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Keywords:

  • biocontrol;
  • bioinoculant formulations;
  • fluorescent pseudomonad;
  • Piriformospora indica;
  • tomato

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  Fluorescent pseudomonads are widely used as bioinoculants for improving plant growth and controlling phytopathogenic fungi. Piriformospora indica (Pi), a symbiotic root endophyte, also has beneficial effects on a number of plants. The present study focuses on the improvement of growth yields of tomato plants and control of Fusarium wilt using inorganic carrier-based formulations of two fluorescent pseudomonad strains (R62 and R81) and Pi.

Methods and Results:  The inorganic carrier-based formulations of pseudomonad strains and Pi were tested for plant growth promotion of tomato plants under glass house and field conditions. In controlled glass house experiments, 8·8-fold increase in dry root weight and 8·6-fold increase in dry shoot weight were observed with talcum powder-based consortium formulation of R81 and Pi. Field trial experiments ascertained the glfass house results with a considerable amount of increase in plant growth responses, and amongst all the treatments, R81 + Pi treatment performed consistently well in field conditions with an increase of 2·6-, 3·1- and 3·9-fold increase in dry root weight, shoot weight and fruit yield, respectively. The fluorescent pseudomonad R81 and Pi also acted as biocontrol agents, as their treatments could control the incidence of wilt disease caused by Fusarium oxysporum f.sp. lycopersici in tomato plants under glass house conditions.

Conclusions:  The culture broths of pseudomonads R62, R81 and Pi were successfully used for development of talcum- and vermiculite-based bioinoculant formulations. In controlled glasshouse experiments, the talcum-based bioinoculant formulations performed significantly better over vermiculite-based formulations. In field experiments the talcum-based consortium formulation of pseudomonad R81 and Pi was most effective.

Significance and Impact of the Study:  This study suggests that the formulations of pseudomonad strains (R62 and R81) and Pi can be used as bioinoculants for improving the productivity of tomato plants. The application of such formulations is a step forward towards sustainable agriculture.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The demand for safer agricultural products and food is increasing all over the world. Beneficial microorganisms applied as biofertilizers/biopesticides play an important role in today’s agriculture by improving soil fertility and crop productivity. In this context, rhizobacteria that have the capabilities to aggressively colonize the roots and positively affect the plant growth are of considerable interest (Haas and Défago 2005; Lugtenberg and Kamilova 2009). Amongst plant growth promoting rhizobacteria (PGPR), fluorescent pseudomonads have been exploited for plant growth promotion and suppression of crop diseases (Whipps 2001; Haas and Défago 2005). The pseudomonad strains used in the present investigation were characterized as positive for phosphate solubilization, and for production of indole-3-acetic acid, siderophores (hydroxamate type) and 2,4-diacetyl phloroglucinol (DAPG), which makes them potential PGPR (Gaur et al. 2004; Roesti et al. 2006; Sarma et al. 2009; Mäder et al. 2011). Piriformospora indica (Pi), a symbiont root endophytic fungus infests roots of a broad range of mono- and di-cotyledonous plants (Varma et al. 1999). A perusal of current literature shows that Pi has enormous potential for growth promotion of plants by colonization of roots (Varma et al. 1999; Schäfer et al. 2007). The fungus also provided induced systemic resistance (ISR) via jasmonate signalling associated with JA-regulated defence genes and cytoplasmic functioning of nonexpressor pathogenesis-related gene, NPR1 (Stein et al. 2008).

Fusarium wilts are soil-borne diseases, which affect many economically important crops worldwide. Biological control methods, based on the use of beneficial microorganisms isolated from suppressive soils, represent an alternative for protection of plants against Fusarium wilts (Alabouvette et al. 1993). In the current study, fluorescent pseudomonad R81 and Pi formulations were tested for their efficacy against the Fusarium wilt.

