Alcaligenes faecalis: Identification and study of its antagonistic properties against Botrytis cinerea

by

Dagoberto Rodriguez Gonzalez, B. Sc.

A Thesis

submitted to the Department of Biological Sciences

in partial fulfillment of the requirements

for the degree of Master of Science

October, 1998

Brock University St. Catharines, Ontario Canada

© Dagoberto Rodriguez Gonzalez, 1998 2

Abstract

A Gram negative aerobic flagellated bacterium with fungal growth inhibitory properties was isolated from a culture of Trichoderma harzianum. According to its cultural characteristics and biochemical properties it was identified as a strain of Alcaligenes (aeca/is Castellani and Chalmers.

Antisera prepared in Balbc mice injected with live and heat-killed bacterial cells gave strong reactions with the homologous immunogen and with ATCC 15554, the type strain of A. taeca/is, but not with Escherichia coli or Enterobacter aerogens in immunoprecipitation and dot immunobinding assays.

Growth of Botrytis cinerea Pers. and several other fungi was significantly affected when co-cultured with A. taeca/is on solid media. Its detrimental effect on germination and growth of B. cinerea has been found to be associated with antifungal substances produced by the bacterium and released into the growth medium.

A biotest for the antibiotic substances, based on their inhibitory effect on germination of B. cinerea conidia, was developed. This biotest was used to study the properties of these substances, the conditions in which they are produced, and to monitor the steps of their separation during extraction procedures.

It has been found that at least two substances could be involved in the antagonistic interaction. One of these is a basic volatile substance and has been identified as ammonia. The other substance is a nonvolatile, dialysable, heat stable, polar compound released into the growth medium.

After separation of growth medium samples by Sephadex G-10 column chromatography a single peak with a molecular weight below 700 Daltons exhibited inhibitory activity. From its behaviour in electrophoretic separation in agarose gels it seems that this is a neutral or slightly positively charged 4

Acknowledgments

To my supervisor Dr. M. S. Manocha for giving me the opportunity to pursue this study and for his support and advice.

To Dr. A. Castle, Dr. L. Stobbs, and Dr. D. Battista for their many valuable comments and advice.

To Ismail AI-Jourmy and J. Pinder for being always helpful in so many ways.

To all the people at Brock University and Vineland Research Station who have helped me. They have contributed to make my stay at Brock an enjoyable experience. 5

Table of Contents

Page Abstract 2

Acknowledgments 4

Table of Contents 5

List of Tables 6

List of Illustrations 7

Introduction 9

Review of literature 11 Botrytis cinerea 11 The genus Alcaligenes 15 Bacteria in biocontrol 18

Materials and Methods 21 Cultures 21 Isolation and identification 25 Serological tests 27 Effects of A. faecalis on B. cinerea germination and growth 29 Biotests for fungal inhibitors 29 Biocontrol tests 30 Inhibitory substances produced by A. faecalis 31

Results 34 Isolation and identification 34 Serological tests 39 Effects of A. faecalis on B. cinerea germination and growth 48 Biocontrol tests 55 Inhibitory substances produced by A. faecalis 61 The nature of the inhibitory substance 67 Purification of the inhibitory substance 72

Discussion 75

References 82 6

List of Tables

Page

Table 1. List of Botrytis cinerea isolates used in this study 22

Table 2. Compound tested as sole source of carbon 26

Table 3. Serological reactions of antisera prepared against Alcaligenes faecalis (SF) 40

Table 4. Effect of Alcaligenes faecalis on colony growth of several fungi 58

Table 5. Relationship between the inhibitory activities and optical densities of several 144 h old Alcaligenes faecalis cultures in different media 66 7

List of Illustrations

Page Figure 1. Electron micrograph of a sample of a 48 h old nutrient broth culture of Alcaligenes faecalis SF showing flagellated cells 37

Figure 2. Dot immunobinding assay of Alcaligenes faecalis 42

Figure 3. Growth curves of Alcaligenes faecalis at different temperatures 45

Figure 4. Growth curves of Alcaligenes faecalis in different media 45

Figure 5. The pH of shaken cultures of Alcaligenes faecalis at 37°C as influenced by different media 47

Figure 6. Effect of concentrated suspensions of Alcaligenes faecalis on germination of conidia of Botrytis cinerea 50

Figure 7. Effect of a 1:8 dilution of a suspension of Alcaligenes faecalis on germination of conidia of Botrytis cinerea 50

Figure 8. Effect of the dilution of liquid culture of Alcaligenes faecalis on Botrytis cinerea germ tubes 52

Figure 9. Effect of washed cells of Alcaligenes faecalis on Botrytis cinerea germ tube elongation in water 52

Figure 10. Effect of Alcaligenes faecalis on Botrytis cinerea germ tubes, formation of phialides and microconidia 54

Figure 11. Effect of Alcaligenes faecalis on colony growth rate of Botrytis cinerea 57

Figure 12. Relationship between inhibitory effect of Alcaligenes faecalis and colony growth rate of Botrytis cinerea 57

Figure 13. Partial protection of grapes by Alcaligenes faecalis against Botrytis rot 60

Figure 14. Treatment of grape berries with Alcaligenes faecalis prevents damage caused by Botrytis cinerea 63

Figure 15. Effect of Alcaligenes faecalis on Botrytis cinerea induced damage in fresh cabbage leaf fragments 63 8

Figure 16. Filtrated liquid from one week old cultures of Alcaligenes faecalis completely inhibited germination of Botrytis cinerea conidia 65

Figure 17. Changes in time of the inhibitory activity and optical density of the cultures of Alcaligenes faecalis in MBC 69

Figure 18. Botrytis cinerea does not grow in a flask where Alcaligenes faecalis grew for one week 69

Figure 19. Effect of ammonia on germination of conidia of Botrytis cinerea 71

Figure 20. The inhibitory activity of cultures of Alcaligenes faecalis in MBC against Botrytis cinerea was removed by dialysis but not by 15 minute heat treatment between 55 and 100°C or lyophilization 71

Figure 21. The inhibitory activity of cultures ofAlcaligenes faecalis in MBC after separation in a column of Sephadex G-1 0 was localized in a single peak 74 9

Introduction

Botrytis cinerea is a fungus which produces important diseases in fruits, vegetables and other crops (Coley-Smith, 1980; Verhoeff, 1992). It often causes serious losses in grapes under the humid conditions of southern Ontario and other countries (Northover, Ker and Leuty, 1990). Control of diseases caused by B. cinerea is achieved primarily with the fungicide Iprodione in the USA and Canada and with Vinclozolin and Captan in other countries, but the pathogen seems to develop resistance to these fungicides (Harman et aI., 1996).

Increasing concerns on the ecological and toxicological impact of chemicals and the risk of development of resistance to fungicides has focused attention on using biocontrol agents to fight fungal diseases (Chet and Inbar, 1994). Biological control could replace all or part of the requirements for chemical pesticides (Harman et aI., 1996). Most of the work in biological control of Botrytis has been done with Trichoderma spp. (Chet, 1987; Dubos, 1992; Gullino, 1992a; Harman et aI., 1996; Samuels, 1996; Tronsmo, 1991).

In an early experiment in our laboratory, in which a culture of Trichoderma harzianum was inoculated on malt extract agar, only bacterial colonies developed. Growth of both T. harzianum and Botrytis cinerea Pers. was significantly affected when co-cultured with these bacteria on solid media, hence antagonism was suspected.

Antagonistic bacteria are presently under scrutiny as means of biological control (Punja, 1997). A better understanding of the antagonistic properties of microorganisms which might help to develop new biocontrol agents has been suggested by Deacon (1991). Several aspects of the interaction between the fungus and the control agent should be clarified. The stage of the development of the fungus at which the biocontrol is effective, its mode of action and the conditions in which it is more effective, are important elements of the information required towards the development of a microbial biocontrol agent as a 10 commercial .product for disease control. Screening methods for laboratory evaluation of activity and methods for isolation of strains are two important aspects that must also be addressed (Punja, 1997).

The present study was undertaken with the bacterium isolated from T. harzianum culture in our laboratory which showed antagonistic effect against T. harzianum and B. cinerea. The objectives of this study were as follows: (1) identification of the bacterium; and (2) determination of its mode of action against B. cinerea. 11

Literature review

Botrytis cinerea

Botrytis cinerea is a well studied fungus. According to Coley-Smith (1980) it was first described in 1729 by Micheli. Since then, it has been the object of many studies, principally because it is responsible for many important diseases in fruits, vegetables and other crops (Jarvis, 1977; Coley-Smith, 1980; Verhoeff, 1992). Northover, Kerr and Leuty (1990) reported that it causes serious losses in grapes in Ontario,.

Botrytis cinerea and Botryotinia fuckeliana are considered to be the asexual (anamorphic) and sexual (teleomorphic) stages, respectively, in the life cycle of this fungus. The structure and properties of these forms has been reviewed by Hennebert (1973) and Jarvis (1977, 1980).

Reproduction

B. cinerea reproduces asexually from conidia, mycelia (Verhoeff, 1980) and sclerotia (Coley-Smith, 1980). It is also known to reproduce sexually. Most strains are hermaphrodites and form both male (microconidia) and female (sclerotia) gametes. Sclerotia are the site for plasmogamy. Asclerotial strains cannot function as females, but they may still function as males (Lorbeer, 1980). After conditioning for about one month at 0 °C in the dark, sclerotia undergo carpogenic germination when fertilized by spermatia and incubated at 10 °C. One or two months later apothecia are formed (Faretra and Antonacci, 1987).

Drayton (1937) and Whetzel (1945) considered that the only function of microconidia is as male spermatia and that they cannot be induced to germinate. Others considered that microconidia can germinate and give rise to normal mycelium. Brierley (1918) gave detailed descriptions and pictures of germination of microconidia. Hino (1929) reported that microconidia germinated successfully 12

"only in a few rare cases". Harrison and Hargreaves (1977) found that freshly harvested B. fabae microconidia did not germinate and that only 5 % of those harvested after 112 days germinated after 24 h in nutrient broth but not in water. They found that some microconidia germinated after 25-57 days of cold exposure. Faretra and Grindle (1992) considered that microconidia might be a good sources of mutants if they could be induced to germinate, because they are uninucleate. It seems that they give some hope to this possibility.

Variability

Colonies of B. cinerea in agarized cultures frequently show sectors with distinct morphology. Some sectors produce many spores, others form mainly sclerotia and still others, produce only mycelia. Even when the starting isolate may be of one particular type, it often gives rise to the other types. This was explained by Hansen and Smith (1932) considering that B. cinerea cells contain several different types of nuclei. For some reason, a particular type of nucleus predominates in a given branch. This phenomenon is known as heterokaryosis and is fairly frequent in many other fungi as well.

Heterokaryosis is believed to be the main source of variation in B. cinerea and has been used to explain the lack of reproducibility and the contradictions between reported results for many studies of Botrytis cinerea (Lorbeer, 1980). Working with monoconidial cultures does not solve the problem because a single conidium contains in average about 5 nuclei (Shirame et aI., 1989). Buttner et al. (1994) found that field isolates of B. cinerea differ considerably in their DNA content per nucleus indicating that aneuploidy and or polyploidy is a widespread phenomenon. This can explain the phenotypic instability of many field isolates and the difficulty in recovering stable recessive laboratory mutants. 13

Diseases and their control

B. cinerea causes important diseases in many crops. It has a very wide host range including at least 235 plant species (Jarvis, 1977). Our knowledge of the diseases caused by B. cinerea has been hampered by the fact that in nature, only the asexual multinucleate conidia are normally found and isolates usually show phenotypic variations when subcultured. This has made it impossible to compare results from one region to another. Epidemiological studies are difficult because it is impossible to establish whether populations differ between crops or geographical regions (Verhoeff, 1992).

