Microbial consortium–mediated reprogramming of defence network in pea to enhance tolerance against Sclerotinia sclerotiorum

Authors


Harikesh Bahadur Singh, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India. E-mail: hbs1@rediffmail.com

Abstract

Aims:  To evaluate the potentiality of three rhizosphere microorganisms in suppression of Sclerotinia rot in pea in consortia mode and their impact on host defence responses.

Methods and Results: Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 from rhizospheric soils were selected based on compatibility, antagonistic and plant growth promotion activities. The microbes were used as consortia to assess their ability to trigger the phenylpropanoid and antioxidant activities and accumulation of proline, total phenol and pathogenesis-related (PR) proteins in pea under the challenge of the soft-rot pathogen Sclerotinia sclerotiorum. The triple-microbe consortium and single-microbe treatments showed 1·4–2·3 and 1·1–1·7-fold increment in defence parameters, respectively, when compared to untreated challenged control. Activation of the phenylpropanoid pathway and accumulation of total phenolics were highest at 48 h, whereas accumulation of proline and PR proteins along with activities of the antioxidant enzymes was highest at 72 h.

Conclusions:  The compatible microbial consortia triggered defence responses in an enhanced level in pea than the microbes alone and provided better protection against Sclerotinia rot.

Significance and Impact of the Study:  Rhizosphere microbes in consortium can enhance protection in pea against the soft-rot pathogen through augmented elicitation of host defence responses.

Introduction

Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan necrotrophic fungal pathogen, attacking more than 500 species of higher plants (Willetts and Wong 1980) and causes numerous soft rots of horticultural and agricultural crops. Severe outbreaks of soft rot occur where very humid and cool conditions prevail in fields. Damage from soft rot is a cause of concern in many countries including India. The fungus survives in soil as sclerotia, which are hardened structures composed of compact fungal mycelium. Sclerotia germinate carpogenically to produce apothecia, which release ascospores to serve as the primary source of inoculum for infection to develop in crops.

Epidemics of outbreaks of Sclerotinia rot have been consistently reported in pea production areas worldwide (Kim et al. 2006). The pathogen can infect all above-ground foliage, and lesions produced from infection are characterized by a white cotton-like mass of mycelium growing on the surface of stem, leaf or pod tissue. Infected tissue becomes soft and slimy with a water-soaked appearance. The persistence of sclerotia of the pathogen in the soil for long periods and its wide host range prevent any management of this disease using crop rotations. Microbe-mediated suppression of the pathogen in the rhizosphere can be a suitable management strategy (Whipps 2001). Biological control has received considerable attention because of the unavailability of resistant sources in most crops and increasing concerns over fungicide resistance in populations of S. sclerotiorum and fungicide residues in the environment (Pan 1998; Gossen et al. 2001). Therefore, use of biocontrol agents (BCAs) appears as the most economically feasible practical strategy to reduce the inoculum potential of Sclerotinia. Although several BCAs have been studied for controlling S. sclerotiorum rot in different crops (Fernando et al. 2007; Abdullah et al. 2008; Zhang and Xue 2010), little information is, however, available on biocontrol of Sclerotinia rot of peas. Among the BCAs, Coniothyrium minitans, commercialized as ‘Contans’, was reported to have good potentiality to suppress Sclerotinia rot (Vrije et al. 2001; Dillard and Cobb 2002).

An emerging trend in using these BCAs is to apply a mixture of compatible but diverse group of microorganisms. Multiple organisms in a biocontrol preparation can be superior to product of single organism to suppress plant disease, provide enhanced efficacy, consistency and reliability from field to field (Stockwell et al. 2011). Combining microorganisms increases the possibility of synergistic action of the multiple antagonistic and stress tolerant traits of the released BCAs. Interestingly, several researchers have observed increased plant growth and improved disease control using microbial consortia comprising of various biocontrol organisms such as Trichoderma, Pseudomonas, Bacillus spp., etc. in wheat, radish, chickpea, tomato, pepper, Arabidopsis and pigeon pea (Duffy et al. 1996; Rudresh et al. 2005; Jetiyanon 2007; Kannan and Sureendar 2009; Srivastava et al. 2010).