A formulation containing one or more beneficial bacterial strains (or species) in an easy-to-use and economical carrier material has been studied for effective delivery of bioinoculants (Vidhyasekaran et al. 1997). Usually the carriers are classified into soils (peat, coal and clays), plant waste materials (compost, soybean meal and farm yard manure) and inert materials (talcum powder, vermiculite, perlite, alginate and other inorganic minerals) (Bashan 1998). Here, two inorganic carriers, talcum powder and vermiculite, were used for preparing the bioinoculant formulations due to their advantages described by Bashan (1998) and Vidhyasekaran et al. (1997). Here, we report that fluorescent pseudomonad strains R62 and R81, and root endophyte Pi can be used as effective bioinoculants for growth promotional and disease resistance activities for tomato plants.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Microorganisms

Fluorescent pseudomonad strains R62 and R81, isolated from the rhizosphere of wheat (variety UP 2338) from Budaun, Uttar Pradesh and India were used in this study. These two strains were obtained from Dr A. K Sharma of Department of Biological Sciences, CBSH, GB Pant University of Agriculture and Technology (GBPUAT), Pantnagar, India. Bacterial cultures were maintained as 50% glycerol stocks at −20°C in King’s B medium (King et al. 1948). Piriformospora indica was provided by Prof. Ajit Varma, Amity Institute of Herbal and Microbial Studies, Noida, India. Piriformospora indica, isolated by Verma et al. (1998) from sandy desert soils of Rajasthan (India), was maintained on Kafer’s medium (Prasad et al. 2005). All chemicals (extra pure grade) used in this study were obtained from Merck (Mumbai, India).

Culture conditions

The fluorescent pseudomonad strains R62 and R81 were grown in 500 ml Erlenmeyer flasks using 100 ml of modified Schlegel’s medium. The modified medium contained glycerol (10·0 g l−1), succinic acid (0·5 g l−1), Na2HPO4 (4·20 g l−1), KH2PO4 (1·30 g l−1), NH4Cl (0·32 g l−1), urea (0·35 g l−1), KCl (0·35 g l−1), NaCl (0·65 g l−1), MgSO4.7H2O (0·50 g l−1), ammonium ferric citrate (150 μg l−1), CaCl2.2H2O (0·9 g l−1) and trace elements solution (0·9 ml l−1) of composition as in Schlegel’s original medium (Aragno and Schlegel 1991). For induction of DAPG production by the fluorescent pseudomonad R81, the medium was separately amended with 0·2 mmol l−1 ZnSO4 7H2O, 0·1 mmol l−1 (NH4)6 Mo7O24 and 800 μg l−1 of ammonium ferric citrate. The final pH of the medium before sterilization was adjusted to 7·1. The cultivation was carried out for 30 h at 28°C in an orbital shaker (Scigenics Biotech, Chennai, India) at 240 rev min−1. Piriformospora indica was cultured on potato dextrose broth for preparation of inoculum and on Kaefer medium (Prasad et al. 2005) for spore production. The cultivation for the production of fungal spores was carried out for 8 days at 30°C in the orbital shaker at 200 rev min−1 in 500 ml Erlenmeyer flask containing 100 ml working volume with initial pH of 6·5.

Estimation of cell growth

The cell growth of pseudomonads was estimated turbidimetrically using Helios Thermo Spectronic spectrophotometer (Thermo Electron Corp., Madison, WI, USA). The sample was diluted, if required, with 0·2% saline to get cell optical density in range of 0·1–0·4 at 600 nm. The CFU of the pseudomonads was determined using standard serial dilution method by plating in King’s B agar medium. The growth of Pi and the quantification of the spores were carried out as described by Kumar et al. (2011).

Quantification of DAPG production by pseudomonad strains

For estimation of DAPG, the bacterial cultures were centrifuged after 36 h of growth and the supernatants of respective strains were pooled. The pH of the supernatant was adjusted to 2·0 by adding 4 N HCl and the samples were extracted twice with equal volume of ethyl acetate. The extracts, which contained DAPG, were evaporated to dryness in vacuo. The extracted crude antibiotic was dissolved in methanol and stored in −20°C for further purification and analysis. Purification was performed by column chromatography on a silica gel C-200 (22 mm × 200 mm) (Himedia, Mumbai, India). The crude extract dissolved in methanol was re-dissolved in 20 ml of ethyl acetate and applied to the column. After washing with 50 ml of ethyl acetate, the column was eluted with 300 ml of toluene–acetone (4 : 1, v/v). Five millilitre fractions were collected and examined for the presence of the antibiotic by thin-layer chromatography in UV detection chamber against the standard antibiotic. Camag HPTLC instrument (Anchrom, Mumbai, India) was used for quantitative analysis of the antibiotic. The concentration of the antibiotic in the sample was estimated by using a standard curve between the peak area and the concentration (10–80 mg l−1) of the standard antibiotic (Toronto Research Chemicals Inc., Ontario, Canada).