Infection of plants by B. cinerea is usually conceived as the result of germination of conidia on the surface of the plant, penetration of germ tubes through the epidermis and expansion of fungal hyphae inside the host tissue. Factors affecting the germination of conidia of B. cinerea include the temperature, the viability of conidia, the availability of nutrients, the presence of endogenous and exogenous inhibitors and the concentration of conidia. (Blakeman, 1980; Kamoen, 1992).

Penetration of plant tissues by B. cinerea takes place with the aid of an infection peg which may result from an appressorium or the tip of a germ tube (Kamoen, 1992). The molecular mechanisms involved and the relative importance of mechanical pressure and enzymatic degradation in fungal penetration of plants has been reviewed by Hamer and Holden (1997) and Schafer (1994). Factors involved in penetration of B. cinerea through wounds have been reviewed by Staples and Mayer (1995).

Diseases caused by B. cinerea can sometimes be controlled by means of fungicides (Gullino, 1992b). Control of diseases caused by B. cinerea is achieved primarily with the fungicide Iprodione in USA and Canada and with Vinclozolin and Captan in other countries (Harman et aI., 1996). However, because of the rapidity of infection from conidia, little warning can be obtained 14 from meteorological data and fungicides are often applied at an incorrect time. Many crops become infected by B. cinerea just before harvest and attention must be paid to the levels of residues that can be tolerated in foods and in grape must. Storage diseases can often be controlled by careful control of storage environment and certain treatments (Jarvis, 1977).

One of the main problems with chemical control of B. cinerea is probably the development of resistance to fungicides. The once highly effective benzimidazoles became of limited use after 2-4 years in most areas. The introduction of dicarboximides in the 1980's could not solve the problem and B. cinerea also developed resistance to this new type of fungicide (Gullino, 1992b). Recent concerns on the impact of chemicals on the environment has focused attention on using biocontrol agents to fight fungal diseases (Chat and Inbar, 1994). Biological control could replace all or part of the requirements for chemical pesticides (Harman et aI., 1996). The integration of chemical treatments with cultural management systems, epidemiological models and biological control agents, is now becoming a realistic and rational approach for management of diseases caused by B. cinerea (Gullino, 1992b).

Genetics

Despite the economic importance of diseases caused by B. cinerea, studies on the genetics of this fungus are scarce compared to other fungi. Only recently has advances in methodology injected new impetus in the field (Bergmans and Van Kan, 1992; Faretra and Grindle, 1992; Faretra and Antonacci, 1987; Faretra et aI., 1988a and 1988b; Faretra and Mayer, 1992; Kusters-van Someren et aI., 1992; Van Kan et aI., 1992; Van der Vlugt-Bergmans et aI., 1993).

Faretra et al. (1988b) studied the sexual compatibility of 213 field isolates of B. cinerea. They concluded that most of them are heterothallic. Sexual compatibility is controlled by a single mating type gene with two alleles MAT1-1 and MAT1-2. Sclerotia from isolates carrying the MAT1-1 allele can be fertilized with 15 spermatia from MAT1-2 isolates but not with those from MAT1-1 isolates. Spermatia from MAT1-1 isolates can fertilize MAT1-2 sclerotia, but not their own MAT1-1 sclerotia. Fertilized sclerotia can form apothecia with asci and ascospores (Faretra et aI., 1988a).

MAT1-1 allele has been conventionally assigned to isolate SASS6 and MAT1-2 to isolate SAS40S characterized by Faretra and coworkers at the University of Bari, Italy. They have been used as reference strains in genetic studies by many researchers around the world (Van Kan et aI., 1992; Kusters-van Someren et aI., 1992; Bergmans and Van Kan, 1992; Van der Vlugt-Bergmans et aI., 1993). Buttner et al. (1994) have demonstrated that isolate SASS6 is probably polyploid and have obtained haploid derivatives that should provide a good basis for classical and molecular genetic studies.

The structure of the mating type and the genes controlling resistance to dicarboximides and benzimidazole fungicides is now being elucidated by analysis of ordered tetrads (Faretra and Pollastro, 1991; 1996).

The genus Alcaligenes

The genus Alcaligenes is formed by some common, apparently saprophytic bacteria. They seem to be ubiquitous and have been found in soil and water, dairy products, rotten eggs, and the intestinal tract of vertebrates where they probably live saprophytically (Kersters and De Ley, 1984).

According to Mitchell and Clarke (1965) the peritrichous flagellation of this genus has aroused controversy since the original description in the last century. When A. faecalis was described as the type species of the genus Alcaligenes no reference was made to the strong fruity odor. Later a Gram-negative organism was isolated from human faeces and called "Bacterium alcali-aromaticum". These bacteria were motile by peritrichous flagella and grew in broth with 16 intense fruity odor which disappeared after a week, to be replaced by a cheesy odor. The broth reached pH 8.0 in 7 to 10 days. These bacteria did not use a wide range of carbohydrates.

Malek et al. (1963) reported that during the Second World War they isolated a microorganism from clinical specimens in Czechoslovakia, which was readily recognized by its odor. This bacterium was first described as Pseudomonas odorans. Later, it was revised and compared with several other strains isolated from the larvae of insects and nematodes and the new name Alcaligenes odorans was proposed (Malek et aI., 1963). Mitchell and Clarke, between 1952 and 1964 compared many strains isolated from pathological material and concluded that they were very similar to A. odorans, except for the capacity to hemolyze blood agar and their resistance to some antibiotics. They proposed those strains to be considered as A. odorans var. viridans (Mitchell and Clarke, 1965).

The taxonomic situation of the genus Alcaligenes has always been confusing. In the eighth edition of Bergey's Manual only 4 species were included: A. faecalis, A. aquamarinus, A. eutrophus and A. paradoxus. The ninth edition included only two authentic species: A. faecalis and A. denitrificans, leaving A. eutrophus, A. paradoxus, A. latus, A. aestus, A. aquamarinus, A. cupidus, A. pacificus and A. venustus as species incertae sedis (Kersters and De Ley, 1984). Holt (1993) listed A. (aecalis, A. eutrophus, A. paradoxus, A. latus, in Alcaligenes, but also included A. piechaudii, and renamed A. denitrificans as A. xylosoxidans. The names A. aquamarinus, A. aestus, A. cupidus, A. pacificus and A. venustus were reclassified in the genus Deleya. The classification of A. denitrificans and Achromobacterxylosoxidans as subspecies of A. xylosoxidans has been recently reconsidered and it seems that it is appropriate to consider these two taxa distinct species of the genus Alcaligenes (Vandamme et ai, 1996). Foss et al. (1998) have recently described several strains of Alcaligenes defragrans Spa nov. which use monoterpenes as the sole carbon source. 17

The genus Alcaligenes is presently characterized by rods, coccal rods, or cocci, 0.5-1.0 X 0.5-2.6, which usually occur singly. Resting stages are not known. Gram staining reaction is negative, motility is by peritrichous flagella. These bacteria are generally obligately aerobic with respirative type of metabolism and oxygen as the terminal electron acceptor. Some strains are capable of anaerobic respiration in the presence of nitrate or nitrite. Optimum temperature for growth is 30-37 °C. Colonies on nutrient agar are nonpigmented. Other characteristics include: positive catalase and oxidase tests; negative cellullase, gelatin, indole, acetyl methyl carbinol, 3-ketolactose and starch tests; chemoorganothrophic, using a variety of organic acids and amino acids as carbon sources; alkali is produced from several organic salts and amides and carbohydrates are usually not utilized (Holt, 1993). Most strains of Alcaligenes faecalis produce a characteristic aromatic fruity odor and form colonies with a thin spreading irregular edge. Carbohydrates are not utilized as the sole carbon source (Kersters and De Ley, 1984).

Mitchell and Clarke (1965) found no pathogenic role for A. odorans var. viridans. Malek et al. (1963) found A. odorans to be only weakly pathogenic to mice through intraperitoneal injection. Alcaligenes spp. occasionally cause opportunistic infections in humans (Holt, 1993). Obi et al. (1995) found A. (aecalis in mixed infections with other bacteria in 6.2 °Al of 350 patients with chronic otitis media. Alcaligenes species, most often A. xylosoxidans, often colonize the respiratory tract of intubated children and of patients with cystic fibrosis (Dunne and Maisch, 1995). Endotoxins of A. 'aecalis represent a potential risk of inflammatory lung reaction and respiratory disease for agricultural workers inhaling organic dusts contaminated with these organisms (Skorska et aI., 1996).

Alcaligenes spp. are presently the subject of intensive research as a source of biopolymers (Shinomiya at aI., 1998; Sim et aI., 1996; Yoon et aI., 1996) and for the biodegradation of toxic compounds (Field et aI., 1994; Fukumori and 18

Hausinger, 1993; Leisinger and Cook, 1994; Nishio at aI., 1994). Sasaki, et al. (1998) have used nitrite reductase from Alcaligenes faecalis in a biosensor system for the measurement of nitrite in natural waters.

Bacteria in biocontrol

The origin of research on biocontrol of fungal diseases by bacteria can be traced back to the 1920's. It was soon realized that several components of the normal soil microflora serve naturally to regulate the activities of the pathogens. This is now known as general soil suppressiveness. Pseudomonads playa major role in specific suppressiveness (Deacon, 1991).

The most extensively studied bacterial agents for biocontrol on vegetable crops are Pseudomonads, Bacillus subtilis, and Enterobacter cloacae. They have been used to control diseases caused by Pyfhium spp., Rhizoctonia solani, Sclerotium rolfsii, Aphanomyces euteiches, Fusarium oxysporum and Thielaviopsis basico/a (Punja, 1997). Debao (1994) has studied the biocontrol of bacterial blight of rice (Xanthomonas oryzae pv. oryzae) with many isolates of different bacteria and found that Bacillus spp. has promising prospects.

Leifert et al. (1995) have studied in vitro antibiotic production by Bacillus subtilis and B. puml1us in different media. Antibiotic production was found to depend on the growth medium. Antibiotic activity was found to depend on pH and nutrient concentration in the assay medium. Bacillus brevis produces the antibiotic gramicidin S which is fungicidal to B. cinerea in vitro and protects cabbage against damage by B. cinerea, but the mode of action in planta is associated with reduction of leaf wetness. The antibiotic binds to the leaf and is inactive in this form (Edwards and Seddon, 1992).

Bacillus spp. are known to produce antifungal antibiotics, biosurfactants, chitinases and other wall-degrading enzymes, volatiles and compounds which elicit plant resistance mechanisms. Some of these substances have been 19 implicated in biocontrol (Leifert et aI., 1995). Brown rot of peach (Monilinia fructicola) has been effectively controlled with Bacillus subtilis (Pusey and Wilson, 1984).

Benhamou et al. (1996) have studied the effect of Pseudomonas fluorescens on pea root rot caused by Pythium ultimum and Fusarium oxysporum f. sp. pisi. Pseudomonas cepacia and P. fluorescens were used to control Pythium damping-off and Aphanomyces root rot of peas (Parke, 1990; Parke et aI., 1991). Agrobacterium radiobacter has long been used commercially for control of crown gall (A. tumefaciens) of fruit trees and other dicotyledoneous crops (Deacon, 1991). Enterobacter cloacae has been used to control Rhizopus rot of peach (Wilson et aI., 1987) and rots of seeds and seedling incited in cucumbers, peas, and beets by Pythium (Hadar et aI., 1983, Nelson et aI., 1986).