Plant defence responses are typically triggered by the contact with phytopathogenic microorganisms, but they can also be stimulated by beneficial microbes (Shoresh et al. 2010). Induce systemic resistance (ISR) has gained considerable attention as an important phenomenon that occurs when plants develop enhanced defensive capacity in response to an appropriate elicitor, rendering spatially distant plant parts with resistance to the pathogens. These ISR elicitors are perceived by plants to ultimately give rise to an exaggerated immune response. ISR is associated to reprogramme and mobilize defence-related enzymes, such as those pathogenesis-related (PR) proteins, phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), superoxide dismutase (SOD) (Chen et al. 2000; Jetiyanon 2007; Magnin-Robert et al. 2007), and induce accumulation of proline and phenols. The increased production of enzymes and phenolic compounds may be of key importance in the resistance process observed in plants (Mandal and Mitra 2007).

Very limited knowledge is available regarding use of microbial consortium for management of S. sclerotiorum. Therefore, the main objective of this study was to evaluate the potential of BCAs viz., isolates of Trichoderma harzianum ARS culture collection number NRRL 30596, Bacillus subtilisJN099686 and Pseudomonas aeruginosaJN099685 in consortium for management of Sclerotinia rot in pea under greenhouse conditions and to understand the host defence responses mediated by the microbial consortium. Detection of any activation of ISR would indicate the ability of microbes in single and consortium mode to reprogramme plant gene expression as a primary method of pathogen control.

Materials and methods

Isolation and multiplication of the pathogen

An isolate of S. sclerotiorum was obtained from an infected pea plant uprooted from the Agricultural Farm, Banaras Hindu University, Varanasi. The sclerotium was surface sterilized in 70% ethanol for 2 min, rinsed twice in sterile distilled water, and bisected, and one of the two sclerotial halves was transferred to potato dextrose agar (PDA, MO96; Himedia, Mumbai, India) in a Petri dish with the freshly cut surface towards the agar and incubated at 25 ± 2°C in darkness for 4 days. The isolate, developed from a single mycelial PDA plug, was stored at 4°C.

Isolation of antagonistic microbes

Antagonistic microbes viz., fluorescent Pseudomonas and Bacillus were isolated from rhizospheric soil samples collected from various agroclimatic regions in India (Dehradun, Jaipur, Jaunpur, Hyderabad, Patna and Varanasi). Rhizosperic soil (1 g) from different crops (Abelmoschus esculentus, Capsicum annum, Cicer arietinum, Phaseolus vulgaris, Pisum sativum, Raphanus sativus, Solanum melongena, Solanum lycopersicum) was suspended separately in 10 ml of sterile distilled water and vortexed for 5 min. For isolation of Bacillus spp., the suspension was held at 75°C for 20 min to kill all vegetative cells. The suspension was further serially diluted. Fluorescent pseudomonads were isolated by the pour plate method on King’s B medium (King et al. 1954), while Bacillus was blotted by spread plate method, using nutrient agar (NA; Himedia, MV001), and Trichoderma was isolated on selective medium (Elad et al. 1981). Some Trichoderma isolates were also obtained from National Botanical Research Institute, Lucknow. The bacterial isolates were tentatively identified on the basis of their morphological characteristics. Pure cultures of these organisms were maintained on respective agar slants and stored at 4°C for further use.

Detection of antagonistic activity in vitro assay

Evaluation of potential biocontrol bacteria (Pseudomonas and Bacillus) and Trichoderma against S. sclerotiorum was carried out in vitro using dual-culture plate assay. The bacterial isolates were streaked individually with sterile inoculating loop 10 mm from the edge of the plates containing PDA. Similarly, Trichoderma was screened by inoculating a 5-mm mycelial plug. In the same Petri dishes perpendicular to the bacterial streak or Trichoderma fungal disc, a 5-mm mycelial disc of 5-day-old pathogen culture was placed 6 cm away at the opposite side of the Petri dish. Plates were incubated at 27 ± 2°C, and growth of the pathogen mycelia towards the bacterial colony or fungal disc was observed, and inhibition zones were measured 5 days after incubation. The experiment was repeated thrice.

Selection of compatible microbes

Compatibility test between Bacillus and fluorescent Pseudomonas isolates was performed on NA. Bacillus colonies were streaked on the centre of the plate, followed by spraying of 48-h-old culture of fluorescent Pseudomonas using an atomizer. Compatible bacterial isolates showed no zone of inhibition and were further used to check compatibility with Trichoderma isolates on PDA. A 5-mm-diameter mycelial plug from actively growing culture of Trichoderma was kept at the centre of the plate and Bacillus and Pseudomonas isolates were streaked on either side. Growth of Trichoderma was recorded after 5 days. Any overgrowth of Trichoderma on bacterial streaks without a zone of inhibition was considered compatibility of Trichoderma with bacterial isolates. Based on antagonistic activity and compatibility, bacterial strains PJHU15 and BHHU100 were selected, and 16S rDNA sequence results confirmed the strains to be Ps. aeruginosa (GenBank accession: JN099685) and B. subtilis (GenBank accession: JN099686), respectively. Pseudomonas aeruginosa and B. subtilis were isolated from rhizosphere of P. sativum (Jaipur) and P. sativum (Hyderabad), respectively. The Trichoderma isolate TNHU27 selected for the experiments was priorly identified as Trichoderma harzianum (ATCC no. PTA-3701) and was isolated from an agricultural farm (Pantnagar).

