Agglutination potential of Pseudomonas fluorescens in relation to energy stress and colonization of Macrophomina phaseolina

Soil Biology & Biochemistry 32 (2000) 511±519

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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 di€er signi®cantly …P ˆ 0:05† from sclerotia incubated with Agg+, Aggl or Aggÿ (a non-agglutinable Tn5 mutant of wild type 12). Most of the C lost from stressed sclerotia was evolved as 14CO2 (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, Aggÿ cells 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 of P. ¯uorescens was directly correlated with germination repression …r ˆ ÿ0:89to ÿ 0:96; P ˆ 0:05). Greater colonization of sclerotia by Agg+ was observed compared to Aggl or Aggÿ isolates. Our ®ndings clearly demonstrate the existence of a great diversity of P. ¯uorescens isolates in natural soils in respect to their agglutination potential on M. phaseolina sclerotia. 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. 7 2000 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; Hyakumachi and Arora, 1998) and their germination is regulated 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 * Corresponding author. Fax: +91-542-317-074/313-965. E-mail address: [email protected] (D.K. Arora).

and Lockwood, 1989; Mondal et al., 1996; Mondal and Hyakumachi, 1998). The stress imposed on fungal propagules by di€erent 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 surfaces and di€erent 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

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 8 0 - 7

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microorganisms to a fungal host or pathogen surface or to one another, is a feature of antagonistic interactions (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, agglutination of antagonists on a fungal host appears to be one of the important phenomena as it also enables retention of antagonists on pathogenic fungal propagules (Inbar and Chet, 1997). Though antagonistic potential 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 (Srivastava et al., 1996a) has already been established, no detailed study has been done to elucidate the agglutination between fungal pathogens and bacterial antagonists, i.e. fungal±bacterial systems in general and M. phaseolina and Pseudomonas ¯uorescens in particular. We have demonstrated that soil contains a large number of parasites of M. phaseolina sclerotia which potentially reduce the host population in soil (Srivastava et al., 1996a). The e€ects of nutritional and growth factors on the production of M. phaseolina agglutinin and its response towards agglutination of P. ¯uorescens have been evaluated (Srivastava et al., 1996b). However, the potential of agglutinable (Agg+), less agglutinable (Aggl) or non-agglutinable (Aggÿ) isolates of P. ¯uorescens to impose competitive energy stress on fungal propagules and its subsequent e€ect on viability and colonization has not been investigated. Our aim was (i) to isolate P. ¯uorescens strains from chickpea rhizosphere and to evaluate their agglutination potential on the sclerotia surface and agglutinin produced by M. phaseolina, (ii) to assess the ability of Agg+, Aggl and Aggÿ Tn5 mutant generated from Agg+ wild type, to impose energy stress in M. phaseolina sclerotia and its subsequent e€ect on their germinability, (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 sclerotia by Agg+, Aggl or Aggÿ isolates. 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. phaseolina (Tassi) Goid. were obtained from the Applied

Mycology Culture Collection, Banaras Hindu University (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; 1Ci ˆ 37GBq; Bhaba Atomic Research Centre, Bombay, India). Strains of P. ¯uorescens were isolated from rhizosphere soils of 10 di€erent chickpea ®elds. Diluted soil samples (10ÿ7 to 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 morphological and biochemical tests used for identi®cation were reaction pro®les on API test strips (API Laboratory Products, Canada). In brief, all isolates were examined for oxidase reaction, Gram's reaction, motility 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-inositol, benzylamine and 2,3-butanediol (Molin and Ternstrom, 1982). From 1470 isolates, 172 were selected as Table 1 Percent agglutination of di€erent isolates of Pseudomonas ¯uorescens to crude agglutinin and on the surface of washed sclerotia of Macrophomina phaseolina Isolate No.

12 17 15 19 149 99 29 51 147 75 98 18 111 22 1 123 6 30 Tn5 Aggÿ isolate 12 a

Crude agglutinin

Sclerotia

agglutination (%)a

ratingb

agglutination (%)

rating

7322.0 5321.8 5121.8 4923.1 4922.3 48.24.3 4423.2 4121.5 3022.4 2822.0 2621.4 2621.5 2521.3 2222.1 2021.4 1822.3 1421.4 1221.8 0

3 2 2 2 2 2 2 2 2 2 1 1 1 2 1 1 1 1 0

5722.7 3921.4 3823.4 3623.5 3023.0 3523.9 4021.8 3222.1 1921.3 1522.0 1521.4 1422.1 1421.8 1721.7 1321.9 1722.4 1121.8 1022.0 0

