IDENTIFICATION AND DIFFERENTIATION OF BACTT,I,US SUBTTLIS - -- -

AND A STUDY OF ITS AUTECOLOGY IN ONION RHIZOSPHERE.

Dominic Chung Wah Wong

B.Sc.(Hons.)Simon Fraser University 1977

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

in the Department

of

Biological Sciences

0 Dominic Chung Wah Wong 1981

SIMON FRASER UNIVERSITY

August 1981

All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy

or other means, without permission of the author.

Approval

Name : Chung Wah (Dominic) Wong

Degree : Master o f S c i e n c e

T i t l e of T h e s i s : I d e n t j f i c a t i o n and d i f f e r e n t i a t i o n o f B a c i l l u s

s u b t i l i s and a s t u d y of i t s au teco logy i n

onion r h i z o s p h e r e

Exam i n i n g Conun i t t e e :

Cha irman : D r . Robert C . Brooke

D~~P&= Rahe, S e n i o r ~@~erpA!sor -

-- D r . -~%hn H . Borden

__1----

Dr'. A. C. Oeh lsch lager

-- - -- - D r . Robert S . Vernon, Research S c i e n t i s t ,

A g r i c u l t u r e Canada, Vancouver, B . C . , Publ. i c Examiner

Date approved: 4 September 1981 -- - -

PARTIAL COPYRIGHT LICENSE

I hereby g ran t t o Simon Fraser U n i v e r s i t y t h e r i g h t t o lend

my thef3is, p r o j e c t o r extended essay ( t h e t i t l e o f which i s shown below)

t o user s o f t'hc Simon Fraser U n i v e r s i t y L i brary, and t o make p a r t i a l o r

s i n g l e c o p i e s on ly f o r such users o r i n response t o a request from the

l i b r a r y o f any o the r u n i v e r s i t y , o r o the r educat ional i n s t i t u t i o n , on

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w i t h o u t niy w r i t t e n permission.

T i t l e o f Thesis/Project/Extended Essay

I d e n t i f i c a t i o n and d i f f e r e n t i a t i o n o f B a c i l l u s s u b t i l i s and a s t u d y - of i t s a u t e c o l o g y i n on ion r h i z o s p h e r e

Author:

Chung Wah (Dominic) Wong

ABSTRACT

Six bacterial isolates antagonistic to Sclerotium cepivorum

Berk., were confirmed to be strains of Bacillus subtilis

Ehrenberg on the basis of standard morphological and biochemical

criteria.

Several biochemical tests known to produce variable

reactions by strains of B. subtilis did not permit -

differentiation of the six isolates. Differentiation of the six

isolates was possible on the basis of cultural characteristics

either on nutrient agar or on 10% skim milk agar, thereby

suggesting that the isolates were distinct strains.

Vegetative and spore antigens were developed for the strain

B2 of - B. subtilis. The vegetative antiserum was found to be

species-specific when tested against 10 Bacillus species, eight

B. subtilis strains and 50 unidentified bacteria. The spore -

antiserum was less specific. It cross reacted with spores of

Bacillus coagulans Hammer and Bacillus pumilis Meyer and

Goltheil.

A fluorescent antibody technique was used to assess the

populations of vegetative and spore cells of - B. subtilis in

onion root rhizosphere of bacterized and unbacterized seeds. The

population survey was conducted over several weeks, after

planting the seeds in the following conditions: (i) sterilized

and non sterilized muck soils under greenhouse conditions, and

iii

(ii) muck soil in the field.

Generally higher - B. subtilis populations were associated

with seeds treatment. Significant differences occurred during

germination and early stages of plant growth under greenhouse

conditions and persisted longer in sterilized than in

nonsterilized soil. In the field trial only one sample period

showed a significantly greater number of - B. subtilis cells

zssociated with seed treatment.

ACKNOWLEDGEMENTS

The i n v e s t i g a t i o n s r e p o r t e d i n t h i s t h e s i s we re c a r r i e d o u t

u n d e r t h e g u i d a n c e o f D r . J . E . Rahe f o r whose encou ragemen t and

a d v i c e I am t r u l y g r a t e f u l .

I t h a n k D r . R . S . U t k h e d e , D r . J . H . Borden and D r . A . C .

O e h l s c h l a g e r f o r t h e i r v a l u a b l e a s s i s t a n c e d u r i n g t h e c o u r s e o f

t h i s work .

F i n a n c i a l a s s i s t a n c e f r o m t h e D e p a r t m e n t o f B i o l o g i c a l

S c i e n c e s , S . F . U . , A g r i c u l t u r e Canada and t h e N a t i o n a l R e s e a r c h

C o u n c i l of Canada i s g r a t e f u l l y acknowledged .

A p p r e c i a t i o n i s a l s o e x t e n d e d t o t h e f a c u l t y and s t a f f of

t h e D e p a r t m e n t o f B i o l o g i c a l S c i e n c e s , S.F.U.

TABLE OF CONTENTS

Approval ...................................................... ii Abstract .................................................... iii Acknowledgements .............................................. v List of Tables ............................................. viii List of Figures ............................................... x

Introduction to the thesis ........................... 11 Chapter 1 .

Identification and differentiation of six bacterial isolates antagonistic to Sclerotium cepivorum ......................................... 4

Introduction .......................................... 4

Methods and Materials ................................. 5 Results ............................................... 8

............................ Discussion and Conclusion 27

Chapter 2 . A quantitative autecological study of Bacillus subtilis in the onion root rhizosphere ........... 28

Introduction ......................................... 28 Methods and Materials ................................ 31 A . Fluorescent antibody preparation and evaluation

of specificity .................................... 31 B.Population studies .................................. 35

1.Pot trials under greenhouse conditions .......... 35 (a).~hizosphere soils: ........................ 35 (b) Nonrhizosphere soils ...................... 38 ..................................... 2.Field trial 38

Results .............................................. 40 A.Specificity of - B . subtilis antisera ................. 40

.................................. B.Population studies 40 1.Pot trial under greenhouse conditions ........... 40

(a).~terilized soil ........................... 40 (b).~onsterilized soil ........................ 44

2.Field trial ..................................... 54 C.Antagonistic bacteria from sterilized and

................................ nonsterilized soil 54

Discussion ........................................... 65 Conclusion ........................................... 71 General discussion conclusion and recommendation . . . . . 73

List of References ........................................... 77

vii

LIST OF TABLES

Chapter 1.

Table 1. Sources of Bacillus subtilis isolates and known strains. 6 . . . . . . . . . . . . . . . . . . . . . . . .

Table 2. Cultural characteristics of six Bacillus - - -- subtil-is - . - isolates grown on nutrient agarl . . . . . . . . . . . . 9

Table 3. Cultural characteristics of Bacillus subtilis isolates -- ----- grown on 10% skim milk agar1.. . . . . . . . . . . . . . 10

Table 4. Cultural characteristics of six Bacillus -- subtilis isolates grown on potato dextrose agar with Sclerotium - . . . . . . . . . . . . . . . . . . . . . cepivorum 1 .

Table 5. Biochemical characteristics for isolates and known strains of Bacillus subtilis.. . . . . . . . . . . . . --

Table 6. Sugar fermentation patterns for isolates and known strains of Bacillus subtilis.. . . . . . . . . . . . . -

Chapter 2.

Table 7. Cross reactivity of Bacillus subtilis (B2) antibodies with flagellar(~). somatic(0) and spore antigens of - B. subtilis strains.

