protectant for the seed. Morven, Nurelle, and Septigard did not remain stable on seed and their activities were lost after a relatively short period of time.

Effect of Chemical Treatments on Germination of Cauliflower Seed

In attempting to control a plant disease with chemicals, the possibility of injurious effects of these chemicals on plants or plant parts must be considered. Since a seed treatment with chemical solutions was an object of this study, the effect of the chemicals on cauliflower seed germination was evaluated by germination tests.

Separate lots of cauliflower seeds were treated at 250, 500, and 1000 ppm with the various chemical solutions for a period of 30 minutes. The treated seeds, after a drying period of 24 hours, were plated on moistened filter paper in 12 x 6 inch glass plates. Twenty replications were made for each treatment with 25 seeds per replica­ tion. Seeds soaked in distilled water for 30 minutes were used as 23 controls. Plates were held in room temperature for a period of 15 days. Germination data for each treatment were taken each day.

These are presented in Figures 3, 4, and 5.

In general there was no marked impairment of seed germina­ tion at concentrations of 25 0 or 5 00 ppm when seeds were treated with EP-166, Morven, and Septigard. Nurelle treated seed at concentrations of 1000 ppm inhibited germination up to 87 per cent.

In tests to determine whether any of the chemical treatments retarded seed germination, it was shown that the germination of un­ treated control seed was complete within 3 days after plating, but on seed treated with chemicals the germination was delayed. At all concentrations used,'germination of EP-166, Morven, and Septigard treated seeds was complete within the first 3 days, although the number of germinated seeds treated with Morven and EP-166 at a concentration of 1000 ppm, was lower on the first 2 days than on the third day. Germination of Nurelle treated seed at concentrations of 25 0 and 5 00 ppm was delayed until 5 and 9 days, respectively, and at concentrations of 1000 ppm only 10 per cent of Nurelle treated seeds were germinated 4 days after plating.

Reduction of per cent germination usually increased with increase in concentration of the chemical solutions. Duration of treatment in­ creased the amount of time required for germination. Chlorosis also occurred on some of the germinated seedlings. EP-166 treated seeds 24 8 E P -1 6 6 f c NURELLE MORVEN SEPTIGARD DA Y 5 Y DA 4 chemicals at a concentration of 250 ppm. - 90i 10 2 0 . lOtf 80. 6 0 . 4 0 .

NO/1 VN/W&30 ■/■ Figure 3. Retardation of germination of cauliflower seed treated with Figure 4. Retardation of germination of cauiiflow er seed treated with with treated seed er cauiiflow of germination of Retardation 4. Figure

* * GERM/NA T/ON 0 3 0 4 \oa 50 0 6 60 90 . j . chem icals at a concentration of 500 ppm. Keys for the the 3. for Keys Figure in ppm. given 500 are of ents treatm concentration a various at icals chem 2 3 4 DAYS 5 6 7 -_S, 25 I 2 3 4 5 6 DAYS

Figure 5. Retardation of germination of cauliflower seed treated with chemicals at a concentration of 1000 ppm. Keys for the various treatments are given in Figure 3. ’ 27 showed neither reduction of germination nor chlorosis on the seedlings, even at higher concentrations.

Effect of Chemical Seed Treatment on Root and Stem Growth of Cauliflower Seedlings

To determine whether these seed treatments affected the growth of seedlings, seeds treated for 30 minutes with the various chemicals at 5 00 ppm were placed on filter paper moistened with distilled water in 12 x 6 inch glass plates. Root and stem measurements taken 7 days after treatment, are given in Table 3.

Marked inhibition of root and stem growth was caused by

Septigard and Morven. Stem growth was inhibited when Nurelle was used. EP-166 caused considerable increase in root growth.

Effect of Chemical Solutions on Cabbage Seedlings Grown from Treated Seed

Observations of phytotoxic effects to seedlings grown from seeds treated with chemicals were made on greenhouse-grown cabbage seedlings. Seeds were treated for 30 minutes at concentrations of 250,

500, 1000, and 2500 ppm and allowed to dry for 24 hours after which the seeds were planted at a rate of 100 seeds per flat. The planting mixture contained sterile soil, sand, and peat moss in a ratio of 4:1:2.

The first readings were made 7 days after seedling emergence, and ratings were made. The phytotoxic effects of the chemical solu­ tions on cabbage seedlings grown from treated seeds are shown in 28

Table 3. Effect of chemical seed treatments on root and stem growth of cauliflower seedlings.

Number of roots and stems Average lenqth (mm) Treatment* measured Roots Stems

Distilled water 174 31.8 38.3

EP-166 183 39.9 37.5

Morven 177 23.7 30.5

Nurelle 189 30.8 30.1

Septigard 181 25.8 28.7

*Chemical solutions of 5 00 ppmx 30 minute treatment. 29

Table 4. At a concentration of 25 0 ppm some reduction in germina­ tion occurred with Morven. At concentrations of 500 and 1000 ppm a noticeable reduction in germination of Morven and Nurelle treated seeds occurred. Septigard treated seeds showed reduced germina­ tion at 1800 and 25 00 ppm. Complete inhibition of germination was recorded on Nurelle treated seeds at a concentration of 25 00 ppm.

A vigorous growth comparable to that of the control was obtained with seeds treated with EP-166 at all concentrations used.

The number of stunted plants grown from seed treated with various chemicals was not large, but there was an increase in the number of stunted plants with an increase in concentration of the chemical solutions.

Phytotoxicity to seedlings in the cotyledonary stage generally appeared in the form of chlorosis. On cotyledons of cabbage this was a general whitening or yellowing in the case of severe phyto­ toxicity. In some cases a purple discoloration occurred on the

seedlings. A slight chlorosis was evident on seedlings grown from

seed treated with EP-166, while chlorosis was more pronounced on

seedlings grown from Morven, Nurelle and Septigard treated seeds.

As with stunting and reduction in germination, the amount of chlorosis t increased with increasing concentration.

Affected plants generally recovered their normal green color, but the period required for recovery seemed to be related to the 30

Table 4. Phytotoxic effect of various chemical seed treatment to emergent cabbage seedlings.

Description of phytotoxicity o//o % % °//o chlorotic Rate of stunted dead Chem icals ppm emergence plants chlorosis* plants seedlings

Check 78 0.0 0.0 0.0 0.0

EP-166 250 79 2.3 + 6.3 0.0 500 79 4.0 + 6.3 2.7 1000 74 4 .0 ++ 5.4 1.3 2500 75 6.6 ++ 10.6 2.6

Morven 25 0 67 4.4 ++ 4 .4 0.0 500 56 5.4 +++ 7.1 1.8 1000 38 13.1 +++ 23.6 4.7 2500 35 17.1 +++ 28.5 5.7

Nurelle 25 0 78 5.1 + 9.1 0.0 5 00 28 14.2 +++ 17.8 7.1 1000 25 20.0 +++ 32.0 12.0 2500 0 - - - -

Septigard 800 72 5.5 + 11.1 4.1 1000 70 8.5 ++ 12.8 2.8 1800 58 17,2 13.8 5.1 2500 57 14.0 +++ 15.8 7.0

*Ratings were as follows: +++ = cotyledons completely chlorotic ++ = moderate, partial chlorosis of both cotyledons + = slight, marginal chlorosis of one or both cotyledons 0 = no phytotoxicity.

1 severity of chlorosis. Slightly chlorotic plants recovered more rapidly than did severely affected ones. Severely chlorotic seedlings

died without becoming normal in color. Chlorosis of the first true leaf

and successive leaves did not occnr in any case regardless of the

chemical concentrations used. The mortality rate of the seedlings varied with different chemicals, but was not appreciable in any case. DISCUSSION

The primary objective of this study was to attempt to find a bactericide capable of destroying the black rot organism in and on contaminated seed without injuring the seed.

The black rot bacteria can contaminate the seeds in either of two ways. They can be present on the surface of the seed, in which case control by disinfestation of the seed is a relatively simple matter, or the seeds can be infected internally, usually be­ neath the seed coat or between the cotyledons. In the latter case, control is a much more difficult problem, and several factors must be considered when attempting to find an agent capable of controlling the disease. The toxicity of the agent to the bacteria and its ability to penetrate into the internal parts of the seed where the bacteria are located, without appreciably affecting the ability of the seed to germinate and produce a normal plant should be the foremost of these considerations.

In vitro tests showed that all of the chemicals tested possessed activity against X. campestris at concentrations as low as 25 ppm, although the activity of the chemicals varied greatly. Inhibition by

Morven and Nurelle increased with increasing concentration, up to the highest level tested. Septigard showed a low level of activity

32 33 at all concentrations tested. EP-166 had a much greater activity than the other chemicals, at low concentrations„ With both EP-166 and Septigard, however, a concentration was reached (4000-5000 ppm) at which no significant increase in antibacterial activity occurred, despite increases in concentration.