Inorganic carriers

Talcum powder and vermiculite were obtained from Starke & Co. Pvt Ltd, New Delhi, India and Sri Ramamaruthi Vermiculite Mines, Chennai, India, respectively. The average sizes of the particles were 50–80 μm and 40–70 μm for talcum and vermiculite, respectively. One hundred gram of the carrier powder with 20% moisture was autoclaved at 121°C for 20 min. The sterilization was repeated on second and third consecutive days with an overnight incubation in between. The sterile powders were later used for preparation of bioinoculant formulations.

Shelf life assessment of bacterial viability

Both the carriers were tested for the shelf life of the fluorescent pseudomonads over a period of 1 year by standard serial dilution method on King’s B (King et al. 1948) agar plates. The bioinoculant formulations, made using sterilized carriers, were stored at 28°C. The viable cell count was determined on King’s B agar plates at the end of 0, 70, 110, 200, 250 and 360 days of incubation using serial dilution method. The viable bacterial count in the formulations during storage was made by suspending 1 g of formulation in 9 ml of 0·2% sterile saline solution containing 0·1% Tween-80. Plating was done in triplicates and the average of three readings was used to represent viable bacterial count.

Compatibility test

The bacterial cultures were investigated for their compatibility with each other and with Pi by cross-streak assay method on King’s B agar medium + potato dextrose agar (PDA), prepared in 1 : 1 ratio. Piriformospora indica mycelial disc of 8 mm size was placed in the middle of the agar plates and were incubated at 28°C for 4 days. After robust growth of the fungus radially, the other cultures were streaked vertically to the test organism. The plates were incubated at 28°C for two more days for observation.

Formulation preparation

The broth containing 1·5–3·5 × 1011 CFU ml−1 of R62 and R81 strains was used for the preparation of formulations. To 98 ml culture broth, 1 ml glycerol (1·0 g ml−1) and 1 ml carboxymethylcellulose (CMC, 1·0 g l−1) were added. Glycerol served as a carbon source for keeping the cells viable, whereas CMC acted as an adhesive. The broth, containing the additives, was mixed uniformly on a vortex mixer. To make 100 g of inorganic carrier-based formulations, 80 g of three-time sterilized carrier and 20 ml of culture broth with additives were mixed under sterile conditions. Following the method of Vidhyasekaran and Muthamilan (1995), the product was shade-dried to reduce the moisture content to c. 18% and then packed in UV-sterilized polythene bags and sealed. The talcum powder formulations contained 2·5 × 1010 CFU g−1 and 6·3 × 1010 CFU g−1 of R62 and R81 strains, respectively, whereas vermiculite-based formulations contained 3·0 × 1010 CFU g−1 and 2·4 × 1010 CFU g−1 of R62 and R81 strains, respectively. Aseptic conditions were maintained throughout the process. To prepare the formulations of Pi, the fungus was harvested after 8 days when the dry cell weight was 9·5 g l−1 and chlamydospore density was very high (c. 8·0 × 107 ml−1). One hundred millilitres of this broth were mixed with 1 ml CMC solution (1·0 g l−1) for preparing formulations. Twenty millilitres of this broth were used to prepare the fungal formulations in a similar way as described above for bacterial formulations. Both carrier-based formulations contained c. 8 × 106 fungal spores per gram carrier. For preparing 100 g of consortium formulations, the amount of the carrier and volume of the culture broth of the three microorganisms were mixed according to Table 1.

Table 1.   Preparation of 100 g carrier-based consortium formulations of pseudomonads (R62 and R81) and Piriformospora indica (Pi)
Consortium formulations containingAmount of CarrierVolume of culture broth (ml) of
R62*R81*Pi†
  1. *The values in parentheses are the bacterial cell counts in CFU ml−1.

  2. †The values in parentheses are the dry cell weight of Pi in g l−1.