Table 8. Cross reactivity of Bacillus subtilis (B2) flagellar(~) and spore(^) antibodies with H , 0 and?? antigens of other Bacillus species.. -- . . . . . . . . . . . . . . . . . . . 42

Table 9. Cross reactivity of Bacillus subtilis(B2) flagelLar(H) and spore(S) antibodies with bacterial isolates from , - soil.. . . . . . . . . . . . . . . . . . . . . . . . . . 4 3

Table 10. Populations of vegetative(^) and spore(S) cells of Bacillus subtilis in rhizos here of onions grown in -- sterilized muck sail. (x 10-8/10 plants). . . . . . . . . 4 7

Table 11. Populations of Bacillus - subtilis in nonrhizosphere and rhizosphere soils of onions grown in sterili.zed muck soil,(x 10-5/gm soil). . . . . . . . . . . . . . . . . . 50

Table 12. Populations of vegetative(^) and spore(^) cells of

viii

Bacillus subtilis in rhizospheres of onions grown in - -- 5 3 nonsterilized muck soil,(x 10'4/10 plants).. . . . Table 13. Comparison of populations of Bacillus subtilis in

rhizosphere and nonrhizosphere soils of onions grown in . . . . . . nonsterilized muck soil,(x 1.0-5/gm soil).. 57

Table 14. Populations of vegetative(V) and spore(S) cells of Bacillus subtilis in rhizosphere of onions grown under

60 field conaitions,(x l0-5/10 plants). . . . . . . . . . . Table 1 5 . Meteorological data for the 1980 growing season: mean

daily temperature and rainfall for 10 day periods prior 6 3 to field sampling at indicated days after planting.. . . Table 16. Summary of meteorological data for field trial area

for 1976 through 1980 growing seasonsl.. . 64

Table 1 7 . Streak tests for antagonistic bacteria from sterilized 66 and nonsterilized soilsl.. . . . . . . . . . . . . . . .

LIST O F F I G U R E S Chapter 1.

Figure 1. Bacillus subtilis isolate BK on nutrient agar on the 7th day after inoculation and incubation at 22-24 C. 1 3 . .

Figure 2. Bacillus subtilis isolate BO on nutrient agar on the 7th day after inoculation and incubation at 22-24 C. 15 . .

Figure 3. Bacillus subtilis isolate Bl on nutrient agar on the 7th day after inoculation and incubation at 22-24 C. 17 . .

Figure 4. Bacillus subtilis isolate B2 on nutrient agar on t h ~ 7th day after inoculation and incubation at 22-24 C. 19 . .

Figure 5. Bacillus subtilis isolate B4 on nutrient agar on the 2, 7th day after inoculation and incubation at 22-24 C. . .

Figure 6 Bacillus subtilis isolate B8 on nutrient agar on t-he 7th day after inoculation and incubation at 22-24 C. 2 3 . .

Chapter 2.

Figure 7 (a). Bacillus --- subtilis and Proteus vulgaris under --- ---- tungsten illumination. (b). Same field of view showing fluorescent Bacillus subtilis among Proteus vu1gari.s using epifluorescence-. . . . . . . . . . . . . . . .34

Figure 8 Rhizosphere populations of vegetative cells of -- Bacillus . . subtilis on onions grown in sterilized muck soil.. .46

Figure 9 Rhizosphere populations of spore cells of Bacillus . . subtilis on onions grown in sterilized muck soil.. .49

Figure 10 Rhizosphere populations of vegetative cells of Bacillus subtilis on onions grown in nonsterilized muck soil.. . . . . . . . . . . . . . . . . . . . . . . . . . 5 2

Figure 11 Rhizosphere populations of spore cells of ------ Bacillus subtilis on onions grown in nonsterilized soil.. 56 . . . .

Figure 12 Rhizosphere populations of vegetative cells of Bacillus subtilis on onions grown in muck soil under field conditions. 59 . . . . . . . . . . . . . . . . . . .

Fisure 13 Rhizosnhere po~ulations of sDore cells of Bacillus a - * & -- subtilis on onions grown in muck soil under field conditions. 6 2 . . . . . . . . . . . . . . . . . . . . . .

In t roduc t ion t o t h e t h e s i s . --

Sclero t ium cepivorum Berk., t h e c a u s a l agent of white r o t

of onion and o t h e r Allium spp. , i s of long s t a n d i n g , world wide

d i s t r i b u t i o n and of considerable economic importance (41 ,43) .

The pathogen e x i s t s i n s o i l a s dormant s c l e r o t i a which can

survive f o r many years i n t h e absence of t h e h o s t p l a n t ( 4 3 ) .

The d i s e a s e was f i r s t recorded i n t h e F r a s e r Valley of

B r i t i s h Columbia i n 1970. Since 1974 p rov i s ions of t h e \

Prov inc ia l P l a n t P ro tec t ion Act have p r o h i b i t e d f u r t h e r onion

production on known i n f e s t e d f i e l d s . These measures have not

h a l t e d t h e advance of t h e d i s e a s e , and a t t h e p r e s e n t time

v i r t u a l l y a l l farms producing commercial onions i n t h e Fraser

Valley a r e i n f e s t e d . " L i t e r a t u r e sugges ts ( 3 , 9 , 2 9 ) t h a t white

r o t may become a major l i m i t a t i o n t o onion product ion i n t h e

Fraser Val ley wi th in a few years i f t h e p r e s e n t i n f e s t a t i o n s

cannot be checked" ( 4 0 ) .

Chemicals can be used t o c o n t r o l so i l -borne p l a n t pathogens

by e l i m i n a t i n g t h e pathogen from t h e s o i l o r by reducing t h e

inoculum l e v e l t o allow economic crop product ion . However, no

p r a c t i c a l t r ea tmen t has y e t been produced t h a t w i l l e r a d i c a t e

t h e S . cepivorum fungus from t h e s o i l . Calomel (mercurous -

c h l o r i d e ) g i v e s economic con t ro l of t h e d i s e a s e b u t i s hazardous

and its use is no longer permitted. The fungicide Ronilan is

used in Europe, but is not registered on onions in North \

America. The need for an effective, cheap and safer alternative

is emphasized (22). Resistant varieties would be useful but at

present none are known (41).

Many cultural practices have come into use which tend to

reduce the progress of diseases. Liming of soil to bring the pH

up to about 7.0 reduced the incidence of white rot but did not

give complete control (38). Walker (43) studied the relationship

of soil temperature and moisture to the disease. He found that

the pathogen was most devastating at cool temperatures (10 to 20

C) and in fairly dry soil conditions (45% of the water holding

capacity). Avoidance of these conditions under field situations

is not practical.

Stimulation of germination of sclerotia of - S. cepivorum in

the absence of host plants, thereby reducing the level of

pathogenic inoculum, is an approach based on the research by

Coley-Smith and his co-workers (8). Field tests conducted by

Merriman -- et al. who injected onion oil in soil before sowing

have demonstrated the potential of this method for control of

white rot in dry bulb onions. However, introduction of this type

of disease control into commercial practices still requires

improvements in the methods, timing of application of treatment,

and availability of effective germination stimulants at

acceptible cost. Various attempts at biological control of white

+

rot have been reported and will be referred to in Chapter 1. The

subject of this thesis is an evaluation of the nature and

autecology of bacterial antagonists to S. cepivorum that have - provided significant control of white rot when applied to onions

as seed treatments.

Chapter 1.

Identification and differentiation of six bacterial isolates

antagonistic to Sclerotium cepivorum

Introduction

In a greenhouse experiment, Ahmed and Tribe (1) used

pycnidial dust prepared from Coniothyrium minitans as soil

treatments and as seed dressing to protect onion seeds grown in

S. cepivorum infested soil. They found that the control afforded -

by C. minitans protected the plants from white rot as well as

calomel dressing. C. minitans was recommended for development

for the practical control of white rot.

More recently, Ayers and Adams (2) using Sporidesmium

sclerotivorum demonstrated in moist sand, steamed soil and

natural soils that the mycoparasite was able to infect and

destroy the sclerotia of - S. cepivorum. They recommended the

mycoparasite for biological control by direct seeding of the

field to increase parasitism of the natural population of

sclerotia. However, its effectiveness may be limited by its

apparent inability to survive saprophytically and/or infect the

hyphae of - S. cepivorum.