Bacteria are supposed to reduce disease losses caused by fungi by production of antibiotics, toxins and lytic enzymes, direct parasitism of fungal structures, competition for infection sites and nutrients, induction of host defense mechanisms and reduction of aggressiveness or virulence through mycovirus transmission (Punja, 1997). Soil suppressiveness to Fusarium was strongly correlated with the level of siderophores produced by fluorescent Pseudomonads and not with total bacterial biomass of fine sandy loam (Sneh et aI., 1984). Iron bound siderophores, cyanic acid, and antibiotics are involved in suppression of black root rot of tobacco (Thielaviopsis basicola) by a Pseudomonas fluorescens strain (Ahl et aI., 1986).

Bacteria in biocontrol of B. cinerea

Most of the work in biological control of Botrytis has been done with Trichoderma spp. (Chet, 1987; Dubos, 1992; Gullino, 1992a; Harman et aI., 1996; Samuels, 1996; Tronsmo, 1991) but it is well known that bacteria might be a good alternative for control of Botrytis. Bacteria with in-vitro antagonistic effect against B. cinerea have been known for a long time (Jarvis, 1977; Blakeman and Fraser, 20

1971; Blakeman, 1972; Blakeman, 1980; Edwards and Seddon, 1992) but control in the field often falls below expectancies probably because of poor knowledge of the mechanisms of action.

Several members of the genera Bacillus and Pseudomonas have been used in biological control of diseases caused by B. cinerea in grapes (Dubos, 1992). Species of Pseudomonas, Enterobacter and Bacillus, highly inhibitory to B. cinerea, were isolated from compost extracts which reduced infection of grapevine by B. cinerea (Ketterer et aL, 1992). Biological control of apple diseases with microorganisms has been studied by Gullino et al. (1992). They found that several strains of yeast and bacteria were effective against Botrytis rot of apple.

Pyrrolnitrin, a metabolite of Pseudomonas cepacia has been reported as a new fungicide with good activity against B. cinerea in strawberries in Belgium (Creemers, 1992). Spoilage of cabbage by B. cinerea could be prevented by Pseudomonas and Serratia antagonists (Leifert et aI., 1992).

Alcaligenes as antagonists

Alcaligenes spp. have been previously reported as antagonists (Jackson et aL, 1991) but their effect on B. cinerea and its mechanisms of action has not been published. They found that the four most active isolates against Sclerotium cepivorum, which causes white rot of Allium, were Alcaligenes or Pseudomonas spp.

Strain ATCC 15246 of A. (aecalis has been patented for the production of penicillin derivatives (U. S. Pat. 3 682 777, listed herein as P. aeruginosa). 21

Materials and Methods

Cultures

The strain SF of A. faecalis used in this study was isolated from a culture of T. harzianum kindly supplied by Dr. V. Govindsamy (Department of Biological Sciences, Brock University). A. faecalis type strain 15554 was purchased from the American Type Culture Collection (ATCC). Three strains of T. harzianum were supplied by Dr. A. Castle (Department of Biological Sciences, Brock University). Strains of other fungi and of Bacillus cereus, B. coagulans, B. subtilis, Enterobacter aerogens and Escherichia coli were obtained from the culture collection of the Biology Department at Brock University.

Sixteen isolates of Botrytis cinerea, kindly supplied by Dr. J. Northover and Dr. A. Svircev (Agriculture Canada, Vineland Research Station, Vineland, On, Canada) and a culture of B. cinerea isolated from grape rachis in a field near Thorold and listed here as BcU were used in this study. Several monoconidial isolates (MCI) were obtained from the original isolates Bc1, Bc2 and Bc3. Three of these, one from each original isolate, were also used in this study (Table 1).

Media

Potato Dextrose Broth (PDB) was prepared as follows: 200 g of freshly sliced potatoes were boiled in approximately 300 ml of tap water, the liquid was filtered through cheese cloth, then 10 g of Bacto Dextrose was added and the volume was brought to 1 L. Peptone enriched POB (PDPB) was prepared by adding 0.5

9 of Bacto Peptone to 1 L of PDB. The clear liquid had a pH of 6.5-7.0 before sterilization. Nutrient Broth (NB) was prepared by dissolving 8.3 9 of commercial dry powder (Difco Laboratories, Detroit, Michigan, USA) in 1 L of distilled water. Nutrient Agar (NA), Potato Dextrose Agar (PDA), peptone enriched PDA (PDPA) and Water Agar (WA), were prepared by adding 15 9 of Bacto Agar to 1 L of NB, 22

Table 1. List of Botrytis cinerea isolates used in this study.

List Description* Origin Bc1 Be1 Vineland** Bc2 Be2 Vineland Be3 Be3 Vineland Bc4 Be4 Vineland Be5 RE3.5.1 Vineland Be6 RC?4 Vineland Be? RC3.4.1 Vineland Be8 RA7.8.1 Vineland Be9 RBF20 Vineland Bc10 VR/HRIO.15 Vineland Bc11 RBNE.1 Vineland Be12 RBNE.4 Vineland Be13 RBHRIO.8 Vineland Bc14 RBHRIO.13 Vineland Be15 RB/F3 Vineland Bc16 RB/HRIO.15 Vineland Be1490 MCI from Bc1 This work Bc2502 MCI from Bc2 This work Be350? MCI from Bc3 This work BcU Isol. grapes This work

Notes: * Description on the plate when received ** Agriculture Canada, Vineland Research Station, Vineland, On, Ca. MCI Monoconidial isolate. 23

POS, POPS or water respectively. lonoagar (tA) was prepared by adding 15 9 of lonoagar to 1 L of distilled water.

NB and NA were routinely used as culture media for bacterial cultures. Palleroni's (1984) synthetic basal medium (PBM) with 0.1 % of the appropriate carbon source was used for some experiments. An alternative basal medium

(MB) was prepared with 5 ml of concentrated H3P04, 5 9 of KCI and 1 9 of

MgS04 and adjusted to 5 L with water. MBC was prepared by adding 10 9 of tri-sodium citrate to 1 L of MB medium.

Acetate medium (MA) was prepared with 2 9 of ammonium acetate, 2 9 of

K2HP04, 0.2 9 of MgS04, 50 IJI of a solution of CaCb (0.005 gIL), 20 IJI of a solution of ferrous citrate (0.5 gIL) and adjusting to 1 L with distilled water. Acetate-ethanol medium (MAE) is 1 % (v/v) ethanol in MA.

All media were adjusted to pH 6.8 with NH40H and were sterilized by autoclaving 20 min at 121 °C unless otherwise indicated.

Agarized media were allowed to cool below 50 °C before 10 ml aliquots were pipetted onto 100 X15 mm sterile plastic Petri dishes (Fisher Scientific, Co.) and were left to solidify. Plates were stored at 4 °C if not used immediately.

Inoculations

Mycelial inoculations: 8-10 mm discs were cut from the periphery of colonies of actively growing fungi in PDA plates with a sterile cork borer and transferred to a new plate with a sterile needle. Alternatively, small portions of colonies were cut with a flame sterilized loop and transferred to the surface of a new plate.

Suspension drops: 5-10 IJI drops of conidial suspensions of 1 X 105 or 1 X 107 conidia/ml were deposited on the surface of the plate according to the previously marked positions on the bottom of the dish with a template. 24

Suspensions in molten media: Samples of 100 JJI of conidial suspensions of 1 X 107 conidia/ml were pipetted onto a sterile Petri dish. Then 10 ml of molten medium at about 40°C was added and thoroughly mixed with the inoculum.

Cultural conditions

Unless otherwise stated, agar cultures were incubated in the dark, in incubators maintained at 20 °C for fungi and 37°C for bacteria. Plates were sealed with Parafilm strips or kept inside sealed plastic bags and were incubated in an inverted position. Liquid cultures were grown in an incubator shaker at 37°C. When plates or tubes were taken out for measuring or evaluation they were returned to the incubators as soon as possible.

Preparation of monoconidial cultures

Monoconidial cultures were prepared by inoculating PDA plates with 10 IJI samples of a suspension of approximately 3 X 103 conidia/ml and marking the places where single conidia were growing. These colonies were then transferred to individual plates and incubated until sporulation.

Preparation of conidial suspensions

Conidia from sporulated plates were suspended in 10 ml of distilled water and filtered through cheesecloth to separate hyphal fragments. Three washing cycles were conducted in the cold room by centrifuging the conidia at 13, 000 g for one minute and resuspending again in water. The suspensions were kept in Eppendorff tubes in the cold room and were briefly vortexed before use. Cell concentration was measured with a counting chamber and diluted to the desired concentration with sterile distilled water. 25

Isolation and identification

Isolation and identification of bacteria was made according to the procedures of Holding and Collee (1971). Isolation was performed by the streak and serial dilution methods. Individual colonies were selected, suspended in 1 ml of distilled water and tested for fungal growth inhibitory effect (Leifert et aI., 1995). Briefly, 10 ~I drops of the test sample were deposited in wells made in PDA plates seeded with fungal conidia. After 24-48 h incubation at 20°C for B. cinerea or 27°C for T. harzianum, the plates were visually evaluated. Only samples that showed the inhibition halo around the wells were selected for further study.

The ability to grow using certain carbon sources was tested in PBM with 0.1 % of the appropriate carbon source (Table 2). Routine biochemical tests were conducted according to Holding and Collee (1971) using known strains as references. Prepared Difco media were used for the following microbiological tests: phenol red, nitrate broth, MR-VP, phenylalanine deaminase, litmus milk, Simmons' citrate, starch, and spirit blue.

Heat treatment at 80°C for 10 min was used to isolate sporogenic forms of bacteria (Parker and Duerden, 1990).

Cell morphology was studied in unstained samples by phase contrast microscopy and by bright field light microscopy in Gram stained smears.

Some samples were studied by transmission electron microscopy as follows: A formvar filmed copper grid was floated for 15 min on a drop of a bacterial suspension. The grid was then briefly touched on a piece of filter paper to drain the liquid, stained for 1 min with 2 % uranyl acetate and examined in a Phillips 300 electron microscope at 60 kV. 26

Table 2. Compounds tested as sole source of carbon for Alcaligenes (aeca/is. Each compound was dissolved in Palleroni's basal medium (PBM) to give a final concentration of 0.1 % (w/V) , filtered through a 0.22 IJm membrane filter and dispensed in 5 ml aliquots into sterile culture tubes. Two tubes of each carbon compound in PBM and two tubes of PBM alone were inoculated with 10 ~I of a suspension of A. f8eca/is of approximately 106 celis/mi. One tube of each compound in PBM was left as uni,noculated control. Growth in each medium was evaluated after 4 days of incubation at 37°C in incubator shakers at 100 rpm.

Compound Compound Compound acetate D-ribose L-malate acetone D-trehalose L-methionine benzene D-xylose L-ornithine cellulose ethanol L-phenylalanine citrate formaldehyde L-proline D-arabinose fumarate L-sorbose D-cellobiose glucose-5-phosphate L-tryptophan D-fructose glycerin L-valine D-galactose glycogen maltose D-gluconate glyoxal methanol D-glucose inositol n-hexane dioxane L-alfa-alanine pyruvate D-Iactose L-arginine B-alanine DL-Iactate L-aspartate starch D-Iyxose L-cysteine succinate D-mannitol L-glutamate sucrose D-mannose L-Iysine xylitol 27

To test the effect of high temperature upon A. (aeca/is survival, two replicate tubes containing 10 IJI of a bacterial suspension (106 cells/ml) in 5 ml of PBMC were incubated at each of 50, 55, 60, 65, 70, 75, 80 and 85°C in a water bath for 10 min. The tubes were then cooled in cold water and incubated at 37°C in incubator shakers at 100 rpm. The experiment was repeated later with three replicates for the last three temperatures. Growth was evaluated after 24 and 72 h of incubation by measuring the optical density of the suspensions at 590 nm in 1 cm cuvettes in a Bausch & Lomb spectrophotometer.