R62 + R8180 g10 ml (2 × 1011)10 ml (2 × 1011)NA
R62 + Pi80 g10 ml (2 × 1011)NA10 ml (9·5)
R81 + Pi80 gNA10 ml (2 × 1011)10 ml (9·5)
R62 + R81 + Pi80 g6·7 ml (2 × 1011)6·7 ml (2 × 1011)6·6 ml (9·5)

Sterilization and bacterization of seeds

Seeds of tomato (cv: Nutech, PUSA RUBY; National Seed Centre, Indian Agricultural Research Institute, Pusa Complex, New Delhi, India) used for glasshouse and field experiments were surface-sterilized with 0·1% sodium hypochlorite for 3 min followed by repeated washing with sterile distilled water. Later, 50 g of tomato seeds were treated with 0·5 g of bioinoculant carrier formulations. For uniform treatment of seeds with bioinoculant formulations, the flasks were kept in an orbital shaker at 500 rev min−1for 2 h. The average bacterial counts on the seeds were about 1 × 104 CFU per seed for talcum powder-based formulations and 1 × 103 CFU per seed for vermiculite-based formulations. The formulations of fungal mycelia rich in chlamydospores were coated on the seeds similarly.

Glasshouse studies

The soil used for both the glasshouse and field studies is sandy loam having pH 6·81, EC 0·3 dS m−1, organic matter 0·81%, total P 12 kg ha−1, total N 95 kg ha−1, Zn 0·545 ppm, Mn 23·1 ppm, Fe 16·81 ppm, Cu 1·01 ppm, and S 9·5 ppm. The soil used for glasshouse experiments was sterilized under moist heat conditions of 121°C for 1 h and the cycle was repeated on two consecutive days with overnight incubation. The seeds were coated only with the sterile inorganic carrier, but without bioinoculants served as control. Six seeds were sown per pot and 64 (8 treatments × 4 replicates × 2 carriers) such pots of 750 ml capacity were maintained in a completely randomized design in the glasshouse. The glasshouse was maintained at 28 ± 4°C. Total 20 replicates (5 plants per pot × 4 pots) of each treatment were considered for analysis after the harvest. The soil was amended with farm yard manure at the rate of 6 t ha−1 considering 2 × 106 kg soil ha−1.

Field studies

The farming system trial was set up at the Organic Farming Block of GBPUAT, Pantnagar, India in July 2008. Initially nursery of tomato plants was raised using bioinoculant-coated seeds in the adjacent field with same soil conditions. After 1 month, the plantlets were uprooted and the roots were dipped in the poly bags of respective bioinoculant formulation with the same dosage as used for seed coating, and then placed in a randomized manner in the actual field. The field experiment was set up in a randomized block design with four replications. Each plot was of 16 m2 (4 m × 4 m). In each plot, there were 64 plants (8 rows × 8 columns) with a distance of 0·5 m between each plant. Totally, there were 28 such plots (7 treatments × 4 replicates) with each plot being a replicate. Total 80 replicates (20 plants per plot × 4 plots) of each treatment were considered for analysis after the harvest. After the harvest of fruits, the remaining crop from all the plots was collected, washed under tap water and assayed for biometric observations. Each plot was fertilized with a farm yard manure at the rate of 6 t ha−1.

Biometric observations

In case of glasshouse pot experiments, the plants were harvested at the end of 45 days to record biometric observations. To observe the effect on growth parameters, root weights and shoot weights were quantified at the end of 45 days. The shoots and roots of the plants were separated and dry weight of shoots and roots was estimated after drying in an oven at 70°C for 48 h. In case of field experimentation, the tomato plants were harvested after 60 days from the day of transplantation of nursery plantlets in the field. Shoots and roots were rinsed in distilled water, dried at 70°C for 48 h and were later grounded using mortar and pestle to make them fine enough to pass through a 0·5 mm screen and digested in conc. H2SO4. The total nitrogen of the digest was measured using Kjeldhal method. The P and K content in the digest were determined chemically and by using flame photometry, respectively (Jackson 1973; Chapman and Pratt 1978). The N, P and K content of the roots and shoots were pooled and reported as mg per gram of plant.

Minimum inhibitory concentration (MIC) of DAPG for inhibition of fungal plant pathogens

The inhibitory effect of DAPG produced by pseudomonads R62 and R81 on growth of Fusarium semitectum, Fusarium graminearum, Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii was assessed on PDA plates. The assay was performed by spreading the plates with various concentrations (50, 100, 150 and 200 mg l−1) of purified DAPG dissolved in methanol on PDA plates. The control plate was spread with the same volume of methanol alone. Later, a 0·6 cm plug from each of the actively growing fungi on PDA plate was placed at the centre of the agar plates. The plates were incubated at 28°C and fungal inhibition was assessed after 14 days by measuring the distance of the edges of the fungal growth from the centre of the plate. The inhibition was expressed as percentage of growth inhibition with respect to the control. The experiment was done in triplicate for all the fungi and for all concentrations of DAPG. The MIC of DAPG is defined as 100% inhibition in the growth of test fungus.

