Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue4.2000:
Soil Biology & Biochemistry 32 (2000) 511±519
www.elsevier.com/locate/soilbio
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 P 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 ÿ0:89to ÿ 0:96; P 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: dkarora@banaras.ernet.in (D.K. Arora).
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 surfaces 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
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
512
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 eects 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 eect 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 eect 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 37GBq; Bhaba Atomic Research
Centre, Bombay, India). Strains of P. ¯uorescens were
isolated from rhizosphere soils of 10 dierent 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 dierent 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
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 (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: 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-
514
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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.
2.6. 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. Eect on germination
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 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).
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 Pfeer'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 Agg , 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
Sclerotia incubated for 60 d in buer 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 dierent 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 dierent 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 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 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-
516
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 agglutination 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 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
buer 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 dierent treatments did not
dier 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 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-
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 ÿ0:89 to ÿ 0:96;
P 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 dierence (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 germination of sclerotia (30-d-old) in buer 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 eort was made to evalu-
518
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 eciency 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 of M. phaseolina (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 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 dierent amount of C loss by Agg+, Aggl
and Aggÿ can be attributed to dierence in sink eciencies 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 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 propagules also depends upon the nature and properties of
dierent soils. Besides these, size, age and physiological state of fungal propagules could also in¯uence sink
eciencies 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 ÿ0:86 to ÿ 0:96; P 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 eciency 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
dierent ecological niches and competitive soil environments.
Acknowledgements
One of the authors (T.K.J.) thanks the University
Grants Commission, New Delhi for ®nancial assistance.
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
References
Anderson, A.J., Tari, P.H., Tepper, C.S., 1988. Molecular studies on
the role of a root surface agglutinin in adherence and colonization by Pseudomonas putida. Applied and Environmental
Microbiology 54, 375±380.
Arora, D.K., Filonow, A.B., Lockwood, J.L., 1983. Exudation from
14
C-labeled fungal propagules in the presence of speci®c microorganisms. Canadian Journal of Microbiology 29, 1487±1492.
Arora, D.K., Filonow, A.B., Lockwood, J.L., 1985. Decreased
aggressiveness of Bipolaris sorokiniana conidia in response to
nutrient stress. Physiological and Molecular Plant Pathology 26,
135±142.
Arora, D.K., 1988. Eect of microorganisms on aggressiveness of
Bipolaris sorokiniana. Canadian Journal of Botany 66, 242±246.
Benyagoub, M., Jabazi-Hare, S.H., Chamberland, H., Charest, P.M.,
1996. Cytochemical and immuno-cytochemical investigation of
the mycoparasite between Stachybotrys elegans and its host
Rhizoctonia solani (AG-3). Mycological Research 100, 79±86.
Bin, L., Knudsen, G.R., Eschen, D.J., 1991. In¯uence of an antagonistic strain of Pseudomonas ¯uorescens on growth and ability of
Trichoderma harzianum to colonize sclerotia of Sclerotinia sclerotiorum in soil. Phytopathology 81, 994±1000.
Buell, C.R., Whetton, R., Tari, P.H., Anderson, A.J., 1993.
Characterization of cell surface properties in agglutinable and
non-agglutinable mutants of Pseudomonas putida. Canadian
Journal of Microbiology 39, 787±794.
Bull, C.T., Weller, D.M., Thomashow, L.S., 1991. Relationship
between root colonization and suppression of Gaeumannomyces
graminis var. tritici by Pseudomonas ¯uorescens strain 2-79.
Phytopathology 81, 945±959.
Elad, Y., Chet, I., 1983. Possible role of lectins in mycoparasitism.
Journal of Bacteriology 154, 1431±1435.
Epstein, L., Lockwood, J.L., 1984. Eect of soil microbiota on germination of Bipolaris victoriae conidia. Transaction of the British
Mycological Society 82, 63±69.
Expert, J.M., Digat, G., 1995. Biocontrol of Sclerotinia wilt of sun¯ower by Pseudomonas ¯uorescens and Pseudomonas putida
strains. Canadian Journal of Microbiology 41, 685±691.
