Symbiosis

DOI 10.1007/s13199-015-0313-7

The growth of tomato seedlings inoculated with co-cultivated

Piriformospora indica and Bacillus pumilus
K. N. Anith & Anjana Sreekumar & J. Sreekumar

Received: 17 June 2014 / Accepted: 15 January 2015

# Springer Science+Business Media Dordrecht 2015

Abstract A novel co-culture method using the beneficial root 1 Introduction

endophytic fungus Piriformospora indica and a plant-growth
promoting rhizobacterial strain of Bacillus pumilus to promote The production of good quality seedlings with high seedling
the growth of tomato seedlings in plug trays, is described. vigour is one of the important factors in production systems
Coconut water, a waste product from the coconut industry, for vegetables. The use of healthy seedlings with well
was used as medium for co-cultivation of the two biological established root systems helps to avoid transplantation shock
agents. During the co-culture with the fungus, the bacterial when the seedlings are transferred to the field from the nurs-
strain showed a similar growth rate to that in monoculture in ery. Raising seedling using plug trays (pro-trays) allows the
fresh autoclaved coconut water. In contrast, co-culture in po- grower to establish seedlings with optimal spacing and uni-
tato dextrose broth (PDB), a medium routinely used for culti- form physiological status prior to transplanting (Vavrina
vation of the endophytic fungus, did not support bacterial 1998). Plug tray transplants are commercially used for crops
growth. Inoculation with the co-culture or a mixture of like chilli, tomato, brinjal, cauliflower, cabbage, broccoli, cel-
P. indica and B. pumilus promoted tomato seedling growth ery, cucumber etc. The use of biological agents at the nursery
significantly when compared with individual application of production stage is advantageous as root colonization by ben-
the two biological agents. No difference was observed with eficial microbes, both fungi and bacteria results in the transfer
respect to the degree of root colonization in tomato seedlings of Bbio-primed^ seedlings to the field. Plant growth promot-
whether it was done with a monoculture or a co-culture with ing rhizobacteria usually colonize the root system, both exter-
rhizobacterium. The co-culture system developed reduces the nally as well as endophytically. They positively affect plant
cost of inoculum production as it uses coconut water, a cheap health, leading to increased growth and improved yields
and locally available waste product and a single fermentation (Kloepper et al. 1980; Hass and Defago 2005). Several of
vessel. Further research is required to know the endophytic these growth promoters possess additional capabilities such
nature of the rhizobacterial strain and its possible role in help- as the biological suppression of plant diseases and insect pests
ing the fungus in root colonization. (Lugtenberg and Kamilova 2009). Biological amendment
with PGPR inoculants for the production of vegetable seed-
ling have been reported by several workers (Gagne et al. 1993;
Keywords Co-culture . Piriformospora indica . PGPR . Nemec et al. 1996; Kokalis-Burelle et al. 2002; Russo 2006a,
Bacillus pumilus . Coconut water . Plant-growth promotion . b; Russo and Perkins-Veazie 2010). The survival and root
Tomato colonization pattern in tomato of several rhizobacteria in
soil-less growth medium was studied by Yan et al. (2003),
K. N. Anith (*) : A. Sreekumar
while the value of the fungus Trichoderma harzianum at the
Department of Agricultural Microbiology, College of Agriculture,
Kerala Agricultural University, Vellayani, Thiruvananthapuram 695 nursery stage has been studied by Chowdappa et al. (2013).
522, Kerala, India Piriformospora indica, a fungus belonging to the
e-mail: [email protected] Sebacinales in the Basidiomycota is a root colonizing endo-
phyte with a wide host range and plant growth-promoting
J. Sreekumar
Central Tuber Crops Research Institute, Sreekaryam, ability. The fungus can be cultivated on complex or minimal
Thiruvananthapuram 695 017, Kerala, India media (Verma et al. 1998; Weiss et al. 2004). This fungus may
 K.N. Anith et al.

