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Agglutination potential of

Pseudomonas ¯uorescens

in relation to

energy stress and colonization of

Macrophomina phaseolina

T.K. Jana

a

, A.K. Srivastva

a

, K. Csery

b

, D.K. Arora

a,

*

a

Laboratory of Applied Mycology, Centre of Advanced Study in Botany, Banaras Hindu University, P.O. Box 5020, Varanasi 221 005, India

b

Sopron University, Sopron, Hungary

Accepted 25 September 1999

Abstract

Agglutination potential of 172 isolates of Pseudomonas ¯uorescens, isolated from the rhizosphere soil of chickpea plants, was evaluated in crude agglutinin (CA) of Macrophomina phaseolina and on sclerotia and hyphae surfaces. Eighteen such isolates varied signi®cantly in their agglutination potential (10±73%). Isolates 12 (Agg+) and 30 (Aggl) showed maximum (73%) and minimum (10%) agglutination, respectively. Total loss of endogenous C reserve did not dier signi®cantly P0:05 from sclerotia incubated with Agg+, Agglor Aggÿ(a non-agglutinable Tn5 mutant of wild type 12). Most of the C lost from stressed sclerotia was evolved as14CO2 (40%), whereas 5% C was lost in the form of sclerotial exudate (residual C). The total C loss

was in the order: Agg+> Aggl> Aggÿ> unsterilized soil. Germination of sclerotia incubated with Agg+, Aggl, Aggÿcells or in soil was suppressed both in the presence or absence of C source and such sclerotia retained a greater portion of their viability even after 60 d. Loss of C from the sclerotia incubated with isolates ofP. ¯uorescenswas directly correlated with germination repression r ÿ0:89toÿ0:96;P0:05). Greater colonization of sclerotia by Agg

+

was observed compared to Agglor Aggÿ isolates. Our ®ndings clearly demonstrate the existence of a great diversity ofP. ¯uorescensisolates in natural soils in respect to their agglutination potential onM. phaseolinasclerotia. Irrespective of the agglutination potential of isolates, they can invariably impose energy stress on sclerotia resulting in accelerated loss of C and also elevating the nutrient requirement for sclerotia germination.72000 Elsevier Science Ltd. All rights reserved.

Keywords:Agglutination; Energy stress;Pseudomonas ¯uorescens;Macrophomina phaseolina

1. Introduction

Fungal propagules in soil are subjected to numerous abiotic and biotic stresses (Lockwood, 1992; Hyaku-machi and Arora, 1998) and their germination is regu-lated by the loss of endogenous energy-yielding compounds to the microbial `nutrient sink' in soil (Lockwood, 1992). Fungal propagules exposed to energy stress, lose endogenous C by respiration and exudation resulting in energy (nutrient) stress, with demand for nutrients during germination, viability loss and decreased pathogenic aggressiveness (Hyakumachi

and Lockwood, 1989; Mondal et al., 1996; Mondal and Hyakumachi, 1998). The stress imposed on fungal propagules by dierent soil microorganisms also results in accelerated loss of C (Arora et al., 1983; Epstein and Lockwood, 1984; Arora, 1988).

Recognition by microorganisms to the appropriate host surface is a specialized event of cell adhesion (Savage and Fletcher, 1985; Manocha and Sahai, 1993). Several agglutination assays have been done between animal host cells and a variety of bacteria (Tomita et al., 1994; Ofek et al., 1995), plant root sur-faces and dierent soil microorganisms (Anderson et al., 1988; Glandorf et al., 1994) or fungal host±fungal antagonist (Benyagoub et al., 1996; Inbar and Chet, 1997) in order to unravel the interactive mechanism of cellular recognition. Agglutination of antagonistic

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* Corresponding author. Fax: +91-542-317-074/313-965.