Biocontrol test

The efficacy of powder formulations of fluorescent pseudomonad R81 and Pi in controlling the wilt disease of tomato caused by F. oxysporum was assessed under the glasshouse conditions. The tomato seeds were sown in pots containing sand-soil sterile mixture, incorporated with F. oxysporum culture grown on potato dextrose broth. Fifty millilitre of microconidial suspension of F. oxysporum containing 103 conidia ml−1 were added uniformly to the soil of each pot (Ramamoorthy et al. 2002). The fungal suspension was added once before sowing the seeds, once after germination and finally after 24 days’ growth of tomato plants. The control pots with sterilized soil were taken utmost care from contamination of test fungus by placing the pots away from the treated ones. Six seeds were sown per pot and four pots per replication were maintained. All four replications and pots were arranged in a randomized manner. The wilt incidence of the tomato plants was observed at an interval of 10 days. The disease development in various treatments was monitored every 10 days during three time infestation. The disease intensity was normalized to the scale from 0–9, where 9 indicate the death of plant due to wilt disease. Wilt development on each plant was rated using the following scale, which was the modified form of the scale of Bora et al. (2004): 0 : no symptoms; 1: <10% of leaves with symptoms; 2: 11–15% of leaves with symptoms; 3: 16–25% of leaves with symptoms; 4: 26–40% of leaves with symptoms; 5: 41–55% of leaves with symptoms; 6: 56–70% of leaves with symptoms; 7: 71–85% of leaves with symptoms; and 8: 86–100% of leaves with symptoms. The entire trial was done twice, and data are represented as the means of two trials, each containing four replicates.

Statistical analysis

The experiments were carried out in a completely randomized design for glass house studies and randomized block design for field studies. The experimental data were analysed statistically using anova. Duncan’s multiple range test was used to separate group mean values when anova were significant at P < 0·05 (Gomez and Gomez 1984).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Compatibility amongst the bioinoculants

It was observed from the in-vitro agar plate assay that all the three bioinoculants used in the present study were compatible with each other in the cross-streak assay, as no inhibition zones were observed (Fig. 1).

image

Figure 1.  Compatibility test of fluorescent pseudomonads R62 and R81 (streaked towards the edges) against the fungus Piriformospora indica (radial growth).

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Shelf life of bioinoculant formulations

The shelf life of the formulations was studied at 28°C. The viable CFU g−1 of carrier was enumerated for both R62 and R81 in the bioinoculant formulations. Figure 2 shows the decrease in CFU g−1 with time when strain R81 formulations were stored at 28°C in an incubator for 1 year. Vermiculite was found to have better shelf life in comparison with the talcum powder, as the viable CFU g−1 decreased from 2·4 × 1010 to 6·3 × 107, when compared with 6·3 × 1010 to 4·5 × 106 in case of talcum powder-based formulations. In case of R62, a similar profile was observed (data not shown). In the current studies, inorganic carriers viz. talcum powder and vermiculite increased the shelf life of the PGPR strains to a great extent, as they could be kept for about a year until their application. The viable cell count in the formulations, which was 1·0 × 107 CFU g−1 carrier or more for 6 months, was sufficient to use them for treatment of crops. The shelf life of the bacterial bioinoculants without the carriers was less than 1 month (data not shown).

image

Figure 2.  Variations of the logarithm of CFU per gram carrier vs time during storage of fluorescent pseudomonad R81 inorganic carrier-based formulations, talcum powder (inline image, log10 CFU g−1) and vermiculite (inline image, log10 CFU g−1) at 28°C. The error bars on the y-axis indicate value ± SD (n = 3). The initial CFU per gram carrier was 6·3 × 1010 CFU g−1 and 2·4 × 1010 CFU g−1 for talcum powder and vermiculite, respectively.