The use of bacterial antagonists for the biological control

of onion white rot has met with some success (40). Utkhede and

Rahe (40) demonstrated that the levels of protection provided by

some of the six bacterial isolates obtained from - S. cepivorum

sclerotia were comparable to those provided by chemical

treatments. They concluded that these antagonists represented

practical potential for the field control of onion white rot.

The isolates were tentatively identified in 1977 as strains

of Bacillus subtilis Ehrenberg by students in Bioscience 303 at

Simon Fraser University

The objective of the research reported in this chapter was

to confirm the identity of the isolates and to differentiate

among them, if possible.

Methods and Materials

Six test isolates were investigated (Table 1). Stock

cultures were maintained on nutrient agar at 25 C. Twenty

four-hour-old cultures grown at 37 C were used as inocula for

morphological, cultural and biochemical studies.

Standard procedures (17) were followed for morphological

studies, Gram stain, Voges-Proskaur test for production of

acetylmethylcarbinol, utilization of carbohydrates, succinate

and citrate, hydrolysis of starch and casein, production of

Table 1. Sources o f B a c i l l u s s u b t i l i s i s o l a t e s and known s t r a i n s .

Source

Kelowna, B .C.

Olympia, Washington.

Burnaby , B . C.

Ca ro l i na B i o l o g i c a l

Supply Co.

urease and catalase, and the demonstration of possible anaerobic

growth utilizing an overlay of sterile mineral oil over

inoculated 18 glucose nutrient broth.

Growth in glucose ammonium salts medium and hydrolysis of

hippurate were tested according to Cowan and Steel (11).

Growth on nutrient agar slopes at 25 C and 60 C at pH 6 . 0 ,

and retardation effects due to 1% glucose on nutrient agar were

tested according to Wolf and Barker (45).

To test for antagonism against - S. cepivorum each bacterial

strain was applied as a single streak onto potato dextrose agar

(PDA) containing a freshly transferred plug from a day old

culture of S. cepivorum grown on PDA (the plug was cut with a -

sterile 4mm dia., cork borer). The antagonism test plates were

kept at 22-24 C under the normal light conditions of the

laboratory and evaluated five days after inoculation. This

antagonism study was conducted several times over a number of

different occassions.

Photographs of the six isolates on nutrient agar were taken

at the seventh day after inoculation.

Results

Morphological characteristics:

All of the six isolates formed rods whose size ranged from

0.8 x 2.4 to 0.8 x 3.2pm. The spores were oval and centrally

located. The typical spore size ranged from 0.3-0.5 x 1.0-1.6

Pm' Cultural characteristics:

The cultural characteristics for all six isolates on

various media are shown in Tables 2-4 and Figures 1-6.

In nutrient broth all isolates formed a pellicle two days

after inoculation and incubation at 37 C. Turbidity was slight

or lacking. The sediment was slight also.

In cultivation of the bacterial isolates with - S. cepivorum

on potato dextrose agar ( P D A ) , inhibition zones were observed

between the bacterial colony and mycelial growth by the second ,

day after inoculation and incubation under laboratory conditions

(Table 4). On the third day after inoculation the fungal

mycelium took on a brown colour and became black with time. By

the fifth day white precipitation bands appeared in the

1

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Figure 1. Bacillus subtilis isolate BK on nutrient agar on the 7th day after inoculation and incubation at 22-24 C, ( 7 x).

Figure 2. Bacillus subtilis isolate BO on nutrient agar on the 7th day after inoculation and incubation at 22-24 C, (5 X I .

Figure 3. Bacillus subtilis isol.ate B1 on nutrient agar on the 7th day after inoculation and incubation at 22-24 C, ( 6 X I .

Figure 4. Bacillus subtilis isolate B2 on nutrient agar on the 7th day after inoculation and incubation at 22-24 C, (6.5 x).

Figure 5. Bacillus subtilis isolate B4 on nutrient agar on the 7th day after inoculation and incubation at 22-24 C, ( 8 X I -

Figure 6. Bacillus subtilis isolate B8 on nutrient agar on the 7th day after inoculation and incubation at.22-24 C, (7 XI.

inhibition zones for some of the bacterial strains. These

precipitation bands became more conspicuous with time. Isolates

B2 and BK consistently displayed the heaviest bands (Table 4 ) .

Biochemical Characteristics.

All six isolates were Gram positive, catalase producing,

aerobic spore-forming rods (the three main identifying

characteristics of the genus Bacillus ) , and on the basis of

standard biochemical criteria (19,45) all of the isolates were

identified as B. subtilis. Their identities were further

substantiated by the comparison of sugar fermentation patterns

and other biochemical characteristics with those reported for - B.

subtilis by other authors(15,23,30).

Biochemical characteristics other than sugar fermentation

patterns (Table 5) showed no distinguishing characteristics for

any of the six isolates. Sugar fermentation patterns (Table 6)

disclosed that isolate B2 showed weak acid production in

D-glucose and none in D-mannitol, whereas all other isolates

produced acid from these sugars. Isolate BO produced a slightly

acid solution in salicin whereas the other strains did not. The

remainder of the isolates exhibited no distinguishing

Table 5. Biochemical c h a r a c t e r i s t i c s f o r i s o l a t e s and known s t r a i n s

of B a c i l l u s s u b t i l i s .

Tes t BK,BO,Bl,BZ,B4,B8, C h a r a c t e r i s t i c s of n i g e r , g l o b g i i . B a c i l l u s s u b t i l is.

Acid d e t e c t e d i n g lucose ammonium s a l t . Glucose r e t a r d o r y o r n o n i n h i b i t o r y . Urease product ion . Methly r ed r e a c t i o n . C i t r a t e u t i l i z a t i o n . S u c c i n a t e u t i l i z a t i o n Aerobic growth a t 60 C, pH 6 on n u t r i e n t aga r . Casein h y d r o l y s i s . S t a r c h hyd ro lys i s . Product ion o f a c e t y l - methylcarb inol . Hippura te h y d r o l y s i s . Reduction of n i t r a t e t o n i t r i t e . Anaerobic growth in n u t r i e n t a g a r . Anaerobic growth i n g lucose b r o t h . Growth i n 7% NaC1. P e l l i c l e i n n u t r i e n t b ro th .

a l l p o s i t i v e p o s i t i v e

a l l n o n i n h i b i t o r y non i n h i b i t o r y a l l grew;growth r e t a r d e d v a r i a b l e

a l l n e g a t i v e a l l n e g a t i v e

v a r i a b l e v a r i a b l e

a l l n e g a t i v e v a r i a b l e

a l l n e g a t i v e a l l p o s i t i v e a l l p o s i t i v e

a l l p o s i t i v e a l l n e g a t i v e

a l l p o s i t i v e

a l l n e g a t i v e

a l l n e g a t i v e a l l p o s i t i v e

a l l p o s i t i v e

n e g a t i v e p o s i t i v e p o s i t i v e

p o s i t i v e n e g a t i v e

p o s i t i v e

n e g a t i v e

n e g a t i v e p o s i t i v e

p o s i t i v e

1 A s r epo r t ed i n r e f e r e n c e s 15, 19, 23, and 4 5 -

Tab le 6. Sugar f e r m e n t a t i o n p a t t e r n s ' f o r i s o l a t e s and known s t r a i n s

o f B a c i l l u s s u b t i l i s .

Sugar B K 80 B1 B2 84 88 n i g e r g o l g i i

D g lucose + Sucrose + D rnanni t o 1 + D x y l o s e - L rharnnose - D a rab inose - Lactose - Sal i c i n - D s o r b i t a l -

- - -

1 p a t t e r n s i n d i c a t e d by: + = a c i d b u t no gas, + = weak, - = - nega t i ve .

characteristics.