Correlations between the concentrations at which these chemicals were capable of disinfesting artificially inoculated seed without impairing germination or showing phytotoxic effects on the seedlings indicated that the majority of them have a limited useful­ ness. Septigard, although it showed very little activity in the agar disc tests, completely disinfested artificially inoculated seeds at concentrations as low as 5 00 ppm. The disparity of these results may have been due to its inability to diffuse into the agar media.

This compound showed little phytotoxicity to cabbage and cauliflower when tested at concentrations capable of disinfesting the seeds, although it was quite phytotoxic at slightly higher concentrations.

The activity of Nurelle, as measured by the paper disc method, was quite low at concentrations below 5 00 ppm. Seeds were dis­ infested at a concentration of 500 ppm, but phytotoxicity, measured as reduction in germination, was great. Morven, although notice­ ably more active than Nurelle in the paper disc tests, particularly at concentrations below 1000 ppm, did not completely disinfest the seeds at concentrations as high as 1000 ppm. A reduction in the 34

amount of contamination, as compared with the check, did occur, but some phytotoxicity occurred at concentrations as low as 250

ppm. EP-166 showed activity at 5 0 ppm comparable to that of the other chemicals at 1000 ppm or higher. With EP-166, disinfesta­ tion of seeds was obtained at 5 00 ppm, and no significant phyto­ toxicity was observed at this concentration. Treatment of the seeds with concentrations as high as 25 00 ppm resulted in very little

phytotoxicity.

Attempts to determine whether any of these chemicals could

control the bacteria on the internal seed parts yielded negative

results. Attempts to produce infected seeds by means of inoculating

the peduncle of flowering cabbage plants with a virulent suspension

of X. campestris were unsuccessful. Samples of infected cabbage

and broccoli seeds obtained from other sources contained such low

percentages of infected seeds that it was impossible to use them

in testing uptake of the chemicals. Consequently, an indirect

approach, that of assaying the internal parts of treated seeds after

careful removal of the seed coats, had to be used. Although some

of the seed centers showed activity after 30 minutes treatment when

tested by this method, the percentage was so small that it could

probably be accounted for by the presence of small cracks in the

seed allowing the chemicals to come through. Treating the seed for

longer periods of time increased the number of seed centers showing 35 activity, but loosening and splitting of many of the seed coats also occurred, indicating that this was probably the reason for the increase. It is also possible that some of the chemicals may be absorbed by the seed, but not in sufficient quantities to be de­ tected by this method, or that they may be absorbed and then tied up by the seed, thus inactivating them. At any rate, the results of the tests to determine if uptake of the chemicals occurred can only be regarded as negative.

On the basis of these results, it is clear that the chemicals

Morven and Nurelle should not be considered as seed treatments for

Crucifer seed, as the concentrations inducing phytotoxicity are lower than those capable of disinfesting the seed„ Septigard may be of some use as a disinfestant but the range between phytotoxicity and control is small0 In addition this compound does not remain stable on the seed. EP-166 shows considerable promise as a dis­ infestant for Crucifer seed. At concentrations which disinfested the seed, virtually no phytotoxicity was noticed, and plants often appeared to be stimulated. In addition, the activity of this compound against a wide range of bacteria and fungi combined with its stability on treated seeds for long storage periods, indicates that it may be of use in treatment for Crucifer seeds.-

The usefulness of EP-166 as a means of controlling X. campestris in Crucifer seed is worthy of consideration. In any given 36 lot of diseased seed, both types of contamination, infestation and infection, can probably be found. The relative amounts of these types of contamination will depend to a great extent on the type and stage of infection occurring in the seed producing plants, which in turn will be dependent upon environmental and cultural factors. In general, seeds from plants which become systemically infected at an early stage are more likely to be internally infected, while seeds from plants in which the seed pods are locally infected are more likely to be infested on the surface of the seed coat.

Although a considerable amount of internal infection can also take place from local seed pod infections, a great many of these render the seed incapable of germination, and hence minimize its role as a possible source of secondary infection.

As with almost any plant disease, a reduction in the amount of primary inoculum will generally increase the amount of time re­ quired for the secondary spread of the disease. Consequently, a treatment capable of reducing the amount of infected seed would be of some value. With the black rot disease, however, secondary spread can be so rapid that even a few infected seeds are capable of initiating a devastating epidemic, particularly in the seed bed.

With these facts in mind, the use of a chemical such as EP-166 should probably only be considered when complete disinfection, by means of the hot water treatment, is not possible. SUMMARY

Aqueous solutions of EP-166 (9- (p-n-hexyloxyphenyl)-10- methyl -acridinium chloride), Morven (sodium 2,4,5-trichlorophenate),

Nurelle (2,4,5-trichlorophenol) and Septigard 25 (Alkyl tolyl methyl- trimethyl ammonium chloride) were tested as 30 minute seed treat­ ments in the laboratory and greenhouse for control of the black rot disease of Cruciferae caused by the bacterium Xanthomonas campestris.

In vitro tests showed that all of the chemicals tested possessed activity against X. campestris at concentrations as low as 25 ppm, although the activity of the chemicals varied greatly. EP-166 had a greater activity than the other chemicals at low concentrations.

Septigard showed a low level of activity at all concentrations tested.

With both EP-166 and Septigard, however, a concentration was reached

(4000-5 000 ppm) at which no significant increase in antibacterial activity occurred, despite increases in concentration. Inhibition by

Morven and Nurelle increased with increasing concentration, up to the highest level tested.

Complete disinfestation of seeds artificially inoculated with

X. campestris was obtained with EP-166, Nurelle and Septigard at concentrations of 5 00 ppm. A few bacterial colonies developed from

37 38 seqds treated with Morven at concentrations of 500 and 1000 ppm

8 days after plating. Colony development was observed as early as on the third day following plating in the case of nontreated artificially inoculated seed.

Assay of internal seed parts, by means of a bioassay on agar plates seeded with X. campestris. for evidence of chemical absorp­ tion by seed treated with these chemicals yielded negative results, except in a few cases which can probably be attributed to minute cracks in the seed coats. However, the number of EP-166 treated seeds showing inhibition zones when plated after removing seed coats was greater than that of other chemicals, indicating there may be some uptake of EP-166. It is possible that the chemicals are absorbed by the seed but not in sufficient amounts to be detected by the technique used, or that they may be tied up by the seed.

Since naturally infected seeds were not available for use in these tests, it is not possible to state definitely that these chemicals can not disinfect the seeds.

The effect of chemical seed treatments on percentage germina­ tion and on root and stem growth of cauliflower seedlings was studied.

No marked reduction in seed germination occurred with EP-166, Morven, and Septigard at 1000 ppm but Nurelle treated seeds at the same concen­ tration caused an 87 per cent reduction in germination. Retardation of germination occurred on Nurelle and Septigard treated seeds at 39 concentrations of 500 ppm. Retardation and reduction of germination were more pronounced at higher concentrationsRoot and stem growth was inhibited by Nurelle, Marven and Septigard, but EP-166 caused considerable increase in root growth. Phytotoxicity, manifested as chlorosis, stunting, and mortality rate of seedlings grown from seeds treated with chemicals was generally not great in the case of EP-166 treated seeds, but Morven, Nurelle, and Septigard showed consider­ able phytotoxic effects.

The ability of EP-166 to disinfest the seed, combined with its low level of phytotoxicity to seeds and seedlings and its stability on treated seeds indicate that it may be of some use as a disinfestant and seed protectant. Its apparent inability to penetrate the seeds limits its usefulness as a seed treatment for black rot, however. LITERATURE CITED

Ark, P. A. 1947. Effect of crystalline streptomycin on phyto- pathogenic bacteria and fungi. Phytopathology 37: 842. (Abst.)

______. 1949. Use of streptomycin in agriculture. Jn_ Streptomycin S. A. Waksman. Ed. Williams Wilkins Co., Baltimore, pp. 607-617.

Arndt, C. H. 1953. Evaluation of fungicides as protectants of cotton seedlings from infection by Rhizoctonia solani. Plant Disease Reptr. 37: 397-400.

Arndt, C. H ., Chairman. 1947. Summary of cooperative tests of cotton seed treatments. Plant Disease Reptr. 31: 204-211.

Brian, P. W. and W. G. Hemming. 1945. Gliotoxin, a fungi­ static metabolic product of Trichoderma viride. Ann. Appl. Biol. 32: 214-220.

Chupp, C. and A. F. Sherf. 1960. Vegetable diseases and their control. The Ronald Press Co., New York. 692 pp.

Clayton, E. E. 1925. Second progress report of black rot in­ vestigations on Long Island seed infection and seasonal development. Phytopathology 15: 48-49 (Abstr.).

______. 1929. Studies of the black rot or blight disease of cauliflower. New York (Geneva) Agric. Exp. Sta. Bulletin, 576, 44 pp.

Cook, H. L. 1937. Spinach and cabbage seed treatment. Va. Truck Exp. Sta. Bulletin 96: 1491-1510.

