Induced Systemic Resistance and Evaluation of Bio-control Agents for Management of Pigeonpea Wilt Caused by Fusarium udum

B.M. Ravikumara1,2*, M.K. Naik2, Mamta Sharma1, Gururaj Sunkad2Ayyan Gouda Patil3, S. Muniswamy3 and K.P. Viswanath3

1Legumes Pathology Division, International Crops Research Institute for the Semi- Arid Tropics, Patancheru- 502324, India.
2Department of Plant Pathology, University of Agricultural Sciences, Raichur-584104, India.
3Department of Genetics and Plant Breeding, University of Agricultural Sciences, Raichur-584104, India.

ABSTRACT

The performance of the four fungal and two bacterial bioagents were evaluated for the bicontrol efficacy and ability to induce systemic resistance against Fusarium udum causing wilt of pigeonpea which is posing a serious threat to pigeonpea growing regions of India. Among the six isolates, maximum mycelial inhibition was noticed in Trichoderma harzianum (Th- R) as compared to other biocontrol agents. Among contact fungicides, maximum inhibition (> 75 %) of mycelium was recorded in Mancozeb and capton at 0.20 and 0.3 % concentrations. More than 90% inhibition was recorded among the systemic fungicides at all the all the concentrations except thiophanate methyl which recorded 53.67 % inhibition at 0.05 % concentration. Among different treatment combinations of biocontrol agents, the highest vigour index was recorded in P. fluorescense (RP- 46) + P. putida (RP- 56) treated seeds in both the cultivars (Moderately resistant and susceptible). The level of expression of defense related enzymes (PO, PPO & PAL) was more in moderately resistant cultivar(BSMR- 736) rather than susceptible one(ICP- 2376). In glass house experiment seeds treated with P. flourescens (RP- 46) + P. putida (RP-56) recorded least wilt incidence as compared to other treatments. In both Kharif seasons of 2013/14 and 2014/15 recorded significantly lowest wilt incidence and highest yield in soil drenching with 0.2 % Carbendazim fungicide. Among the biocontrol agents, seed treatment @4 g / kg seeds + soil application of PGPR (P. flourescens & P. putida) consortium @ 25kg/ ha in FYM @ 50 kg/ ha, recorded least wilt incidence and highest yield.

Keywords: Pigeonpea; Fungicide; Biocontrol; Induced systemic resistance; Fusarium udum; Pseudomonas; Trichoderma; PGPR.

Introduction

Pigeonpea is an important pulse cum grain legume crop and the area under the crop is increasing due to the productivity, favorable conditions and economics of the crop. India is a principal pigeonpea growing country contributing nearly 90% of total world production. Currently, it occupies an area of 5.2 million ha with an annual production of 4.2 million tonnes1. Pigeonpea is attacked by more than 100 pathogens including fungi, bacteria, viruses, nematodes, and mycoplasma-like organisms, but only a few of them cause economic losses2. The diseases of considerable economic importance at present are fusarium wilt (Fusarium udum), sterility mosaic (Sterility mosaic virus), and phytophthora blight (Phytophthora dreschsleri f. sp. cajani). Among them, wilt caused by F. udum is considered the most important soil borne pathogen of pigeon pea3.
The disease is typically soil borne and the pathogen perpetuates in soil for several years by means of chlamydospores4. Chemical control of a soil borne plant pathogen is frequently ineffective because of the physical and chemical heterogeneity of the soil, which may prevent effective concentration of the chemical from reaching the pathogen. Hence, the best alternative measure is to look for bio control agents, which colonize the rhizosphere, the site requiring protection and leave no toxic residues, as opposed to chemicals. A multitude of microbes has been implicated to be biocontrol agents of plant pathogens sometimes with excellent documentations5- 8. Hence, experiments were carried out to find out the efficacy of some fungal and bacterial bio control agents against F. udum on pigeonpea and were also tested for induction of systemic resistance. Fluorescent Pseudomonas and Trichoderma species are important groups of plant growth-promoting microorganism reported to protect plants against pathogens by evolving various mechanisms such as antagonism, competition and Induced systemic resistance (ISR) 9- 11.
Induced systemic resistance (ISR) triggered by plant growth-promoting fungi (PGPFs) and Plant growth promoting rhizobacteria (PGPR) confers a broad-spectrum resistance that is effective against different types of pathogens12. PGPR produce phytohormones that are believed to be related to their ability to stimulate plant growth. Indole-3-acetic acid is a phytohormone which is known to be involved in root initiation, cell division, and cell enlargement13. This hormone is very commonly produced by PGPR14. Most commonly, IAA-producing PGPR are believed to increase root growth and root length, resulting in greater root surface area which enables the plant to access more nutrients from soil. Cytokinins are a class of phytohormones which are known to promote cell divisions, cell enlargement, and tissue expansion in certain plant parts13. Cytokinin is produced by Pseudomonas fluorescens isolated from the rhizosphere of the soybean15.
Compared to the use of individual PGPR strains, mixtures of several strains may result in a more stable rhizosphere community, provide several mechanisms of biological control, and may suppress a broader range of pathogens16. Compatible mixtures of certain biocontrol strains with antagonism as the main mechanism of action have provided a greater disease suppression than that used individually17- 22. In one of the study used PGPR that elicit ISR, indicated that mixtures of PGPR provided synergistic activity against a broader range of pathogens on one host22. The aim of the present experimentation is to evaluate the performance of Trichoderma and two Pseudomonas isolates for their bio control efficacy and ability to induce systemic resistance against F. udum causing pigeonpea wilt.