also confer resistance against several biotic and abiotic stress- Dextrose Broth (PDB; pH 6.5) or Potato Dextrose Agar
es including root and leaf fungal pathogens, as well as pro- (PDA; pH 6.5) under conditions described earlier (Anith
moting early flowering or enhanced seed production et al. 2011). During the course of the present study, cultivation
(Oelmüller et al. 2009; Franken 2012; Varma et al. 2012). was also performed on coconut water agar (CWA) and
Root colonization by the fungus induced improved secondary autoclaved coconut water (ACW) providing similar incuba-
metabolite production in the medicinal plant Centella asiatica tion temperature as in the case of PDA and PDB. For this,
(Satheesan et al. 2012) and also increased gel content, phenol fresh coconut water was procured from a local coconut pro-
content, aloin content and radical scavenging capacity in Aloe cessing facility and filtered through muslin cloth to get rid of
vera (Sharma et al. 2014). Finally, P. indica has been reported the suspended particles and debris. 100 ml of coconut water
to act as a growth promoter for tomato (Fakhro et al. 2010). were transferred to a 500 ml Erlenmeyer flask, the pH was
The efficiency of bio-inoculants can be increased by using adjusted to 6.5 and the solution was sterilized by autoclaving
more than one biological agent that can work together thereby at 121 °C for 20 min. For preparing CWA, 2 % agar were
increasing the action spectrum (Janisiewicz 1988; Pierson and added before autoclaving.
Weller 1994; Vidhyasekarn and Muthamilan 1995; Schisler Bacterial cultures used in the study included PGPR strains
et al. 1997; Slininger et al. 2010; Schisler et al. 2011). P. indica of Bacillus pumilus, Bacillus subtilis, Bacillus
has been applied with beneficial bacterial strains such as amyloliquefaciens and Pseudomonas fluorescens available at
PGPR as a mixed inoculum for mung bean and tomato. This the Department of Agricultural Microbiology, College of Ag-
resulted in improved growth response and disease suppression riculture, Vellayani (India). Bacillus strains were grown on
ability (Sarma et al. 2011; Kumar et al. 2012). Improved plant Nutrient Agar/broth (Anith 2009) and the Pseudomonas strain
growth in chick pea has also been reported for the combined on King’s medium B agar/broth (Anith et al. 2002) at 28 °C.
inoculation involving P. indica and the phosphate solubilizing Broth cultures were incubated in a shaker at 150 rpm for 48 h.
bacterium Pseudomonas striata (Meena et al. 2010). Positive
effects of dual inoculation of P. indica and the mycoparasitic 2.2 Growth of P. indica on coconut water based media
fungus Trichoderma harzianum have been found on the
growth of black pepper in tissue culture (Anith et al. 2011). For finding out the suitability of coconut water based media
Co-culture systems containing a combination of a plant- for the growth of P. indica, the fungus was cultivated on CWA
growth promoting/plant-defense enhancing fungus and a bac- and in ACW. For measuring the growth of P. indica on CWA,
terium have not been reported to date. In the present study, it a mycelial disc (8 mm dia) of P. indica from freshly grown
was decided to employ a plant-derived organic substrate for PDA plate was cut out with a sterile cork borer, placed in the
the cultivation of the microbial agents to minimize costs. Co- center of a CWA plate and incubated at 28 °C. The diameter of
conut water in the intact coconut is free from microbial con- the radial growth of the fungus was measured at regular inter-
tamination and is highly nutritious, rich in many amino acids, vals. In parallel, the growth of the fungus was analyzed on
vitamins and minerals (Nandakumar 1990). Coconut water PDA plates. Ten replicates were analyzed for each medium.
from the mature coconut is a waste product from processing Fungal growth in liquid medium (ACW) was measured by
the nuts, though it has been used for growing microbes and in quantifying the mycelial wet and dry weight after an incuba-
plant tissue culture (Anandaraj and Sarma 1997; Gopal et al. tion period of 15 days under shaking at 90 rpm. To 100 ml of
2006; Survase et al. 2007; Unagul et al. 2007; Prabhakaran ACW a mycelial disc (8 mm dia) of P. indica was aseptically
et al. 2008). It is an excellent multiplication medium for plant inoculated and incubated with agitation (90 rpm). After
growth promoting rhizobacteria (Anith 2009). In the present 15 days of growth, the mycelium was collected by filtration
paper we describe an efficient co-cultivation protocol involv- through a filter paper (Sartorius No. 292) and the fresh weight
ing the fungus P. indica and the plant-growth promoting of the mycelium was determined. The collected mycelium
rhizobacterial strain of Bacillus pumilus in autoclaved coconut was dried in an oven at 55 °C till it achieved constant weight
water and the effect of the co-cultured inoculum on the growth and the dry weight was determined. The results were com-
of tomato seedlings. pared with fungal growth in PDB under the same conditions.
Five replicates were examined per type of medium.