Discussion and Conclusion.

Morphological, cultural and biochemical tests confirm the

identity of the six isolates as true strains of B. subtilis. The

behaviour of the strains in the various biochemical tests does

not allow for differentiation among them. While it is possible

to differentiate the strains on the basis of growth patterns on

nutrient agar, 10% skim milk agar, or PDA, it is not practical.

This impracticality is due to the subjective nature of growth

patterns and their variability in relation to factors such as

composition and age of nutrient medium, time, temperature, age

of culture, possible change in cultural characteristics with

repeated transfers and probably many other variables. It is

obvious that more reliable, efficient and less time consuming

methods are still needed for the differentiation of - B. subtilis

strains.

Chapter 2.

A quantitative autecological study of Bacillus subtilis in the

onion root rhizosphere.

Introduction

Plants and microorganisms coexist in a state of constant

interaction. Some specific interactions are of such obvious

importance that they attract intense research. The benefits of

symbiotic nitrogen fixation in the Rhizobium-legume interaction

are documented extensively (36). Another example of beneficial

interaction between plant and microbe is the mycorrhizal

(literally, fungus root) association. It is an effective

mechanism for increasing water and nutrient absorption by roots

(16). Marx (25) showed that mycorrhizal fungi protected roots of

shortleaf pine, Pinus echinata Nemnich, seedlings from infection

by Phytophthora cinnamomi Rands.

Bacterization of seeds for biological control of plant

diseases is well documented (6). Nevertheless, relatively little

progress has been made towards the long range goal of increasing

world food production via bacterization for biological control

(36). The goal of achieving biological control of plant

pathogens by manipulation of the microbial community in which

the damaging relationship is initiated remains remote.

Biological control of harmful interactions and exploitation

of beneficial associations requires a "bird's eye view" of

microbial ecology. Before any kind of plant-microorganism

association is attained, the microbe must first interact with

the microbial community of the soil. It must survive competition

in the rhizosphere until the interaction with the plant host is

consolidated. Data dealing with a specific microbe in a

rhizosphere before establishment of an association with a plant

host are virtually nonexistent (36).

The reason for this paucity of data is one of methodology.

Imrnunofluorescence is one of the few experimental procedures to

offer a method to study the autecolgy of a specific

microorganism in a natural rhizosphere (14,20,24,34,37,). The

technique requires facilities which are still not widely

available (36). Hence application of the method for examining

rhizosphere populations has been minimal.

One of the problems involved in enumerating fluorescent

antibody-stained bacteria is that the field of view is extremely

small and only a limited amount of soil can be tolerated in each

field. Consequently, populations must be high in order to

encounter a reasonable number of cells. The problem in aquatic

systems is less severe since microorganisms can be concentrated

on a membrane filter (18,37).

Since the development of an acceptable system for bacterial

enumeration in soil by Schmidt (34) several papers on this

subject have appeared. Reyes and Schmidt (32) used a fluorescent

antibody (FA) technique to enumerate the population of a

particular strain of Rhizobium japonicum Kirchner, in the

rhizosphere of its soybean host. More recently, Jones and Morley

(20) used a fluorescent enzyme linked immunosorbent assay

(ELISA) technique to study the effect of pH on host plant

'preference' for strains of Rhizobium trifolii Dangeard.

Malajczuk -- et al. (24) used a soil sieving technique together

with immunofluorescence to assess spore populations of - P.

cinnamomi in the rhizosphere and nonrhizosphere soils of tree

grass, Xanthorahoea australis Haeck.

The long dormant interest in the autecology of plant

rhizospheres is awakening in response to new experimental

methods (36).

Utkhede and Rahe (40) reported that the inoculation of

onion seeds with liquid cultures of several - B. subtilis strains

provided varying degrees of the biological control of onion

white rot. I am not aware of any published autecological study

of - B. subtilis in the onion root rhizosphere. This study was

undertaken to (i) evaluate the effect of onion seed

bacterization on rhizosphere populations of - B. subtilis, and

(ii) to determine whether immunofluorescence would differentiate

the six newly identified bacterial strains (Chapter 1).

This chapter describes the preparation of antisera against

the strain of B. subtilis ( ~ 2 ) that was most effective in

controlling onion white rot (40). The specificities of the

antisera were tested against diverse bacterial isolates from

soil and laboratory cultures. Gamma immunoglobulins (1g) from

the antisera were isolated and purified for use in the FA

technique. Immunofluorescence was then used to assess - B.

subtilis populations in onion root rhizosphere soils under

various conditions.

Methods and Materials.

A. Fluorescent antibody preparation and evaluation of

specificity.

Antisera against flagellar(H), somatic(0) and spore ( S )

cells of - B. subtilis strain B2 were prepared from white New

Zealand rabbits according to procedures described by Norris and

Wolf (28). Gamma immunoglobulins (Ig) of high titer antisera

were isolated and purified by precipitating three times with

ammonium sulphate and desalting by dialysis in phosphate

buffered saline(^^^) pH 7.2 (14). These Ig were used for all

subsequent FA studies.

An indirect FA technique was used to evaluate the

specificity of the antibodies. This technique requires two

binding reactions. Firstly, rabbit (primary) antibacteria Ig are

bound to the bacteria. Secondly, antirabbit

fluorescein-conjugate goat (secondary) Ig are bound to the

rabbit proteins (Figure 7). A brighter fluorescence is attained

with this method than with the direct method (21)(in the direct

technique the fluorescein molecule is conjugated directly to

rabbit antibacteria 1g). Caprine antirabbit Ig fluorescein

isothiocyanate was obtained in lyophilized form from BBL,

Cockeysville MD, USA.

For the specificity studies bacteria were heat fixed onto

glass slides. This was followed by seperate 30 min incubations

with primary and secondary antibodies. The incubations were

performed under moist conditions in the dark at room

temperature. Preparations were rinsed at the end of each

incubation with PBS pH 7.2. The preparations were dried with

tissue and covered with a drop of Difco FA mounting fluid. A

coverslip(#=l 1/2, 22mm sq.) was then gently lowered onto the

slide. FA-stained preparations were viewed with a Zeiss

microscope equipped with epiillumination at lOOOx magnification.

Illuminiation was provided by a HBO 50 mercury source , an

exciter filter BP450-490, chromatic beam splitter FT510 and

barrier filter LP520. Reactions of the fluorescing bacteria were

rated visually: -(none), +(dim), ++(medium bright), +++(very

F i g u r e 7 ( a ) . B a c i l l u s s u b t i l i s and P r o t e u s v u l g a r i s under t u n g s t e n i l l u m i n a t i o n . ( b ) . Same f i e l d of view showing f l u o r e s c e n t B. s u b t i l i s among - P. v u l g a r i s u s i n g e p i f l u o r e s c e n c e , (1000 x ) .

bright).

B.Population studies.

1.Pot trials under greenhouse conditions.

(a) Rhizosphere soil.

B. subtilis populations in rhizosphere soils of -

bacteria-treated and untreated onion seeds (cv. Autumn Spice)

were compared at 5, 10, 14, 21 and 42 days after planting. At

zero time, onion seeds were coated with bacteria by immersing

them in a 10-day-old potato dextrose broth culture of B2 for 5

min. Three samples of ten seeds each were transferred into

Erlenmyer flasks containing 30 ml of 0.85% saline and mixed

thoroughly for 3 min. Appropriate volumes were filtered through

Nucleopore filters (25 mm dia., 0.4um pore) and bacterial counts

were obtained using the FA technique.

Soil for this experiment was collected from a - S.

cepivorum-infested commercial vegetable farm in Burnaby, British

Columbia.

For sterilization, soil was placed in metal trays, covered

with aluminum foil, and kept wet for 30 min at 120 C.