3 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 0

Data are means of 10 replicates2S.D. Mean of 100 counts; agglutination rating was calculated as described in Section 2. 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 of M. 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 medium 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 colonies 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 agglutination, growth rate and ¯uorescence under UV radiation. 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 following 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 plasmid and rifampicin resistant Agg+ isolate 12 (recipient) 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 transconjugates, 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 e€ective killing rate of 83 to 89% was obtained. Tn5 mutagenesis resulted in 1360 transconjugates. All

513

transconjugates were screened for agglutination ability in CA of M. 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 subcultures. 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 nutritional 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; MgSO4  7H2O, 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 ®ltrate (CF) was collected by vacuum ®ltration, centrifuged (5000  g for 5 min; 48C) and dialyzed for 48 h against 4  2000 ml of phosphate bu€er saline (PBS; pH 7) at 48C using dialysis membranes with a molecular mass cut o€ of 10±12 kDa (Sigma). The dialyzed CF was lyophilized and stored at ÿ208C. Prior to agglutination assay the lyophilized CF was resuspended in PBS to obtain a ®nal protein concentration of 2 mg mlÿ1 and served as crude agglutinin (CA). 2.4. Agglutination assay Isolates of P. ¯uorescens and mutant Tn5 Aggÿ isolate 12 (Table 1) were grown to exponential phase (A550; 0.15±0.18) and washed twice with PBS (pH 7) to yield 105 cells mlÿ1. To test the agglutination of Agg+, Aggl or Aggÿ cells, 5 ml 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: 0 ˆ no clumps; 1 ˆ 1±4 clumps, each containing approximately 25 to 50 cells; 2 ˆ 5±15 clumps (ca. 50 to 100 cells) and 3 ˆ 16±20 clumps (ca. >100 cells). The percent agglutination was determined by growing the isolates to exponential phase in KB broth. Cells were washed and suspended in PBS …O:D: ˆ 0:9, A550). A cell suspension (2 ml) was mixed with an equal volume of CA and vortexed for 1 min. The absorbance (0.6±0.62, A550) of the mixed suspension was recorded and the tubes were kept for 30 min to permit agglutination. The absorbance was recorded and % agglutination was calculated as: initial absor-

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banceÿabsorbance after agglutination/initial absorbance  100. In control treatments PBS was added instead of CA. The agglutination of sclerotia by Agg+, Aggl and Aggÿ isolates 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 agglutination observed under a phase contrast microscope. The % agglutination on sclerotia surface was calculated as: number of sclerotia agglutinated/total number of sclerotia per microscopic ®eld  100. Agglutinating 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.

14

C 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 containing 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 diameter  45 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 incubation, 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 containing ethanolamine cocktail were replaced every 6 h for 60 d. The residual 14C loss was assessed by measuring the radioactivity by oxidizing the bu€er 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 bu€er 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, sclerotia 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 surface of sclerotia (30-d-old) served as control. 2.7. E€ect on germination Out of the remaining four replicates used for the 14C experiment, sclerotia from one replicate were again used for a germination assay. Membrane ®lters containing labeled sclerotia were removed from each treatment 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 Pfe€er's salt solution (PSS; pH 6; without C source) or potato-dextrose broth (PDB; pH 6; with C source) at 28±308C

Table 2 Percent agglutination of Agg+ and Aggl isolates in crude agglutinin and Macrophomina phaseolina sclerotial surface previously stressed with Agg+, Aggl or Aggÿ isolates or in unsterilized soila Sclerotia incubated withc

Days of incubation controlb

Agg+ Crude agglutinin Sclerotia surface Aggl Crude agglutinin Sclerotia surface Aggÿ Crude agglutinin Sclerotia surface Soil Crude agglutinin Sclerotia surface

60

A

B

A

B

7622.5 5624.1

1221.2 1122.0

722 2.2 5221.0

1022.1 920.9

7523.5 5323.0

1221.7 1022.1

7022.1 4822.9

1021.6 921.5

7522.0 5824.2

1221.4 922.3

7022.8 5122.0

1021.3 821.2

7423.1 5522.0

1221.8 1021.6

6022.5 4823.3

1021.9 921.7

a A ˆ Agg‡ , B ˆ 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 Sclerotia incubated for 60 d in bu€er containing Agg+, Aggl, Aggÿ cells or in non-infested soil.