Cook, A. A., R. H. Larson, and J. C. Walker. 195 2. Relation of the black rot pathogen to cabbage seed. Phytopathology 42: 316-320.

Cunningham, H. S. and E. G. Shamelle. 1940. Organic seed protectants for lima beans. Phytopathology 30: 4. (Abstr.)

Gilbert, W. W. and M. W. Gardner. 1918. Seed treatment, control, and overwintering of cucumber angular leaf spot. Phytopathology 8: 229-233. 41

13. Gorman, H. 1891. A,bacterial disease of cabbage. Bot. Gaz. 16: 265.

14. Grove, D. C. andW. A. Randall. 1955. Assay methods of antibiotics. Medical Encyclopedia, Inc., New York. 238 pp.

15. Haskell, R. J. and S. P. Doolittle. 1940. Vegetable seed treatments, U. S. D. A. Farmers Bulletin 1862. 9 pp.

16. Henery, A. W , , E. A. Paterson, R. L. Millar, and J. S. Harricks. 1951. Control of covered smut of oats by seed treatment with an antibiotic. Science 113: 390.

17. Katzuelson, H. and M. D. Sutton. 1961. Inhibition of plant pathogenic bacteria_in vitro by antibiotics and quaternary ammonium compounds. Canadian J. Bot. 29: 270-278.

18. Kellerman, W. A. and W. T. Swingle. 1890. Preliminary experi­ ments with fungicides for stinking smut of wheat. Kansas Agric. Exp. Sta. Bulletin 12: 27-50.

19. Klisiewicz, J. M. and G. S. Pound. 1961. Studies on control of black rot of crucifers by treating seeds with antibiotics. Phytopathology 51: 495-5 00.

20. Kreitlow, K. W. 1940. Seed treatment for the control of bacterial bean blight. Phytopathology 30: 14. (Abstr.)

21. Leukel, R. W. 1947. Relative effectiveness of certain fungi cides as seed protectants and disinfectants. Plant Disease Reptr. 31: 476-478.

22. Martini, H. 1959. The scientific principles of crop protection. Edward Arnold, Ltd., London. 359 pp.

23. Monteith, J. R. 1921. Seed transmission and overwintering of cabbage black rot. Phytopathology 11: 53-54.

24. Pammel, L. H. 1892. Bacteriosis of rutabaga. Iowa Agric. Exp. Sta. Bui. 27: 130.

25. Peterson, S., W. Gauss, und E. Dusbschat. 1955. Synthese einfacher chinonderivate mit fungiziden, bakteriostatischen odes cytostatischen Eigenschaften. Angew. Chem. 67: 217-230.

26. Rose, G. J. 1955. Crop protection. Leonard Hill, Ltd., London, 223 pp. 42

27. Russell, H. L. 1899. A bacterial rot of cabbage and allied plants. Wis. Agric. Exp. Sta. Bui. 65: 38 pp.

28. Smith, E. F. 1899. The black rot of cabbage. U. S. D. A. Farmers Bulletin 68. 22 pp.

29. ______. 1911. Bacteria in relation to plant diseases. Vol. 2. Washington, D. C ., Carnegie Institution of Washington.

30. Sutton, M. D. and W. Bell. 1954. The use of aureomycin as treatment of swede seed for the control of black rot fXanthomonas campestris), Plant Disease Reptr. 38: 547-552.

31. Walker, J. C. 1952. Diseases of Vegetable Crops. McGraw- Hill Book Company, Inc., New York. 529 pp. PART II

INTRODUCTION

Following the work of Fleming, Flory, Waksman and many other pioneers showing that antimicrobial substances present in lower plants have important biological significance, an intensive effort in investi­ gating the vast field offered by higher plants has been made. The presence in higher plants of substances with antibacterial properties has been reported by many investigators (27, 32, 35, 40, 49, 51).

In the course of a study carried on to determine the absorption and uptake of various chemicals by Brassica seeds, it was found that cauliflower seeds, when soaked in sterile distilled water and plated on nutrient agar medium seeded with Xanthomonas campestris

(Pammel) Dowson, inhibited the growth of this bacterium.

This paper presents the results of further experiments to determine the spectrum of activity of the substance present in the seeds, and to attempt to determine some of its physical and chemical properties.

43 REVIEW OF UTERATURE

Our earliest knowledge of the antimicrobial properties of

microorganisms and various other natural materials can be traced to folklore (62). It is only with the beginning of the fourth quarter of the last century that we find specific references to this subject.

According to Goldberg (21), Gratia and Dath were among the

first to begin a systematic search for antagonistic organisms from

natural sources. They extracted a lytic agent from a mold and used

it in successful treatment of staphylococcal skin infections. Such work progressed rapidly among soil and plant microbiologists of the

day, and reports emerged confirming antagonism by fungi. The

occurrence of antagonistic properties of molds thereby became an

accepted fact, culminating in the discovery of penicillin by Fleming

in 1929.

Since the discovery of penicillin and the subsequent demon­

stration of its antibacterial activity in vivo, considerable interest

has been centered on various lower and higher plants in an attempt

to discover other antimicrobial agents. The results of Osbom*s study

(45) indicated that plants containing antibacterial substances are widely distributed in nature and offer a fertile field for investigations.

The practical use of antibacterial substances in green plants,

44 45 especially those commonly used as food, in the prevention and cure of diseases had remained in the speculative stage until a group of Russian workers (5) demonstrated that volatile sub­ stances, especially those emitted from fresh onion or garlic pastes, were highly effective in the treatment of infected wounds in rabbits and humans. There are now available the results of a considerable number of studies pertaining to the bacteriostatic and bactericidal action of substances contained in one or more parts of green plants

(26, 51).

McKnight and Lindergren (40) and W alton et a l. (60) showed that the vapors from freshly crushed garlic were germicidal to certain organisms including strains of acid-fast and nonacid-fast

Mycobacterium leprae. Later Lovell (39) reported that onion vapors were also bactericidal but to a lesser extent than garlic vapors. The volatile substances responsible for the bactericidal action were later determined to be crotonaldehyde and acrolein by Ingersol et al. (27) and Richard et^cH. (49). Walker and Link (59), and James and Higgins

(32) also are given credit for their reports on the growth inhibiting properties of onion juice toward several species of bacteria and fungi.

For centuries, plant drugs have been used in all parts of the world as folk remedies. While some drugs have been accepted for medical purposes, most of them have remained in obscurity and their employment has often been regarded as superstitious (42). Lewis and 46

Lucas (35) thought that some of these plants might contain sub­ stances adverse to the development of undesirable microorganisms in the human body. They examined the root systems of plants men­ tioned in the folklore of numerous countries as being beneficial in their application in the case of bacterial infections. Their con­ clusion was that some higher plants contain antibacterial substances at certain stages of their development. The applicability of some of their findings to plant pathological problems, particularly in connec­ tion with the treatment of seed-borne diseases is envisaged by some investigators (35). They support the idea that the resistance of some plants to disease is due to the presence in the host cell of distinct chemical substances which are antagonistic to specific pathogens.

In recent years the occurrence of possible antifungal substances in plants has been investigated by several workers (4, 34, 48). Rela­ tively greater resistance to certain diseases has long been known in those varieties of onion in which red or yellow pigments are produced in the bulb scales as they approach maturity. It is especially notice­ able in the case of smudge caused by Colletotrichum circinans and neck rot caused by Botrytis spp. The nature of this resistance was first studied by Walker (55, 56). It was found that in colored onions a water-soluble toxic substance occurred in the outer scales of the bulbs. This substance inhibited the growth of the smudge and neck rot organisms. 47

Further studies (24, 27, 59) showed that the extracts of colored onion scales were toxic not only to the smudge and neck rot organ­ isms but also to several other fungi tested. Link and Walker and their co-workers (2, 33, 36, 37, 38) isolated a water-soluble toxic substance from the outer scales of colored onions and identified it as the phenolic acid commonly known as protocatechinic acid.

Ranker (48) tested filtrates from various parts of corn plants against Ustilago zeae. He found that filtrates of the husk tissue of resistan t varieties such as Silver King No. 67, Silver King No. 72 and Rustler No. 35 markedly inhibited growth of this fungus, while the juice from the leaves of Salmon Silk No. 14, a moderately re­

sistant variety, possessed the greatest relative inhibitory action.

The results showed that some resistant varieties of corn contained

soluble substances in the tissue juice which inhibited the growth of the fungus. In some of the lines the substance was more inhibiting in the husk juice, as in Silver King No. 87, while in other lines the leaf juice was relatively most inhibiting to the fungus growth, as in Salmon Silk No. 14.

Extensive work has been done in the Biochemical Institute of

Finland on isolation of antifungal substances from plants and the determination of their chemical structure. Arturii and his co-workers

(4) reported that the pressed juice of green rye seedlings possessed

strong antifungal activity and the active part of the juice was 48 determined to be 2(3)-benzoxyazolinone. This substance inhibited the growth of Fusarium nivale, Sclerotinia trifolio rum and others.