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microorganisms to a fungal host or pathogen surface or to one another, is a feature of antagonistic inter-actions (Manocha and Sahai, 1993). The colonization of a fungal host by the potential antagonist may involve molecular interactions between the pathogen and the antagonist surface to promote attachment (Manocha, 1991). From a biocontrol view point, ag-glutination of antagonists on a fungal host appears to be one of the important phenomena as it also enables retention of antagonists on pathogenic fungal propa-gules (Inbar and Chet, 1997). Though antagonistic po-tential of ¯uorescent pseudomonads against sclerotial pathogens such as Rhizoctonia solani (Gupta et al., 1995),Sclerotinia sclerotiorum (Bin et al., 1991; Expert and Digat, 1995) and Macrophomina phaseolina (Sri-vastava et al., 1996a) has already been established, no detailed study has been done to elucidate the aggluti-nation between fungal pathogens and bacterial antag-onists, i.e. fungal±bacterial systems in general and M.

phaseolina and Pseudomonas ¯uorescens in particular.

We have demonstrated that soil contains a large num-ber of parasites ofM. phaseolinasclerotia which poten-tially reduce the host population in soil (Srivastava et al., 1996a). The eects of nutritional and growth fac-tors on the production ofM. phaseolinaagglutinin and its response towards agglutination of P. ¯uorescens

have been evaluated (Srivastava et al., 1996b). How-ever, the potential of agglutinable (Agg+), less agglu-tinable (Aggl) or non-agglutinable (Aggÿ) isolates of

P. ¯uorescens to impose competitive energy stress on

fungal propagules and its subsequent eect on viability and colonization has not been investigated.

Our aim was (i) to isolateP. ¯uorescensstrains from chickpea rhizosphere and to evaluate their aggluti-nation potential on the sclerotia surface and agglutinin produced byM. phaseolina, (ii) to assess the ability of Agg+, Aggl and Aggÿ Tn5 mutant generated from Agg+ wild type, to impose energy stress in M. phaseo-linasclerotia and its subsequent eect on their germin-ability, (iii) to evaluate the agglutination response of

P. ¯uorescens on the stressed sclerotial surface and the

agglutinin produced by the sclerotia and (iv) to assess the role of agglutination in the colonization of sclero-tia by Agg+, Agglor Aggÿisolates.

2. Materials and methods

2.1. Soil and microorganisms

A sandy loam soil (sand 70%, silt 17%, clay 10.5% and organic matter 2.5%) was obtained from ®elds where chickpea (Cicer arietinum L.) had been grown over the past 7 years. Soil was sieved (4 mm) and stored moist at 4±68C until use. Six isolates of M.

pha-seolina (Tassi) Goid. were obtained from the Applied

Mycology Culture Collection, Banaras Hindu Univer-sity (Srivastava et al., 1996a). The pathogen was grown on carrot agar (pH 5.6; 25±288C) for 30 d. Sclerotia were scraped from the surface of culture plates, dried for 24 h over laminar ¯ow and stored at ÿ208C until use. 14C-labeled sclerotia were obtained from the cultures supplemented with 14C-glucose (12.5

mCi mMÿ1; 1Ci37GBq; Bhaba Atomic Research Centre, Bombay, India). Strains of P. ¯uorescenswere isolated from rhizosphere soils of 10 dierent chickpea ®elds. Diluted soil samples (10ÿ7to 10ÿ4) were plated on Kings B medium (KB, Kings et al., 1954) and Sand's ¯uorescent pseudomonad medium at 28±308C (Sands and Hankin, 1975). After 72 h, plates were examined under UV radiation (365 nm). All isolates were characterized according to Bergey's Manual of Systematic Bacteriology (Palleroni, 1984). The mor-phological and biochemical tests used for identi®cation were reaction pro®les on API test strips (API Labora-tory Products, Canada). In brief, all isolates were examined for oxidase reaction, Gram's reaction, moti-lity and production of catalase. Pseudomonas isolates were further examined for production of ¯uorescent pigment, hydrolysis of gelatin, levan production from sucrose, utilization of saccharate, trehalose,meso -inosi-tol, benzylamine and 2,3-butanediol (Molin and Tern-strom, 1982). From 1470 isolates, 172 were selected as

Table 1

Percent agglutination of dierent isolates ofPseudomonas ¯uorescens to crude agglutinin and on the surface of washed sclerotia of Macro-phomina phaseolina

Isolate No. Crude agglutinin Sclerotia

agglutination

Data are means of 10 replicates2S.D.

b

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these signi®cantly inhibited germination of sclerotia and colony growth in an in vitro test (results not shown). Out of these isolates, 154 isolates exhibited very poor agglutination (<4%), whereas 18 isolates showed strong (40±73%) or less agglutination (9±15%) in crude agglutinin (CA) or on the surfaces of sclerotia and hyphae ofM. phaseolina(Table 1). An isolate that showed strong agglutination (Agg+; isolate 12) and another showing much less agglutination (Aggl; isolate 30) in CA were used in all experiments (Table 1). These isolates were maintained and stored on KB med-ium at 48C.