2 Materials and methods 2.3 Testing for in vitro antagonism between P. indica
and bacterial strains
2.1 Cultivation of fungal and bacterial strains
Dual culture plate assays were conducted on PDA and CWA
The endophytic fungus Piriformospora indica, obtained from as described elsewhere to assess whether the bacteria
Dr. Ajit Varma, Professor emeritus at Jawaharlal Nehru Uni- displayed antagonism against P. indica (Nair and Anith
versity, New Delhi, India was routinely cultivated on Potato 2009). A mycelial disc (8 mm dia) from a freshly grown
The growth of tomato seedlings inoculated with co-cultivated Piriformospora

P. indica culture on PDA was placed in the centre of a Petri strain grown separately in ACW was also included for com-
dish with agar medium and incubated at 28 °C for 5 days. parison. The control treatment involved no inoculation. The
Single colonies of the Bacillus strains and of Pseudomonas experiment was designed in CRD with three replicates of 10
fluorescens were grown on Nutrient Agar and King’s medium seedlings each.
B, respectively. After 5 days of incubation of the P. indica Fungal mycelium was incorporated into the planting medi-
plates, a heavy inoculum from a single colony of the bacterial um before filling it in the plug trays as described elsewhere
strain was applied with an inoculation loop as a band of 1.5 cm (Anith et al. 2011). For this the mycelium of P. indica grown
length equidistantly on two opposite edges of the agar plate in PDB and ACW in 100 ml medium in 500 ml flasks for
with the fungus, so that two independent measurement on 15 days was collected by filtering the suspension through
fungal growth inhibition could be taken from a single plate. muslin cloth. It was weighed and mixed thoroughly with ster-
Four plates were examined for each of the bacterial strains. ile vermiculite so as to get a 1 % (w/v) final concentration of
The plates were incubated at 28 °C and observations on the the fungal mycelium in the vermiculite.
inhibition of fungal growth by the bacterial strains were made For inoculation with bacteria alone, B. pumilus was used
by measuring the zone of inhibition 5 days after the inocula- after growth in ACW for 48 h at 28 °C with agitation to a
tion with the bacteria. Plates containing the fungus alone density of 8×108 cfu/ml. No incorporation of the bacterial
served as the control. culture into the planting medium was done prior to planting.
However, one ml of the bacterial culture was added to the
2.4 Co-culture experiment planting hole in the vermiculite after depositing the seeds.
Co-culturing of the fungus and the bacteria was done as
Since Bacillus pumilus showed no antagonism against described earlier. Forty-eight hours after the addition of the
P. indica on both CWA and PDA medium, it was selected bacteria to the flask containing the fungus grown in ACW,
for the co-cultivation experiment. P. indica was initially the mixture was filtered through muslin cloth and the fungal
grown in PDB or ACW for 15 days as described above. mycelium was collected and its fresh weight was determined.
B. pumilus was streaked out for single colonies on a nutrient The incorporation of the fungal-bacterial mixture in vermicu-
agar plate. Cells from a single colony were pooled in one ml of lite (1 % w/v) was performed as described above. No further
sterile distilled water (population density of 2×106 cfu/ml) supplementation of bacterial culture was done, as the co-
and 200 μl of the bacterial suspension was aseptically added cultured fungal mycelium contained bacterial cells at a popu-
to flasks of PDB and ACW wherein P. indica had been grow- lation level of 9.23×108 cfu/g (on a wet weight basis).
ing since 15 days. The initial number of the bacteria added to Mixed inoculation of the fungus and bacteria was per-
the flasks was determined by dilution plating on nutrient agar formed as follows. Mycelium of the fungus grown in ACW