Seeds for three replications per treatment were placed in

holes 0.5 cm dia., spaced 2 to 3 cm apart in soil in 15 x 13 x 5

cm plastic trays. Three seeds per hole were sown. There were

t h r e e t r a y s p e r t r ea tmen t . The t r a y s were t hen p laced i n a

growth chamber a t 18 C w i t h 12 h photoper iod ( ~ a d i a n t f l u x 96.4

uE m-2 s ec - I PAR measured a t p l a n t h e i g h t wi th L1-cor Quantum

sensor t y p e L1-190s coupled t o a L1-185 Quantum m e t e r ) . S o i l

mois ture was mainta ined below f i e l d c a p a c i t y .

Samples c o n s i s t e d of t e n p l a n t s p e r r e p l i c a t i o n . Whole r o o t

systems were dug c a r e f u l l y and s u b j e c t e d t o g e n t l e shaking. The

s o i l s t i l l adhe r ing was des igna t ed a s rh i zosphe re s o i l .

Dislodged s o i l was d i s c a r d e d . Roots and adher ing s o i l from t e n

p l a n t s (compr is ing a sample) were detached and a g i t a t e d f o r 10

sec i n a minimal amount o f s o l u t i o n c o n t a i n i n g 0.85 % s a l i n e , 10

ug/ml polymyxin B s u l p h a t e and 10 ug/ml cycloheximide. To

i s o l a t e and de te rmine t h e n a t u r e of any b a c t e r i a a n t a g o n i s t i c t o

S. cepivorum , l o o p f u l s o f t h e suspension were t hen s t r e a k e d - o n t o PDA p l a t e s c o n t a i n i n g a c t i v e l y growing c u l t u r e s o f - S.

cepivorum. B a c t e r i a a n t a g o n i s t i c t o - S. cepivorum were examined

morphologica l ly and immunologically.

A m o d i f i c a t i o n of Schmid t ' s procedures (34 ) was used t o

o b t a i n d i r e c t coun t s of FA-reacting b a c t e r i a i n t h e onion r o o t

rh i zosphe re a s w e l l a s i n nonrhizosphere s o i l . Addi t iona l

d i l u a n t (0 .85 8 s a l i n e , 0.05% Tween 80) was added t o t h e r o o t

suspension which was then blended f o r 3 min us ing a S o r v a l l

Omni-mixer a t a 3.5 s e t t i n g . Th i s mix ture was poured i n t o a

graduated c y c l i n d e r , b rought up t o 100 m l w i t h a d d i t i o n a l

d i l u a n t and s t i r r e d thoroughly f o r 2 min. A f t e r 30 min of

s e t t l i n g , appropr ia te volumes of superna tan t were p r e f i l t e r e d

using a 47 mm d i a . Nucleopore polycarbonate f i l t e r (2 .0 um pore

s i z e ) . For t h e f i e l d samples a f u r t h e r 1:10 d i l u t i o n was

necessary f o r 10-week and o lde r p l a n t s because t h e undi luted

suspension was too th ick t o p r e f i l t e r . B a c t e r i a were then

c o l l e c t e d on to an India ink-pre t rea ted Nucleopore f i l t e r ( 2 5 rnrn

d i a . 0.4 um p o r e ) . The e f f e c t i v e f i l t r a t i o n s u r f a c e was t r e a t e d

s e q u e n t i a l l y with 0.1 m l of gelatine-rhodamine conjugate ( t o

e l imina te nonspeci f ic b i n d i n g ) , primary and secondary

an t ibod ies . Each t reatment was incubated f o r 30 min i n dark

moist cond i t ions followed by washing wi th copious amounts of PBS

pH 7 . 2 . The f i l t e r s were then placed onto g l a s s s l i d e s with a

drop of Difco FA mounting f l u i d and covered wi th a #=1 1 / 2 , 22mm

sq. cover s l i p . Fluorescing b a c t e r i a i n 30 f i e l d s per f i l t e r

were enumerated. One f i l t e r each f o r v e g e t a t i v e and spore c e l l s

per sample was counted. The number of b a c t e r i a pe r t e n p l a n t s

was es t imated using t h e formula:

where N=average # of b a c t e r i a per f i e l d . A=effect ive f i l t e r i n g a r e a . a=area of microscopic f i e l d . D=dilut ion f a c t o r .

Prel iminary s t u d i e s had shown t h a t t h e r e was no c o r r e l a t i o n

between t h e number of b a c t e r i a with e i t h e r a i r - d r i e d s o i l o r a i r

d r i e d p l a n t r o o t weight. Therefore, t h e number of b a c t e r i a per

t e n p l a n t s was chosen a s t h e a r b i t r a r y b a s i s f o r p resen ta t ion of

data, except for the comparison of levels of B. subtilis between -

rhizosphere and nonrhizosphere soils where the number of

bacteria is expressed per gram of air dried soil. Rhizosphere

soil was collected by filtration through a Whatman # 1 filter

(preweighed) which was then air dried at 22-24 C for four days.

(b ) Nonrhizosphere soils.

At 5, 14 and 42 days after planting approximately 2 cc

volumes of nonrhizosphere soil from each of three replications

of sterilized and nonsterilized soil were scooped from between

plants at a depth of 2 to 4 cm below the soil surface. These

samples were then subjected to the procedures described for the

quantification of B. subtilis in the onion root rhizosphere.

Bacteria inhibitory to - S. cepivorum were isolated by the

streaking technique and examined morphologically and

immunologically.

2 .Field trial.

The field trial was conducted beginning May 2, 1980 in

Burnaby, British Columbia on the commercial vegetable farm from

which soil for the pot trials was obtained. Onions, cv. Autumn

Spice, were grown on raised beds containing 5 rows 1.75 m long,

spaced 25 cm apart and double seeded at 7 cm spacing. Plants for

sampling were taken at random from the three middle rows. Each

bed constituted a replication of which there were three for each

treatment. The beds were prepared parallel with every alternate

bed being planted with treated seeds.

During the growing period samples were taken at 28, 49, 70,

91, 112 and 1 3 3 days after planting and subjected to procedures

already described for enumeration of bacteria.

All results were transformed x'= r( x + 0.5) before analysis by ANOVAR and the Newman-Keul's test at 5% level of

significance.

Results

A.Specificity of - B. subtilis antisera.

Vegetative antibodies, flagellar(~) and somatic(^), cross

reacted with antigens of each other but not with spores (Table

7). Spore antibody did not cross react with either flagellar or

somatic antigens (Table 7).

Flagellar and somatic antibodies of B. subtilis B2 did not - cross react with any of the flagellar or somatic antigens of the

other Bacillus species tested able 8). The spore antibody

cross reacted with spores of - B. pumilis and - B. coagulans(Tab1e

8)

With the exception of one bacterial isolate from the soil

there was no cross reactivity shown by either flagellar or spore

antibodies (Table 9). This one exception was later identified as

a strain of B. subtilis. -

B. Population studies.

1.Pot trial under greenhouse conditions.

(a).Sterilized soil.

Populations of the vegetative cells of - B. subtilis

T a b l e 7. Cross reactivity o f Bacillus subtilis 8 2 antibodies with1 f l a g e l l a r ( ~ ) , s o m a t i . m d - ) antigens o f B.subtilis strains .

Strains Antigens ~ n t ibodiesl

+ + -

ti-

+ -

+++ +++ -

ttt

+++ -

+++ +++ -

+ + -

1 Visual rating indicated by:

+++ = very bright, ++ = medium bright, + = d i m , - = none.

Table 8. C r o s s r e a c t i v i t y o f B a c i l l u s s u b t i l i s ( ~ 2 ) f l a g e l l a r ( H ) , s o m a t i c ( 0 ) and s p o r e ( S ) a n t i b o d i e s w i t h H , 0 and S

a n t i g e n s o f o t h e r B a c i l l u s s p e c i e s .