2.2. Isolation of mutant and antibiotic resistant isolates

A spontaneous rifampicin resistant (Rifr) strain of Agg+ isolate 12 was obtained by transferring the colo-nies to KB plates containing 50, 100, 150, 200 or 250

mg rifampicin mlÿ1 (Sigma). The strain was double marked with streptomycin (250 mg mlÿ1; Hi Media, India). Agg+ (Rifr±Strr) were obtained by transferring the colonies on KB plates containing 50±250 mg of streptomycin mlÿ1 in addition to rifampicin (250 mg mlÿ1). The resistant colonies were tested for aggluti-nation, growth rate and ¯uorescence under UV radi-ation. Similarly, Aggl isolate 30 was selected for resistance to tetracycline (Tetr) by transferring to plates containing tetracycline (50±250 mg mlÿ1; Hi Media, India). There was no evidence for reversal by these antibiotic resistant isolates to the parent type fol-lowing more than 15 generations. Rifampicin and kanamycin resistant (Rifr Kanr) mutants of isolate 12 were generated by transposon mutagenesis (Anderson et al., 1988). Transposon mutagenesis involved Tn5 insertion with the suicidal vector system of Escherichia coli SM (Kanr) obtained from IACR-Rothamsted, UK. Biparental matings were conducted between E. coli SM (donor) containing Tn5 on the suicidal plas-mid and rifampicin resistant Agg+ isolate 12 (recipi-ent) from cultures grown overnight with appropriate antibiotics. Donor (8 ml) and recipient (4 ml) cells were mixed, harvested by centrifugation, resuspended in Luria-Burtani broth (LB; lÿ1: tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; pH 7) and were spotted (200

ml) onto a HAWP membrane (0.45 mm; Millipore, USA) on LB agar. After growth at 28±308C for 18 to 24 h, the bacteria were scraped from the ®lter and resuspended in 10 ml of LB broth. To select transcon-jugates, diluted cell suspension (10ÿ7±10ÿ4) was plated with appropriate antibiotics and grown at 28±308C for 48 h. Antibiotic resistant transconjugants (Rifr±Kanr) were picked and recombinants selected twice by single colony isolation. The ability of recombinants to grow on LB medium containing antibiotics was tested and an eective killing rate of 83 to 89% was obtained. Tn5 mutagenesis resulted in 1360 transconjugates. All

transconjugates were screened for agglutination ability in CA ofM. phaseolina (Srivastava et al., 1996b). Only one transconjugate did not show agglutination in CA and was selected for further study. Though we have not done any genetic analysis of this Tn5-derived mutant, no agglutination in CA or on the surface of sclerotia was observed even after 20 repeated subcul-tures. The mutant (Aggÿ) also showed growth rate and ¯uorescence in UV-light similar to the wild-type isolate 12 on KB broth and agar indicating that nutri-tional de®ciency was not a factor in agglutination. The resistant strains Agg+ (Rifr±Strr), Aggl (Tetr) and Aggÿ(Rifr±Kanr) were used throughout our study and hereafter referred to as Agg+, Aggl and Aggÿ unless stated otherwise.

2.3. Production of agglutinin

The inoculum of M. phaseolina (ca. 1 sclerotium mlÿ1) was added to 100 ml of synthetic medium (SM; lÿ1: K2HPO4, 0.9 g; MgSO47H2O, 0.2 g; KCl, 0.2 g; NH4NO3, 1 g; glucose, 15 g; Mn2+, 2 mg; Fe2+, 2 mg; Zn2+, 2 mg; thyamine hydrochloride, 0.1 mg; pH 6) and grown on a shaker for 15 d (28±308C). Culture ®l-trate (CF) was collected by vacuum ®ltration, centri-fuged (5000g for 5 min; 48C) and dialyzed for 48 h against 42000 ml of phosphate buer saline (PBS; pH 7) at 48C using dialysis membranes with a molecu-lar mass cut o of 10±12 kDa (Sigma). The dialyzed CF was lyophilized and stored at ÿ208C. Prior to ag-glutination assay the lyophilized CF was resuspended in PBS to obtain a ®nal protein concentration of 2 mg mlÿ1and served as crude agglutinin (CA).