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Influence of the bioinoculants on growth responses of tomato plant under glasshouse conditions

All the treatments were delivered as uniform coating around the sterile tomato seeds, which were later, sowed in the pots. A two-way anova was performed to see the effects of treatments and carriers on plant growth responses. There was, in general, significant increase in the growth of the plant in comparison with the control, when these bioinoculants were used (Table 2). Amongst all the treatments R81 + Pi consortium performed extremely well with an increment of 8·8-fold in dry root weight and 16·8-fold in dry shoot weight when talcum was used as the carrier and 8·6-fold in dry root weight and 12·4-folds in dry shoot weight when vermiculite was used as the carrier. To our knowledge, this magnitude of growth was noteworthy and has not been reported previously in the literature. In the case of consortium, of all the three organisms (R62 + R81 + Pi), there was 4·5- and 8·7-fold increase in dry root weight and dry shoot weight when talcum was used as carrier and 5·0- and 9·6-fold increment in dry root weight and dry shoot weight when vermiculite was used as carrier (Table 2). As seen from Table 3a,b, there was a significant main effect of treatments on the dry root and shoot weights of tomato plant as F (7, 48) = 19·368 and F (7, 48)  = 20·720, P < 0·001, respectively. There was also a significant main effect of carriers on the dry root and shoot weights of tomato plant as F (1, 48) = 24·289 and F (1, 48) = 18·232, P < 0·001, respectively. Both talcum powder and vermiculite contained above 109 CFU g−1 of carrier at the time of application, but talcum powder yielded better results in pot experimentation and was selected for the field trials. Moreover, the CFU count per seed was 10 times more in case of talcum powder-based formulations in comparison with vermiculite-based formulations. The compatibility of bioinoculant carrier formulation with the test crop’s seed cannot be ruled out when rest of the additives were maintained constant for both the carriers. This also suggests that texture of the seeds and lattice structure of the carrier are related. Further, R62 + Pi combination was excluded in field trials due to its lower performance when compared with R62 alone in glasshouse conditions (Table 2).

Table 2.   Effect of bioinoculant formulations on growth responses of tomato plants under glasshouse conditions
Treatments withTalcum powder-based formulationVermiculite powder-based formulation
Dry root Weight (g plant−1) Dry shoot weight (g plant−1)Dry root weight (g plant−1)Dry shoot weight (g plant−1)
  1. The growth responses were analysed at the end of 45 days. The values represent the mean of 20 replicates (±SD). Means followed by the same letter in a column are not significantly different from each other according to Duncan’s multiple range test.

None (control)0·020a0·063a0·016a0·059a
R620·087bc0·460bc0·043bc0·387c
R810·103c0·733cde0·066cde0·508cd
Pi0·105c0·934e0·073cde0·568d
R62 + R810·090bc0·882de0·055bcd0·410c
R62 + Pi0·047ab0·226ab0·031ab0·215b
R81 + Pi0·177d1·061e0·137e0·732e
R62 + R81 + Pi0·090bc0·551bcd0·081d0·567d
Table 3.   Two-way anova table between talcum powder and vermiculite carrier for dry root weight estimate. Dependent variable: (a) Dry root weight. (b) Dry shoot weight
SourceSum of squaresd.f.Mean squareFSig.
(a)
 Carriers (A)0·01510·01524·2890·000
 Treatments (B)0·08670·01219·3680·000
 (A × B)0·00470·0010·8600·545
 Error0·031480·001  
 Total0·53064   
 Corrected total0·13663   
(b)
 Carriers (A)0·53510·53518·2320·000
 Treatments (B)4·26070·60920·7200·000
 (A × B)0·50870·0732·4710·030
 Error1·410480·029  
 Total24·19464   
 Corrected total6·71363   

Influence of the bioinoculants on growth responses of tomato plant under field conditions

The delivery of bioinoculants under field conditions was done in two stages. Here, again all the treatments significantly promoted the growth responses in comparison with the control. The consortium of R81 + Pi performed significantly well amongst all the treatments (Fig. 3a,b); there was an increase of 2·6- and 3·1-fold on dry root and dry shoot weight, respectively, when compared with the control treatment. The number of branches and maximum fruit yield was also increased by 3·1- and 2·9-fold, respectively, when R81 + Pi treatment was used (Fig. 4a,b). The increase in growth responses was not as marked as in glasshouse-controlled conditions, indicating the complex soil and environmental dynamics in field conditions.

image

Figure 3.  Effect of talcum powder-based formulations of fluorescent pseudomonad strains R62, R81 and Piriformospora indica (Pi) on (a) dry root weight (g plant−1) and (b) dry shoot weight (g plant−1) of tomato plant under field conditions. The growth responses were analysed at the end of 60 days. The values represent the mean of 80 replicates (±SD). Bars labelled with the same letter represent nonsignificantly different mean values, according to Duncan’s multiple range test (P < 0·05), after anova.