Materials and Methods

Bio- control agents
The bio-agents Trichoderma viride(Tv-R),Trichoderma harzianum(Th- R), Pseudomonas fluorescens(RP-46) and Pseudomonas putida (RP- 56) used in this study were obtained from the collection at the Department of Plant Pathology, University of Agricultural Sciences, Raichur, India and the two isolates, Trichoderma spp (ICRISAT- T) and Trichoderma spp (GLB- I) were obtained from rhizosphere of pigeonpea from Sick plot (BIL- 17), ICRISAT, Hyderabad, India and sick plot, ARS, Kalaburgi, Karnataka, India respectively. The Trichoderma isolates were maintained on a Trichoderma specific medium and the Pseudomonas isolates were maintained on King’s B medium23.
Isolation of the Pathogen
The wilted pigeonpea plant sample collected from the field and the infected tissues from the stem/ collar region were cut into small bits of 1- 2 mm in size, surface sterilized in 1 per cent sodium hypochlorite solution for a minute and then washed three times in sterile distilled water to remove any traces of sodium hypochlorite. They were subsequently blotted dry and plated on sterile Potato Dextrose Agar [PDA] plate and incubated at 26 ± 2ºC and alternate cycles of 12 h light and 12 h darkness. Culture is purified by using single spore isolation and proved Koch’s postulate by using susceptible cultivar (ICP 2376). The fungal isolates were preserved in PDA slants for subsequent uses. Total of 111 isolates of F. udum were collected from 397 farmers’ fields in Karnataka, Madhya Pradesh, Maharashtra, Tamil Nadu and Telangana states. However, dual culture test and evaluation of bio-control agents under glass house and field condition was carried out using only one local isolate of F. udum (FU- 37) from ARS Kalaburgi, Karnataka.
In-vitro evaluation of bio-agents by Dual culture test
Bio-control agents (Fungal and Bacterial isolates) and the F. udum(FU- 37) isolate were placed side by side on a single Petri-dish containing solidified PDA. There were three replications for each isolate with one control each of the pathogen and the bio-control agent. They were incubated at 26 ± 2º C and grown for 6-8 days. The diameter of the colony of both the bio-control agent and the pathogen was measured in two directions and the average was calculated. Percent inhibition of growth of the test pathogen was calculated24. In-vitro evaluation was done three times with three replications.
The percentage of inhibition was estimated using the following formula:
I= (C – T)/C* 100