Resistance to salt was tested as follows: three replicate tubes containing 10 tJl of a bacterial suspension (106 cells/ml) in 5 ml of PBMC were incubated at 37°C in incubator shakers at 100 rpm with NaCI concentrations ranging from 0.5 % to 7.5%.

Serological tests

Antisera against the isolated strain of A. (aeca/is were obtained from 4 Balbc female mice injected with immunogens prepared from live and heat-killed cells and tested for sensitivity and specificity. The antigen was prepared with the cells of a one-week old bacterial culture in MBC. The cells were collected by centrifugation, washed three times with water and resuspended in 2 ml of water. One ml was separated to be used as live cell immunogen. The other half was heated for 15 min at 100°C in a boiling water bath and the cells were collected by centrifugation, washed three times with water and resuspended in 1 ml of water. A sample of pre-immune blood was taken from one of the mice. The other three mice were injected subcutaneously with the first dose of 0.1 ml of an emulsion formed by shaking the immunogen with the same volume of complete Freund's adjuvant. Three weeks later a blood sample was taken from the tip of the tail of one of the mice and the presence of antibodies was tested by immuno­ precipitation. An intraperitoneal boost injection was then given with 0.1 ml of the immunogen in incomplete Freund's adjuvant. After three weeks the mice were 28 anesthetized and sacrificed for blood collection. The serum was separated from the clot and stored at -30 °C until needed.

Immunoprecipitation

Antisera were tested for antibodies by immunoprecipitation with homologous antigen. One microlitre drops of the antigen and the dilutions of the antisera (1:2 to 1:2048) were mixed on glass slides and incubated in sealed Petri dishes for 6 hours at 20 ac. Immunoprecipitation was evaluated under a Wild-Leitz Diaplan phase contrast microscope at 250 X. Homologous antibody titre was calculated as the reciprocal of the highest dilution that still produces obvious agglutination as compared with control tests with normal serum at the same dilution. Antisera were tested as above for heterologous reactions against washed cells of A. (aeca/is type strain ATCC 15554, B. subti/is, E. coli and E. aerogens.

Dot immunobinding assay

Three colloidal gold markers were prepared to test if the antisera could be used in a Dot Immunobinding assay. A 20 nm colloidal gold stabilized reagent was first prepared according to Geoghegan and Ackerman (1977). Portions of 20 ml of this reagent were then conjugated with Protein A or with bovine serum albumin (BSA). Three microlitre samples of the antigen were spotted on nitrocellulose strips (Immobilon, Sigma Chemicals) and dried for 30 min at room temperature. After blocking for 1 h in 3 % gelatin in TBS (20 mM Tris, 500 mM sodium chloride, pH 7,5) and washing three times for 10 min in 0.05 % Tween-20 in TBS, the strips were incubated for 4 h in 1:1000 diluted antiserum containing 1 % gelatin and were washed again. The samples were then treated for 4 h with the Protein A marker, washed again and let dry. Positive reactions were characterized by pink to red spots while negative reactions resulted in no visible spots on the strips. 29

Effect of A. 'aeea/is on B. cinerea germination and growth

Effect of A. (aeca/is on B. cinerea colony growth was tested as follows: Ten millimeter discs were cut from the periphery of actively growing fungi in PDA and were deposited on the surface of fresh medium or cultures of A. (8eca/is pre-incubated for 24 h at 37°C and then incubated at 20 °C for different periods of time. Colony diameter was measured with a ruler. Plates were held against a dark background with oblique illumination to get maximum contrast from light diffused by the colony. Growth rate was calculated as an increase in diameter in a given time and expressed in mm per day.

In some experiments drops of conidia were placed on dialysis membranes placed on A. (aeca/is cultures or drops of A. faeca/is were placed on dialysis membranes placed on PDA plates seeded with B. cinerea conidia to test if the inhibitory effect could be expressed through membranes.

Unless otherwise expressed, all experiments on the effect of A. faeca/is on B. cinerea germination and growth were performed in incubators at 20°C.

Biotests for fungal inhibitors

Samples of different cultures of A. faeca/is were tested for their effect on germination of conidia and germ tube elongation of B. cinerea in the following manner: 10 IJI drops of the test samples were deposited in Petri dishes and mixed with 10 IJI of a suspension of 105 conidia/ml of B. cinerea in water or in 5 0/0 glucose. The dishes were incubated at 20°C for different periods of time. The first 100 conidia were evaluated at 100 X magnification under the microscope and considered germinated if an emerging germ tube could be seen. This method is a slight modification of the glass slide method traditionally used to test fungicides (Torgeson, 1967). Germ tube dimensions were measured by direct comparison against a calibrated ocular micrometer or by measuring images projected from negatives with a Durst enlarger. 30

Biocontrol tests

The in vivo antagonistic effect of A. (aeca/is on B. cinerea was studied in red and white seedless grapes, bean leaves and cabbage leaves purchased at a local market. All the biocontrol experiments were conducted at 20°C for one week.

Bunches of grapes were immersed in a one-week old NB culture of A. (aeca/is, a suspension of T. harzianum of 2 X 107 conidia/ml or in tap water for the controls, and let dry overnight at room temperature. Then they were immersed in a suspension of 105 conidia/ml of B. cinerea in 5 % glucose, placed in plastic trays, sealed with polystyrene film and incubated.

Individual detached grape berries in a plastic holder were inoculated as above and punctured with a needle. The holder was incubated in a sealed box with a small sponge impregnated in water to keep moisture.

Pinto bean leaves were detached from two week old plants grown in the laboratory, placed on a tray lined with wet filter paper and inoculated with ten microlitre drops of a one-week old MBC culture of A. (aeca/is, or with MBC for the controls. Subsequently, 10 IJI of a suspension of 105 conidia/ml of B. cinerea was added and the tray was sealed and incubated.

Pieces of cabbage leaves of approximately 20 cm2 were inoculated as above and incubated in plastic Petri dishes lined with wet filter paper.

The experiments with bunches of grapes, bean and heat-treated cabbage leaves consisted of six replicates per treatment and were not repeated. The experiments with detached grape berries and fresh cabbage leaves consisted of six replicates and were repeated three times. 31

Inhibitory substances produced by Alcaligenes faecalls

In order to test if the antagonistic effect was due to the bacterial cells or some substances released into the medium, cells were separated from the medium and tested separately for the inhibitory effect.

A sample of a one-week old culture of A. faecalis in MBC was distributed in six Eppendorff tubes and centrifuged for 10 min at 4 ac. The liquid was passed through a 0.22 IJm Millipore membrane filter. The cells were collected and washed three times with distilled water and resuspended in 10 ml of distilled water. Inhibitory activity was measured in cells and filtered liquid with the biotest for fungal inhibitors described above. Washed cells and supernatant liquid of E. coli and E. aerogens cultures were used as controls.

To test if volatile inhibitory substances were also produced by A. faecalis in liquid cultures, ten microlitre drops of a suspension of 105 conidia/ml of B.

cinerea in 5 % glucose were deposited on glass cover slips glued on the inside surface of rubber stoppers. Then the cap of the flask with 250 ml of a one-week old culture of A. faecalis was quickly replaced with the rubber stopper carrying the coverslip and the drop of conidia facing the interior of the flask. Another flask with medium alone was used as a control in the same way_ After one week of incubation at 20°C the drops were compared in both flasks. The experiment was repeated once with NB and twice with MBC.

The effect of different concentrations of ammonia on the germination of conidia of B. cinerea was tested with a slight modification of the procedure described by Candole and Rothrock (1997). Briefly, drops of conidial suspensions were placed on the inner surface of the lid of a Petri dish and 5 ml of a dilution of

NH40H was placed in the other part of the dish. Then 1 ml of 1 N NaOH was

added to the NH40H, quickly covered with the lid, sealed with Parafilm and incubated until the evaluation for germination and germ tube length. 32

The effect of different media on the production of inhibitory substances was studied with MBC, MA, MAE and basal medium pius ethanol (MBE). Culture samples of A. faeca/is in each medium were taken at different times and the optical density at 595 nm was measured in 1 cm light path tubes in a Bausch & Lomb spectrophotometer. The pH was measured by a Beckman pH meter. Inhibitory activity was measured with the biotest for fungal inhibitors described above.

The effect of lyophilization, heat treatment and dialysis upon the stability of the inhibitory substances was studied as follows: 10 ml samples of the supernatant of one-week old culture of A. faeea/is in MBC were frozen at -30°C, lyophilized, resuspended in their original volume of distilled water and filtered through a 0.22 IJm Millipore filter as described above or kept at -30°C until needed. Fresh filtered samples of one-week old MBC culture of A. faeca/is were dispensed in 0.1 ml aliquots into Eppendorff tubes and heated for 15 min in a water bath at 55 °C, at 80°C or at 100 °C in boiling water. The other two aliquots of 0.1 ml were dialyzed against distilled water at 4 °C for 6 h and at room temperature overnight, respectively. Inhibitory activity was measured with the biotest for fungal inhibitors described above and compared to the activity of the untreated culture. Samples of the lyophilized material were extracted with methanol, ethanol, acetone and chloroform to determine if the inhibitory substances were soluble in any of these solvents. After separation from the insoluble residue, the liqUids were evaporated to dryness at 50°C in a vacuum rotoevaporator and resuspended in water. The insoluble residue was dried to evaporate traces of solvents, resuspended in water, and tested for inhibitory substances as described earlier.

Exclusion chromatography was performed in order to establish a range of molecular weights and to try to separate the inhibitory substances from contaminants. One cm diameter columns were filled with Sephadex G-10 to a height of 25 cm and were equilibrated with sterile distilled water. Blue Dextran 33 was used to determine the exclusion volume. Samples of 400 ~I of the culture liquid were eluted with water and 0.5 ml fractions were collected in weighed tubes. The tubes were then dried in a desiccator, weighed to calculate the amount of substance in each tube, resuspended in 1 ml of water and tested for inhibitory activity.

The fractions that showed inhibitory activity were pooled and dinitrosalicylic acid test for reducing sugars (Wang et aI., 1997), ninhydrin test for amino acids (Moore and Stein, 1948) and absorption spectra in the visible and UV range were performed. Tests for sugars and amino acids were repeated after overnight hydrolysis in 6 N Hel.

Electrophoretic mobility of the inhibitory substance was tested in 0.8 % agarose gels in 0.1 % ammonium acetate that does not interfere with the test for inhibitory activity. Five hundred microlitres of the inhibitory liquid were transferred to a well cut in the middle of the gel and the electrodes were connected to a power supply at 100 V and 40 mA over 1 h. Then the liquid remaining in the well was drawn with a pipette, the gel was cut in strips of 1 cm wide and each strip was identified according to its position relative to anode or cathode. After a brief rinse the strips were transferred to 25 ml centrifuge tubes and centrifuged at 10,000 9 for ten min. The liquid extracted from the gels was collected and tested for inhibitory activity.

Some samples were tested for the presence of chloride ions. One microlitre of the sample was mixed in a cover slip with one microlitre of 0.1 % silver nitrate and after 4 h incubation at 20°C in a sealed Petri dish, the drop was monitored under the phase contrast microscope for silver chloride granules.