2.4. Agglutination assay

Isolates of P. ¯uorescens and mutant Tn5 Aggÿ iso-late 12 (Table 1) were grown to exponential phase (A550; 0.15±0.18) and washed twice with PBS (pH 7) to yield 105cells mlÿ1. To test the agglutination of Agg+, Aggl or Aggÿ cells, 5ml of CA was mixed with equal volume of cell suspension on acid-washed glass slides and agglutination was examined after 30 min under phase contrast. Agglutination was rated by scale 0±3:

0no clumps; 11±4clumps, each containing

ap-proximately 25 to 50 cells; 25±15clumps (ca. 50 to 100 cells) and 316±20clumps(ca. >100 cells).

The percent agglutination was determined by grow-ing the isolates to exponential phase in KB broth. Cells were washed and suspended in PBS O:D:0:9,

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banceÿabsorbance after agglutination/initial absor-bance 100. In control treatments PBS was added instead of CA. The agglutination of sclerotia by Agg+, Aggl and Aggÿisolates was tested by exposing the washed sclerotia (105 mlÿ1) to the suspension of Agg+, Aggl or Aggÿ for 30 min. Following exposure, sclerotia were washed gently with sterile PBS and ag-glutination observed under a phase contrast micro-scope. The % agglutination on sclerotia surface was calculated as: number of sclerotia agglutinated/total number of sclerotia per microscopic ®eld100. Agglu-tinating clumps ranged approximately between 4 to 20 containing not less than 10±15 cells. The agglutination rating on sclerotial surfaces was determined as described before.

2.5.14C loss from M. phaseolina sclerotia

The loss of C from labeled M. phaseolina sclerotia incubated separately in a cell suspension of Agg+, Aggl, Aggÿ or unsterilized soil was measured as respired 14CO2 and residual 14C (Arora et al., 1985). Nuclepore membrane ®lters (25 mm pore size; 25 mm diameter) containing labeled sclerotia (ca. 104 ®lterÿ1) were ¯oated on sterile stainless steel planchets contain-ing 5 ml suspension each of Agg+ or Aggl or Aggÿ (108 cells mlÿ1) or buried in 5 g of unsterilized soil. Soil moisture was maintained at ÿ5 kPa by adding pre-determined amounts of water to each plate every 48 h. Planchets (six replicates for each treatments) were placed in airtight glass chambers (40 mm diam-eter45 mm deep; 1 planchet chamberÿ1), connected with a CO2-free moist air (50 ml minÿ1) source and an exit tube for collecting 14CO2. During 1±60 d of incu-bation, the evolved 14CO2 was collected in 15 ml of 15% ethanolamine scintillation cocktail (ethanolamine, 150 ml; ethylene glycol, 70 ml and basic scintillation cocktail, 780 ml). Basic scintillation cocktail contained 5 g PPO (2,5 diphenyl oxazole; Sigma), 50 mg POPOP (1,4 bis-5-phenyl oxazolyl benzol; Sigma) in 250 ml methanol and 50 ml toluene. Scintillation vials con-taining ethanolamine cocktail were replaced every 6 h for 60 d.

The residual14C loss was assessed by measuring the radioactivity by oxidizing the buer containing Agg+ or Aggl or Aggÿ or soil in biological oxidizer (Arora et al., 1983; Hyakumachi et al., 1989). 14C loss from labeled sclerotia represents the sum of 14C respired and that remaining in the buer containing bacteria or soil. Total 14C in the sclerotia before incubation, was estimated by summing the 14C loss and that remaining in the sclerotia at the end of the experiment (Arora, 1988).

2.6. Agglutination response of P. ¯uorescens isolates on stressed sclerotia

Out of the 6 replicates for the 14C experiment, scler-otia from 2 replicates per treatment were used to assess the agglutination potential of Agg+ or Aggl cells (Table 2). Crude agglutinin from sclerotia, stressed for 60 d in the presence of Agg+, Aggl or Aggÿ, were obtained by incubating washed sclerotia in 100 ml of SM (ca. 1 sclerotium mlÿ1) for 15 d. Agglutination of Agg+ and Aggl cells on the sclerotial surfaces or in CA produced from stressed sclerotia was assayed as described before. Agglutination in CA or on the sur-face of sclerotia (30-d-old) served as control.