Since the benzoxyazolinone content of the lower leaves of rye seedlings is less than that of the stem and apical leaves, fungi can destroy them in spring without subsequent destruction of the seed lin g s.

The same laboratories also reported antimicrobial substances from young wheat, maize, alfalfa, red clover and potato leaves. In potato leaves, chlorogenic acid was found to occur in a concentration high enough to inhibit the late blight organism (Phytophthora infestans).

Fusarium wilt, caused by Fusarium oxysporum f. lycopersici

(Snyder and Hansen), is one of the most prevalent and damaging diseases of tomatoes in many regions of the United States. The mechanism of the wilting caused by this vascular parasite is not definitely known, but wilting of tomato plants infected with Fusarium oxysporum f. lycopersici can be correlated with the presence in the tracheal fluid of the host of a toxin which is presumably elaborated by the fungus.

Since tomato varieties vary in their susceptibility to Fusarium wilt it might be postulated variously; (l) that certain varieties are wilt resistant because they are able to produce a substance or sub­ stances which either neutralize the toxin directly or inhibit the growth of the fungus; (2) that certain varieties are susceptible because they produce a substance or substances that promote or make possible growth of the parasite; or (3) that differences in susceptibility or resistance are due to differences in amounts of such substances common to both categories of plants. Research has been carried out by many investigators concerning determination of an antifungal substance in resistant tomato varieties. Fisher (17) and Gottlieb

(23) obtained evidence to indicate that the expressed juice from tomato plants retards growth of F. oxysporum f. lycopersici in culture in proportion to the wilt resistance of the tomato varieties tested. Porte and Walker (47) showed that the expressed juice of

Pan American tomato plants (highly wilt resistant variety) possessed marked fungistatic activity toward the wilt fungus. This antibiotic agent, designated "lycopersicin," was shown to be stable for at least 1 hour at 100°C and withstood autoclaving for at least 15 minutes at 15 pound pressure. It was dialyzable, adsorbed from aqueous solution at pH 5.5 on charcoal (activated Norite A), soluble in water and methanol, partially soluble in ethanol, and insoluble in chloroform, acetone, ethyl acetate, ether, petroleum ether and benzene.

Irwing and Fontaine (28) did further research on the activity of lycopersicin and reported that of the different susceptible and resistant tomato varieties tested all contained the inhibitor in various concentrations. Lycopersicin activity, while absent in the seed, 50 appears in seedlings germinated in the dark and in the plant within

8 days after planting. The concentration of lycopersicin varies with the age of the plant and considerably with the plant part assayed.

Extensive work has since been done in regard to the partial purifica­ tion of tomatin (previously lycopersicin) and determination of its properties. It has been found that tomatin has an inhibitory effect upon a considerable number of microorganisms, especially human dermatophytic fungi such as Microsporum. Epidermatophyton,

Trichophyton, and Achorion and several other fungi (18, 19). Bacteria

are also inhibited by this substance (19, 29).

Bishop and Russel (9) in Canada screened 209 plants for anti­ bacterial substances. Solvents such as distilled water, alcohol,

ether, acetone, and benzene were used to extract substances from

each plant. They showed that 33.4 per cent of the plants tested

contained one or more substances inhibitory to Staphylococcus aureus

and/or Escherichia coli. From 940 extractions made using various

solvents 15 per cent were active against_S_. aureus and 4.6 per cent

against E. coli. The highest incidence of activity was obtained with

the ether extractions. These workers also reported that in many

cases the extracts of certain plants caused stimulation rather than

inhibition of the test organisms and concluded that stimulatory sub­

stances may reduce the effect of any inhibitory substances present.

A similar investigation was conducted in the Philippines. 51

Three hundred and twenty nine species of higher plants from 95 families were tested for the presence of substances inhibitory to

S_. aureus and E. coli. The results showed the presence of anti­ bacterial substances in some of the plants tested, according to

Victoria et a l. (5.4).

Studies by Carlson and Douglas (11) indicated that the use of certain solvents resulted in the detection of antimicrobial sub­ stances not evident in pressed juice extracts. Reports by Sanders and his co-workers (50) on 120 plants collected in India added further information concerning the presence of antibacterial substances in various plants from different regions.

Ardeth et al. (3) showed the in vitro activity of root extracts of Solanum pseudocapsicum against Mycobacterium tuberculosis.

Subsequently, the active principle was also found in the leaves and

stems and was identified as the alkaloid solanocapsine. This alkaloid had first been characterized by Barger and Fraenkel-Conrat (7).

Further studies showed that crude as well as purified preparations of the alkaloid base had moderate to considerable activity against a number of microorganisms (10).

Werner and associates (63) reported antimicrobial activity in extracts of Clematis dioscoreifolia, and the purification of the active principle. The active principle was shown to be identical with

"anemonin" by analysis, color reactions and mixed melting point. 52

Anchel (l) employed a simple method for isolation of the antibiotic principle from the leaves of Cassia reticulata and rhubarb root.

The antibiotic substance which was previously named "cassic acid," was identified as 4,5-dichydroxyanthraquinone-2-carboxylic acid.

Carlson and Douglas (12) reported the antibiotic activity of

an oil fraction separated from the root of Leptolaenia dissecta de­ termined on 62 strains and species of bacteria and fungi. The

active substance was heat stable and bactericidal for G+ bacteria

at 10“4 dilution and at 10~3 dilution for G~ bacteria.

Twenty-three genera belonging to 15 families of plants were

screened for the presence of possible antibacterial substances by

Huddleson and his co-workers (26). Three different plants, two of which belonged to the same genus, containing considerable amounts

of antibacterial substances, were selected for chemical studies to

determine therapeutic possibilities in the treatment of brucellosis in the guinea pig. It was found that the substances present in

selected plants could be used as preventives of infectious diseases through ingestion.

Several previous reports have been made regardint the oc­

currence of antimicrobial activity in plants of the family Cruciferae.

Sherman and Hodge, at Cornell University, in 1946 demonstrated a relatively small but distinct action in raw juices pressed from the heads of cabbages and from the roots of turnips. They stated that the magnitude of this "germicidal action" was somewhat greater than that found in freshly drawn milk but not nearly as great as that of fresh normal blood. No positive results were obtained in an attempt to demonstrate a similar property in juice pressed from carrots, cucumbers and parsnips (51). Pederson and Fisher (46) followed the work of Sherman and Hodge (51), and identified the more common organisms usually present on cabbage. They tested the bactericidal effect of cabbage juice upon these organisms as well as those causing the natural fermentation of cabbage.

They also compared the effect of other vegetable juices with the effect of cabbage juice on these bacteria. In confirmation of

Sherman and Hodge*s work, they stated that the bactericidal sub­ stances in cabbage tissue caused a marked reduction in the number of bacteria which are commonly present upon the surface of a leafy vegetable such as cabbage. Osborn (45) showed that inhibitory action of the extract of cabbage seeds was much greater than that of the expressed juices from heads of cabbage.

Horvath and IvanovicB (25) reported the presence of antibacterial substances in different plants belonging to the family Cruciferae. Later these authors isolated an antibacterial substance from radish (Raphanus sativus) seed, and named it Raphanin. Aqueous extracts of the seeds were very effective in preventing the growth of bacteria (Staphylococcus aureus, Bacillus coli). Since the active part was heat stable and 54 dialyzable and no activity was obtained when the seeds were boiled

15 minutes before crushing them, it was assumed that the seeds contained an enzyme capable of converting an inactive precursor into an antibiotic. The physical, chemical, antibiotic and toxic properties of Raphanin have been studied, but its exact structure has not been demonstrated (30, 31).

Foter and Golick (20) stated that the vapors from crushed horseradish roots have a strong inhibitory effect on Escherichia coli.

Bacillus subtilis, Mycobacterium phlei, and M. tuberculosis var. ho minis. This inhibitory effect is greater than that of garlic and onion but is more quickly exhausted than that of garlic, when tested under similar conditions.

A number of saturated and unsaturated fatty acids and related compounds were tested for their antimicrobial activity by Clark, in

1899 (13). Later other workers (44, 52) revealed many facts about the properties of these compounds. Research in this area is still in progress.

Gooding (22) found that certain of the a,B-unsaturated mono- carboxylic aliphatic acids, including sorbic and crotonic acids are good fungistatic agents for food and food wrappers. Sorbic acid is particularly well suited as a fungistatic agent for foods. It may be used to prevent mold growth in food products such as cheese, fish, pickles, gum, sugar solutions, citrus products, fruit purees and 55 pharmaceutical preparations. The advantages of using sorbic acid rather than sodium benzoate and sodium propionate are: (1) a smaller quantity of sorbic acid would be required to protect foods,

(2) a, B-unsaturated fatty acids are readily metabolizable, while benzoic acid can not be so utilized. Today this compound is com­ monly used in food protection against mold infections (8, 14, 15, 41,

61).