Seed treatment with bioagent

The bacterial strains PJHU15 and BHHU100 were grown on NA for routine use and maintained in nutrient broth (NB; Himedia M002) with 20% glycerol at −80°C for long-term storage. Single colony of B. subtilis and Ps. aeruginosa was transferred to 500-ml flasks containing 200 ml of NB and were grown on a rotating shaker (150 rev min−1) for 48 h at 27 ± 2°C. The bacterial suspension was centrifuged at 6000 g for 10 min at 4°C and washed twice with sterile distilled water. The final pellet was resuspended in a small quantity of sterile distilled water, and the final concentration was adjusted to 4 × 108 CFU ml−1 using ‘Thermo Scientific UV 1’ spectrophotometer. Similarly, T. harzianum was grown on PDA for 6 days at 27 ± 2°C, and the spores were harvested and brought to a final concentration of 2 × 107 CFU ml−1.

Greenhouse experiment

Soil mixture containing sandy soil, vermicompost and farmyard manure (2 : 1 : 1) was sterilized in an autoclave at 15 lbs pressure for 30 min on three consecutive days, and 1·5 kg of the mixture was filled in each plastic pot (15 × 10 cm). The following treatments were examined: (i) B. subtilis (BHHU100), (ii) T. harzianum (TNHU27), (iii) Ps. aeruginosa (PJHU15), (iv) BHHU100 + TNHU27, (v) BHHU100 + PJHU15, (vi) TNHU27 + PJHU15 and (vii) BHHU100 + TNHU27 + PJHU15. Seeds of pea (Pisum sativum L. cv. Arkel) were surface sterilized with 1% sodium hypochlorite for 30 s, rinsed twice with sterile distilled water and dried under a sterile air stream. The seeds were coated with B. subtilis, Ps. aeruginosa and T. harzianum either singly or in dual or triple combinations with the suspensions of the organisms prepared in 1% CMC (carboxymethyl cellulose), used as adhesive. For coating, seeds were soaked in their respective suspensions for 10 h [in the case of consortia, equal amount of suspension (v/v) was mixed]. Then, the microbial suspension was drained off and the seeds were dried overnight in sterile Petri dishes. Two sets of untreated control plants were maintained. For each treatment, five pots were used and six seeds were sown in each pot. The pots were placed in the greenhouse, and irrigation was provided as required or at 2 days interval till partial saturation. A cycle of 10 h dark/14 h light and temperature of 22 ± 2°C were maintained in the greenhouse. The experimental set up was completely randomized.

The pathogen was multiplied on bajra (Pennisetum typhoides Pers.) seed meal-sand medium (bajra seed 250 g, washed white sand 750 g, distilled water 250 ml) at 25 ± 2°C for 15 days (Sarma et al. 2007). Colonized culture was blended well prior to use as inoculum and inoculated on the collar region of the plants in all the treatments at the rate of 50 g per pot after 4 weeks of sowing. One set of control plants was left unchallenged. A cycle of 10 h dark/14 h light and temperature of 18 ± 2°C were maintained in the greenhouse. The total number of infected plants was recorded after 10 days postinfection, and a second reading after 20 days of the first reading was taken to determine per cent mortality. Mortality of the plants was indicated by withering of the leaves and stems following soft, watery rot with white, mouldy growth on the stems. The whole experiment was repeated once, and these data were pooled.

Sample collection for biochemical analysis

From each treatment, randomly five plants were uprooted (one from each pot) carefully without causing any damage at 24 h intervals after the pathogen inoculation up to 96 h. Nodal leaves (3rd to 5th nodes) from the bottom were collected as samples. Collected leaves were washed in running tap water, dried with blotting paper and stored in a deep freezer (−80°C) until used for biochemical analysis.