Filonow, A.B., Lockwood, J.L., 1983. Mycostasis in relation to the
microbial sinks of ®ve soils. Soil Biology & Biochemistry 15, 557±
565.
Glandorf, D.C.M., Sluis, I.V.D., Anderson, A.J., Bakker, P.A.H.M.,
Schippers, B., 1994. Agglutination, adherence and root colonization by ¯uorescent pseudomonds. Applied and Environmental
Microbiology 60, 1726±1733.
Gupta, S., Arora, D.K., Srivastava, A.K., 1995. Growth promotion
of tomato plants by rhizobacteria and imposition of energy stress
on Rhizoctonia solani. Soil Biology & Biochemistry 27, 1051±
1058.
Hyakumachi, M., Lockwood, J.L., 1989. Relation of carbon loss
from sclerotia of Sclerotium rolfsii during incubation on soil to
decreased
germinability
and
pathogenic
aggressiveness.
Phytopathology 79, 1059±1063.
Hyakumachi, M., Loer, H.J.M., Lockwood, J.L., 1989. Methods
for determination of carbon loss from fungal propagules incubated in soil. Soil Biology & Biochemistry 21, 567±571.
Hyakumachi, M., Arora, D.K., 1998. The response of fungal propagules to competitive energy stress in soil. In: Csermely, P. (Ed.),
Stress of Life from Molecules to Man. Annals of the New York
Academy of Sciences, New York, pp. 152±162.
Inbar, J., Chet, I., 1997. Lectins and biocontrol. Critical Reviews in
Biotechnology 17, 1±20.
King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for
519
demonstration of pyocyanin and ¯uorescein. Journal of
Laboratory Clinical Medicine 44, 301.
Kuo, K., Hoch, H.C., 1996. Germination of Phyllostcia ampelicida
pycnidiospores: prerequisite of adhesion to the substratum and
the relationship of substratum wettability. Fungal Genetics and
Biology 20, 18±29.
Lockwood, J.L., 1990. Relation of energy stress to behaviour of soilborne plant pathogens and to disease development. In: Hornby,
D. (Ed.), Biological Control of Soilborne Plant Pathogens. CAB
International, Oxon, pp. 127±214.
Lockwood, J.L., 1992. Exploitation competition. In: Carrol, G.C.,
Wicklow, D.T. (Eds.), The Fungal Community: its Organization
and Role in the Ecosystems. Marcel Dekker, New York, pp. 243±
264.
Manocha, M.S., 1991. Physiology and biochemistry of biotrophic
mycoparasitism. In: Arora, D.K., Rai, B., Mukerji, K.G.,
Knudsen, G.R. (Eds.), Handbook of Applied Mycology, vol. I.
Marcel Dekker, New York, pp. 273±300.
Manocha, M.S., Sahai, A.S., 1993. Mechanism of recognition of
necrotrophic and biotrophic mycoparasites. Canadian Journal of
Microbiology 39, 269±274.
Mercure, E.W., Kunow, H., Nicholson, R.L., 1994. Adhesion of
Colletotrichum graminicola conidia to corn leaves: a requirement
for disease development. Physiological and Molecular Plant
Pathology 45, 407±420.
Molin, G., Ternstrom, A., 1982. Numerical taxonomy of psychotrophic pseudomonads. Journal of General Microbiology 128,
1249±1264.
Mondal, S.N., Kageyama, K., Hyakumachi, M., 1996. Decreased
germinability and virulence of oospores of Pythium aphanidermatum in relation to loss of endogenous carbon during incubation
on soil. Soil Biology & Biochemistry 28, 545±553.
Mondal, S.N., Hyakumachi, M., 1998. Carbon loss and germinability of chlamydospores of Fusarium solani f. sp. phaseoli after exposure to soil at dierent pH levels, temperatures and matric
potentials. Phytopathology 88, 148±155.