Recently Novak and Clark (43) at Louisiana State University i studied the antimicrobial activity of some ricinoleic and oleic acid derivatives. Two bacteria--Micrococcus pyogenes, Escherichia coli, several yeasts —Saccharomyces cervisiae, Candida stellatoidea, and several molds—Neurospora spp., Alternaria spp., Mucor sp.,

Penicillium sp. and Aspergillus sp ., were used as test organisms.

Spoehr and Smith (52) were of the opinion that autotrophic organisms offer some advantages, in that their culture requires only water, simple nutrient elements, air and sunlight, as compared with the culture of molds and bacteria, which require organic nutrient media. Using a particular technique, these workers isolated the fatty acids from plants such as Chlorella sysenoides, Umbellularia californica and others in large amounts. The development of anti­ bacterial properties in the fatty acid extracts through photo-oxidation suggested that unsaturated fatty acids or related compounds, were involved in this phenomenon. In this investigation various isolated fatty acids were tested against some of the Salmonella, Mycobacterium, and Staphylococcus species. MATERIALS AND METHODS

Seeds used in all experiments were obtained from the Reuter

Seed Company, New Orleans, Louisiana. Xanthomonas campestris was isolated from field infected cauliflower plants. Cultures of

X. vesicatoria, X. phaseoli, Pseudomonas phaseolicola, Erwinia carotovora and E. aroideae were obtained from Dr. R. S. Dickey of

Cornell University. Species of Corynebacterium were obtained from

Dr. M. P. Starr of the University of California. Cultures of

Escherichia coli, Staphylococcus aureus. Aerobacter aerogenes,

Bacillus cereus and Corynebacterium insidiosum were supplied by the L.S.U. Bacteriology Department.

Nutrient agar was used in culturing all bacteria except

Corynebacterium in which case .1 per cent yeast extract was added to potato dextrose agar. Nutrient agar contained the following in­ gredients: tryptose 10 g, beef extract 3 g, dextrose 10 g, sodium

chloride 5 g, agar 15 g, and distilled water, 1 1. The potato dextrose

agar contained: potato 200 g, dextrose 20 g, agar 15 g, and distilled water 1 1. To this 0.1 per cent yeast extract was added. The cul­ tures were incubated at room temperature. In preliminary tests for

determining the inhibitory effect of cauliflower seed on the growth of

X. campestris. seeds with low germination percentage were surface

57 58 sterilized with 0.3 per cent sodium hypochlorite for 3 minutes, and washed several times with sterile, distilled water; Whole seeds, seed coats and seed centers were then plated separately on nutrient agar seeded with bacteria.

Test plates were prepared by adding 4 ml of bacterial suspen­ sion to 100 ml melted nutrient agar at 45-5 0°C. After shaking thoroughly, 10 ml of the agar suspension was poured into 10 cm petri plates, and allowed to harden.

Bacterial suspensions were made by adding one loopful of bacteria from an agar slant to 10 ml of sterile distilled water.

Whenever comparative studies were made, the bacterial suspensions were adjusted to 65 per cent light transmission at 625 mu by means of a Bausch and Lomb colorimeter.

Extracts of the antibacterial substance were prepared by sur­ face sterilizing cauliflower seeds as described above. One gram of seed was soaked in 5 ml of sterile distilled water and this was kept in the refrigerator for 10 hours. Seeds were then ground in a sterile mortar and pestle, strained through cheesecloth, and the residue squeezed out in a hand press. The resulting extract was filtered through a Sietz filter into a sterilized 25 0 ml filter flask and then transferred into a sterilized 125 ml Erlenmeyer flask. This filtrate was used in testing for antibacterial activity. Whenever small quantities of the extract were needed, the extract was filtered by 59 means of a Swinny filter and the filtrate collected in a sterile test tube.

The filter paper disc-agar diffusion method was used for testing extracts for antibacterial activity. Filter paper discs 12.7 mm in diameter (made by Schleicher and Schull Company) were placed in test tubes and autoclaved for 20 minutes at 15 pounds pressure and then dried in the oven for 4 hours at 65°C. Discs were then dipped into the test extracts aseptically, and the excess liquid was allowed to drain by allowing the lower edge of the disc to touch the side of the tube. Three such discs were prepared and carefully placed on the surface of the seeded agar in such a way that each of the 3 discs was in the center of a quadrant. A fourth disc, wet with sterile distilled water, was placed as a control in the middle of the fourth quadrant. To distinguish the control disc from those dipped in the extract, the outer surface of the petri dish under the control disc was marked "C" with a glass pencil (Figure 1). The test plates were incubated at room temperature, and the diameter of the zone of inhibition was measured to the nearest 1 mm by means of a celluloid millimeter ruler. A magnifying glass was used when needed.

In conducting experiments to determine the effectiveness of the antibacterial substance on the growth of different bacteria, standardization of the various bacterial cultures was carried out in 60

Figure 1. Inhibitory effect of the cauliflower seed extract on growth of X. campestris, as tested by filter paper disc method. 61 the following manner: A loopful from a 24-hour old bacterial culture was suspended in 10 ml of sterile distilled water. All bacterial sus­ pensions were adjusted to the same optical density by use of a colorimeter; the number of bacteria at this optical concentration was determined by the dilution plate technique. On the basis of this determination, the optical readings of the suspensions were re­ adjusted in order to obtain an equal number of bacteria in all of the suspensions. One-half ml of each suspension, containing about 180 million organisms per ml was added to each petri plate containing

10 ml of melted nutrient agar at 45-50°C. After shaking the plates gently, they were allowed to harden. The extract was then tested for antibacterial activity against the various bacteria by the filter paper disc method.

In tube agglutination tests, bacterial suspensions were made by washing X. campestris from an agar slant with sterile distilled water. This heavy suspension was diluted to 65 per cent light trans­ mission (optical density 0.20) by means of a colorimeter. The seed extract used was partially purified as will be described later. A series of two-fold dilutions of the extract was then made. A control tube containing the extract was included in the test. One ml of the bacterial suspension was then added to each tube containing 4 ml of the extract. The tubes were shaken thoroughly, and then allowed to stand for 5 minutes, after which the amount of agglutination in each 62

tube was recorded.

To determine the solubility of the antibacterial substance,

water, 80 per cent ethanol, acetone, and ether were used as solvents.

When water and ethanol were used as solvents, one part of seed was

used with 5 parts of extractant. In the case of acetone and ether,

20 ml extractant was used for one part of seed. Ethanol, acetone

and ether were evaporated under vacuum, and the residues were

brought to the volume of the original sample (1:5) with distilled

w ater.

The effect of the extract on germination of various plant seeds,

including those of cauliflower itself, was also studied. Seeds were

placed on filter paper moistened with different concentrations of the

extract and incubated at room temperature in petri plates. Seeds

plated on distilled water moistened filter papers were used as controls.

For investigating the effect of temperature on the activity of

crude extracts, a thermostatically controlled water bath was used.

Two ml aliquots of the extract were placed in test tubes with a

diameter of 10 mm and kept in the bath at constant temperature for

10, 20 and 30 minutes. The level of water was such that the en­

closed extracts were wholly immersed. Upon being removed from the

water bath, the samples were cooled to room temperature by placing

them in cold water. The activity of each sample was determined by’

the filter paper disc method, using X. campestris as a test organism. 63

Freshly prepared extract was used as a control.

Determinations of pH were made by means of a Beckman pH meter and adjustments in pH were made by addition of N solutions of hydro­ chloric acid or sodium hydroxide. Extracts at different pH levels, as well as the original untitrated sample, were bioassayed using the filter paper disc method.

For dialysis studies 20 ml of the crude extract was placed in dialysis tubing with a diameter of 40 mm, and placed in a 1000 ml beaker containing distilled water. The water was stirred by placing the beaker on a magnetic stirrer. The extract was dialyzed for 48 hours. During this period the water was changed every 5 hours.

The dialysis was performed in a refrigerator in order to keep the multiplication of possible contaminants at a low level.

Crude extracts were fractionated by descending paper chroma­ tography. The apparatus used was a Reco Chromatocab (Harshaw

Scientific Co.), Watman No. 1 filter paper was used throughout.

Solvents used were either Butanol-acetic acidfwater (4:1:1) or isopropanol-water (4:1). The temperature of the cabinet was kept constant throughout these experiments (28°C). In these experiments

5 or 10 ul quantities of the extract were spotted successively at a distance of 10 cm from the edge of the 53 x 3 cm strip of filter paper by means of a micropipette. After spotting the extract on the paper and drying it, the paper was placed in the cabinet for equilibration 64 with the atmosphere of the cabinet for 12 hours. The atmosphere in the cabinet was fully saturated with the solvent by placing it at the bottom of the cabinet in large petri dishes. The chromato­ graphs were developed when the solvent front was at 50 cm. EXPERIMENTAL RESULTS

Antibacterial Effect of Cauliflower Seed

In preliminary tests, it was noted that some seeds of cauliflower

exhibited an inhibitory effect on the growth of X.. campestris (Figure 2).