Phenylalanine ammonia-lyase (PAL) assay

Leaf sample of 0·1 g from each of the treatments was homogenized in 2 ml of 0·1 mol l−1 sodium borate buffer (pH 7·0; 4°C) containing 1·4 mmol l−1β-mercaptoethanol and centrifuged at 16 000 g at 4°C for 15 min. The supernatant was used as enzyme source. To the reaction mixture containing 0·2 ml of enzyme extract, 0·5 ml of 0·2 mol l−1 borate buffer (pH 8·7) and 1·3 ml of water were added. The reaction was initiated by the addition of 1 ml of 0·1 mol l−1l-phenylalanine (pH-8·7) and incubated for 30 min at 32°C. The reaction was terminated by addition of 0·5 ml of trichloroacetic acid (TCA, 1 mol l−1). PAL (EC 4.1.3.5) activity was measured following the formation of trans-cinnamic acid at 290 nm as described by Brueske (1980) and was expressed in terms of μmol l−1 TCA per g fresh weight (FW).

Total phenolic content (TPC)

The TPC was determined following the method of Zheng and Shetty (2000). Leaf tissue (0·1 g) was placed in 5 ml of 95% ethanol and kept at 0°C for 48 h. The samples were homogenized individually and centrifuged at 13 000 g for 10 min. To 1 ml of the supernatant, 1 ml of 95% ethanol and 5 ml of sterile distilled water and 0·5 ml of 50% Folin–Ciocalteau regent were added, and the content was mixed thoroughly. After 5 min., 1 ml of 5% sodium carbonate was added, the reaction mixture was allowed to stand for 1 h and the absorbance of the colour developed was recorded at 725 nm. Standard curves were prepared for each assay using various concentrations of gallic acid (GA; Sigma-Aldrich-27645) in 95% ethanol. Absorbance values were converted to mg GA equivalents (GAE) per g FW.

Polyphenol oxidase (PPO) assay

Leaf samples (0·1 g) were homogenized in 2 ml of ice-cold phosphate buffer (0·1 mol l−1, at pH 6·5). The homogenate was centrifuged at 16 000 g for 30 min at 4°C, and the supernatant, thus, obtained was used directly in the enzyme assay. The reaction mixture contained 0·4 ml catechol (1 mmol l−1) in 3 ml of 0·05 mol l−1 sodium phosphate buffer (pH 6·5) and 0·4 ml enzyme extract. Reaction mixture containing only substrate served as control. Catechol was used as substrate for PPO (EC 1.14.18.1), and the increase in absorbance was recorded at 405 nm (Gauillard et al. 1993). The linear portion of the activity curve was used to express PPO enzyme activity as change in OD per min per g FW.

Superoxide dismutase (SOD) assay

SOD (EC 1.15.1.1) activity was assayed following the method of Fridovich (1974) by measuring the ability of enzyme extract from samples to inhibit photochemical reduction of nitroblue tetrazolium (NBT) chloride. Fresh leaves (0·1 g) from each of the treatments were homogenized in 2·0 ml of extraction buffer (0·1 mol l−1 phosphate buffer containing 0·5 mmol l−1 EDTA at pH 7·5) in a prechilled mortar and pestle. The homogenate was centrifuged at 15 000 g for 20 min at 4°C. The reaction mixture contained 200 mmol l−1 methionine, 2·25 mmol l−1 NBT, 3 mmol l−1 EDTA, 100 mmol l−1 phosphate buffer (pH 7·8), 1·5 mol l−1 sodium carbonate and enzyme extract. The final volume was maintained to 3 ml. Reaction was started by adding 2 μmol l−1 riboflavin (0·4 ml), and the tubes were illuminated with two 15-W fluorescent lamps for 15 min. Reaction mixture without enzyme served as control. The reaction was terminated by putting the light off and keeping the tubes in dark until the absorbance was recorded at 560 nm. One unit of the SOD activity was defined as the amount of enzyme reducing the absorbance to 50% in comparison to control lacking enzyme.

Peroxidase (PO) assay

PO (EC 1.11.1.7) activity was assayed by the method of Hammerschmidt et al. (1982), with slight modification. Leaf samples (0·1 g) were homogenized separately in 2 ml of 0·1 mol l−1 phosphate buffer (pH 7·0), at 4°C, centrifuged at 16 000 g at 4°C for 15 min and the supernatant was used as enzyme source. The reaction mixture consisted of 1·5 ml pyrogallol (0·05 mol l−1), 0·05 ml enzyme extract and 0·5 ml H2O2 (1% v/v). Reaction mixture without enzyme served as control. The changes in the absorbance at 420 nm were recorded after 30 s intervals for 3 min. The enzyme activity was expressed as change in the U per min per g FW.