Ofek, I., Goldhar, J., Keisari, Y., Sharon, N., 1995. Nonopsonic
phagocytosis of microorganisms. Annual Review of Microbiology
49, 239±276.
Palleroni, N.J., 1984. Gram-negative rods and cocci: family I
Pseudomonadaceae. genus I Pseudomonas. In: Krieg, N.R., Holt,
J.G. (Eds.), Bergey's Manual of Systematic Bacteriology, Vol. I.
Williams and Wilkins, Baltimore, pp. 141±199.
Sands, D.C., Hankin, L., 1975. Ecology and physiology of ¯uorescent pectolytic pseudomonads. Phytopathology 65, 921±924.
Savage, D.C., Fletcher, M., 1985. Bacterial Adhesion Mechanisms
and Physiological Signi®cance. Plenum Press, London.
Srivastava, A.K., Gupta, S., Arora, D.K., 1996a. Agglutination response of Pseudomonas ¯uorescens and Trichoderma harzianum to
Macrophomina phaseolina under dierent growth conditions.
Microbiological Research 151, 193±200.
Srivastava, A.K., Arora, D.K., Gupta, S., Pandey, R.R., Lee, M.W.,
1996b. Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil. Biology and Fertility of
Soils 22, 136±140.
Tari, P.H., Anderson, A.J., 1988. Fusarium wilt suppression and
agglutinability
of
Pseudomonas
putida.
Applied
and
Environmental Microbiology 54, 2037±2041.
Tomita, S., Matsue, M., Matsuyama, J., Kiyosawa, I., 1994.
Agglutination of bacterial cells of Clostridium inoccum,
Bi®dobacterium longum and Micrococcus luteus by lactoferrin and
ovotransferrin. Bioscience Biotechnology Biochemistry 58, 722±
726.
Tunlid, A., Jansson, H.B., Nordbring-Hertz, B., 1992. Fungal attachment to nematodes. Mycological Research 96, 401±412.
www.elsevier.com/locate/soilbio
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 P 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 ÿ0:89to ÿ 0:96; P 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: dkarora@banaras.ernet.in (D.K. Arora).
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 surfaces 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
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
512
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 eects 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 eect 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 eect 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 37GBq; Bhaba Atomic Research
Centre, Bombay, India). Strains of P. ¯uorescens were
isolated from rhizosphere soils of 10 dierent 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 dierent 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
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 (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: 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-
514
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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.
2.6. 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. Eect on germination
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 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).
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 Pfeer'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 Agg , 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
Sclerotia incubated for 60 d in buer 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 dierent 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 dierent 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 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 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-
516
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 agglutination 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 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
buer 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 dierent treatments did not
dier 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 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-
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 ÿ0:89 to ÿ 0:96;
P 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 dierence (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 germination of sclerotia (30-d-old) in buer 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 eort was made to evalu-
518
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
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 eciency 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 of M. phaseolina (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 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 dierent amount of C loss by Agg+, Aggl
and Aggÿ can be attributed to dierence in sink eciencies 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 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 propagules also depends upon the nature and properties of
dierent soils. Besides these, size, age and physiological state of fungal propagules could also in¯uence sink
eciencies 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 ÿ0:86 to ÿ 0:96; P 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 eciency 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
dierent ecological niches and competitive soil environments.
Acknowledgements
One of the authors (T.K.J.) thanks the University
Grants Commission, New Delhi for ®nancial assistance.
T.K. Jana et al. / Soil Biology & Biochemistry 32 (2000) 511±519
References
Anderson, A.J., Tari, P.H., Tepper, C.S., 1988. Molecular studies on
the role of a root surface agglutinin in adherence and colonization by Pseudomonas putida. Applied and Environmental
Microbiology 54, 375±380.
Arora, D.K., Filonow, A.B., Lockwood, J.L., 1983. Exudation from
14
C-labeled fungal propagules in the presence of speci®c microorganisms. Canadian Journal of Microbiology 29, 1487±1492.