2.7. Eect on germination

Out of the remaining four replicates used for the14C experiment, sclerotia from one replicate were again used for a germination assay. Membrane ®lters con-taining labeled sclerotia were removed from each treat-ment at 1, 5, 10, 20, 30, 45 and 60 d incubation with Agg+, Aggl, Aggÿ strains or in unsterilized soil, washed by centrifugation and again deposited on membrane ®lter (ca. 500 sclerotia ®lterÿ1; 25 mm pore size; 25 mm diameter) and incubated on Pfeer's salt solution (PSS; pH 6; without C source) or potato-dex-trose broth (PDB; pH 6; with C source) at 28±308C

Table 2

Percent agglutination of Agg+_{and Agg}l_{isolates in crude agglutinin}

and Macrophomina phaseolina sclerotial surface previously stressed

with Agg+_{, Agg}l_{or Agg}ÿ_{isolates or in unsterilized soil}a

Sclerotia incubated withc _{Days of incubation}

controlb 60

A B A B

Agg+

Crude agglutinin 7622.5 1221.2 7222.2 1022.1 Sclerotia surface 5624.1 1122.0 5221.0 920.9

Aggl

Aggÿ

Crude agglutinin 7522.0 1221.4 7022.8 1021.3 Sclerotia surface 5824.2 922.3 5122.0 821.2 Soil

a

AAgg_, _B_Aggl_{; stressed sclerotia were washed in PBS and}

used for CA production (see Section 2).

b

Agglutination in CA or on the surface of sclerotia (30-d-old) served as control. Data are mean of 10 replicates2S.D.

c

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for 24 h. After incubation, sclerotia were stained with phenolic rose Bengal and germination was examined under a phase contrast microscope using incident light. The germination of sclerotia, harvested from 30-d-old cultures, in PBS or in sterilized soil (ÿ5 kPa; 24 h) served as control.

2.8. Colonization of P. ¯uorescens isolates on non-agglutinated sclerotia

The colonizing ability of Agg+, Aggl, and Aggÿ strains of P. ¯uorescens on sclerotia was evaluated in soils separately infested with Agg+, Aggl, Aggÿ, Agg++Aggl, Agg++Aggÿ, Aggl+Aggÿ and Agg++Aggl+Aggÿ cells (ca. 8 log CFU gÿ1 soil). Infested soil was equilibrated for 2 d at ÿ5 kPa and placed in small Petri plates (15 g plateÿ1). The sclerotia (30-d-old) were placed in a Millipore membrane ®lter pouch (105 sclerotia ®lterÿ1; 25 mm pore size; 25 mm diameter) and carefully buried in soil with moisture maintained at ÿ5 kPa by adding pre-determined amounts of water at every 48 h. After 60 d of incu-bation, ®lters were removed and sclerotia were brushed o into a glass tube containing 5 ml of PBS. The hom-ogenized sclerotial suspension was plated on KB agar containing: rifampicin and streptomycin (200 mg mlÿ1 each) or tetracycline (200 mg mlÿ1) or rifampicin+ka-namycin (200 mg mlÿ1 each) to determine the coloniz-ing population of Agg+, Aggl or Aggÿ, respectively. The colony number was scored after 24 and 72 h.

2.9. Colonization of P. ¯uorescens isolates on pre-agglutinated sclerotia

The colonization eciency of P. ¯uorescens isolates was evaluated on sclerotia pre-agglutinated separately with cells of Agg+, Aggl or Aggÿ. Sclerotia (30-d-old) were subjected to agglutination with the P. ¯uorescens

isolates by the method described earlier. Agglutinated sclerotia (105®lterÿ1) were placed in a membrane ®lter pouch (25 mm pore size; 25 mm dia.) and buried in soils infested with dierent combinations of Agg+, Aggl and Aggÿ (ca. 8 log CFU gÿ1 soil) or in non-infested unsterilized soil and incubated for 60 d (Table 3). The other conditions for incubation of pre-agglutinated sclerotia in infested or non-infested unsterilized soils were the same as described before. Colonization on sclerotial surface was determined on media amended with appropriate antibiotics.