PR-2 protein assay

For PR-2 protein (β-1,3-glucanase, EC 3.2.1.39) assay, plant samples (0·1 g) were homogenized with 0·05 mol l−1 sodium acetate buffer (2·0 ml; pH 5·0) and centrifuged at 16 000 g for 15 min at 4°C. The reaction mixture consisted of 0·25 ml dialyzed enzyme solution, 0·3 ml sodium acetate buffer (1 mol l−1; pH 5·3) and 0·5 ml laminarin (4%). The reaction was carried out at 40°C for 60 min and stopped by adding 0·375 ml dinitrosalicylic acid, following heating for 5 min in boiling water bath. The content was vortexed, and the absorbance was recorded at 500 nm (Pan et al. 1991). The enzyme activity was expressed as μmol equivalent glucose released per min per g FW.

PR-3 protein assay

For PR-3 protein (chitinase, EC 3.2.1.14) assay, colloidal chitin was prepared by acetylation of glycol chitosan following the method of Trudel and Asselin (1989). The activity was assayed following the method of Boller and Mauch (1988). Plant samples (0·1 g) were homogenized in 2·0 ml of sodium citrate buffer (pH 5·0) and centrifuged at 16 000 g for 15 min at 4°C. The reaction mixture consisted of 0·01 ml enzyme extract and 0·1 ml colloidal chitin (10 mg). After 2 h of incubation at 37°C, the reaction was stopped by centrifugation at 8000 g for 3 min. An aliquot of supernatant (0·3 ml) was taken in a glass reagent tube containing 0·03 ml of 1 mol l−1 potassium phosphate buffer (pH 7·1) and was incubated with 0·02 ml desalted snail helicase. The final mixture was incubated with 2·0 ml dimethylaminobenzaldehyde for 20 min at 37°C, and the absorbance was recorded at 585 nm. N-acetylglucosamine (GlcNac) was used as standard, and enzyme activity was expressed as μmoles GlcNAc equivalent per min per g FW.

Free proline content

Proline content was measured as described by Bates et al. (1973). About 0·1 g of leaf tissues was homogenized with 5 ml of sulphosalicylic acid (3%) in a prechilled mortar and pestle. The homogenate was centrifuged at 10 000 g for 15 min. Two millilitres of the extract was reacted with 2 ml of glacial acetic acid and 2 ml ninhydrin (1·25 g ninhydrin warmed in 30 ml glacial acetic acid and 20 ml 6 mol l−1 phosphoric acid until resolved) in a water bath (100°C) for an hour. The reaction was terminated in an ice bath to stabilize the purple colour of the extract and was brought to room temperature. Four millilitres of toluene was added to each tube and vortexed for 15–20 s. The absorbance of top purple aqueous layer was recorded at 520 nm in a spectrophotometer. The concentration of proline samples was determined according to the standard curve plotted with known concentrations of l-proline.

Statistical analysis

Values from different experiments shown in figures are mean ± standard deviation (SD) of at least five replications of each of the experiments. All the data collected in this study were subjected to analysis of variance (anova). The treatment mean values were compared by Duncan’s multiple range test at  0·05 significance level. The software used for analysis was spss ver. 16 (SPSS Inc., Chicago, IL).

Results

Antagonistic activity of microbial isolates

Among 100 fluorescent Pseudomonas isolates tested, 74 isolates reduced the mycelia growth of S. sclerotiorum significantly at  0·05. Of these Pseudomonas isolates, PJHU15 showed zone of inhibition of 2·7 ± 0·6 cm, followed by isolates PJHU07 and PJHU27, which showed the mycelia inhibition of 2·9 ± 0·4 and 2·0 ± 0·8 cm, respectively. Similarly, the zone of inhibition recorded for Bacillus spp. was maximum (2·3 ± 0·9 cm) for isolate BHHU100 followed by BHHU04 (2·0 ± 0·9 cm). Among the Trichoderma isolates tested, TNHU27 showed maximum inhibition of 73·3% over control followed by PP41 (70·4%).

Compatibility among BCAs

All three microbial isolates (Ps. aeruginosa PJHU15, B. subtilis BHHU100 and T. harzianum TNHU27) were found compatible with one another and used for further experimentation (Fig. 1). Treatment comprising of all the three microbes also showed plant growth promotion traits in greenhouse trial (data not shown).

Figure 1.

 Compatible and incompatible isolates of Trichoderma harzianum and bacterial isolate (left and right, respectively).