Arora, D.K., Filonow, A.B., Lockwood, J.L., 1985. Decreased
aggressiveness of Bipolaris sorokiniana conidia in response to
nutrient stress. Physiological and Molecular Plant Pathology 26,
135±142.
Arora, D.K., 1988. Eect of microorganisms on aggressiveness of
Bipolaris sorokiniana. Canadian Journal of Botany 66, 242±246.
Benyagoub, M., Jabazi-Hare, S.H., Chamberland, H., Charest, P.M.,
1996. Cytochemical and immuno-cytochemical investigation of
the mycoparasite between Stachybotrys elegans and its host
Rhizoctonia solani (AG-3). Mycological Research 100, 79±86.
Bin, L., Knudsen, G.R., Eschen, D.J., 1991. In¯uence of an antagonistic strain of Pseudomonas ¯uorescens on growth and ability of
Trichoderma harzianum to colonize sclerotia of Sclerotinia sclerotiorum in soil. Phytopathology 81, 994±1000.
Buell, C.R., Whetton, R., Tari, P.H., Anderson, A.J., 1993.
Characterization of cell surface properties in agglutinable and
non-agglutinable mutants of Pseudomonas putida. Canadian
Journal of Microbiology 39, 787±794.
Bull, C.T., Weller, D.M., Thomashow, L.S., 1991. Relationship
between root colonization and suppression of Gaeumannomyces
graminis var. tritici by Pseudomonas ¯uorescens strain 2-79.
Phytopathology 81, 945±959.
Elad, Y., Chet, I., 1983. Possible role of lectins in mycoparasitism.
Journal of Bacteriology 154, 1431±1435.
Epstein, L., Lockwood, J.L., 1984. Eect of soil microbiota on germination of Bipolaris victoriae conidia. Transaction of the British
Mycological Society 82, 63±69.
Expert, J.M., Digat, G., 1995. Biocontrol of Sclerotinia wilt of sun¯ower by Pseudomonas ¯uorescens and Pseudomonas putida
strains. Canadian Journal of Microbiology 41, 685±691.
Filonow, A.B., Lockwood, J.L., 1983. Mycostasis in relation to the
microbial sinks of ®ve soils. Soil Biology & Biochemistry 15, 557±
565.
Glandorf, D.C.M., Sluis, I.V.D., Anderson, A.J., Bakker, P.A.H.M.,
Schippers, B., 1994. Agglutination, adherence and root colonization by ¯uorescent pseudomonds. Applied and Environmental
Microbiology 60, 1726±1733.
Gupta, S., Arora, D.K., Srivastava, A.K., 1995. Growth promotion
of tomato plants by rhizobacteria and imposition of energy stress
on Rhizoctonia solani. Soil Biology & Biochemistry 27, 1051±
1058.
Hyakumachi, M., Lockwood, J.L., 1989. Relation of carbon loss
from sclerotia of Sclerotium rolfsii during incubation on soil to
decreased
germinability
and
pathogenic
aggressiveness.
Phytopathology 79, 1059±1063.
Hyakumachi, M., Loer, H.J.M., Lockwood, J.L., 1989. Methods
for determination of carbon loss from fungal propagules incubated in soil. Soil Biology & Biochemistry 21, 567±571.
Hyakumachi, M., Arora, D.K., 1998. The response of fungal propagules to competitive energy stress in soil. In: Csermely, P. (Ed.),
Stress of Life from Molecules to Man. Annals of the New York
Academy of Sciences, New York, pp. 152±162.
Inbar, J., Chet, I., 1997. Lectins and biocontrol. Critical Reviews in
Biotechnology 17, 1±20.
King, E.O., Ward, M.K., Raney, D.E., 1954. Two simple media for
519
demonstration of pyocyanin and ¯uorescein. Journal of
Laboratory Clinical Medicine 44, 301.
Kuo, K., Hoch, H.C., 1996. Germination of Phyllostcia ampelicida
pycnidiospores: prerequisite of adhesion to the substratum and
the relationship of substratum wettability. Fungal Genetics and
Biology 20, 18±29.