Each experiment consisted of a minimum of two replicates and was repeated at least twice unless otherwise expressed. 34

Results

The original sample of the bacteria under investigation was obtained from cultures of Trichoderma harzianum grown on a plate of PDA. The growth of this fungus was poor on this plate, almost no aerial mycelium was present. Bacterial colonies were not evident. However, the inoculation of discs of this material on malt extract agar produced only bacterial colonies. Exuberant bacterial growth began on the disks after 48 h and spread onto the fresh medium in one week.

In one experiment, discs of Botrytis cinerea and the original sample were inoculated 4 cm apart on the same plate. After one week, the mycelium of B. cinerea covered most of the plate, except around the bacterial growth. In another experiment, suspensions of conidia of B. cinerea did not germinate on plates precolonized with the bacteria.

Isolation and identification

Two different types of colonies with strong fungal growth inhibitory effect were obtained from NA plates streaked with the bacteria obtained from the original sample: a dome-shaped colony and a flat colony (SF). The former colony was probably of mixed populations because both Gram positive and Gram negative cells were present. Electron microscopy of samples from the growth obtained after heat treatment of the dome-shaped colony showed rod-shaped cells and spore-like structures suggestive of the genus Bacillus, but they did not produce any noticeable fungal growth inhibitory effect. This isolate was no longer studied. Only the isolate SF was characterized and identified.

Characterization

When mixed with warm molten NA and incubated at 37°C for 24-48 h, SF formed two distinct types of colonies depending on their location. Inside the medium, it formed small (usually less than 0.5 mm in diameter) biconvex colonies up to 1 or 35

2 mm below the surface, that appear yellow to light brown in color under the microscope and with the plane of the colony growing at any angle from horizontal to vertical. Usually a patch of denser material was attached to the surface of the colony. On the agar surface, it formed round, flat, translucent colonies, up to several millimeters in diameter, colorless when viewed from the front but iridescent at certain angles. An almost imperceptible spreading edge could often be seen around the colonies. Both types of colonies were always obtained from either the flat or the biconvex form. They both showed fungal growth inhibitory effect, Gram negative motile rod cells and the same characteristic fruity smell in young cultures.

When inoculated by streaking with a loop on the surface of NA plates or slants, SF produced abundant growth in the center of the streak and small beaded colonies near the edge, sometimes forming a line that extends up to several centimeters away from the point of inoculation. Colonies were nonpigmented to the naked eye.

Growth in NS was observed between 20°C and 40 °C, but not at or above 42 °C. In NA, SF grew, at least, between 20 and 37 °C. A sweet smell was produced during the first 2-4 days of growth, that later changed to an unpleasant odor suggestive of ammonia and amines.

In young NB cultures at 37°C, individual rod cells measuring 0.5-1.0 by 1.0-1.5 JJm and rounded small cells were mainly present. Long chains and big rods (sometimes up to 1.5 by 3.5 tJm) were frequently found in NA cultures. All these types of cells always stained Gram negative. In uranyl acetate stained young cultures, examined with the electron microscope, flagellated cells were frequently observed (Fig. 1).

In stab inoculated cultures in tubes, SF grew well on the surface but no growth was visible below 5 mm, indicating aerobic metabolism. No acid or gas was 36

Figure 1. Electron micrograph of a sample of a 48 h old nutrient broth culture of Alcaligenes faecalis (SF) showing flagellated cells. A formvar coated copper grid was floated for 15 min on a drop of the bacterial suspension. The grid was then briefly touched on a pie·ce of filter paper to drain the liquid, stained for 1 min with 2 % uranyl acetate, and examined with a Phillips 300 electron microscope at 60 kV. 37

Figure 1. 38 produced in phenol red medium from either glucose, fructose, galactose, lactose, sucrose or manitol. SF reacted positively to oxidase, catalase, lipase and nitrate or manitol. SF reacted positively to oxidase, catalase, lipase and nitrate reductase tests, and negatively to methyl red, Voges-Proskauer, starch, gelatin, urease, indole, hydrogen sulphide and litmus milk tests.

SF grew well in PBM using as a the sale source of carbon the following compounds: acetate, citrate, fumarate, pyruvate, succinate, L-aspartate, L-glutamate, L-malate, DL-Iactate, L-tryptophan, L-phenylalanine, L-methionine, L-cysteine, L-valine, L-alfa-alanine, L-proline and ethanol.

SF did not grow in PBM with methanol, acetone, glycerin, formaldehyde, dioxane, glyoxal, n-hexane, benzene, L-arginine, B-alanine, L-Iysine, L-ornithine, L-cysteine, D-ribose, D-fructose, D-mannose, D-glucose, D-gluconate, glucose-5-phosphate, D-arabinose, D-galactose, D-Iactose, maltose, sucrose, D-Iyxose, D-xylose, L-sorbose, D-mannitol, inositol, xylitol, D-cellobiose, D-trehalose, glycogen, starch, or cellulose as the sole carbon source.

When grown in PBM with ethanol as the sole carbon source, BF slightly acidifies the medium from an initial pH of 6.8 to a final pH of 6.1 in 48 h. However, when grown in PSM with citrate it raises the pH from 6.8 to 7.4 in the same period. In both media SF produces the characteristic fruity smell, but in citrate medium the smell later changes to ammonia while in ethanol the frUity smell persists.

SF grew well in PBM with NaCI concentration below 2.0 %, only slowly at 3 OJb and not at all above that level.

SF survived well with temperatures up to 45°C for ten min. At higher temperatures only a small proportion of cells, if any, survived. Over 75°C, no growth was ever obtained. 39

Identification

According to the above information the isolate BF was identified as Alca/igenes (aeca/is (see Discussion Section).

Serological tests

Antisera against the isolated strain of A. (aeca/is (SF) were prepared by inoculating four Balbc female mice with live (L) and heat killed (H) cells immunogens. These antisera were tested for sensitivity and specificity by immunoprecipitation and dot immunobinding assay.

Immunoprecipitation

The four antisera obtained showed positive reaction in immunoprecipitation tests with their respective antigens. Best homologous titre was obtained for the antiserum prepared from the mouse injected with two doses of heat killed immunogen. All four antisera reacted well with their antigens and with cells of A. (aeca/is type strain ATCC 15554 (Table 3). No heterologous reactions were detected by this method against antigens from B. subti/is, E. coli or E. aerogens.

Dot Immunobinding assay

Three colloidal gold markers were prepared to test if the antisera prepared against A. (aeca/is could be used in the dot immunobinding assay. The 20 nm colloidal gold stabilized reagent was shown to react strongly with proteins. Nanogram amounts of BSA spotted on nitrocellulose membranes were easily detected (Fig. 2A). The Protein A marker reacted with undiluted mouse serum and diluted 1:100, but not with 1 % eSA (Fig. 28). The BSA negative control marker did not react with mouse serum, BSA, or any of the bacterial cells. Positive reactions were observed with the Protein A marker for SF and A. (aeea/is type strain ATCC 15554. Weak heterologous reactions were detected for B. subtilis cells. No reaction was detected with E. coli cells (Fig. 2C). Table 3.- Serological reactions of antisera prepared against Alcaligenes faecalis (SF).

One microlitre drops of the antigen and the dilutions of the antisera (1:2 to 1:2048) were mixed on glass slides and incubated in sealed Petri dishes for 6 hours at 20°C. Immunoprecipitation was evaluated under a Wild-Leitz Diaplan phase contrast microscope at 250 X. Titre was calculated as the reciprocal of the highest dilution that still produces obvious agglutination as compared with control tests with normal serum at the same dilution. TITRE Mouse Immunization Sampling Homologous Heterolologous Identification 1st 2nd Days SF Af Ss Ec Ea 1 - - 0 0 Control 1 H - 21 >16 >16 Ns10 1 H - 43 1024 >128 AS11 2 HH 45 2048 >128 As12 3 L L 45 512 >128 As21 4 L L 45 64 NT NT NT NT As22

Notes: L: live cells immunogen. H: heat kii'led cells immunogen. -: negative reaction. NT : Not tested Ea : E. aerogens Af: A. faecalis ATCC 15554 Bs : B. subtilis Ec : E. coli

.,J:::a.. o 41

Figure 2. Dot Immunobinding assay of Alca/igenes (aecalis. Three microlitre samples of the antigen were spotted on nitrocellulose strips (Immobilon, Sigma Chemicals) and dried for 30 min at room temperature. After blocking for 1 h in 3 % gelatin in TBS (20 mM Tris, 500 mM sodium chloride, pH 7,5) and washing three times for 10 min in 0.05 % Tween-20 in TBS, the strips were incubated for 4 h in 1:1000 diluted antiserum containing 1 % gelatin and were washed again. The samples were then treated for 4 h with the Protein A marker, washed again and let dry. Positive reactions were characterized by pink to red spots while negative reactions resulted in no visible spots on the strips. A: reaction of stabilized colloidal gold reagent with a serial dilution of bovine serum albumin (BSA) 1: 10 IJg; 2: 1 IJg; 3: 0.1 IJg; 4: 0.01 IJg; and 5: 0.001 ~g. B: reaction of Protein A marker with normal rabbit serum (1) and 1: 100 dilution (3), but not with BSA (2). C: Protein A marker reacted with A. (aeca/is SF (1) and A. (aeca/is type strain ATCC 15554 (2) after incubation with antiserum AS12 diluted 1:100. E. coli (3) did not react. B. subtilis (4) gave a faint reaction. 42

A I~~:·"~·' .. ~ ,I iii i 1 2 3 4 5 c_---

1 2 3 4

Figure 2. 43

Growth characteristics BF can be grown in NB and in synthetic media PBMC, MBC, MBE, MA and MAE. In agarized media it grew weH in NA and PDPA. In PBMC it grew well at 36 °c reaching maximum biomass in about 21 hours (Fig. 3).

At lower temperatures it reached almost the same biomass but it took a few hours longer. When co-cultured with B. cinerea it also grew on PDA, but sometimes failed to grow on PDA alone. However, SF grew better in MBC, MBE and MAE than in PBMC or MA (Fig. 4).

Growth of A. (aeea/is in a given medium produced changes in the pH of the medium. When SF grew in NB, PBMC, MBC, and MA it increased the pH from 6.8 to about 8.0- 8.9 in 5 days. In MAE the increase in pH was less marked, but it still reached pH 7.75. However, in MBE the pH of the medium decreased to 5.2 in 5 days (Fig. 5).

Biotests for fungal inhibitors.

The biotest for fungal inhibitory substances usually gave clear results. Replicates in the same Petri dish were always very close in percent germination and tube length. The same suspension of conidia, kept at 4 °C, usually gave the same results when tested daily over several weeks. Results from different Petri dishes, even from the same lot were sometimes highly variable. Plastic disposable Petri dishes usually gave better germination than detergent cleaned glass Petri dishes.

Glucose (5 %) strongly stimulated germination of conidia of all Botrytis cinerea strains tested. 44

Figure 3.- Growth curves of Alcaligenes faecalis at different temperatures. Each of the 3 samples of 100 ml of PBMC were inoculated with 100 JlI of A. (aeca/is (SF) in 250 ml flasks with lateral tubes and incubated at 30, 33 or 36°C in incubators at 100 rpm. Optical density was measured at 595 nm at different times. Each point is the average of three measurements and the vertical bars represents 0.05 confidence intervals.