medium immediately after inoculation. The flasks were fur- for 15 days was mixed with vermiculite at the rate of 1 %
ther incubated under agitation (150 rpm) for 72 h and the (w/v) as described above and filled in plug trays. B. pumilus
population of the bacteria was determined at 24 h intervals was grown in ACW for 48 h with agitation in a rotary shaker.
by dilution plating on nutrient agar medium. The bacterial After planting the seeds, one ml of the bacterial culture with a
population from five flasks was independently assessed for density of 8×108 cfu/ml was added to the planting hole.
both growth media. Growth of the bacteria in fresh PDB and Two seeds were planted in a single cavity of the plug tray.
ACW was taken as baseline to determine the efficiency of the After germination, a single seedling was maintained per
co-culture in supporting bacterial growth. cavity. Plants were cultivated in a glass house with 16 h
light at 28±2 °C. Seedlings were irrigated twice daily
2.5 Plant growth experiment with sterile distilled water. Hoagland’s Nutrient Solution
(Douds and Schenck 1990) was provided to the seedlings at
Seeds of the tomato variety Vijay were surface sterilized in a rate of 10 ml per plug tray cavity, once in 10 days, starting
1 % sodium hypochlorite solution for 3 min. and washed 1 week after seeding. Plants were kept for 21 days and quan-
thrice with sterile distilled water under aseptic conditions. Ex- tification of plant height, number of leaves per plant and fresh
foliated vermiculite (particle size 0.5 to 1 mm; pH 6.5) was and dry shoot and root weight of the plants was carried out
used as planting medium for the growth promotion experi- after uprooting the plants.
ment. It was sterilized by autoclaving at 121 °C for 1 h each
for three consecutive days. Plastic plug trays with cavities of 2.6 Colonization assay
5 cm diameter and 5 cm depth were used for maintaining the
plants. Plant growth promotion by the fungus grown in PDB Separate sets of five plants each per treatment described above
or ACW, respectively, by co-cultured fungal-bacterial suspen- were kept for colonization studies under greenhouse condi-
sion in ACW and by the bacterial strain grown in ACW was tions. After 21 days of growth, five plants from each treatment
compared. Dual inoculation of the fungus and the bacterial were analyzed for root colonization by the endophytic fungus
 K.N. Anith et al.

P. indica following procedures described earlier (Anith et al. 14th or 16th day, respectively, for PDA and CWA. There was
2011). The plants were uprooted, washed in running tap water slight difference in the colouration of the fungal mycelium on
to get rid of the planting medium and the root systems were the two media as on PDA the mycelial mat appeared slightly
cut off. The collected roots were cut into pieces of one cm and yellowish while on CWA it was almost white. The mycelial
boiled in 10 % KOH for 5 min., then washed in sterile water fresh and dry weight determined after 15 days of growth in
followed by neutralization with 2 % HCl. Roots were then PDB and ACW, respectively, is shown in Table 1. More my-
stained with 0.5 % trypan blue in lactophenol for a period of celium was produced in PDB than in ACW.
10 min. They were then destained with lactophenol solution
for 15 min to remove excess stain. The stained root bits were
viewed under a compound bright field microscope and the 3.2 In vitro antagonism between P. indica and bacterial strains
presence of chlamydospores in the cortex cells documented
for each root segment. The percentage of colonization was Dual cultivation of the fungus and bacterial strains showed
assessed using the following formula. that B. pumilus had no antagonistic effect on the fungus both
on PDA and CWA (Table 2). The maximum zone of inhibition
% root colonization of fungal growth was observed with Bacillus
Number of root segments colonized  100 amyloliquefaciens both on PDA and on CWA.
¼
Total number of root segments observed