All experiments were repeated more than twice to establish reproducibility, and data on the aggluti-nation, C-loss, germination and colonization were

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jected to standard deviation. The eect of C loss and germination was also subjected to regression analysis.

3. Results

3.1. Agglutination potential of P. ¯uorescens isolates

Dierent isolates of P. ¯uorescens varied with respect to the degree of agglutination to CA and also on sclerotia of M. phaseolina (Table 1). The aggluti-nation eciencies of dierent P. ¯uorescens isolates in CA and on sclerotia ranged from 12 to 73% and 10 to 57%, respectively (Table 1). Isolates 12, 15, 17, 19, 29, 51, 99 and 149 showed more than 40% agglutination in CA. Cells of Agg+ (isolate 12) showed maximum agglutination (73%) in CA and on sclerotial surface (57%) (Table 1), whereas agglutination potential of Aggl (isolate 30) ranged from 10 to 12%. In general, agglutination on hyphal surfaces was greater than on sclerotial surfaces (results not shown).

3.2. Loss of C from sclerotia

Sclerotia incubated with Agg+, Aggl, Aggÿ or in soil lost signi®cant amounts (% of total label) of en-dogenous C (Fig. 1). A rapid increase in 14CO2 evol-ution was observed when sclerotia were incubated with Agg+, Aggl, Aggÿor in soil for up to 5 d, followed by a gradual decline until 7±8 d or a very low and steady rate up to 60 d. For example, sclerotia incubated with Agg+ released 2.9 and 6.5% 14CO2 at d 1 and 5, re-spectively, and thereafter14CO2evolution ranged from 0.2±0.6% (Fig. 1A). Though the rate of 14CO2 evol-ution was less when sclerotia were incubated with Aggl, Aggÿ or in soil, the trend of 14CO2 evolution was more or less similar to that observed with Agg+. The average daily rate of 14CO2 evolution was in the order: Agg+> Aggl> Aggÿ> soil. The cumulative evolution of 14CO2 from sclerotia incubated with Agg+, Aggl and Aggÿ up to 60 d ranged from 37 to 40% which was 1.5-fold greater than 14CO2 evolved from sclerotia incubated in soil (Fig. 1B).

Residual 14C (exudate) released from sclerotia in buer containing Agg+, Aggl or Aggÿ cells or in soil was maximum at d 1, declined rapidly until d 20 and

Fig. 1. Loss of14_{C-labeled compounds from sclerotia of}_{Macrophomina phaseolina}_{incubated with Agg}+₍_*_{), Agg}l₍_Q_{), Agg}ÿ₍_R_{) or in soil (}_T₎

for 1±60 d; (A) daily14_CO

2 evolution, (B) cumulative14CO2 evolution, (C) daily residual14C-loss, (D) cumulative residual14C loss, (E) total

daily14_{C loss (daily}14_{C-loss+daily residual} 14_{C loss) and (F) total cumulative}14_{C loss (cumulative}14_CO

2+residual 14C). Each point is the

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remained almost constant for all the treatments until the end of the experiments (Fig. 1C). The residual14C loss was signi®cantly less in proportion to daily14CO2 evolution. For example, daily cumulative 14CO2 evol-ution from sclerotia incubated with Agg+, Aggl, Aggÿ and in soil up to 60 d was 40, 37.1, 37 and 27.5%, re-spectively, and these values were approximately 7- to 8-fold greater than the residual daily cumulative 14C loss (Fig. 1B and D). Total cumulative 14C (14CO2 plus residual 14C) loss in dierent treatments did not dier signi®cantly as the total 14C loss was 48, 45 and 44.4 % in Agg+, Aggl and Aggÿ treatments, respect-ively (Fig. 1F). Daily total 14C loss was maximum (7%) until d 4, declined until d 10 (0.3±0.47 %), fol-lowed by a steady rate of C loss until d 60 (0.1±0.12 %) (Fig. 1E). Total C loss for all the treatments increased with incubation time. For instance, 2.1± 29.6% of total C was lost during 1±5 d, raised to 14.5±41.1% from 6±20 d or to 28±48 % from 21±60 d; maximum loss occurring within 4±10 d (23.7±38.8%) (Fig. 1F). In general, maximum total C loss from the sclerotia was caused by Agg+, followed by Aggl, Aggÿ and natural soil.