B a c i l l u s A n t i g e n s ~ n t i b o d i e s l

s p e c i e s H 0 S

b r e v i s

c e r e u s

c i r c u l a n s

c o a g u l a n s

mega t e r i u m

pumilis

1 V i s u a l r a t i n g i n d i c a t e d by: t-t. = medium b r i g h t , - = none.

Table 9. Cross r e a c t i v i t y of B a c i l l u s s u b t i l i s B2 f l a g e l l a r ( H )

and spore(S) an t ibod ie s wi th b a c t e r i a l i s o l a t e s

from s o i l .

S o i l i s o l a t e s Antibodies I

24 Gram + r o d s

16 Gram - rods

1 Gram + c o c c i

9 G r a m + r o d s

only 1 + only 1 + - - - - - -

1 + = p o s i t i v e r e a c t i o n , - = nega t ive r e a c t i o n .

increased dramatically in the rhizospheres of plants grown from

bacteria treated seeds during the first few days of growth.

Vegetative cells of - B. subtilis also increased in the

rhizospheres of control plants, but more slowly and to lower

levels. The enhancement of vegetative cell populations

associated with seed treatment was significant for at least ten

days, but at the 14, 21 and 42 day sampling periods rhizosphere

populations for both treatment and control plants had stablized

and were virtually identical (Fig. 8, Table 10).

Spore populations of B. subtilis in the rhizospheres of

treated and control plants showed generally similar trends. A

significant enhancement associated with seed treatment was

observed at 5, 10, 14 , and 21 day sampling periods (Fig. 9,

Table 10). No significant differences in vegetative or spore

populations of B. subtilis between control and treatment were -

observed in nonrhizosphere soil a able 11).

(b) .Nonsterilized soil.

Seed treatment with bacterial strain B2 enhanced the

vegetative cell population of - B. subtilis over control

significantly only at 5 days after planting. The population

trends over time of vegetative cells exhibited by control and

treated seedlings were generally similar to those observed in

sterilized soil (Fig. 10, Table 12). Seed treatment had no

apparent effect on spore populations of B. subtilis and there

were no significant changes in rhizosphere spore populations

Figure 8. Rhizosphere populations of vegetative cells of Bacillus subtilis on onions grown in sterilized muck soil under greenhouse conditions. Bars identified by the same letter do not differ significantly, Newman Keul's test, P

0 . 0 5 .

0 5 10 14 21 4 2 Days after p l a n t i n g

Ta

ble

1

0.

Po

pu

lati

on

s o

f v

eg

eta

tiv

e(V

) an

d

spo

re(S

) c

ell

s o

f B

ac

illu

s s

ub

tili

s i

n r

hiz

osp

he

re

-4

of

on

ion

s gr

own

in s

teri

liz

ed

muc

k s

oil

,(x

10

/l

Op

lan

ts).

Day

s a•

’ te

r V

eg

eta

tiv

e (V

) 1

Sp

ore

(S) 1

v

+s

1

pla

nti

ng

c

on

tro

l tr

ea

tme

nt

co

ntr

ol

tre

atm

en

t c

on

tro

l tr

ea

tme

nt

1

Val

ues

wit

hin

ea

ch

ce

ll t

yp

e f

oll

ow

ed

by

th

e s

ame

lett

er

do

no

t d

iffe

r s

ign

ific

an

tly

,

New

man

K

eu

l's

test,

P

-

< 0

.05

.

Figure 9. Rhizosphere populations of spore cells of Bacillus subtilis on onions grown in sterilized muck soil under greenhouse conditions. Bars identified by the same letter do not differ significantly, Newman Keul's test, P 5 0 .O5 .

10 14 Days af

21 t e r planting

Ta

ble

11.

Co

mp

aris

on

o

f p

op

ula

tio

ns

of

Ba

cil

lus

s

ub

tili

s i

n n

on

rhiz

osp

he

re a

nd

rh

izo

sph

ere

so

ils

of

on

ion

s g

row

n

in s

teri

liz

ed

muc

k s

oil

, (x

1

0-

~/

~m

s

oil

).

Ce

ll

typ

e

Day

s a

fte

r

No

nrh

izo

sph

ere

R

hiz

osp

he

re

pla

nti

ng

c

on

tro

l tr

ea

tme

nt

co

ntr

ol

tre

atm

en

t

Ve

ge

tati

ve

1

5

Sp

ore

1

1

Va

lue

s fo

r e

ac

h c

ell

ty

pe

fo

llo

we

d b

y

the

sam

e le

tte

r d

o n

ot

dif

fer

sig

nif

ica

ntl

y,

New

man

K

eu

l's

test,

P

0.0

5.

Figure 10. Rhizosphere populations of vegetative cells of Bacillus subtilis on onions grown in nonsterilized muck soil under greenhouse conditions. Bars identified by the same letter do not differ significantly, Newman Keul's test, P - 0.05.

0 5 10 14 21 ~ a ~ $ after p l a n t i n g

Ta

ble

1

2.

Po

pu

lati

on

s o

f v

eg

eta

tiv

e(V

) an

d

spo

re(S

) c

ell

s o

f B

ac

illu

s s

ub

tili

s i

n r

hiz

osp

he

re

- 4

of

on

ion

pla

nts

gr

own

in n

on

ste

rili

ze

d m

uck

so

il,(

x 1

0

11

0 p

lan

ts).

Day

s a

fte

r

Ve

ge

tati

ve

(V)

Sp

ore

(S) 1

v

+s

1

pla

nti

ng

c

on

tro

l tr

ea

tme

nt

co

ntr

ol

tre

atm

en

t c

on

tro

l tr

ea

tme

nt

I V

alu

es

for

ea

ch

ce

ll t

yp

e f

oll

ow

ed

by

th

e s

ame

lett

er

do

no

t d

iffe

r s

ign

ific

an

tly

, N

ewm

an

~e

ul

's te

st,

P -

< 0

.05

.

over time for either control or treated plants (Fig. 11, Table

12).

With the exception of one sample(treatment 5 days after

planting) there were no significant differences in vegetative

cell or spore populations between control and treatment samples

of nonrhizosphere soil a able 13).

2.Field trial.

At only one sampling period, 91 days after planting, was

there a significant enhancement of vegetative cell populations

for treatment over control. (Fig. 12, Table 14). The population

trends for both treatment and control increased then remained

steady with time. There were no significant differences in the

spore populations between treatment and control (Fig. 13, Table

14).

Meteorological data for the field trial (~ables 15, 16)show

adverse wet conditions that prevailed for the 1980 growing

season, compared with those of previous years.

C.Antagonistic bacteria from sterilized and nonsterilized soil.

Bacteria antagonistic to - S. cepivorum were isolated with a

higher frequency from treated than from control plantings in

both sterilized and nonsterilized soils. More antagonistic

Figure 11. Rhizosphere populations of spore cells of Bacillus subtilis on onions grown in nonsterilized muck soil under greenhouse conditions. Bars identified by the same letter do not differ significantly, Newman Keul's test, P - < 0.05.

0 5 10 14 21 Days after planting

F i g u r e 1 2 . Rh izosphere p o p u l a t i o n s o f v e g e t a t i v e c e l l s of B a c i l l u s s u b t i l i s on o n i o n s grown i n muck s o i l under f i e l d c o n d i t i o n s . Bars i d e n t i f i e d by t h e same l e t t e r do n o t d i f f e r s i g n i f i c a n t l y , Newman K e u l ' s t e s t , P 5 0.05.

28 49 70 91 112 Days after planting

Ta

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Figure 13. Rhizosphere populations of spore cells of Bacillus subtilis on onions grown in muck soil under field conditions. Bars identified by the same letter do not differ significantly, Newman Keul's test, P - < 0 . 0 5 .