3.3 Co-culture experiment

2.7 Statistical analysis
Since B. pumilus had no antagonistic effect on the fungus, this
Statistical analysis for all the parameters was performed using strain was taken for co-cultivation. When 15 day-old cultures
one way analysis of variance (ANOVA) and the means were of the fungus in ACW and PDB, respectively, were inoculated
compared by Duncan’s Multiple Range Test, using the statis- with the bacteria, it became obvious that the former medium
tical package SAS version 8.1 (SAS Institute Inc., Cary, NC, supported the growth of the bacteria similarly to fungus-free
USA). ACW (Table 3). Both monoculture and co-culture in ACW
resulted in achieving a population of 108 cfu/ml from an initial
inoculum of 105 cfu/ml. On the other hand, co-cultivation in
3 Results PDB led to a decline in bacterial population, in spite of the fact
that B. pumilus did not show significant differences in growth
3.1 Growth of Piriformospora indica on coconut water based in fungus-free PDB or ACW. The initial pH of the ACW
media before the growth of P. indica was 6.19, while after 15 days
of growth of the fungus it has come down to 5.97. Though
The average diameter of the fungal mycelium 10 days after there was a reduction in pH value, the bacterial strain
inoculation was 6.2 and 5.38 cm on PDA and CWA, respec- was able to multiply in it. The final pH after 72 h of
tively (Table 1). The entire surface of the medium in the 10 cm growth of the bacterial strain in the co-culture system was
diameter plate was fully covered by fungal mycelium on the 5.95. Since there was only a slight reduction in pH value even
after bacterial growth, the fungal mycelium was not adversely
affected.
Table 1 Growth of P. indica in coconut water based media

Medium Diameter of mycelial Mycelial fresh Mycelial dry

growth (cm)* weight (g)** weight (g)**
Table 2 Mycelial growth inhibition of P. indica by bacterial strains
Agar medium
Bacterial strain Zone of inhibition (cm)*
CWA 5.38±0.147 – –
PDA 6.20±0.201 – – PDA CWA
Broth
ACW – 3.59±0.401 0.342±0.069 Bacillus pumilus Nil Nil
PDB – 4.82±0.193 0.416±0.039 Bacillus amyloliquefaciens 0.75 0.50
Bacillus subtilis 0.85 0.90
*
Observations taken after 10 days of inoculation. Mean ± SD of obser- Pseudomonas fluorescens 0.65 0.45
vations from ten replications
**
Observations taken after 15 days of inoculation. Mean ± SD of obser- *Mean of eight observations from four dual culture plates. Each plate
vations from five replications represents single replication (n=4)
The growth of tomato seedlings inoculated with co-cultivated Piriformospora

Table 3 Population build-up of Bacillus pumilus in different media and 3.5 Colonization assay
cultural conditions