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image

Figure 4.  Effect of talcum powder-based formulations of fluorescent pseudomonad strains R62, R81 and Piriformospora indica (Pi) on (a) number of branches and (b) fruit yield of tomato plant under field conditions. The growth responses were analysed at the end of 60 days. The values represent the mean of 80 replicates (±SD). Bars labelled with the same letter represent nonsignificantly different mean values, according to Duncan’s multiple range test (P < 0·05), after anova.

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Influence of bioinoculants on nutrient uptake

The digests of harvested root and shoot parts of the tomato plant obtained after various treatments were analysed for N, P and K contents. In line with the field results in terms of dry root and dry shoot weights, the N, P and K per gram plant were also enhanced when the bioinoculant treatments were used. There was 178%, 102% and 126% increase in N, P and K, respectively, when R81 + Pi was used (Fig. 5). These findings clearly depict that the control crop was deprived of enough N-P-K, as there was significant increase in these values when bioinoculants were used.

image

Figure 5.  Effect of talcum powder-based formulations of fluorescent pseudomonad strains R62, R81 and Piriformospora indica (Pi) on nutrient uptake (a) nitrogen (mg g−1 plant) and (b) phosphorus (mg g−1 plant) (c) potassium (mg g−1 plant) of tomato plant after 60 days. The values represent the mean of 80 replicates (±SD). Bars labelled with the same letter represent nonsignificantly different mean values, according to Duncan’s multiple range test (P < 0·05), after anova.

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Antagonistic effect of pseudomonad R81 on soil-borne fungal pathogens

The fluorescent pseudomonad R81 produces the antibiotic DAPG (Gaur et al. 2004). Five soil-borne fungal pathogens viz. F. semitectum, F. graminearum, F. oxysporum, R solani and Sclerotium spp. were used to assay antifungal activity of purified DAPG. The 90% purified DAPG exhibited antibiotic activity against all the pathogens. The increasing concentrations of DAPG were found to decrease the radial growth of the pathogens on potato-dextrose agar plates. The minimum inhibitory concentration of DAPG against all the pathogens was found to be c. 150 mg l−1 of DAPG (results not shown). These results against Fusarium spp. and R. solani were in agreement with the work reported by Keel et al. (1992).

Biocontrol of Fusarium wilt of tomato

Biocontrol assay was performed in glass house conditions. The disease suppression of Fusarium wilt of tomato by the fluorescent pseudomonad R81 and Pi in glasshouse microplots was enumerated. Talcum-based formulations of following organisms were used for biocontrol studies (a) R81 (b) Pi and (c) R81 + Pi. Although all the three treatments significantly showed their efficacy against Fusarium wilt the treatment R81 out-performed the other treatments (Fig. 6). The normalized disease incidence values for all the treatments are shown in Table 4. Fluorescent pseudomonads are well documented as biocontrol agents against fungi causing plant root disease (Keel et al. 1992; O’Sulivan and O’Gara 1992).

image

Figure 6.  Effect of talcum powder-based formulations of fluorescent pseudomonad strains R81 and/or Piriformospora indica (Pi) on Fusarium wilt incidence (a) dry root weight (g plant−1) and (b) dry shoot weight (g plant−1) of tomato plant after 45 days. The values represent the mean of 20 replicates (±SD). Bars labelled with the same letter represent nonsignificantly different mean values, according to Duncan’s multiple range test (P < 0·05), after anova. (inline image) Without Fusarium Oxysporum and (inline image) With Fusarium onysprum.

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Table 4.   Effect of seed treatment with talc-based formulations of fluorescent pseudomonad R81 and Piriformospora indica on disease intensity of Fusarium wilt on tomato plant
Days after sowingDisease intensity in grade values (0–9 scale)
Control (TP)TP + R81TP + PiTP + R81 + Pi
  1. The growth responses were analysed at the end of 45 days. The values represent the mean of 20 replicates (±SD).