3.3. Agglutination response of P. ¯uorescens isolates on stressed sclerotia

The agglutination of Agg+ and Aggl cells on stressed sclerotia or in CA, produced from the sclero-tia previously incubated with Agg+, Aggl and Aggÿ, did not dier signi®cantly with the agglutination on the surface of culture harvested sclerotia or in CA (Table 2). For instance, agglutination of Agg+ in CA produced from sclerotia previously stressed with Agg+ cells, showed 72%, whereas on stressed sclerotia

sur-face it was 52%. A similar trend in the agglutination potential of Agg+ cells was also observed when sclero-tia were stressed with Aggl, Aggÿ cells or in soil (Table 2).

3.4. Germination

Germination of sclerotia, which had been incubated with Agg+, Aggl or Aggÿ for 1±60 d was reduced on both PSS (without C source; data not shown) and PDB (with C source). For instance, germination of sclerotia, previously incubated with Agg+ for 1, 5, 10, 20, 30, 45 and 60 d was 86, 78, 63, 45, 30, 28 and 20% on PDB (Fig. 2). A similar trend of inhibition was also noticed with Aggl and Aggÿ. Loss of C from the scler-otia incubated with Agg+ Aggl, Aggÿ or in unsteri-lized soil was signi®cantly r ÿ0:89to ÿ0:96;

P0:05correlated with germination repression.

3.5. Colonization

In comparison to Aggl and Aggÿ, greater coloniza-tion by Agg+ on the sclerotia of M. phaseolina was observed for all the treatments (Table 3). For instance, colonization of Agg+ on sclerotia in soil infested with a mixed population of Agg++Aggl+Aggÿ was 2.7-and 3-fold higher than colonization of Aggland Aggÿ, respectively. Similarly, sclerotia pre-agglutinated with Agg+ cells also exhibited greater colonization in infested soils compared to Aggl or Aggÿ isolates (Table 3). In general, the colonizing population of Aggl or Aggÿon pre-agglutinated sclerotia was higher than on non-agglutinated sclerotia (Table 3). Aggluti-nation of Agg+ in CA produced from culture har-vested sclerotia and the sclerotia pre-colonized with Agg+ cells was 58 and 55%, respectively, thus amounting to no signi®cant dierence (data not shown).

4. Discussion

Considerable research has been done demonstrating the role of cell surface agglutinin in recognition in `root-bacteria' (Anderson et al., 1988; Glandorf et al., 1994), `fungal host and fungal parasite' (Elad and Chet, 1983; Benyagoub et al., 1996; Inbar and Chet, 1997) and `nematode-fungi' (Tunlid et al., 1992). How-ever, virtually nothing is known about the role of cell surface recognition in `fungal and bacterial' inter-actions. We have shown that soil contains a large number of naturally-occurring parasites of M. phaseo-lina sclerotia (Srivastava et al., 1996a) and the physio-logical and growth conditions of M. phaseolina also greatly in¯uenced its agglutinin production (Srivastava et al., 1996b). However, no eort was made to

evalu-Fig. 2. Germination ofMacrophomina phaseolinasclerotia in potato dextrose broth. Sclerotia were previously incubated with Agg+₍_*_),

Aggl ₍_Q_), _Aggÿ ₍_R₎ _or _in _soil ₍_T₎ _for _1±60 _d;

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ate the agglutination potential of naturally-occurring

P. ¯uorescens isolates and their signi®cance in M.

pha-seolina±P. ¯uorescens antagonistic interactions. We

have demonstrated the diversity ofP. ¯uorescens popu-lations in soil with particular reference to their e-ciency to agglutinate and colonize M. phaseolina

sclerotia (Tables 1 and 3). Though Agg+, Aggl and Aggÿ isolates diered greatly in their ability to react with the sclerotial surface and in agglutinin ofM.

pha-seolina (Table 1), apparently no relationship was

observed between the agglutination potential of P.