Type of inoculation Population of B. pumilus (cfu/ml)* Microscopic examination of the root pieces after staining re-
vealed that in all the treatments that included the endophytic
Time of population assessment fungus, colonization was evident by the presence of chla-
mydospores of the fungus in the cortical region of the plant
0h 24 h 48 h 72 h
roots. Maximum percentage colonization (37.4 %) was observed
B. pumilus alone with plants treated with the co-cultured inoculum (Table 5).
PDB 4.72×105 4.51×107 2.65×108 2.67×108 However, there was no statistically significant difference with
ACW 2.12×105 7.27×108 8.50×108 5.02×108 respect to the percentage root colonization among the different
P. indica and B. pumilus co-culture treatments (p=0.833). When the root colonization by the endo-
PDB 4.48×105 5.5×104 4.03×104 2.95×104 phytic fungus was assessed by examining chlamydospores in the
ACW 4.62×105 1.15×107 2.33×108 1.41×108
root cortex cells of tomato, it was found that colonization per-
centage was similar following application of single, mixed and
*Mean population from five flasks. Each flask represents single replica- co-cultured inocula. However, there was a difference with re-
tion (n=5) spect to the pattern of chlamydospore formation in the cortical
cells (Fig. 1). Whenever a single inoculation using P. indica was
performed, the root cortex cells occupied with chlamydospores
3.4 Plant growth experiment were completely filled with many numbers of large sized spores
of the fungus. Only very few adjoining cells were filled with
Results of the plant growth promotion experiment showed that spores. However, when mixed or co-cultured inoculations, were
there were significant differences in plant growth parameters used, it was observed that almost all the cells in the cortex region
between the effects of the combined application of P. indica in the colonized roots were occupied by chlamydospores, but
and B. pumilus, both as co-culture and as mixed inoculum, with a different distribution pattern. Most of the cells were occu-
and the application of either P. indica or B. pumilus alone pied by single or comparatively small spores. More research is
(Table 4). Inoculation with P. indica alone as well as needed as it is uncertain whether the rhizobacterial strain used
B. pumilus alone also improved the plant growth compared also acts as an endophyte and help the fungus to colonize cortical
to the uninoculated control. Among all plant growth parame- cells. Molecular tagging of the bio-agents and confocal micro-
ters analyzed, the maximum values were obtained in plants scopic studies would be necessary to further confirm this.
treated with either the co-cultured suspension of P. indica and
B. pumilus, or with a mixed inoculum of the two biological
agents. With respect to plant height there was no significant 4 Discussion
difference among the treatments except that the uninoculated
control plants were shorter. Treatments with biological agents, The application of biological agents to seedlings in the nursery
when applied singly or in combination, had a positive impact has several advantages as compared with treatment of the
on the growth of the plants when compared with the uninoc- seedlings in the field. Dipping seedling in a suspension of
ulated control. the formulated product during transplantation is cumbersome

Table 4 Growth parameters of portray seedlings of tomato taken after 21 days of planting

Treatment Biometric observations*

Plant Leaf number Fresh shoot Dry shoot weight Fresh root Dry root weight
height (cm) weight (g/plant) (mg/plant) weight (g/plant) (mg/plant)

P. indica in ACW 8.20±0.25a 4.76±0.29a 0.746±0.03bc 57.13±2.75b 0.117±0.010b 7.69±1.21ab

P. indica in PDB 8.58±0.51a 4.26±0.15b 0.724±0.11bc 57.96±10.55ab 0.127±0.015ab 6.65±1.41bc
Co-cultured P. indica and B. pumilus 9.16±0.20a 4.73±0.15a 0.876±0.01a 67.15±2.10a 0.138±0.005a 8.43±0.98a
Mixed inoculation of P. indica and B. pumilus 9.12±0.77a 4.70±0.20a 0.815±0.07ab 61.60±4.88ab 0.128±0.008ab 7.19±0.63abc
B. pumilus in ACW 9.00±0.65a 4.73±0.23a 0.647±0.02c 43.35±1.33c 0.091±0.002c 5.52±0.32c
Control 6.80±0.50b 3.67±0.23c 0.401±0.05d 30.77±3.35d 0.065±0.006d 3.61±0.29d
*
Mean ± SD of three replications having 10 plants each. Figures in a column followed by same letter do not differ significantly according to Duncan’s
Multiple Range Test (DMRT). p<0.05
 K.N. Anith et al.