Reduction of mortality

Three days after challenge inoculation of pea plants with S. sclerotiorum, soft brown lesions had developed in the collar region in the control plants. All the treatments showed significant reduction in plant mortality because of S. sclerotiorum compared with untreated challenged control (Fig. 2). Mortality was reduced to maximum when all three microbes were used (17·2% mortality) compared with the untreated control plants where 67·7% mortality occurred.

Figure 2.

 Mortality in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatment results taken at the same time interval according to Duncan’s multiple range test at  0·05.

Effect of microbial consortium on TPC in leaves

Phenols accumulation in plants treated with microbial agents were significantly higher compared with untreated plants challenged or unchallenged with S. sclerotiorum (Fig. 3). Small quantities of phenols were also detected in unchallenged healthy control in all the assays. Seeds treated with a consortium of B. subtilis, Ps. aeruginosa and T. harzianum showed higher level of phenols in leaves, which was 1·9 times higher than its corresponding challenged control. However, the level of phenol was 10·3 times higher than untreated unchallenged control at 48 h after pathogen challenge. In general, TPC was found to be maximum in consortia of three organisms, followed by dual species consortia and treatment with single bioagent. A downward trend, however, appeared after maximum induction at 48 h.

Figure 3.

 Total phenolic condent at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatment results taken at the same time interval according to Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Effect of microbial consortium on PAL and PPO activity

PAL level increased significantly with all BCAs up to 48 h, followed by a decline in its activity (Fig. 4). Three-species microbial consortium followed by two-species microbial consortium supported a greater PAL activity at all time intervals as compared to singly treated microbes and untreated challenged control. The three-microbe consortium supported 1·9- and 4·5-fold higher PAL activity at 48 h as compared to their corresponding challenged and unchallenged controls and was significantly higher when compared to all other treatments. A similar increase in PPO activity was also recorded in all the treatments with BCAs as compared to their untreated control counterpart (Fig. 5). The maximum activity of PPO was, however, observed at 72 h in all the treatments and thereafter it declined. The three-microbe mixture recorded 1·6- and 4·6-fold higher PPO activity when compared with pathogen-inoculated and untreated controls, respectively. A small amount of PAL and PPO activity was consistently recorded in unchallenged healthy control.

Figure 4.

 Phenylalanine ammonia-lyase activity at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatments within the results taken at the same time interval according Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Figure 5.

 Polyphenol oxidase activity at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatments within the results taken at the same time interval according Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Effect of microbial consortium on PO and SOD activity

PO and SOD activities increased consistently and attained their maximum levels at 72 h in all primed plants (Figs 6 and 7). After challenge inoculation, an increase in SOD activity was observed until 72 h; maximum activity being supported in three-species microbial consortium viz., 1·8- and 2·4-fold higher than challenged and unchallenged controls, respectively. PO activity reached its maximum level at 48 h in untreated challenged plants and an increase of 2·2- and 2·1-fold in PO activity was recorded in the three-microbe-consortium-treated plants as compared to challenged and unchallenged controls, respectively, at 72 h. The PO activity was found to be significantly higher when compared with other treatments. A gradual decline in PO activity was, however, observed at 96 h in all treatments except untreated pathogen-challenged plants, which showed a sharp decline in its PO activity at 72 h. In healthy control plants, a significantly lower level of PO and SOD activity was detected as compared to other treatments, and it remained nearly unchanged during the entire experimental period.

Figure 6.

 Peroxidase activity at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatments within the results taken at the same time interval according Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Figure 7.

 Superoxide dismutase activity at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatments within the results taken at the same time interval according Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Effect of microbial consortium on PR proteins

Both chitinase and β-1,3-glucanase were found to increase after pathogen challenge in response to BCAs (Figs 8 and 9). The three-microbe consortium supported about 1·4- and 1·8-fold increase in chitinase activity at 72 h as compared to challenged and unchallenged controls, respectively, and was simultaneously found to be significantly higher when compared to other treatments. Similarly, β-1,3-glucanase activity was also found to be 1·4- and 4·6-fold higher in pea leaves treated with three-species microbial consortium at 72 h when compared to challenged and unchallenged controls, respectively. Thereafter, a declining trend in β-1,3-glucanase activity was observed in all the treatments.

Figure 8.

 Chitinase activity at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatments within the results taken at the same time interval according Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; inline image) infected control.

Figure 9.