0 28 49 70 91 112 Days after planting

Tab le 15. Me teo ro l og i ca l da ta f o r the 1980 growing season: mean

d a i l y temperature and r a i n f a l l f o r 10 day pe r i ods p r i o r

t o f i e l d sampl ing a t i n d i c a t e d days a f t e r p l a n t i n g .

Days after

planting Temperature (C ) Rainf a l l (mm d n i ly) .

Table 16. Summary o f me teo ro l og i ca l da ta f o r f i e l d t r i a l area f o r 1976 .

th rough 1980 growing seasons1.

---

1976 1977 1978 1979 1980

---- -- ------ --.----

A i r temperature (c ) 30cm

above ground

a b s o l u t e range 7-27

mean da i 1 y range 14-22

Soi 1 temperature (C) lOcm

below ground

a b s o l u t e range 3 - 3 0

mean da i 1 y range 20-25

R a i n f a l l (mm)

t o t a l 283

d a i l y mean f o r

May 4.8 June 2.3

J u l y 1 .o August 3 . 2

Septembe r 1 . 6

- ----- -- - - -

1 Data f o r t he p e r i o d s 18 May t o 15 September 1976, 3 May t o 15

September 1977, 16 May t o 15 September 1978, 3 May t o 15

September 1979 and 7 May t o 12 Septeniber 1980.

b a c t e r i a were i s o l a t e d from t r e a t e d than from c o n t r o l

rhizosphere samples during t h e e a r l y s t a g e s of p l a n t growth

(Table 1 7 ) .

Discussion

A. S p e c i f i c i t y of Bac i l lus s u b t i l i s a n t i s e r a .

(1) . With s t r a i n s of - B . s u b t i l i s .

S tudies by Norr i s and Wolf ( 2 8 ) on - B . s u b t i l i s and Davies

(12) on B a c i l l u s polymxa Prazmowski, have demonstrated t h a t t h e

f l a g e l l a r ( ~ ) a n t i s e r a can be s t r a i n s p e c i f i c . However, a f t e r

maintaining t h e s e s t r a i n s on n u t r i e n t aga r f o r s e v e r a l months

Davies ( 1 2 ) found t h a t f r e s h a n t i s e r a bound not only t h e

homologous s t r a i n but o t h e r s t r a i n s a l s o . Both groups found t h a t

i n j e c t i o n of H an t igen s t imula ted t h e product ion of somat ic(0)

antibody i n a d d i t i o n t o t h e homologous H antibody. My r e s u l t s

(Table 7 ) confirm t h e i r f ind ings , t h a t i s t h a t t h e H a n t i s e r a

bound t h e homologous 0 ant igen a s we l l a s t h e H an t igens of

o t h e r s t r a i n s . The s i m i l a r degrees of f luorescence exhib i ted by

strains B1, B2, and B 4 a able 7 ) demonstra'te that strains B1 and

B4 share more of the antigenic determinants of strain B2 than do

strains BK, BO and B8. Differentiation among the six strains was

not possible using FA.

The spore antibody did not bind H and 0 antigens and vice

versa a able 7). In agglutination reactions, the spore antibody

was less specific. Spore antibody cross reacted with all of the

B. subtilis strains tested a able 7). No variation in the degree

of fluorescence was found. This is in agreement with the study

by Norris and Wolf (28) who reported that the spores of each of

the 25 B. subtilis strains they tested were agglutinated by a

spore antiserum.

(2). With other Bacillus species.

Data in Table 8 confirm the finding of Norris and Wolf (28)

that both flagellar and somatic antibodies are species-specific.

The spore antibody cross reacted with spores of Bacillus pumilis

Meyer and Goltheil, and Bacillus coagulans Hammer a able 8).

These three bacteria share at least a common antigenic

determinant. The nonspecificity of the spore antibody for spores

of B. pumilis confirms the similar finding of Norris and Wolf

(28).

( 3 ) . With other soil bacteria.

It was essential to deterpine the specificity of the

various antibodies prior to their utilization in a soil

population survey. Of all the 50 soil bacteria tested , only one

bound the H antibody (Table 9). This particular isolate was

later identified as B. subtilis. Data in Tables 7 and 8 combined -

with this data set indicate that the H antiserum has a high

degree of species specificity. The spore antibody cross reacted

with only one of the soil bacteria spores that which was later

identified as B. subtilis. -

B.Population studies.

1. Pot trials under greenhouse conditions.

(a) Sterile soil.

Soil sterilization serves to eliminate competing organisms

that would hamper the initiation of an association between plant

and microbe. The data depicted in Table 10, Fig. 8 and 9

indicated that bacterization enhanced the - B. subtilis population

up to at least ten days. These data suggest that B. subtilis is

not being established in the root rhizosphere of cv. Autumn

Spice despite the initial absence of other microflora.

(b) Nonsterile soil.

Data in Table 12, Fig. 10 and 11 reinforce the finding that

treatment significantly enhanced the B. subtilis population.

However, these significant differences were of a shorter

duration than those that occurred in sterile soil. At ten days

there was no significant difference between treatment and

control. This shorter duration is to be expected when one

considers the competitive nature of nonsterile soil. Existing

microflora can prevent the establishment of an inoculant by

competition and/or antibiosis. Indeed isolations of bacteria

from nonrhizosphere soil and rhizosphere soils gave evidence for

the presence of bacteria antagonistic to S. cepivorum (Table

17). These antagonistic bacteria also displayed diffusible

antibiotics suppressive to the growth of B2 when they were

plated together on PDA.

( c ) Assessment of Bacillus subtilis in nonrhizosphere soils.

Data in Tables 11 and 13 lead to several major

observations. Nonrhizosphere soils from treated and untreated

pot t r i a l s contained both vegeta t ive and spore c e l l s of B . - s u b t i l i s . Secondly, f o r the majori ty of the samples assayed the -

r e s i d e n t populat ion i n t h e rhizosphere of cont ro l p l a n t s was not

dramat ica l ly g r e a t e r than i n nonrhizosphere s o i l . Furthermore,

although t r e a t e d p l a n t s demonstrated a s i g n i f i c a n t l y g rea te r

population a t f i v e days a f t e r p lan t ing , t h e r e was no d i f fe rence

a t 14 days. These a d d i t i o n a l data lend support t o the f inding

t h a t B . s u b t i l i s i s not c lose ly a s soc ia ted with onion r o o t s . -

2 . F ie ld t r i a l s .

Treated seeds exh ib i t ed a s i g n i f i c a n t l y higher vegeta t ive

B. s u b t i l i s c e l l population f o r the 13 t h week sample (Table 14, - Fig. 1 2 ) . This phenomenon coincided with a warm dry t e n day

period p r i o r t o sampling (Table 1 5 ) . I t suggests t h a t c l ima t i c

condi t ions played a r o l e i n determining t h e r e s iden t B . s u b t i l i s - -

populat ion. For t h e f i r s t time i n t h r e e years of f i e l d t r i a l s E32

t reatment of onion seeds did not give s i g n i f i c a n t cont ro l of

J-

onion white r o t ( R.S.Utkhede and J.E.Rahed', personal

communication). This lack of cont ro l a l s o coincided with an

abnormal amount of r a i n f a l l f o r t h e 1980 growing season over

s i m i l a r per iods f o r t h e preceeding t h r e e years able 1 6 )

~ g r i c u l t u r e Canada Research S t a t i o n , Summerland, B . C . a n d ~ i o l o g i c a l Sciences, Simon Fraser Univers i ty , Burnaby, B . C . , r e s p e c t i v e l y .