Plating whole seeds, seed coats, and seeds from which seed coats were removed on nutrient agar seeded with the bacterium, revealed that the antibacterial substance was present in the seed centers, while the

seed coats were found to be devoid of such activity (Table 1). Zones

of inhibition as wide as 10-12 mm were measured around the indivi­

dual seed centers, whereas no inhibition zones were observed around

seed coats. Clear zones of inhibition occurred around whole seed

in some cases. These zones were probably due to tiny cracks in the

seed coat through which the antibacterial substance diffused into the

agar. There was a concentric circle of white diffusate present inside the clear zone of inhibition.

The presence of the antibacterial substance appeared to be

somewhat correlated with viability of the seed. Since seed lots with

low germination percentage((5 per cent) contained more seeds capable

of inhibiting the growth of bacteria than did more viable lots ( ^95%),

an experiment was conducted to determine the possible correlation

of the presence of the antibacterial substance with the viability of

cauliflower seed.

65 66

Figure 2. Inhibitory effect of cauliflower seed on X. campestris.

( 67

Seed lots with low and high germination percentages were

surface sterilized and soaked in sterile distilled water for one hour.

The seeds in each lot were then divided into 3 groups and each group was plated on nutrient agar seeded with X». campestris separately in the following manner.

(1) 100 intact seeds

(2) 100 seeds from which seed coats were removed.

(3) 100 seed coats, and

(4) 100 seeds crushed by means of a sterile forceps.

Although germinated seeds could be easily detected on the

nutrient agar, for obtaining more reliable results aliquots of seed

with low and high germinability were also plated on distilled water

moistened filter paper in petri dishes. One hundred seeds, 20 in

each petri dish, were used in this germination test.

The results of this experiment confirmed that the amount of the

antibacterial substance was greater in seeds of low viability than in

those with a high germination percentage. Clear zones of inhibition

occurred around 5 0 per cent of the seeds where germination was 5

per cent. In the seed lot showing 95 per cent germination, only

12 per cent of the seeds developed clear zones of inhibition. The

inhibitory activity of seeds of low and high viability is shown in

Figure 3. In either case no zones of inhibition were observed around

seed coats. Crushing the seeds to prevent germination did not 68

Table 1. Presence of antibacterial substances in relation to viability of seed and seed parts.*

% % seeds seeds germinated showing inhibition

1. Seeds of low viability

W hole seed 5 4

Seed center 5 50

Seed coat - 0

Crushed seed 0 46

2. Seeds of high viability

Whole seed 95 0

Seed center 95 12

Seed coat - 0

Crushed seed 0 8

♦Average of 3 experiments, in each experiment 100 seeds, 20 in each plate. Figure 3. Comparison of inhibitory activity of seeds of low viability (right), and high viability (left). 70 increase the number of seeds showing inhibitory activity against

X. campestris. Most of the seeds showing zones of inhibition turned to greyish black after incubation. The results of this test are given in Table 1.

Agglutination Tests

Addition of 1 ml of the extract to 4 ml of a water suspension of X. campestris with an optical density reading of 0.20 resulted in an immediate agglutination of the bacterial cells. Since no bacterial colonies developed on nutrient agar plates when the agglutinated suspension was streaked on them after a 12-hour incubation period, it was apparent that the bacterial cells were inactivated by addition of the seed extract. Similar results were obtained when extracts were tested against X. phaseoli and X. vesicatoria, although agglutination was less pronounced. With species of the genera Bacillus. Erwinia, Escherichia. Pseudomonas and Corynebacterium some turbidity occurred when the extract was added to water suspensions of these bacteria, and a precipitate occurred after a 24-hour incubation period. The characteristic agglutination obtained with the Xanthomonas species did not occur, however, and 20 to 24 hours were required for complete inactivation of these bacteria. 71

Antibacterial Spectrum

The antibacterial spectrum of the partially purified cauliflower seed extract was demonstrated by using the filter paper disc method.

Various gram-positive and gram-negative bacteria were used as test organism s.

Filter paper discs with a diameter of 12.7 mm were dipped in the seed extract and plated on nutrient agar seeded with various test organisms. The diameter of the inhibition zones was measured when a uniform growth of bacteria covered the surface of the agar, and clear zones of inhibition had developed. The reaction of water

suspensions of the different bacteria was also tested. The organisms

studied are listed in Table 2, together with the results obtained.

From these results it may be deduced that the antibacterial activity of cauliflower seed extract varies greatly against different bacteria. For the sake of simplicity, the organisms can be divided into 3 groups depending upon their relative sensitivity to the anti­ bacterial agent.

Cauliflower seed extract exhibited the greatest antibacterial activity against the genus Xanthomonas. Among the species of

Xanthomonas tested, the largest zone of inhibition (39 mm) occurred with X. campestris. The diameter of the inhibition zone which oc­ curred with X. phaseoli and X. vesicatoria was also large but not as large as that developed with X. campestris. In addition, the aggluti­ nation reaction occurred only with species of Xanthomonas (Figure 4). 72

Table 2. Activity spectrum of cauliflower seed extract against different bacteria.

Reaction of Diameter of bacterial inhibition Bacteria suspension* zone (mm)**

Xanthomonas campestris +++ 39

X. phaseoli •H- 36

X. vesicatoria -H- 36

Pseudomonas phaseolicola T 28

P. syringae T 20

Erwinia carotovora T 20

E. aroideae T 21

Corynebacterium insidiosum T 21

C. michiganense T 23

Staphylococcus aureus - 15

Bacillus cereus T 16

Aerobacter aeroqenes - -

Escherichia coli T 16

* + = agglutination, relative amount indicated by number of + signs. T = turbidity - = no reaction

**Each value is average diameter of 8 replicate filter paper discs. Diameter of the filter paper discs was 12.7 mm. 73

Figure 4. Reaction of various bacteria in suspension to the cauliflower seed extract. From left to right: Tube 1, Xanthomonas campestris (control); tubes 2-7, extract plus: X. campestris. Pseudomonas phaseolicola, Erwinia aroldeae, Corvnebacterium michiganense. Staphylococcus aureus, Escherichia coli; tube 8, extract only. 74

The organisms included in the second group are species of

Erwinia, Pseudomonas and Corynebacterium in which the diameter of the inhibition zone was considerably less than that developed with the Xanthomonas species. Agglutination did not occur upon addition of the extract to suspensions of these bacteria, but notice­ able turbidity occurred in all cases (Figure 4).

The third group includes those bacteria which are least affected by the antibacterial agent. The organisms Staphylococcus aureus,

Bacillus cereus, Aerobacter aerogenes and Escherichia coli are in this group. In this group the diameter of the inhibition zone was very small, being less than 16 mm in all cases. Aerobacter aerogenes was apparently resistant to the antibacterial agent, as the extract gave no inhibition zone when tested against this organism. Little or no turbidity occurred when the extract was added to suspensions of these bacteria (Figure 4).

With practically all species tested, no bacterial colonies could be observed within the inhibition zone if held at room temperature for

6-8 days. After this time, scattered colonies began to grow within this zone and after several days the zone was often obscured by the growth of these bacteria. This did not occur with the two species of

Erwinia, or with Escherichia coli, however. Test plates held for 14 days or more showed no such growth within the inhibition zone. The extract would thus seem to be bacteriostatic to some species and bactericidal to others. 75

Antibacterial Activity of the Extract at Various Dilutions

The standard solution, prepared from 1 g of seed plus 5 ml

water, and various dilutions of this standard solution, were assayed

repeatedly and the average diameter of the inhibition zones produced

by each concentration of the standard was used in constructing

the standard curve shown in Figure 5.

The diameter of the inhibition zone produced by a given solution

was found to be reproducible within narrow limits. For example, the

diameters obtained in replicate tests on different plates for one of

the solutions used were 24.0, 24.0, 23.5, and 24.5, or an average

of 24 mm„ Similar results were also obtained when the experiment

was repeated.

One of the dilutions of the standard solutions can be arbitrarily

considered to contain one unit of active principle per ml. For this

purpose an amount producing a 14-16 mm wide zone of inhibition

was chosen as an arbitrary unit of activity. The curve in Figure 5

is only representative of a group of curves having the same slope

but in which individual curves may lie slightly above or below the

one shown.

By use of the standard solution and the standard curve described

it is possible to assay seeds of various species of the Cruciferae for

antibacterial activity. When parallel dilution tests were made, using

the agglutination and disc inhibition reactions, it was observed that Diameter of Inhibition Zone 40- 10 0 2 - . iue . niioy fet f eea dltos f qeu cuilwr ed extract seed cauliflower aqueous of dilutions several of effect Inhibitory 5. Figure ngot fX cmeti. vj campestris. X- of growth on 1.0 nt o niatra Substance Antibacterial of Units 2.0 3.0 4.0 cr> 77 the agglutination phenomenon disappeared after several dilutions whereas a slight inhibition zone could be detected at the same dilutions o This indicated that the filter paper disc method was probably more sensitive.