Lockwood, J.L., 1990. Relation of energy stress to behaviour of soilborne plant pathogens and to disease development. In: Hornby,
D. (Ed.), Biological Control of Soilborne Plant Pathogens. CAB
International, Oxon, pp. 127±214.
Lockwood, J.L., 1992. Exploitation competition. In: Carrol, G.C.,
Wicklow, D.T. (Eds.), The Fungal Community: its Organization
and Role in the Ecosystems. Marcel Dekker, New York, pp. 243±
264.
Manocha, M.S., 1991. Physiology and biochemistry of biotrophic
mycoparasitism. In: Arora, D.K., Rai, B., Mukerji, K.G.,
Knudsen, G.R. (Eds.), Handbook of Applied Mycology, vol. I.
Marcel Dekker, New York, pp. 273±300.
Manocha, M.S., Sahai, A.S., 1993. Mechanism of recognition of
necrotrophic and biotrophic mycoparasites. Canadian Journal of
Microbiology 39, 269±274.
Mercure, E.W., Kunow, H., Nicholson, R.L., 1994. Adhesion of
Colletotrichum graminicola conidia to corn leaves: a requirement
for disease development. Physiological and Molecular Plant
Pathology 45, 407±420.
Molin, G., Ternstrom, A., 1982. Numerical taxonomy of psychotrophic pseudomonads. Journal of General Microbiology 128,
1249±1264.
Mondal, S.N., Kageyama, K., Hyakumachi, M., 1996. Decreased
germinability and virulence of oospores of Pythium aphanidermatum in relation to loss of endogenous carbon during incubation
on soil. Soil Biology & Biochemistry 28, 545±553.
Mondal, S.N., Hyakumachi, M., 1998. Carbon loss and germinability of chlamydospores of Fusarium solani f. sp. phaseoli after exposure to soil at dierent pH levels, temperatures and matric
potentials. Phytopathology 88, 148±155.
Ofek, I., Goldhar, J., Keisari, Y., Sharon, N., 1995. Nonopsonic
phagocytosis of microorganisms. Annual Review of Microbiology
49, 239±276.
Palleroni, N.J., 1984. Gram-negative rods and cocci: family I
Pseudomonadaceae. genus I Pseudomonas. In: Krieg, N.R., Holt,
J.G. (Eds.), Bergey's Manual of Systematic Bacteriology, Vol. I.
Williams and Wilkins, Baltimore, pp. 141±199.
Sands, D.C., Hankin, L., 1975. Ecology and physiology of ¯uorescent pectolytic pseudomonads. Phytopathology 65, 921±924.
Savage, D.C., Fletcher, M., 1985. Bacterial Adhesion Mechanisms
and Physiological Signi®cance. Plenum Press, London.
Srivastava, A.K., Gupta, S., Arora, D.K., 1996a. Agglutination response of Pseudomonas ¯uorescens and Trichoderma harzianum to
Macrophomina phaseolina under dierent growth conditions.
Microbiological Research 151, 193±200.
Srivastava, A.K., Arora, D.K., Gupta, S., Pandey, R.R., Lee, M.W.,
1996b. Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil. Biology and Fertility of
Soils 22, 136±140.
Tari, P.H., Anderson, A.J., 1988. Fusarium wilt suppression and
agglutinability
of
Pseudomonas
putida.
Applied
and
Environmental Microbiology 54, 2037±2041.
Tomita, S., Matsue, M., Matsuyama, J., Kiyosawa, I., 1994.
Agglutination of bacterial cells of Clostridium inoccum,
Bi®dobacterium longum and Micrococcus luteus by lactoferrin and
ovotransferrin. Bioscience Biotechnology Biochemistry 58, 722±
726.
Tunlid, A., Jansson, H.B., Nordbring-Hertz, B., 1992. Fungal attachment to nematodes. Mycological Research 96, 401±412.