Figure 4.- Growth curves of Alcaligenes faeca/is in different media. Two 500 ml flasks with 250 ml of each medium were inoculated with 100 JlI of A. faeca/is (SF) and incubated at 37°C in incubators at 100 rpm. Samples were aseptically taken at different times to measure optical density at 595 nm. Each point is the average of three measurements for each replicate. Vertical bars represent 0.05 confidence intervals. 45

1.0 x-x a 30-c ~ I • 33t: X 36t 0.8 b II )(fI~i .-til c 0.6 I Q) I "-«J :i .-...,t.> 0.4 CL !/ 0 0.2 ~1 /D 0.0 D /- 0 10 20 30 40 50 Time (h) Figure 3.

96 120 46

Figure 5.- The pH of shaken cultures of Alcaligenes faecalis at 37°C as influenced by different media. Two 500 ml flasks with 250 ml of each medium were inoculated with 100 J.l1 of A. faeealis (SF) and incubated at 37°C in incubators at 100 rpm. Samples were aseptically taken at different times to measure pH. Each point is the average of three measurements for each replicate. Vertical bars represent 0.05 confidence intervals. 47

pH

.I. MBC • MA + MAE x MOE a PBMC

6.00

48 72 Time (h) Figure 5. 48

Effect of A. faeca/is on B. cinerea germination and growth

A. (aaca/is affected germination of B. cinerea in two different ways, depending on the concentration of the suspension. Concentrated suspensions of actively growing A. faeca/is completely inhibited germination of suspensions of 105 conidia/ml of B. cinerea incubated in 5 % glucose at 20°C for 24 h (Fig. 6). Diluted suspensions (1:4 to 1:16 ) of actively growing A. faeca/is retarded germination of B. cinerea. However the inhibitory effect was transitory and after 24 h most of the conidia had germinated. Dilutions between 1:32 and 1:128 showed no effect on germination (Fig. 7).

Effect on germ tube growth rate

Depending on the concentration of the suspension, A. faeca/is can stimulate or retard B. cinerea germ tube elongation. Diluted suspensions (1 :64) stimulate germ tube growth, while more concentrated suspensions (1 :8) retarded germ tube development (Fig. 8). Heavy suspensions of washed A. (aeca/is cells induced elongation of germ tubes of Bc5 as measured after 12 h at 20°C in water (Fig. 9).

Morphological changes

Two types of morphological changes in the germ tubes were detected when conidia of B. cinerea were germinated in the presence of A. (aeca/is: induction of microconidiation and germ tube deformation.

Conidia of Bc1490, Bc2502 and Bc3507 normally germinated on NA plates within 6.. 12 h at 20°C. When the plates were first pre-incubated with A. faecalis for 24 h at 37°C and then inoculated with B. cinerea conidia and incubated at 20 °C, no germination was observed during the first 3 days. After one week, many conidia produced short phialides and microconidia (Fig. 10). 49

Figure 6. Effect of concentrated suspensions of Alcaligenes faecalis on germination of conidia of B. cinerea. Ten JJI drops of a suspension of actively growing A. (aecalis (BF) were deposited in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of three strains of B. cinerea in 5 % glucose. The dishes were incubated at 20°C for 24 hours. The first 100 conidia were evaluated at 100 X magnification under the microscope and considered germinated if a germ tube could be seen. In the controls without bacteria, about 95 % germination was reached. Vertical bars represent 0.05 confidence intervals.

Figure 7. Effect of a 1:8 dilution of a suspension of Alcaligenes faecalis on germination of conidia of B. cinerea. Ten IJI drops of a 1:8 dilution of a suspension of actively growing A. faecalis were deposited in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of B. cinerea (BC?) in 5 0/0 glucose. The dishes were incubated at 20°C and percent germination was evaluated after 4, 8, 12 and 16 hours. The first 100 conidia of each drop were evaluated at 100 X magnification under the microscope and considered germinated if a germ tube could be seen. Vertical bars represent 0.05 confidence intervals. 50

c 76 0 11 £: .~

8) 50 0) ....c (D U II- 8) 0- 25

Treatment Figure 6.

100 • Control ~! -~ D A. faecalis // I: 75 // 0 i 1/ .5 / /1 E /' I- t» 50 I /"' ....,OJ / or// I: ,.// t» /- U I L. 4D I /" 0- 25 " (.y 1// 1/ 1/ /;' / / /1' / 0 /.- -D" 0 5 10 15 20 25 Time (h) Figure 7. 51

Figure 8. Effect of the dilution of liquid culture of Alcaligenes faecalis on Botrytis cinerea germ tube. Ten JJI drops of 1:8 and 1:64 dilutions of a suspension of actively growing A. faecalis (BF) in MBC were deposited in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of B. cinerea (Bc5) in 5 % glucose. In the controls drops of MBC alone were mixed with drops of the conidia. The dishes were incubated at 20 °C and germ tube length was measured after 8 hours. The first 50 germinated conidia of each drop were measured against a calibrated ocular micrometer at 100 X magnification under the microscope. Vertical bars represent 0.05 confidence intervals.

Figure 9. Effect of washed cells of Alcaligenes faecalis on Botrytis cinerea germ tube elongation in water. Ten IJI drops of distilled water and of a suspension of washed A. faeca/is (BF) cells in water were deposited in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of B. cinerea (Bc5) in water. The dishes were incubated at 20 °C and germ tube length was measured after 12 hours. The first 50 germinated conidia of each drop were measured against a calibrated ocular micrometer at 100 X magnification under the microscope. Vertical bars represent 0.05 confidence intervals. 52

1 : 8 Treatment 1 :64

Figure 8.

100

~ E 75 :::l. ~ J: +'a c .!! 50 (I) .a ::J I- 25

o Water Treatment Figure 9. 53

Figure 10. Effect of Alcaligenes faecalis on Botrytis cinerea germ tubes, formation of phialides and microconidia. Plates were first seeded with the bacteria by spreading 10 ~I drops of A. faecalis (SF) with a sterile glass rod and pre-incubating the plates for 24 h at 37°C. Then the plates were inoculated with 10 tJl drops of B. cinerea (Bc2502) conidia and incubated at 20°C. Phialides and microconidia could be easily seen under the microscope at 100 X magnification. 54

...... 25}Jm

Figure 10. 55

B. cinerea conidia also produced microconidia when placed on poor nutrient media like water agar or ionoagar. When B. cinerea conidia were placed over a piece of dialysis membrane on the plate containing A. faecalis, conidia did not germinate and microconidia were not formed.

When conidia of Bc7 were incubated in 5 % glucose for 7 h at 20°C, about 50 % of the conidia germinated. The germ tubes continued to grow slowly when mixed with drops of concentrated A. faecalis suspensions. The swelling of the tip and bending of the base of the tubes was frequent but no bursting of the tubes was evident.

Colony growth

Colony growth rate of all tested strains of B. cinerea was significantly affected when grown over PDPA plates precolonized by A. faecalis (Fig. 11). Colony growth rate of other fungi were also affected (Table 4). No correlation was found between the inhibitory effect of A. faecalis and the growth rate for sixteen strains of B. cinerea (Fig. 12).

Biocontrol tests

A. faecalis protected grapes and cabbage against damage caused by B. cinerea in different biotests.

In grapes: Bunches of grapes were partially protected against Botrytis bunch rot by immersion in A. faecalis culture in NB. B. cinerea induced extensive bunch rot of white and red grapes when inoculated alone, but less if co-inoculated with A. faecalis or T. harzianum (Fig. 13).

In another experiment, intact individual grape berries were inoculated with drops of Bc? or with mixtures of Bc? plus A. faecalis. Abundant mycelium developed only in absence of A. faecalis, but after 15 days no damage to the grapes was evident. 56

Figure 11. Effect of Alcaligenes (aecalis on colony growth rate of Botrytis cinerea. Ten millimeter discs were cut from the periphery of actively growing B. cinerea in PDA and were deposited on the surface of fresh PDPA or 24 h cultures of A. (aeca/is in PDPA at 37°C and then incubated at 20 °C for 64 h. Colony diameter was measured with a ruler. Plates were held against a dark background with oblique illumination to get maximum contrast from light diffused by the colony. Growth rate was calculated in mm per day.

Figure 12. Relationship between inhibitory effect of Alcaligenes (aecalis and growth rate of Botrytis cinerea. No correlation was found between inhibitory effect of A. (aecalis (SF) on B. cinerea colony growth rate measured as percent of inhibition (Pi) relative to the control and growth rate (Gr) of 16 strains of B. cinerea in mm/day. 57

30

~ .....-u• 20 <. E ,....,.,E

~ •... J: 10 i 0 l- f!)

o 1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 (Strain number) Figure 11.

100

c: o :;:; :c:a .~ ~ &: 50 •U l- 0..•

o o 10 20 30 Growth rate (mm/day) Figure 12. Table 4. Effect of Alcaligenes faecalis (BF) on colony growth of several fungi. Ten mm discs were cut from the periphery of actively growing fungi in PDA and were deposited on the surface of fresh PDPA or 24 h cultures of A. faecalis in PDPA at 37°C and then incubated at 20 °C for 64 h. Colony diameter was measured with a ruler. Plates were held against a dark background with oblique illumination to get maximum contrast from light diffused by the colony. Percent inhibition was calculated relative to the control without bacteria. The identification numbers refer to the Biology Department culture collection code. BcU is a strain isolated in the present work from grapes in a field near Thorold, Ontario.

Identification Fungi Percent inhibition 301 Phycomyces blakesleeanus 90 305 Rhizopus stolonifer 83 306 Rhizopus nigricans 85 307 Sordaria fimicola 58 315 Mortierella hygrophila 13 317 Sordaria fimicola gray 42 319 Sordaria fimicola tan 42 SLEA27 Trichoderma harzianum 29 SLEA21 Trichoderma harzianum 27 SWEL5 Trichoderma harzianum 30 BcD Botrytis cinerea 90

Vl 00 59

Figure 13- Partial protection of grapes by Alcaligenes faecalis against Botrytis rot. Bunches of white and red seedless grapes purchased at a local market were immersed in a one-week old NB culture of A. faecalis (BF), a suspension of Trichoderma harzianum (SLEA 27) of 2 X 107 conidia/ml (T) or in tap water for the controls (W), and let dry overnight at room temperature. Then they were immersed in a suspension of 105 conidia/ml of Botrytis cinerea (Be), placed in plastic trays, sealed with polystyrene film and incubated. C is the uninoculated control. Damage was estimated according to the following scale: Damage Rotten berries o 0 1 Up to 20 % 2 Up to 40 % 3 Up to 60 % 4 Up to 80 0/0 5 Up to 100 % 60

5

4

2

1

Red grapes Treatment Figure 13. 61

Individual detached green grape berries developed gray mold and rot after inoculation by puncture with B. cinerea in the presence of medium plus 5 % glucose, but not if A. faecalis was present. No damage was seen in the control with medium and glucose alone (Fig. 14).

In bean leaves: Conidia of Bc1 germinated in drops deposited on detached bean leaves but did not produce visible mycelium or any damage. Addition of A. faecalis to the drops, completely inhibited germination of conidia.

In cabbage: Drops of Bc5 plus 5 % glucose produced abundant mycelium on fresh cabbage leaf fragments but no damage in one week. Addition of A. faecalis suspension to the drops prevented mycelium development. Bc? induced necrotic lesions in punctured fresh cabbage leaf fragments in one week and completely degraded heat treated cabbage disks. A. faecalis provided protection against damage in both cases (Fig. 15).

Inhibitory substances produced by Alcaligenes faecalis

Cells of A. faecalis separated from the supernatant liquid by centrifugation and washed with water did not inhibit or retard germination of B. cinerea. The inhibitory activity was only found in the filtrate (Fig. 16).