Table 5 Root colonization in tomato by Piriformospora indica (Fakhro et al. 2010; Anith et al. 2011; Sarma et al. 2011;
Treatment Root colonization (%)* Kumar et al. 2012). In the present study, the fungus grew well
on solid CWA, and autoclaved coconut water (ACW) was
P. indica in ACW 35.4a found to be suitable for mass multiplication of P. indica. The
P. indica in PDB 35.8a growth of the fungus in ACW was comparable to that in po-
Co-cultured P. indica and B. pumilus 37.4a tato dextrose broth, a routine medium for its culture. During
Mixed inoculation of P. indica and B. pumilus 36.7a the course of evaluation of ACW as a medium for cultivation
B. pumilus in ACW ND of the endophytic fungus, the mycelial growth was separated
Control ND from the liquid portion by straining through a muslin cloth and
the liquid portion was left behind in an unsterile vessel. It was
*Observation taken 21 days after planting. Mean of five replications noticed on the next day that the clear liquid turned turbid as a
having one plant each (n=5). From each plant 100 root segments were
assayed for determining the presence of chlamydospores in the root cor- result of multiplication of bacterial contaminants. This
tex cells. Figures in a column followed by same letter do not differ sig- prompted us to use the left over coconut water that was used
nificantly according to Duncan’s Multiple Range Test (DMRT). p<0.05 for cultivation of P. indica for 15 days, as a medium for growth
ND Not detected of bacterial biological agents. The liquid portions from several
such flasks were collected aseptically after removing the
and labour intensive. If seedlings are already treated with the P. indica mycelium and evaluated for the growth of PGPR
biological agents while in plug trays, the further multiplication strains in them. All the bacterial strains tested, three Bacillus
of the agents may be expected during crop growth without and one Pseudomonas strain, were able to multiply in the
additional application (Yan et al. 2003; Russo 2006a, b; Russo liquid medium (data not presented). Cultivation of the PGPR
and Perkins-Veazie 2010). The endophytic fungus P. indica is strains along with the endophytic fungus in a co-culture sys-
usually mass multiplied in potato dextrose broth for plant tem was then contemplated after studying their in vitro
growth promotion experiments and inoculum preparations interactions.
Co-culturing of biological agents in the same fermentor
system has been proposed previously (Slininger et al. 2010)
for bacterial antagonists to control diseases in stored potatoes.
Dual culture has been used to understand antagonism between
different biological agents (Li et al. 2002; Anith et al. 2003;
Aravind et al. 2009). The endophytic root symbiont P. indica
induced various reactions with rhizobacterial strains. Many
had a neutral response, but some displayed inhibitory to stim-
ulatory reactions to P. indica when co-cultivated on agar plates
(Varma et al. 2012). In the present study, the selection of the
rhizobacterial strain was done after an in vitro assay for an-
tagonistic interactions against the fungal endophyte on differ-
ent solid media by the dual culture plate method. Since the
PGPR strain B. pumilus showed no antagonism against
P. indica, it was used for co-cultivation. Co-cultivation in
ACW supported the growth of the bacterial strain and bacte-
rial growth was comparable to that in a monoculture in ACW,
even in the presence of the fungal mycelium. It is inferred that
the ACW still contains sufficient amount of nutrients that
could support the growth of a bacterial strain even after sup-
plying nutrients for the growth of the fungus for 15 days. The
initial pH of the ACW before the growth of P. indica was 6.19,
Fig 1 Piriformosproa indica colonization and chlamydospore formation while after 15 days of growth of the fungus it has come down
pattern in the root cortex of tomato seedlings. Upper panel: Root
colonization in plants treated with P. indica as a single inoculum. (a-
to 5.97. Though there was a reduction in pH value, the bacte-
Enlarged view of a single cell filled with many numbers of the rial strain was able to multiply in it.
chlamydospores; b- Root segment showing chlamydospores within the In the present study the two biological agents used were
cells with very few cells occupied by the spores). Lower panel: Root compatible to each other as shown by the dual culture assay.
colonization in plants treated with co-culture of P. indica and Bacillus
pumilus. (c- Enlarged view of cells filled with single chlamydospores; d-
Whenever there is antagonism among the different biological
Root segment showing chlamydospores within almost all the cells, occu- agents it is not advisable to use them for mixed inoculation on
pied singly) crop plants. However, incompatibility of the inoculants could
The growth of tomato seedlings inoculated with co-cultivated Piriformospora

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