108454
208344
307343
407343

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Microbial antagonism against pathogenic agents decreases the frequency of root infections (Schippers et al. 1987). Plant growth promotion can also be ascribed to metabolites such as growth substances, which affect the plant physiology (Glick 1995). Specific bacterial metabolites may elicit defence reactions of the host plant (ISR) (van Loon et al. 1998); consequently, some fluorescent pseudomonads are known to improve plant health and/or growth (Lemanceau and Alabouvette 1993; Gamalero et al. 2004). They have been shown to play a role in the natural suppressiveness of soils to Fusarium wilts (Alabouvette and Lemanceau 1996) and to take-all disease of wheat (Raaijmakers and Weller 1998). The facultative symbiont Pi has tremendous potential to be used as biofertiliser, biopesticide and a tool for biological hardening of micropropagated plants (Varma et al. 1999; Waller et al. 2005; Yadav et al. 2010). The fungus induces systemic disease resistance by enhancing the concentration of antioxidants, ascorbate and glutathione, in the plant body to cope up with the oxidative stress caused by pathogens (Waller et al. 2005; Vadassery et al. 2009; Zuccaro et al. 2009).

In all studies done so far with fluorescent pseudomonad strains R62 and R81 and with Pi, their formulations were not used; only their fresh cultures were either applied as seed coating or directly mixed with the soil (Waller et al. 2005; Roesti et al. 2006). Hence, there was a need for development of bioinoculant formulations for improved shelf life in case of pseudomonads and for easy and controlled delivery of these microorganisms to the host plants. The compatibility of PGPR consortia with mycorrhiza was already established by Roesti et al. (2006). The compatibility of R62 and R81 with Pi was established in this study. The shelf life of pseudomonads was observed to be approximately 1 year in both talcum- and vermiculite-based formulations in comparison with 6 months as observed by Vidhyasekaran and Muthamilan (1995) for talc-based formulations. The moisture retention capacity of vermiculite could be the reason behind its better shelf life. The results obtained in synergy of R81 strain with Pi are promising and noteworthy. Here, the combination of the fluorescent pseudomonad R81 and Pi out-performed all other treatments under both glasshouses as well as under field conditions for tomato plants. The plausible reason is that the niches of roots of the plant where the PGPR and Pi act are quite different. PGPR rigorously colonize the roots, whereas Pi is root endophyte, which symbiotically spreads internally. In previous studies by Waller et al. (2008), the chlamydospores of Pi germinated on the root surface of barley plant, with the growing hyphae closely attached to rhizodermal cell walls and after 24–36 h of inoculation, hyphae penetrated into intercellular spaces between rhizodermal cells. Presumably, the inoculated plants possessed better root density for improved uptake of the nutrients from the rhizosphere. Both the PGPR strains are phosphate solubilizers (Roesti et al. 2006) and Pi produces significant amounts of acid phosphatases for the mobilization of a broad range of insoluble, condensed or complex forms of phosphate, enabling the host plant for the accessibility of adequate phosphorus from immobilized reserves in the soil (Singh et al. 2000). Similar results were observed by Wong et al. (1999) where an increase in dry weight of Brassica chinensis and Zea mays L indicated a better nutrient status in terms of N, P and K of the plants treated with manure compost. Pseudomonas spp. produce several antifungal substances such as pyoluteorin, 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid, pyrrolnitrin and hydrogen cyanide, which contribute to the suppression of the plant diseases (Haas and Défago 2005; Lugtenberg and Kamilova 2009). Induction of defence proteins and accumulation of phenolics by P. fluorescens isolate Pf1 against challenge inoculation with F. oxysporum f.sp. lycopersici in tomato has been reported by Ramamoorthy et al. (2002). It has also been reported that Pi can potentially induce resistance to fungal diseases and tolerance to salt stress in the monocotyledonous barley plant (Waller et al. 2008). These authors also correlated the defence mechanism and overall increase in grain yield of barley plant with an elevated antioxidative capacity of this fungus due to an activation of the glutathione–ascorbate cycle. In the current study, strain R81, which was capable of producing DAPG and hydroxamate type siderophore and Pi, which was able to induce systemic resistance, it seems probable that the combined action of DAPG, siderophores and ISR was responsible for more effective biological control of the Fusarium wilt on tomato plants. Such types of multiple microbial interactions involving bacteria and fungi in the rhizosphere have been shown to provide enhanced biocontrol in comparison with single biocontrol agents (Whipps 2001).

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The authors would like to thank Dr Alok Adholeya, The Energy and Resources Institute, New Delhi, India for useful suggestions and technical help.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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