Conidia of B. cinerea germinated well on NA and PDPA plates in the presence of E. coli, E. aerogens and A. faecalis washed cells. In the presence of E. coli and E. aerogens, the fungus grew almost unaffected by the bacteria. However, germ tube growth and further colony development was severely affected in the presence of A. faecalis. In media like lA, WA and PDA where A. faecalis did not grow well, the inhibitory effect on B. cinerea was less pronounced.

The inhibitory activity of A. faecalis cultures on different media was very variable and seemed to depend more on the nature of the medium than on the age of the culture or the optical density of the culture (Table 5). 62

Figure 14. Treatment of grape berries with Alcaligenes faecalis prevents damage caused by Botrytis cinerea. Individual detached white grape berries in a plastic holder were inoculated with ten microlitre drops of a one-week old MBC culture of A. faecalis (BF), or with MBC for the controls. Subsequently, 10 IJI of a suspension of 105 conidia/ml of B. cinerea (Be?) in 5 OJ'<> glucose was added, and each berry was punctured with a needle. The holder was incubated in a sealed box with a small sponge impregnated in water to keep moisture. Left: Unprotected control (K); Protected by A. faecalis (B) Right: B. cinerea damage in unprotected berry.

Figure 15.- Effect of Alcaligenes faecalis on Botrytis cinerea induced damage in fresh cabbage leaf fragments. Pieces of cabbage leaves of approximately 20 cm 2 were inoculated as above and incubated in plastic Petri dishes lined with wet filter paper. The drop on the left is water. In the middle, a necrotic lesion produced by B. cinerea (Be?). On the right, the drop contains a mixture of B. cinerea and A. faecalis. No damage is evident. 63

Figure 14.

Figure 16. 64

Figure 16.- Filtered liquid from one week old cultures of Alcaligenes faecalis completely inhibited germination of Botrytis cinerea conidia. Washed A. faecalis cells did not inhibit germination. A sample of 10 ml of a one-week old culture of A. faecalis (BF) in MBC was centrifuged for 10 min at 13,000 g at 4°C. The liquid was passed through a 0.22 IJm Millipore membrane filter. The cells were collected and washed three times with distilled water and resuspended in 10 ml of distilled water. Ten IJI drops of the suspension of washed cells and of the filtered liquid were deposited in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of B. cinerea (Bc1, Bc2 and Bc3) in 5 °lb glucose. The dishes were incubated at 20°C and percent germination was evaluated after 16 hours. Vertical bars represent 0.05 confidence intervals. 65

Washed cells Filtered liquid

Figure 16. 66

Table 5.- Relationship between the inhibitory activities and optical densities of several 144 h old Alcaligenes faecalis cultures in different media. Culture samples of A. faecalis (BF) in each medium were taken after 144 h of incubation at 37°C and the optical density at 590 nm was measured in 1 cm light path tubes in a Bausch & Lomb spectrophotometer. Inhibitory activity was determined by placing 10 ~I drops of a serial dilution of the cultures in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of B. cinerea (Bc5) in 5 % glucose. In the controls drops of dilutions of the media alone were mixed with drops of the conidia. The dishes were incubated at 20°C and germination was measured after 4 hours. Titre was calculated as the reciprocal of the highest dilution that completely inhibited germination.

Medium Optical density Titre at 4 h MBC 2.94 16 MBE 2.1 2 MAE 3.5 1 MA 0.26 0 67

However, for a given medium the inhibitory activity seemed to depend on the optical density of the culture (Fig. 17).

The nature of the inhibitory substances

Both volatile and nonvolatile inhibitory substances are produced by A. faecalis. The volatile nature of inhibitory substances produced by A. faecalis in liquid media is evident since Botrytis cinerea did not grow in a flask where Alcaligenes faecalis grew for one week (Fig. 18).

A strong characteristic smell and change to blue color of litmus paper introduced in the flask suggested that ammonia was one of the volatile substances produced by A. faecalis. Depending on the concentration, ammonia can retard or irreversibly inhibit germination of B. cinerea (Fig. 19).

The presence of nonvolatile inhibitory substances was indicated by the observation that inhibitory activity was retained after lyophilization or heat treatment of the liquid where A. faecalis grew. However, dialysis against distilled water completely removed the inhibitory activity (Fig. 20).

The dialysable nature of the inhibitory substances was also evidenced by the observation that conidia of Bc1490, Bc2502 and Bc3507 did not germinate on dialysis membranes over A. faecalis cultures. Drops of A. faecalis cultures deposited over dialysis membranes on PDA plates seeded with B. cinerea conidia inhibited germination of conidia below the membrane. 68

Figure 17.- Changes in time of the inhibitory activity and optical density of the cultures of Alcaligenes faecalis in MBC. Two 500 ml flasks with 250 ml of MBC were inoculated with 100 ~I of A. faecalis (BF) and incubated at 37°C in incubators at 100 rpm. Samples were aseptically taken at different times to measure optical density (00) and inhibitory activity (IA). Inhibitory activity was determined by placing 10 IJI drops of a serial dilution of the cultures in Petri dishes and mixed with 10 IJI drops of suspensions of 105 conidia/ml of B. cinerea (Bc7) in 5 % glucose. In the controls drops of dilutions of the media alone were mixed with drops of the conidia. The dishes were incubated at 20°C and germination was evaluated after 6 hours. Titre was calculated as the reciprocal of the highest dilution that completely inhibited germination. Results shown below are from one of two similar experiments.

Figure 18.- Botrytis cinerea does not grow in a flask where Alcaligenes faecalis grew for one week. Ten microlitre drops of a suspension of 105 conidia/ml of B. cinerea in 5 % glucose were deposited on glass cover slips glued on the inside surface of rubber stoppers. Then the cap of a flask with 250 ml of a one-week old culture of A. faecalis was quickly replaced with the rubber stopper carrying the coverslip and the drop of conidia facing the interior of the flask. Another flask with medium alone was used as a control in the same way. After one week of incubation at 20°C the drops were compared in both flasks. A: B. cinerea does not grow in the flask where A. faecalis grew for one week. B: In the control flask without A. faecalis, abundant growth of B. cinerea is evident. C is a general view of the experiment. 69

4-.0 32 Titre

~3.0 ." C G 1:5 11 2.0 16 .-.....(.) oa. 1.0 8

o 48 96 144 191 240 Time (h)

Figure 17.

BF culture Medium alone

Figure 18. 70

Figure 19. Effect of ammonia on germination of conidia of Botrytis cinerea. Drops of 10 J,JI of suspensions of 1X105 conidia/ml of Bc7 were placed on the inner surface of the lid of a Petri dish and 5 ml of diluted NH40H was placed in the other part of the dish. Then 1 ml of 1 N NaOH was added to the NH40H, quickly covered with the lid, sealed with Parafilm and incubated at 20°C. Percent germination was measured after 20 h.

Figure 20.- The inhibitory activity of cultures of Alcaligenes taeca/is in MBC against Botrytis cinerea was removed by dialysis but not by 15 minute heat treatment between 55 and 100°C or lyophilization. Ten ml samples of the supernatant of a one-week old culture of A. taeca/is in MBC were frozen at -30 °C, lyophilized, resuspended in their original volume of distilled water and filtered through 0.22 IJrn Millipore filters. Two 0.1 ml aliquots of a one-week old MBC culture of A. taeca/is were dispensed into Eppendorff tubes and heated for 15 min in a water bath at 55°C, at 80 °C or at 100°C in boiling water. Other two aliquots of 0.1 ml were dialyzed against distilled water at 4 °C for 6 h and at room temperature overnight respectively. Inhibitory activity was determined by mixing 10 ~I drops of the samples with 10 ~I drops of suspensions of 105 eonidia/ml of B. cinerea (Be7) in 5 % glucose in Petri dishes. The dishes were incubated at 20 °C and germination was measured after 12 hours. 71

100

75 c: 0 I -et: I- G) 50 ....,OJ C •...U CD Q.. 25 *

o o 0.8 1.6 Concentration ofNH3 (ppm) Figure 19.

75 c 0 I .5 E '- G) 50 ....,OJ C G U Ji- G Q.. 25

Treatment Figure 20. 72

Purification of the inhibitory substance

An attempt to purify the substance was made using Sephadex G-10 column chromatography. Separation of a sample of A. faeea/is culture filtrate in a column of Sephadex G-10 with exclusion volume of 7.5 ml, produced three separated peaks A, Band C with elution volumes of 10.5, 12 and 13 ml respectively (Fig. 21). These peaks contained most of the eluted mass. However the activity of the fractions was maximum in tube 12 corresponding to a peak with minimum mass.

Microprecipitation tests of samples of the fractions with silver nitrate produced granules indistinguishable from those of silver chloride only from fractions 13 and 14. The pH of fractions 7 to 11, as determined by pH indicator paper, was slightly basic while the pH of the other fractions was neutral. This suggests that peak C is composed of sodium chloride and that peaks A and B are basic salts, probably phosphate and citrate. Similar experiments with 3 different columns of Sephadex G-10 always produced inhibitory activity after the exclusion volume. This suggests that the inhibitory substance behaves as if it has a MW under 700 Daltons. The fractions with inhibitory activity gave negative reaction with Ninhydrin test for amino acids and with DNS test for reducing sugars. No UV absorption peak was detected between 250 and 300 nm and the samples were colorless.

The inhibitory activity could not be extracted from lyophilized samples of A. faecalis cultures using 10 ml of chloroform or acetone. Methanol extract exhibited 60 % of the inhibitory activity and ethanol less than 50 % in single extractions .

After electrophoresis in 0.8 % agarose gels in 0.1 o~ ammonium acetate, the inhibitory activity could only be detected in the central well and in the first centimeter of the gel in the direction of the cathode. 73

Figure 21.- The inhibitory activity of cultures ofAlcaligenes 'aecalis in MBC after separation in a column of Sephadex G-10 was localized in a single peak. The column was prepared with Sephadex G-10 to a height of 25 em and was equilibrated with water. Blue Dextran was used to determine the exclusion volume. Samples of 400 ~I of the culture liquid were eluted with water and 0.5 ml fractions were collected in weighed tubes. The tubes were then dried in a desiccator, weighed to calculate the amount of substance in each tube, resuspended in 1 ml of water and tested for inhibitory activity. Inhibitory activity was determined by mixing 10 J-II drops of the samples with 10 IJI drops of suspensions of 105 eonidia/ml of B. cinerea (Be7) in 5 % glucose in Petri dishes. The dishes were incubated at 20°C and germination was evaluated after 12 hours. The exclusion volume was 7.5 ml. 74

Blue A .B Dextran ,-, /1 Activity peak J\ II) 10 I ,- II C 10 / \ JI I~ ...... i \I I· = r 1 I I I\ e :).... * \A I\ ..., I ~ ...... ~ i \. .S: E= I .... ~ «I en I en 5 5 I (J ;:: :2:" I ·0 I.) I ~ J Ci3 (

.I • / :Y

0 • 0 0 5 10 15 20 Fraction number Figure 21. 75

Discussion

Since the isolated Alcaligenes faecalis (BF) always formed two distinct types of colonies, it was suspected that two organisms could be present. After many unsuccessful attempts to separate them, it was realized that they are probably two different forms of the same microorganism and it was considered to be a pure culture. Malek et al. (1963) described two types of colonies for A. odorans. Mitchell and Clarke (1965) also reported dual colony morphology for A. faecalis var. viridans. These two organisms are now considered to be strains of Alcaligenes faecalis.