In tests comparing the activity of extracts from cauliflower seeds with high ( ^95%), and low ( ^5%) germinability, a series of two-fold dilutions of the extracts was made and tested for activity » by the agglutination technique. The results showed that the seed": with low viability contained a greater amount of the antibacterial substance than did seeds with high viability. The extract of the seeds with low viability was active up to a dilution of 1:128, whereas the extract of the seeds with high germinability lost its activity at a dilution of 1:16. This correlated well with results obtained in plating the individual seeds.

Inhibitory Effect on Seed Germination

When the effect of cauliflower seed extracts on the germination of different seeds was tested, it was found that the aqueous extract inhibited the germination of seeds in species belonging to several fam ilies.

Cauliflower seed extract was effective in inhibiting the germina­ tion of cauliflower, cabbage, tomato, pepper, radish, oat and rice seeds. The sensitivity of different seeds to the same concentrations of the extract varied greatly, however. Cauliflower seed extract at a 78 dilution of 1:5 wholly inhibited the germination of oat and rice seeds, whereas cabbage, cauliflower, tomato, and radish seeds started to germinate at the same dilution. Of all the seeds tested, it seemed that the germination of radish seed was least inhibited by the extract.

The results are given in Table 3. In addition to retarding germina­ tion, cauliflower seed extract also caused inhibition and retardation of root and stem growth of all seeds tested.

As with the antibacterial substance, the germination inhibitor was found to be present in the seed centers but not in the seed coats.

Seed coats and seeds from which seed coats were removed were

soaked in sterile distilled water for a period of 24 hours and the diffusates were tested for inhibition of seed germination. It was

found that the diffusate of the seed from which the seed coat had been removed possessed highly inhibitory activity whereas the diffusate of the seed coats was devoid of activity.

Antibacterial Activity of Seeds of Various Cruciferous Plants

Several species and varieties of cruciferous plants were tested

for the presence of the antibacterial principle in the seeds by the

filter paper disc and agglutination tests, using X. campestris as a test organism. Two lots of cauliflower seeds (vars. Master Original

and Early Snowball) with low viability were also included in this test.

The results of this experiment are shown in Table 4. The

extract of cauliflower seed (var. Master Original), with low viability 79

Table 3, Effect of cauliflower seed extract on seed germination.

No. of seeds germinated No. of U ndiluted Diluted Seeds seeds Control Extract Extract 1:5

Brassica oleracea var. capitata 100 58 16 48

Brassica oleracea var. botrytis 11 92 4 60

Raphanus sativus " 86 32 82

Lycopersicon esculefttum " 98 0 12

Capsicum annum " 80 4 24

Avena sativa " 75 0 0

Oryza sativa " 94 0 0 80

Table 4. Inhibitory effect of seed extracts of different species and varieties of cruciferous plants on X. campestris.

Diameter of % inhibition Seed tested germination Agglutination* zone (mm)**

Cabbage (var. Globe) 90 ++ 14.5

Cauliflower (var. Master Original) 95 +++ 28.0

Cauliflower (var. Master Original) 5 +++ 40.0

Cauliflower (var. Early Snowball) 5 +++ 41.0

Broccoli (var. (Waltham) 95 ++ 16.0

Radish (var.

Champion) 87 - 20.0

* + = agglutination, relative amount indicated by number of +.

** Diameter of the filter paper discs was 12.7 mm. 81 showed the largest inhibition zone in agar plate tests and also gave distinct agglutination. Equal activity was obtained for cauliflower seeds (var. Early Snowball) with low viability. Although character­ istic agglutination of the bacterial suspension occurred with the extract of seeds of cauliflower (var. Master Original) with high germinability, the diameter of the inhibition zone was smaller than that produced by the less viable seed of the same variety.

Cabbage (var. Globe), and broccoli (var. Waltham) showed the least activity against X. campestris. Radish (var. Champion) did not give distinct agglutination with the bacterial suspension whereas the diameter of the inhibition zone was between that produced by cabbage and broccoli, and that of seeds of cauliflower with high viability. The results indicated that among plants tested, cauliflower seeds exhibit the greatest antibacterial activity and this activity is more pronounced in seeds with low viability. Seeds of other plants had lower levels of antibacterial activity.

Physical and Chemical Characteristics

Stability. When water extracts were prepared from 1 g cauli­ flower seed in 5 ml sterile distilled water, the pH of the extract was

5.4-5.6. Neutral or slightly acid (pH 5.6) aqueous extracts of the cauliflower seed showed no detectable change in activity after storage for 2 l/2 months at a temperature of 8 to 10°C. At room temperature there was a loss of 4-5 per cent of the activity after a 2-week storage 82 period.

The extract was inactivated in the high alkaline range of pH while it was more stable under slightly acid conditions.

Cauliflower seeds were extracted in different buffer solutions and the pH determined; the solutions were then heated at various temperatures for 10, 20, and 30 minutes. The activity of the solu­ tions was determined by the filter paper disc method and the results are shown in Table 5.

At the pH range from 4.2 to 6.1 there was some loss of activity but the extract was still active after heating at 100°C for 30 minutes.

Heating at 80°C for 20 minutes at higher pH destroyed the active principle.

Solubility. The solubility of the antibacterial substance in different solvents was established in preliminary tests. The active principle proved to be completely water soluble, slightly soluble in

80 per cent ethanol and insoluble in acetone and ether.

Although the ether extract gave slight inhibition when concen­ trated, no agglutination was detected, and when the extract was brought up to the original volume no activity was observed. This inhibitory effect of the ether extract can probably be accounted for by the presence of fatty acids in the seed, which show activity when concentrated. The antibacterial activity of such compounds has been reported in the literature. Table 5. Antibacterial activity of cauliflower seed extract as influenced by temperature and pH.*

Temperature i 60 80 100 M inutes Buffer pH 10 20 30 10 20 30 10 20 30

Citrate-phosphate 4.2 80 75 70 75 70 65 60 60 50

Citrate-phosphate 6.1 90 90 80 70 70 70 65 50 50

Phosphate 7.1 100 75 70 65 35 0 35 0 0

Borate -pho sphate 8.7 50 40 35 50 0 0 0 0 0

Pho sphate -sodium hydroxide 10.5 50 50 50 50 25 0 40 0 0

*Values are expressed in per cent of original activity. 84

When the aqueous extract was dialyzed against distilled water, it was found that the extract inside the cellophane membrane was devoid of activity after 48 hours dialysis. Since nondialyzed extracts maintained under the same conditions retained their activity, this indicated that the active substance obtained from ground seeds could diffuse through the cellophane membrane (was dialyzable).

Partial Purification. Cauliflower seeds with low viability were extracted with hydrogen peroxide-free ether. The residue ob­ tained was extracted with 80% ethanol, and the supernatant was discarded. The residue was then extracted with distilled water.

This time the residue was discarded and the supernatant was tested againstjf, campestris and other bacteria. This extract was found to have even greater activity than the original water extract, indicating that substances inhibitory to its activity may have been removed.

This extract is referred to as partially purified. The partially purified extract is slightly yellowish in color and darkens on storage at room temperature and even in the refrigerator. The darkening is not associated with any noticeable loss in antibacterial activity.

Addition of Biuret*s and Millon*s reagents to this extract gave posi­ tive reactions, indicating the presence of proteins and phenols. The

extract failed to reduce Fehling*s solution when heated.

Boiling the partially purified extract in a test tube containing

a strip of lead acetate paper resulted in darkening of the paper, 85 indicating the presence of sulfur in the extract. Addition of 15 ml of 40 per cent lead acetate to the partially purified extract resulted in the formation of a precipitate which was soluble in nitric acid.

The resultant supernatant showed a 50 per cent reduction in activity.

Chromatography

The partially purified extract was fractionated by descending paper chromatography. The chromatograms were cut crosswise into strips 2 cm wide and bioassayed by plating them on nutrient agar seeded with X. campestris. The results showed that part, of the chromatogram with Rf value ranging from 0.64 to 0.73 gave an in­ hibition zone on the agar plates. When the chromatogram was sprayed with ninhydrin, and ferric chloride, no reaction occurred indicating that the active spot on the chromatogram was not an amino acid or phenol, although addition of Buret*s and Millon's reagents to the partially purified extracts, indicated the presence of proteins and phenols. DISCUSSION

This preliminary investigation indicates the presence of an antibacterial substance in the seed of cauliflower plants.

In view of the results obtained it seems that the antibacterial activity of seed with low viability is greater than that of seed of high germinability. Physiological changes during the dormancy of the

seed may bring about formation of a substantial amount of the anti­ bacterial substance o

The antibacterial activity does not seem to be limited to any particular group of bacteria. G+ and G- bacteria are inhibited by the antibacterial substance. Among the bacteria tested, the anti­ bacterial principle was more active against the species of the genus

Xanthomonas than any other bacteria.