I n o r d e r f o r t h e inoculum t o c o n t r o l t h e d i sease it m u s t

mul t ip ly around t h e emerging r o o t . I f it d e c l i n e s o r d i e s a

permanent c o n t r o l i s un l ike ly . I have a s c e r t a i n e d t h a t t h e B. -

s u b t i l i s populat ion i n t h e onion r o o t rh izosphere showed no

d i f f e r e n c e between t reatment and c o n t r o l . I f Utkhede and Rahe

had obta ined s i g n i f i c a n t c o n t r o l of onion whi te r o t during t h e

1980 f i e l d t r i a l it could suggest t h a t t h e r e s i d e n t s t r a i n s of

B. s u b t i l i s were d i f f e r e n t i n t r e a t e d p l a n t s than i n c o n t r o l s ,

and t h a t t h i s s t r a i n more e f f e c t i v e l y modified t h e microflora

which i n t u r n e f f e c t e d t h e con t ro l of onion whi te r o t .

Unfortunately t h e s e n s i t i v i t y of t h e a n t i s e r a developed could

not r e a d i l y d i f f e r e n t i a t e among t h e s t r a i n s of - B . s u b t i l i s .

Attempts t o r e i s o l a t e and i d e n t i f y t h e inocu la ted B 2 s t r a i n were

success fu l only i n t h e e a r l y s t a g e s (up t o 10 days a f t e r

p l a n t i n g ) of p l a n t growth, when i t s popula t ion was high a able

1 7 ) .

Conclusion

Fluorescent antibody(irnmunof1uorescence) techniques provide

a new and unique capaci ty t o i d e n t i f y and enumerate s p e c i f i c

b a c t e r i a d i r e c t l y i n a n a t u r a l environment. The method has been

used for strain detection and enumeration of several bacteria in

soi1(13,18,20,21,24,32,34,37). For these reasons several

antisera for - B. subtilis strain B2 were developed.

The cross reactivity studies demonstrated several points:

(i) the vegetative antibody did not cross react with spore

antigens, (ii) it could not be used to differentiate among the

six - B. subtilis strains, (iii) it was species specific, and (iv)

the spore antibody did not cross react with the vegetative

antigens(H and 0). It was less specific because it bound not

only - B. subtilis spores but also those of - B. pumilis and - B.

coaaulans.

This is apparently the first study concerning the direct

enumeration of populations of - B. subtilis in rhizosphere soil of

developing onion roots. In the rhizosphere of onion roots - B.

subtilis multiplied during the early stages of germination and

seedling development, but the population then declined to levels

not significantly different from those in untreated seed.

Results indicated no close relationship between - B. subtilis and

onion roots. In order to maintain a high population of a

desirable - B. subtilis strain an occasional spraying of onion

plants with the bacterium is recommended. It is also important

that future studies evaluate conditions such as strain type, age

of inoculum, soil fertility, pH, moisture, etc., that can affect

the survival and proliferation of bacterial antagonists. Some of

these studies are now feasible with the advent of the FA

t e c h n i q u e .

G e n e r a l d i s c u s s i o n c o n c l u s i o n and recommendat ions .

I t s b r o a d s p e c t r u m o f a c t i v i t y , i t s a b i l i t y t o a d a p t t o

d i f f e r e n t e n v i r o n m e n t a l c o n d i t i o n s and i t s c a p a c i t y t o i n c r e a s e

y e i l d i n some c r o p s ( 2 7 , 4 0 ) s h o u l d make - B. s u b t i l i s an

a t t r a c t i v e b i o l o g i c a l c o n t r o l a g e n t f o r f u r t h e r i n v e s t i g a t i o n

and d e v e l o p ~ e n t . A number o f d i f f e r e n t ways o f a p p l y i n g t h e

a n t a g o n i s t o n t o s e e d s such t h a t t h e i n o c u l u m remains v i a b l e and

i n optmium numbers s h o u l d b e e v a l u a t e d . I t h a s been shown t h a t

t h e amount o f inoculum a p p l i e d d o e s i n f l u e n c e t h e e s t a b l i s h m e n t

o f t h e a n t a g o n i s t and i t s a b i l i t y t o c o n t r o l t h e pa thogen .

P e n i c i l l i u m o x a l i c u m a t 5 x l 0 / s e e d d i d n o t p r o t e c t p e a s from

s e e d l i n g b l i g h t , b u t a t 6 x l 0 / s e e d g a v e as good a c o n t r o l as

d i d s e e d t r e a t m e n t w i t h Cap tan ( 4 4 ) . Brown -- e t a l . ( 1 7 ) found

t h a t 1 0 t o 1 0 b a c t e r i a A z o t o b a c t e r s p . w e r e r e q u i r e d f o r

r h i z o s p h e r e e s t a b l i s h m e n t .

O t h e r methods f o r a p p l y i n g b a c t e r i a o n t o s e e d s t o f a v o u r

e s t a b l i s h m e n t i n t h e r h i z o s p h e r e o f h o s t p l a n t s would b e t o

i n c l u d e a n t i b i o t i c s , n u t r i e n t s , o r o t h e r a n t a g o n i s t s , d i r e c t l y

or p e l l e t e d o n t o t h e s e e d . These s h o u l d a l s o b e e v a l u a t e d . U n t i l

r e c e n t l y no e f f i c i e n t means were a v a i l a b l e t o s t u d y t h e e f f e c t

o f t h e s e d i f f e r e n t t r e a t m e n t s . Such s t u d i e s a r e now f e a s i b l e

with the FA technique.

In the population studies of the vegetative cells of - B.

subtilis only the 13th week sample showed any significant

population difference between bacteria-treated and/-untreated

seeds. A study is recommended to determine whether this

difference is real or due to chance since most of the other data

seem to indicate that there was no establishment of B. - subtilis

in the onion rhizosphere for any long period of time. The study

should try to mimic the conditions of moisture and temperature

that accompanied the time period at which the significant

difference occurred. Should this difference prove to be real

then bacterial treatment had something to do with it, even

though the FA technique did not reveal enhanced populations at

earlier sampling periods.

Coley-Smith and King (10) hypothesized that bacteria "act

in soil as onion rhizosphere colonists and metabolize the

germination stimulants for - S. cepivorum produced from onions".

Sclerotial germination would thus be reduced resulting in a

reduced percentage of infection. A second possible mechanism is

one of antibiosis. Utkhede and Rahe (40) have demonstrated that

B. subtilis strains produce antibiotics inhibitory to mycelial -

growth of - S. cepivorum on PDA. However no evidence for such a

mechanism in soil has been obtained. Both explanations are

possible and could act in concert. These two hypotheses depend

upon the presence of bacterial antagonists in the rhizosphere.

My study of - B. subtilis in onion root rhizosphere has

demonstrated that for unknown reasons B2 did not readily become

established in the roots of Autumn Spice. Biotic and abiotic

factors are probably involved.

Jones and Morley (20) demonstrated that soil pH affected

the association of two strains of Rhizobium trifolii Dangeard,

with their white clover host. Brown et al. (7) inoculated wheat --

grains with Azobacter cultures of different ages. They found

that the populations established in the rhizosphere varied with

the culture age. Viability of young inoculum decreased rapidly

in one day and declined slowly over eight weeks, after which no

cells were found. Old cultures, consisting entirely of encysted

bacteria gavs better rhizosphere establishment. Rakhno and Ryys

(31) stated that it was futile to add bacteria to soils that

would not support these organisms. Soils rich in minerals,

organic matter, a pH near neutrality and moisture conditions

favouring plant and microbial growth would give best results.

Therefore, the immediate problem is to find conditions necessary

for the survival and proliferation of these bacterial

antagonists before any kind of mechanism is investigated. The FA

technique provides an excellent vehicle for such studies.

Intensive research to control plant diseases using

bacterial and fungal biocontrol agents is a recent development.

The number of examples of successful biological control of plant

disease is still minimal. However, the fact that some do exist

should provide stimulus for researchers to develop better

control agents and methods for plant protection. The biological

hazards of chemicals and their rising costs coupled with the

need for a steady and reliable food supply make continued

efforts for development of alternative methods such as microbial

antagonists essential.

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