¯uorescens isolates and their eciency to impose

energy-stress on sclerotia. For example, depletion of total endogenous C reserves from sclerotia by Agg+, Aggl and Aggÿranged from 45 to 48%. This indicates a general non-speci®city of Agg+, Aggl or Aggÿ iso-lates to impose energy stress on sclerotia through excessive loss of endogenous C. This also suggests involvement of a common mechanism presumably via the establishment of a `nutrient sink' (Lockwood, 1992). The dierent amount of C loss by Agg+, Aggl and Aggÿ can be attributed to dierence in sink e-ciencies of these isolates (Hyakumachi and Arora, 1998). Arora (1988) demonstrated that C loss from conidia of Bipolaris sorokiniana was signi®cantly greater in unsterilized soil than soil infested with a soil isolate ofP. ¯uorescens. However, in our study greater C loss from M. phaseolina sclerotia was recorded in the soil infested with P. ¯uorescens isolates than non-infested unsterilized soil (Fig. 1). This variation in result could be due to dierences in sink eciencies of dierent soils or to dierent strains or isolates of P.

¯uorescens. Filonow and Lockwood (1983) reported

that relative strength and the eciency of microbial nutrient sink to impose energy stress on fungal propa-gules also depends upon the nature and properties of dierent soils. Besides these, size, age and physiologi-cal state of fungal propagules could also in¯uence sink eciencies of soils or speci®c microorganisms (Lock-wood, 1990).

Signi®cant germination repression of sclerotia, that had been incubated with Agg+ or Agglor Aggÿ cells, was observed in PDB (with C source). A direct negative correlation was recorded between loss of C and germination repression in PDB r ÿ0:86to ÿ0:96;P0:05). Stressed sclerotia were

able to recover a greater part of their germinability with increased incubation time, i.e. for 7 d in the pre-sence of C source (PDB) (T.K.J., 1998, unpublished Ph.D. thesis, Banaras Hindu University), suggests that germination was possibly delayed due to elevation of nutrient requirement of stressed sclerotia. Gupta et al. (1995) demonstrated that sclerotia of R. solani incu-bated with aP. ¯uorescens isolate retained their viabi-lity even after a substantial loss of endogenous C. There are other reports that fungal propagules, even

after prolonged exposure to stress conditions in soil, were able to retain their C reserves and also their via-bility and biological competence (Hyakumachi and Arora, 1998).

Previous studies elucidated the role of surface bind-ing properties of ¯uorescent pseudomonads in coloni-zation of roots and disease suppression (Bull et al., 1991; Buell et al., 1993). However, no work has been done to evaluate the roles of agglutination eciency of antagonistic bacteria in colonization of pathogenic fungal propagules. In our study, though Agg+, Aggl and Aggÿisolates were able to colonize the sclerotia in soil, a signi®cantly greater colonization was recorded only with the Agg+isolate compared to Agglor Aggÿ (Table 3). Greater colonization of Agg+ pre-aggluti-nated sclerotia was also observed as compared to Aggl or Aggÿ in infested soil (Table 3). Therefore, the potentiality of agglutinable bacterial isolates could be viewed as an important characteristic in colonizing the fungal propagules in soil. Other studies also suggested that the agglutination interaction is important for securing the initial attachment of P. putida cells to bean roots and thereafter the colonization process could be dependent on other factors operating around the root system (Anderson et al., 1988; Tari and Anderson, 1988). In contrast, Glandorf et al. (1994) reported that root agglutinins can only be involved in the short-term adherence of ¯uorescent pseudomonads but do not play a decisive role in root colonization. The importance of adhesion of fungal spores to host surface and its in¯uence on disease initiation has also been investigated in detail suggesting that spore attach-ment is required for a compatible host±pathogen inter-action (Mercure et al., 1994; Kuo and Hoch, 1996).

In conclusion, our ®ndings demonstrate that soils contain a large number of Agg+, Aggl and Aggÿ P.

¯uorescens strains. The ability to impose energy stress

on sclerotia by these isolates of P. ¯uorescensis not re-lated to the agglutination potential. Agglutinable iso-lates play a signi®cant role in colonization of M.

phaseolina sclerotia and could be important for

bio-logical control. Further investigation is needed to understand the role of agglutination in colonization of sclerotia by Agg+, Aggl or Aggÿ of P. ¯uorescens in dierent ecological niches and competitive soil en-vironments.

Acknowledgements

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