The characteristic agglutination which occurs when the extract is added to water suspensions of species of Xanthomonas. might

suggest that the agglutination is responsible for antibacterial activity.

Since in some instances, there is inhibition of growth when no dis­ tinct agglutination occurs, it does not seem probable that the agglu­ tinating properties of the extract can account for all of the antibacterial

effect noted. However, it is apparent that the antibacterial action of the extract is greater when there is agglutination.

8 6 The presence of antibacterial activity in plants belonging to the crucifer family has been reported by several investigators, but there are conflicts in the results obtained by different groups.

This disparity in results is probably due partly to differences in the methods used, and plant or plant parts tested. Sherman and

Hodge (51), using the juice expressed from heads of cabbage, re­ ported relatively mild but distinct bactericidal action against

Staphylococcus aureus and Escherichia coli. This active principle was destroyed at 60°C within 10 minutes. Pederson and Fisher (46) als'o reported on bactericidal activity in heads of cabbage toward G- bacteria, especially those usually present upon the surface of cabbage leaves. This bactericidal substance was also inactivated by heating. Osborn (45) contradicted the report of Sherman and

Hodge by demonstrating that the bactericidal property was present in cabbage seed but not in heads of cabbage. Foter and Golick (20) reported the presence of a volatile antibacterial substance in crushed horseradish. This substance was more active against various bacteria at 37.5°C than those obtained from garlic and onions, but was more rapidly inactivated (20, 60). The stability of the active principle of cauliflower seed extract at room temperature for a period of more than 2 weeks, and its relative resistance to in­ activation by high temperatures, suggest that the active principle of cauliflower seed extract differs from the substances obtained 8 8 from cabbage, horseradish, garlic, and onion by these workers.

Ivanovics and Horvath (31), and Horvath and Ivanovics (25) isolated a substance from radish seed and reported that the seed contained an enzyme capable of converting an inactive precursor into an antibiotic. For proof of this, crushed seeds were extracted with 80% ethanol, and the extracts were concentrated and freed from alcohol in vacuo (extract A). Another portion of the crushed seeds was extracted with water and dialyzed in a cellophane bag against tap water (extract B). Both extracts were entirely devoid of any antibacterial activity. However, when the extracts were mixed and the mixture allowed to stand for one hour, a potent inhibitor resulted when tested against S^. aureus and E. coli. When cauliflower seed extract was prepared in the same manner as mentioned above and tested against S. aureus and E. coli, it was found that the mixture of extracts "A" and l!B!! had the same degree of activity as extract

"A" alone. This indicated that the active principle of cauliflower seed extract was not formed as a result of enzymatic action. In addition, cauliflower seed extract prepared by grinding seeds with water, when tested against X# campestris, showed a large inhibition zone and distinct agglutination with the bacterial cells in suspension, whereas radish seed extract prepared in the same manner exhibited only a slight inhibition zone against X. campestris. and no distinct agglutination was detected when it was added to the bacterial suspension. The »'

89 activity of radish seed extract was lost -.within 24 hours, if the extract was incubated with the crushed seeds, according to Ivanovics and Horvath, whereas cauliflower seed extract did not show any marked loss of activity after incubating with crushed seeds for 3 days o

According to Foter (20), allyl isothiocyanate and related mustard oils isolated from some cruciferous plants possess an antibacterial action, a statement not borne out by other observa­ tions (31, 45). Analytical reagent allyl isothiocyanate obtained from Fisher-Scientific Co. exhibited antibacterial action up to dilu­ tions 1:100, but the characteristic agglutination did not occur when added to suspensions of X. campestris. The allyl isothiocyanate may be present in some cruciferous plants, but whether it is present in sufficient amounts to inhibit the growth of bacteria has not been conclusively demonstrated.

Cauliflower seed extract also showed inhibitory activity against seed germination. The presence of germination inhibitors in the seeds of various plants has been reported by many investigators (6, 16, 53).

Kockemann (6) reported the presence of a class of natural inhibitors, which play a very important part in the biology of the seed, and he named these substances "blastocholines " (germination inhibiting substances). These substances are simple molecules of diverse chemical affinities all possessing a property of preventing germination 90 at relatively low concentrations. These substances usually occur in fruits or seed coats. Sroelov (53) showed the presence of a germina­ tion inhibitor of Sinapis alba seed in the fruit valves and beak. Later it was reported to be mustard oil. Germination inhibitors have been reviewed recently by Evenari (16).

Cauliflower seed coat extract did not show any inhibitory action toward seed germination, while the extract from seed centers strongly inhibited the seed germination. Since the inhibitors previously described were mainly associated with the seed coat, it would seem that a different substance was involved here.

The results of qualitative tests would indicate that the active

substance in the cauliflower seed extract is not an aldehyde or carbohydrate, free amino acid or phenol. The determination of the chemical nature of the active principle requires further investigation. SUMMARY

The presence of an antibacterial substance in cauliflower seed has been reported, and certain of its properties have been determined.

The cauliflower seed extract was tested against 13 different bacteria including plant pathogenic bacteria. The greatest antibacterial activity of the cauliflower seed extract was exhibited against species of the genus Xanthomonas. A distinct agglutination of bacterial cells occurred when the extract was added to suspensions of Xanthomonas species. The growth of species of Pseudomonas, Erwinia,

Corynebacterium, Bacillus, Escherichia and Staphylococcus was also inhibited by the cauliflower seed extract, but to a lesser extent than that of Xanthomonas species. Although some turbidity occurred when the extract was added to water suspensions of these bacteria, the characteristic agglutination obtained with Xanthomonas species did not occur.

Testing the extracts obtained from seed coats and seed centers against X. campestris, revealed that the antibacterial substance was associated with the seed centers.

A greater percentage of seeds with low viability ( ^5 per cent) contained antibacterial agent than did seeds with high germination percentage ( ^95 per cent). This was demonstrated by plating the

91 92 centers of cauliflower seeds with low and high germinability on nutrient agar seeded with X. campestris. and observing the per­ centage of seed showing inhibitory activity.

In tests comparing the activity of extract from cauliflower seeds with high and low germinability, a series of two-fold dilu­ tions of extract was made and tested for activity by the agglutination technique. The extract of seed of low viability was found to be about

8 times more active than that from seed of high viability.

Seeds of various cruciferous plants were tested for presence of the antibacterial principle in the seeds by the filter paper disc and agglutination test using X. campestris as a test organism. The results indicated that cauliflower seeds (vars. Master Original and

Early Snowball) with low viability possessed the greatest activity, followed by those of high viability. Cabbage (var. Globe) and broccoli (var. Waltman) showed the least activity against X. campestris . Radish (var. Champion) did not give distinct agglutina­ tion of the bacterial suspension but the diameter of the inhibition zone on agar plates was between that produced by cauliflower seeds with high viability and cabbage and broccoli seeds.

Cauliflower seed extract also exhibited inhibitory action toward seed germination. Of the seeds tested, rice and oats were completely inhibited, whereas radish seeds were not affected greatly. The germi­ nation inhibitor was also associated with seed centers, and seed coats 93 were devoid of activity.

The antibacterial substance was stable after storage for 2 l/2 months at 8-10°C. At room temperature there was a loss of 4-5 per cent of the activity after a 2-week storage period. The extract was more stable under slightly acid conditions. At the pH range of

4.2-6.1 the extract remained active after heating at 100°C for 30 minutes, although some loss of activity occurred. At higher pH, heating at 80°C for 20 minutes destroyed the active principle. The antibacterial substance was completely water soluble, partially soluble in 80 per cent ethanol, and insoluble in acetone and ether, and was dialyzable.

The results of qualitative tests indicated the presence of protein, phenols and sulfur and lack of the aldehyde or carbohy­ drates in a partially purified extract. When chromotographed, however, the area of the paper showing antibacterial activity gave negative results when tested for the presence of phenols and amino a c id s . LITERATURE CITED

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/ VITA

Fereydoon Malekzadeh was bom on December 5, 1933, in

Tabriz, Iran. He received his elementary and secondary school education in Tahriz. In 1953 he graduated from Ferdowsi High

School. In the same year he enrolled in the University of Teheran,

Faculty of Science, where he received his B.Sc. in June 1956, and M.Sc. in June 1958. He worked as an instructor in the

College of Science, University of Teheran from 1956 to 1960. He has attended Louisiana State University since September 1960. At present he is a candidate for the degree of Doctor of Philosophy at

Louisiana State University.

100 EXAMINATION AND THESIS REPORT

Candidate: Fereydoon Malekzadeh

Major Field: Plant Pathology

Title of Thesis: Evaluation of Several Bactericides as Seed Treatments for the Control of Black Rot of Crucifers and Studies on an Antibacterial Substance from Cauliflower Seed Approved:

Major Professor and Chairman

Dean of the Graduate School

EXAMINING COMMITTEE:

Date of Examination:

July_30,_1962