0 1.920.3 0 1.720.3 0 1.820.3 1.420.2 0

0.620.7 0.520.2 2.820.4 0.520.2 1.620.2 2.420.2 1.020.2 0.420.1

2.8. Colonization of P. ¯uorescens isolates on nonagglutinated sclerotia

0.320.1 2.720.3 0.520.2 1.820.2 0.420.1 2.120.3 1.320.3 0.220.1 a

b

A, B, and C=population of Agg+, Agg1, and Aggÿ, respectively. Data are means of 10 replicates2S.D. Sclerotia harvested from 30-d-old cultures. c Sclerotia had been pre-agglutinated with Agg+, Agg1 or Aggÿ, and buried in infested or in non-infested unsterilized soil. d Colonization of sclerotia (log CFU per 100 sclerotia) after 60 d of incubation in di€erent treatments. e Unsterilized non-infested soil.

3.823 0 0 3.020.3 2.620.2 0 2.820.2 0 0 0 2.320.3 0 1.620.3 1.920.3 1.220.2 0 0 1.920.3 0 1.620.4 0 1.820.2 1.320.3 0 3.820.2 1.220.2 0.920.30 2.320.4 2.620.3 0.720.2 2.020.4 0.520.3 0 0 3.120.3 0 1.320.2 1.620.1 0.920.1 0 0 2.020.1 0 1.720.2 0 1.920.3 1.020.2 0 4.020.3d 0 0 3.020. 3.020.7 0 2.720.6 0 Agg+ Agg1 Aggÿ Agg++Agg1 Agg++Aggÿ Agg1+Aggÿ Agg++Agg1+Aggÿ Soile

515

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.

0 0 3.020.3 0 2.020.3 2.520.3 1.520.2 0

3.320.2 0 0 2.920.2 2.520.2 0 2.720.2 0

C B A A B

C A

B

C

Aggÿ Agg1 Agg+ C B A

Sclerotia pre-agglutinated withc Culture harvested non-agglutinated sclerotiab Sclerotia incubated in soil infested with

Table 3 Colonization of Agg+, Agg1 and Aggÿ strains of Pseudomonas ¯uorescens on sclerotia of Macrophomina phaseolina which had been non-agglutinated or pre-agglutinated with these strains and then buried in soil infested with single or di€erent combinations of Agg+, Agg1, Aggÿ or in non-infested unsterilized chickpea ®eld soila

T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519

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 + l ÿ Agg +Agg +Agg 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 incubation, ®lters were removed and sclerotia were brushed o€ into a glass tube containing 5 ml of PBS. The homogenized sclerotial suspension was plated on KB agar containing: rifampicin and streptomycin (200 mg mlÿ1 each) or tetracycline (200 mg mlÿ1) or rifampicin+kanamycin (200 mg mlÿ1 each) to determine the colonizing 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 preagglutinated sclerotia The colonization eciency 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 di€erent combinations of Agg+, Aggl and Aggÿ (ca. 8 log CFU gÿ1 soil) or in noninfested unsterilized soil and incubated for 60 d (Table 3). The other conditions for incubation of preagglutinated 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 agglutination, C-loss, germination and colonization were sub-

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T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519

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

3. Results

3.1. Agglutination potential of P. ¯uorescens isolates Di€erent isolates of P. ¯uorescens varied with respect to the degree of agglutination to CA and also on sclerotia of M. phaseolina (Table 1). The agglutination eciencies of di€erent 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 endogenous C (Fig. 1). A rapid increase in 14CO2 evolution was observed when sclerotia were incubated with Agg+, Aggl, Aggÿ or 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, respectively, and thereafter 14CO2 evolution ranged from 0.2±0.6% (Fig. 1A). Though the rate of 14CO2 evolution 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 bu€er containing Agg+, Aggl or Aggÿ cells or in soil was maximum at d 1, declined rapidly until d 20 and

Fig. 1. Loss of 14C-labeled compounds from sclerotia of Macrophomina phaseolina incubated with Agg+ (*), Aggl (Q), Aggÿ (R) or in soil (T) for 1±60 d; (A) daily 14CO2 evolution, (B) cumulative 14CO2 evolution, (C) daily residual 14C-loss, (D) cumulative residual 14C loss, (E) total daily 14C loss (daily 14C-loss+daily residual 14C loss) and (F) total cumulative 14C loss (cumulative 14CO2+residual 14C). Each point is the mean of 3 replicates.