 β-1,3-glucanase activity at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatments within the results taken at the same time interval according Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Effect of microbial consortium on free proline content

Proline accumulation increased significantly in all the treatments up to 72 h (Fig. 10). Maximum proline content was found in the two-microbe consortium of T. harzianum + Ps. aeruginosa, followed by the three-microbe consortium. Both of these treatments were nonsignificant when compared to each other, but was significantly higher than all the other treatments. The three-microbe consortium recorded 2·3- and 1·9-fold increase in proline content at 72 h as compared to challenged and unchallenged control. In unchallenged healthy control plants, little proline content was detected, which remained nearly unchanged during the experimentation period.

Figure 10.

 Free proline concentration at different time interval in pea raised from seeds treated with Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 either singly or in combination and challenged with Sclerotinia sclerotiorum. Results are expressed as means of five replicates, and vertical bars indicate standard deviations of the means. Different letters indicate significant differences among treatment results taken at the same time interval according to Duncan’s multiple range test at  0·05. (inline image) Untreated control; (inline image) BHHU100; (inline image) TNHU27; (inline image) PJHU15; (inline image) BHHU100 + TNHU27; (inline image) BHHU100 + PJHU15; (inline image) TNHU27 + PJHU15; (inline image) BHHU100 + TNHU27 + PJHU15; (inline image) infected control.

Discussion

Plants contain an array of cellular mechanisms to defend themselves against invading pathogens. Beneficial microbes may alter the cellular mechanisms and reprogramme metabolism, in response to specific stimuli, which would be visible upon subsequent challenge with a pathogen (Van der Ent et al. 2009). Plants treated with BCAs become primed to respond faster and show stronger activation of cellular defence responses after pathogen challenge compared with unprimed plants (Conrath et al. 2006). Such cellular responses also include an earlier oxidative burst and a stronger upregulation of defence genes (Ahn et al. 2007). Results from the present investigation indicated that the pea plants pretreated with bioagents in consortia exhibited higher activities of defence-related enzymes and accumulated phenols in leaves upon challenge inoculation with S. sclerotiorum compared with control. Interestingly, the enzyme activities and phenol accumulation were even higher (1·4- to 4·6-folds) in the triple-compatible microbial consortium compared with their individual and dual consortia effort. Microbial consortium, consisting of the three BCAs, viz., Ps. aeruginosa, B. subtilis and T. harzianum, together with host plant and pathogen apparently interact in a way that mimics the interaction that occurs in rhizosphere under natural conditions. Higher induction of defence proteins and enzymes in the microbe-treated plants in the present study can be correlated as a defence response triggered against S. sclerotiorum invasion in pea. Inhibitory activities of the three microbial species used in the present study against S. sclerotiorum are also reported in several previous studies (Fernando et al. 2007; Abdullah et al. 2008). However, none had combined all the three BCAs together to test their efficacy against the pathogen. Similar observations were made by Fernando et al. (2007), and they showed that bacterial antagonists Pseudomonas chlororaphis PA-23 and Bacillus amyloliquefaciens BS6 significantly reduced stem rot on canola petals caused by S. sclerotiorum under field conditions by inhibiting ascospore germination and triggering plant defence enzymes.

PAL is the entry point enzyme in the phenylpropanoid biosynthesis pathway leading to synthesis of phytoalexins or phenols, which have defence functions in plants, such as reinforcenment of plant cell wall (Nicholson and Hammerschmidt 1992), antimicrobial activity and synthesis of signalling compounds such as salicylic acid (Wen et al. 2005). Maximum PAL activity in the present study was detected in the plants treated with consortium of the three BCAs in challenge-inoculated plants compared with other microbial combinations and unchallenged control plants. Similarly, a significant increase in phenolic content was also observed in the plants treated with the consortium of the three BCAs in the similar way of PAL activities after challenge with the pathogen. Increase in phenolic content is positively related to the degree of plant’s resistance against pathogens as phenols play a key role in antimicrobial defence arsenal of plants (Shoresh and Harman 2008; Abo-Elyousr et al. 2009). Moreover, induction of phenols is also linked with induced PAL activity, which catalyses the first step in synthesis of phenols. Increased PAL activity and phenol accumulation in the present study may, thus, be correlated with enhanced defence response by the microbial consortium as indicated by the plant mortality data.