Considering that the isolated bacterium was aerobic, Gram negative and has flagellated rod shaped cells of 0.5-1.0 by 1.0-1.5 Jjm, it should be placed in Section 4 of Bergey's Manual (Krieg and Holt, 1984). Bacteria in the following families of this section can be eliminated by some of their differential characteristics, for example: Azotobacteraceae (big cells with diameter over 2 IJm); Halobacteraceae (require 12 % NaCI for growth); Neisseriaceae (not motile); Acetobacteraceae (oxidase negative); Rhizobiaceae (utilize sugars); Legionellaceae (require cysteine for growth) and Methylococcaceae (grow in methanol). The isolate SF does not grow in media containing 12 % NaCI, is motile, does not require cysteine for growth, and cannot utilize sugars or methanol as the sole source of carbon.

Only the family Pseudomonadaceae is not eliminated. However, three of its four genera (Frateuria, Xanthomonas and Zooglea) can also be excluded by differential characteristics like growth at pH 3.6 or the requirement of some growth factors (Palleroni, 1984). The genus Pseudomonas is not eliminated directly, however most of the Pseudomonas species are excluded because they can utilize sugars. Two exceptions listed in Bergey's Manual that do not utilize sugars (Pseudomonas testosteroni and P. alcaligenes) can also be excluded 76 because P. testosteroni does not grow in ethanol and P. alcaligenes does not grow in aspartate as sole source of carbon (Palleroni, 1984) while SF does.

The following genera not assigned to any family can be eliminated by some differential characteristics: Beijerinckia, Derxia, Alteromonas, Halomonas, Janthinobacterium, Xanthobacterium, and Haemophilus all of which can utilize some sugars; Thermus and Thermomicrobium, are thermophiles, Bordetella and Brucella, require growth factors: while Flavobacterium, Francisella, Paracoccus, and Lampropedia are nonmotile. Serpens can be excluded for its distinctive morphology of big spiral cells.

Only Alcaligenes (Kersters & De Ley, 1984) is not excluded. The isolate SF shares the following differential characteristics of this genus: Gram negative, motile with peritrichous flagellation, oxygen requirement, colony color, optimum temperature range, positive catalase and oxidase tests. Cellullose, gelatin, indole, acetyl methyl carbinol, 3-ketolactose and starch tests are all negative. No growth with carbohydrates as sole carbon source. Growth occurs on acetate, succinate, fumarate, lactate, malate, citrate, L-alanine, L-aspartate, L-glutamate, L-proline and L-phenylalanine and production of alkali from carboxylic salts. According to these characteristic SF can be assigned to the genus Alcaligenes.

Two authentic species (Alcaligenes faecalis and A. denitrificans, the later with two subspecies: denitrificans and xylosoxydans) and eight species incer/ae sedis: A. eutrophus, A. paradoxus, A. latus, A. aestus, A. aquamarinus, A. cupidus, A. pacificus and A venustus are included in this genus. Only A. faecalis, A. denitrificans subsp. denitrificans and A. eutrophus cannot use D-Glucose as sole source of carbon. A. eutrophus can be distinguished because it can use fructose but not ~-alanine as carbon source (Kersters and De Ley, 1984). Most A. denitrificans subsp. denitrificans strains grow using D-gluconate, ~-alanine or glycerol as the sole carbon source while A. faecalis (Kersters and De Ley, 1984) and SF do not. 77

SF resembles A. faecalis in more than 40 biochemical tests: Aerobic metabolism. Lack of acid or gas production in Phenol red medium from either glucose, fructose, galactose, lactose, sucrose or manitol. Positive reaction to oxidase, catalase, and lipase tests, and negative to methyl red, Voges-Proskauer, starch, gelatin, urease, indole, hydrogen sulphide and litmus milk tests. They both can grow in synthetic media using as the sole carbon source citrate, acetate, fumarate, succinate, L-malate, DL-Iactate, pyruvate, L-aspartate, L-glutamate, L-tryptophan, L-phenylalanine, L-methionine, L-cysteine, L-valine, L-alfa-alanine, L-proline and ethanol. They do not growth in methanol, acetone, glycerine, formaldehyde, dioxane, glyoxal, n-hexane, benzene, L-arginine, r1-alanine, L-Iysine, L-ornithine or L-cysteine, D-fructose, D-mannose, D-glucose, D-gluconate, glucose-5-phosphate, D-ribose, D-arabinose, D-galactose, D-Iactose, maltose, sucrose, D-Iyxose, D-xylose, L-sorbose, D-mannitol, inositol, xylitol, D-cellobiose, D-trehalose, glycogen, starch, or cellulose as the sole carbon source.

In the earlier tests, when young isolated SF reduced nitrate from Difco nitrate medium, no nitrite was detected. Both nitrate and nitrite reductase activity should have been present at that time. However, after several months of subculturing in the laboratory it had lost this ability. At this moment it does not reduce nitrate or nitrite from Difco nitrate medium and it does not grow in any of them as sole nitrogen source in synthetic medium.

A. faecalis lacks the ability to reduce nitrate, but it can reduce nitrite (ROger and Tan, 1983 ; Kersters and De Ley, 1984). The type strain of A. odorans CCES 554 (previously named P. odorans and later considered as a subjective synonym of A. faecalis) reduced nitrate to nitrite but not to nitrogen. However, the original isolate of P. odorans and 3 other strains did not reduce nitrate or nitrite (Malek et aI., 1963). Nitrate and nitrite utilization seems to be variable characters in the species A. faecalis as presently defined. Besides that, anaerobic respiration in the presence of nitrate may have been lost on subculturing (Hendrie et aI., 78

1974). The ability to denitrify is dependent on the composition of the test medium (Ruger and Tan, 1983) and tests for nitrate reduction and denitrification showed fairly low reproducibility levels when performed with the same strains in different laboratories (Sneath and Collins, 1974).

Most A. faecalis strains form colonies in NA with a thin spreading edge and some (previously named A. odorans) produce fruity odor in young cultures that later changes to cheesy or ammonia. These were considered typical characteristics for this organism (Malek et aI., 1963 ; Mitchell and Clarke, 1965). The organism isolated in the present study closely resembles A. faecalis in these properties.

The reaction of antisera raised against the SF strain isolated in this work with A. faecalis type strain AlCC 15554 in immunoprecipitation and dot immunobinding assays in conditions in which other bacteria do not react, implies close serological relatedness. Malek et aI., (1963) found that four strains of A. faecalis (A. odorans) were serologically heterogeneous, but two of them were related. Bacteria belonging to A. faecalis are usually considered to be difficult to identify because they do not possess specific physiological or biochemical characteristics (Kersters and De Ley, 1984). However, considering all of the above evidence, we feel that SF should be identified as a strain of Alcaligenes faecalis Castellani and Chalmers.

Effect of A. faecalis on B. cinerea.

The biotest for fungal inhibitory substances usually gave satisfactory results but occasionally inexplicable low germination was found in the control experiments. Highly variable results from different Petri dishes, even from the same lot suggest the presence of fungal inhibitors in the dishes. Perhaps residues of substances used in the manufacture process are present in inhibitory levels in some dishes. Germination of conidia of B. cinerea is affected by many volatile compounds (Brown, 1922). 79 somed'ishes.Germinationofconidiaof ·B.cinerea is affected b.yma·ny volatile compounds (Brown, 1922).

Bacteria ha·ve been 10'n9 known to suppress fu'ngal growth (See review section). Jackson et al., (1991) have reported that unidentified isolates of Alcaligenes spp. were amon'Q th·e most active against Scleroliumcepivorum, but to th·ebest of our knowledge, this seems to be the first report of fungal growth inhibitory activity by A. faeea·lis. We have fo,und that A.. fa·ecalis affects growth a·nd developme·nt of B. cinerea and other fungi. The inhibitory effect of A. (aeca/is on B. cinerea is due, at least to two different typ,e,sofsubstances.Oneof them j,svolatile, probably ammonia. The otherisnonvolatiJe and will be discussed later.

Evi'dence' for th'e inhibltory effect of ammoni-a in, OU-f work ca·me· from on-e' experiment (Figure 18) in which B. cinerea (Bc7) did not grow in an atmosphere where A. faeealisgrew for one week. 1n the control experi'mentwith'out A. (seea/is, abundant growth ofB'c7 was evident. Characteristic smell and change of I·itmus papa., introduced in the flask to b'lue· color suggests that ammo'nia is one of the volatile substances produced by A. (seea/is. Ammonia has been -reported as anin'hlbitorysubsta'nce for fungal growth. Its taxi-city has been attributed to its ability to penetrate and disrupt caU membranes (Can-dole a,nd Rothro'ck, 1997).

Depen-d'i'ng on th·e· concentration, ammonia· ca-n retard or i'rreve'rsibly' i'nhibit germi'nation of B. cinerea (Fig. 19). A similar pattern of action was found for clamy'dosporeg'erminationof Thielaviopsis basicola (Cand-ole and Rothrock, 1997). Concentrations of A. faecalis that c'ompl-etely inhi-bited germination of B. cinerea con·i'dia, only reduced the growth rate of the' myceli'um. This is ion agreement with earlier reports that fungal spores are in general more susceptible to ammonia and tosoitfun,g-istasis than fu'n,galmycelia (Candol~e and Rothrock, 1997). 80 nonvolatile inhibitory substance. This is not surprising since many bacteria produce antibiotics. One strain of A. 'aeca/is has been patented for production of antibiotics (US. Pat. 3 682 777).

The identity of the nonvolatile substance could not be established. Its polar nature is evidenced by the fact that it is extractable in polar solvents like water, ethanol and methanol and not in acetone or chloroform. Its low molecular weight is anticipated from experiments in which inhibitory activity is completely lost after dialysis and by gel filtration chromatography through Sephadex G-10.

When germinated B. cinerea conidia were mixed with inhibitory suspensions of A. faeca/is, the germ tubes continued to grow slowly and swelling of the tip was evident. Brown (1922) also found that germ tubes in the presence of plant volatiles were shorter but much stouter than in the control.

The reason for the formation of phialides with microconidia, when conidia of B. cinerea were placed directly over A. faeca/is colonies is not known. We have found that B. cinerea conidia produced microconidia when placed on poor media like water agar or ionoagar. Harrison and Hargreaves (1977) found that conidia of B. (abae buried in soil produced microconidia and they suggested that production of microconidia occurs when the viability of conidia is low. Harrison (1988) considered that microconidia of both B. cinerea and B. (abae are produced as a response to prolonged conditions unfavorable to fungal growth. Perhaps microconidiation is an unspecific reaction to some adverse conditions.

A. 'aeea/is protected cabbage and grapes against damage caused by B. cinerea in different biocontrol tests. It seems logical to suppose that A. faeca/is inhibited B. cinerea germination and colony development in vivo by the combined action of ammonia and other inhibitory substances. However, this is not always the case, for example, Baci//us brevis is known to produce the antibiotic gramicidin S which is fungicidal to B. cinerea in vitro. In field trials B. brevis protects cabbage against damage by B. cinerea, but the mode of action in p/anta was shown to be 81 associated with reduction of leaf wetness. The antibiotic binds to the leaf and is inactive in this form (Edwards and Seddon, 1992).

A. faecalis seems to be a good candidate for the development of biocontrol agents to fight B. cinerea and other fungi. It is an ubiquitous organism with no known deleterious effect on humans, plants or animals. The laboratory screening for inhibitory activity is easy. A. faecalis' ability to grow on simple synthetic media should facilitate the isolation of strains from nature, and the reproduction in large scale.

Further research is needed (i) to characterize the nonvolatile inhibitory agent; and (ii) to evaluate if A. faecalis is efficient to control fungal diseases in the field, the methods of its formulation and application and whether it is commercially viable. 82

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