T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519

remained almost constant for all the treatments until the end of the experiments (Fig. 1C). The residual 14C loss was signi®cantly less in proportion to daily 14CO2 evolution. For example, daily cumulative 14CO2 evolution from sclerotia incubated with Agg+, Aggl, Aggÿ and in soil up to 60 d was 40, 37.1, 37 and 27.5%, respectively, 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 di€erent treatments did not di€er signi®cantly as the total 14C loss was 48, 45 and 44.4 % in Agg+, Aggl and Aggÿ treatments, respectively (Fig. 1F). Daily total 14C loss was maximum (7%) until d 4, declined until d 10 (0.3±0.47 %), followed 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 sclerotia previously incubated with Agg+, Aggl and Aggÿ, did not di€er 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-

517

face it was 52%. A similar trend in the agglutination potential of Agg+ cells was also observed when sclerotia 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 sclerotia incubated with Agg+ Aggl, Aggÿ or in unsterilized soil was signi®cantly …r ˆ ÿ0:89 to ÿ 0:96; P ˆ 0:05† correlated with germination repression. 3.5. Colonization In comparison to Aggl and Aggÿ, greater colonization 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 2.7and 3-fold higher than colonization of Aggl and 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 Aggÿ on pre-agglutinated sclerotia was higher than on non-agglutinated sclerotia (Table 3). Agglutination of Agg+ in CA produced from culture harvested sclerotia and the sclerotia pre-colonized with Agg+ cells was 58 and 55%, respectively, thus amounting to no signi®cant di€erence (data not shown). 4. Discussion

Fig. 2. Germination of Macrophomina phaseolina sclerotia in potato dextrose broth. Sclerotia were previously incubated with Agg+ (*), Aggl (Q), Aggÿ (R) or in soil (T) for 1±60 d; …W† ˆ germination of sclerotia (30-d-old) in bu€er served as control. Values are means of 10 replicates.

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). However, virtually nothing is known about the role of cell surface recognition in `fungal and bacterial' interactions. We have shown that soil contains a large number of naturally-occurring parasites of M. phaseolina sclerotia (Srivastava et al., 1996a) and the physiological and growth conditions of M. phaseolina also greatly in¯uenced its agglutinin production (Srivastava et al., 1996b). However, no e€ort was made to evalu-

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ate the agglutination potential of naturally-occurring P. ¯uorescens isolates and their signi®cance in M. phaseolina±P. ¯uorescens antagonistic interactions. We have demonstrated the diversity of P. ¯uorescens populations 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 di€ered greatly in their ability to react with the sclerotial surface and in agglutinin of M. phaseolina (Table 1), apparently no relationship was observed between the agglutination potential of P. ¯uorescens isolates and their eciency to impose energy-stress on sclerotia. For example, depletion of total endogenous C reserves from sclerotia by Agg+, Aggl and Aggÿ ranged from 45 to 48%. This indicates a general non-speci®city of Agg+, Aggl or Aggÿ isolates 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 di€erent amount of C loss by Agg+, Aggl and Aggÿ can be attributed to di€erence 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 of P. ¯uorescens. However, in our study greater C loss from M. phaseolina sclerotia was recorded in the soil infested with P. ¯uorescens isolates than noninfested unsterilized soil (Fig. 1). This variation in result could be due to di€erences in sink eciencies of di€erent soils or to di€erent strains or isolates of P. ¯uorescens. Filonow and Lockwood (1983) reported that relative strength and the eciency of microbial nutrient sink to impose energy stress on fungal propagules also depends upon the nature and properties of di€erent soils. Besides these, size, age and physiological state of fungal propagules could also in¯uence sink eciencies of soils or speci®c microorganisms (Lockwood, 1990). Signi®cant germination repression of sclerotia, that had been incubated with Agg+ or Aggl or 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 ˆ ÿ0:86 to ÿ 0:96; P ˆ 0:05). Stressed sclerotia were able to recover a greater part of their germinability with increased incubation time, i.e. for 7 d in the presence 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 incubated with a P. ¯uorescens isolate retained their viability 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 viability and biological competence (Hyakumachi and Arora, 1998). Previous studies elucidated the role of surface binding properties of ¯uorescent pseudomonads in colonization 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 eciency of antagonistic bacteria in colonization of pathogenic fungal propagules. In our study, though Agg+, Aggl and Aggÿ isolates were able to colonize the sclerotia in soil, a signi®cantly greater colonization was recorded only with the Agg+ isolate compared to Aggl or Aggÿ (Table 3). Greater colonization of Agg+ pre-agglutinated 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 attachment is required for a compatible host±pathogen interaction (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. ¯uorescens is not related to the agglutination potential. Agglutinable isolates play a signi®cant role in colonization of M. phaseolina sclerotia and could be important for biological control. Further investigation is needed to understand the role of agglutination in colonization of sclerotia by Agg+, Aggl or Aggÿ of P. ¯uorescens in di€erent ecological niches and competitive soil environments.

Acknowledgements One of the authors (T.K.J.) thanks the University Grants Commission, New Delhi for ®nancial assistance.

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