The enzymes SOD and PO work together with other enzymes of the ascorbate–glutathione cycle to promote scavenging of free radicals (Hernandez et al. 2001). In the present study, analysis of plants after pathogen infection indicated that pea seeds treated with the three-microbe consortium followed by two-microbe consortium of B. subtilis and Ps. aeruginosa exhibited maximum activities of PO and SOD compared with the plants treated with single BCAs and untreated control. Similar results were also obtained in a previous study, where microbial mixture of Bacillus strains IN937a and IN937b was found to induce maximum SOD and PO activity compared with untreated control (Jetiyanon 2007). A proteomic approach to identify Trichoderma-induced enzymes also showed increased levels of antioxidant and other detoxifying enzymes in maize leaves (Shoresh and Harman 2008). These reports confirm the role of the three microbial species in triggering activities of PO and SOD in plants when applied as seed treatment and strengthen the results obtained in the present study.

Enhanced PPO activities against disease and insect pests have been reported in several beneficial plant–microbe interactions. An increased level of PPO was recorded on co-inoculation of rhizobia with Bacillus cereus BS03 and Ps. aeruginosa RRLJ04 in pegion pea under the challenge of the Fusarium wilt pathogen (Dutta et al. 2008). Maximum PPO activity in the present study was observed in pea plants treated with the consortium of all the three microbes. Our results are also in agreement with Harish et al. (2009), who showed that PO and PPO activities increase in plants treated with mixtures of P. fluorescens Pf1 and Bacillus spp. EPB22 and challenge with the Banana bunchy top virus. The increased level of phenolics recorded in the triple-microbe-treated plants could also be correlated with increased PO and PPO levels as phenols also serve as substrate for oxidative reactions catalysed by PPO and/or PO (Lattanzio et al. 2006).

PR proteins are host-coded proteins induced by different types of pathogens and abiotic stresses, and their synthesis and accumulation have been reported to play an important role in plant defence (Van Loon 1997). In vitro experiments have shown that chitinases from various plant sources inhibit the growth of fungal pathogens in synergism with β-1,3-glucanases (Mauch et al. 1988). Besides the direct antifungal activity, they may also be involved in inducing host resistance by helping in releasing the oligomers from microbial cell walls, which behave as active elicitors of other defence responses (Côté and Hahn 1994). Microbial consortia comprising of the three microbes followed by B. subtilis and T. harzianum consortium showed maximum induction of chitinases and β-1,3-glucanases in the present study. Similar in line, a study involving application of Pseudomonas fluorescens and Trichoderma viride in combination with chitin, also showed increased accumulation of phenols and activities of chitinase and β-1,3-glucanase in coconut palm compared with other treatments and control (Karthikeyan et al. 2006).

Proline acts as an osmoregulant and helps to maintain the water potential of plant to extract water from soil (Hanson et al. 1979). Proline is also known to act as an antioxidant agent apart from its well-known role in osmoprotection (Schobert and Tschesche 1978). Free proline accumulation was significantly high in plants treated with the two-microbe consortium consisting of T. harzianum and Ps. aeruginosa and was nearly equal in the three-microbe consortium after pathogen challenge in the present study. Microbe-mediated proline accumulation in leaf tissues of Arabidopsis thaliana, treated with Pseudomonas syringae pv. tomato avirulent strains, was also reported by Fabro et al. (2004). A similar increase in proline content was also demonstrated by Sarvanakumar et al. (2011) in green gram plants bacterized with Ps. fluorescens Pf1 followed by B. subtilis EPB22. Increased accumulation of proline was also found in the plants inoculated with Pseudomonas mendocino alone or in combination with either of the selected AM fungi under severe stress (Kohler et al. 2008). Therefore, increased proline content in the BCA-treated plants in the current study may be correlated to an early response of pea towards the challenging pathogen in managing the oxidative stress induced by S. sclerotiorum.

Colonization by the three beneficial microorganisms leading to significant increase in the activities of defence-related enzymes especially PAL, PO, PPO, SOD, glucanases and chitinase, and accumulation of proline and phenols in the present study suggests that these parameters are regulated by the signals released by the microbes. The consortium-mediated host physiological responses can also be associated in disease resistance as the triple-microbe-treated plants showed least plant mortality. Further understanding of the mechanisms of action adopted by compatible beneficial microbes in consortium would open new doors to design strategies for improving the efficacy of BCAs, as they have the opportunity to act without competing with each other by being spatially separated in the rhizosphere (Duffy et al. 1996). From the results of the present study, it can be concluded that microbial consortium comprising of all three microbes increased plant’s resistance against infection by S. sclerotiorum, most likely through communicating in a synergistic manner, providing the plant with a powerful capacity to regulate its immune response against the pathogen.

Acknowledgements

A.J. is grateful to Department of Science and Technology, Govt. of India, New Delhi, for financial assistance under AORC scheme as INSPIRE-JRF.

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