Where; I= Percentage of inhibition, C= radial growth of the pathogen in control and T= radial growth of pathogen in treatment.
In-vitro evaluation of fungicides by poisoned food technique
Four non- systemic fungicides namely Capton, Chlorothalonil, Mancozeb and Zineb. four systemic fungicides namely Benomyl, Carbendazim, Thiophanate methyl and Carbendazim 25 % + Mancozeb 50 % were tested against colony growth of F. udum isolate FU- 37. Non-systemic fungicides were used at 0.1%, 0.2 % and 0.3 % and systemic fungicides at 0.05%, 0.1% and 0.20% concentrations in autoclaved PDA medium by poisoned food techniques25. Five mm diameter agar disc of test fungi was cut from 6 day old culture and placed in the center of Petri plates containing different concentration of fungicides. The plates without fungicides served as control. The inoculated plates were incubated at 26 ± 2°C. The radial growth was recorded after 7 to 8 days of incubation when the fungus covered the plates completely in control. The percent inhibition of the fungus over control was calculated24.
Seedling vigour
Seedling vigour of the Pseudomonas spp. and Trichoderma spp. treated seeds was determined by the standard roll towel method (ISTA in 2005). Four replicates of 50 treated seeds were placed at equi-distance on the paper towel and covered with another pre-soaked paper towel, rolled up along with polythene wrapping to prevent drying of the towels. The rolled towels were then incubated in an incubation chamber for 8 days. Paper towels were unrolled after incubation period and number of germinated seeds were counted and represented in percentage. Seedling vigour was analysed using the method of Abdul-Baki and Anderson26. The length of the root and shoot of individual seedlings were measured with different treatment combination to assess the vigour. The vigour index (VI) was calculated using the formula
VI = (Mean root length+ Mean shoot length) x % germination.
Induction of defense mechanisms
Two each of Trichoderma and Psuedomonas isolates were used in the induction of defense reactions in two pigeonpea genotypes, namely BSMR- 736 (Moderately resistant) and ICP 2376 (Susceptible). Fungus treated (Trichoderma) and bacterized (Pseudomonas) seeds were sown in polythene covers filled with sterilized river bed sand. Eight-day-old seedlings were transplanted in plastic pots @ 10 seedlings per pot. Ten days after transplanting, soil application with Trichoderma suspension (3×103 spores/ml) and bacterial suspension (10 mL of suspension containing 108 cfu mL”1) was done. Bacterized plants were divided into two treatments. In the first treatment, bacterized plants were challenge inoculated with F. udum (50 mL of microconidial suspension containing 6×106 conidia/ ml per pot) at one day after soil application of fungal and bacterial suspension and in the second treatment, bacterized plants were not challenged with the pathogen. Plants without prior treatment of Trichoderma and Pseudomonas were inoculated with the pathogen. The plants neither treated with bacterial suspension nor challenged by the pathogen were kept as control. Three replications were maintained in each treatment; each replicate consisted of 10 pots and in each pot six plants were maintained. The experiments were conducted using randomized block design on a greenhouse bench. The humidity in the greenhouse was maintained at RH of 70%. The temperature was adjusted to 25- 27æ%C (day) / 20- 22æ%C (night).
Sample collection for biochemical analysis
Plants were carefully uprooted without causing any damage to root tissues at different time intervals (0, 3, 6, 9 and 12 days after the pathogen inoculation). Six plants were sampled from each replication of the treatment separately (treatments were mentioned in the experimental design) and were maintained separately for biochemical analysis. Fresh roots were washed in running tap water and homogenized with liquid nitrogen in a pre-chilled mortar and pestle. The homogenized root tissues were stored in deep freezer (“80æ% C) until used for biochemical analysis.
Assay of PO
Root samples (1 g) were homogenized in 2 mL of 0.1 M phosphate buffer, pH 7.0 at 4 æ%C. The homogenate was centrifuged at 16 000 g at 4 æ%C for 15 min and the supernatant was used as enzyme source. The reaction mixture consisted of 1.5 mL of 0.05 m pyrogallol, 0.5 mL of enzyme extract and 0.5 mL of 1% H2O2. The reaction mixture was incubated at room temperature (28±2æ%C). The changes in absorbance at 470 nM were recorded at 30 s intervals for 3 min. The enzyme activity was expressed as changes in the absorbance min”1 mg”1 protein27.
Assay of PPO
PPO activity was determined as per the procedure28. Root samples (1 g) were homogenized in 2 mL of 0.1 M sodium phosphate buffer (pH 6.5) and centrifuged at 16 000 g for 15 min at 4 æ%C. The supernatant was used as the enzyme source. The reaction mixture consisted of 200 µL of the enzyme extract and 1.5 mL of 0.1 M sodium phosphate buffer (pH 6.5). To start the reaction, 200µL of 0.01 M catechol was added and the activity was expressed as changes in absorbance at 420 nM min”1mg”1 protein.
Estimation of PAL activity
Root samples (1 g) were homogenized in 3 mL of ice cold 0.1 M sodium borate buffer, pH 7.0 containing 1.4 m M of 2-mercaptoethanol and 0.1 g of insoluble polyvenylpyrrolidone. The extract was filtered through cheese cloth and the filtrate was centrifuged at 16000g for 15 min. The supernatant was used as enzyme source. PAL activity was determined as the rate of conversion of L-phenylalanine to trans-cinnamic acid at 290 nM29. Sample containing 0.4 mL of enzyme extract was incubated with 0.5 mL of 0.1 M borate buffer, pH 8.8 and 0.5 mL of 12 mM L-phenylalanine in the same buffer for 30 min at 30æ%C. The amount of trans-cinnamic acid synthesized was calculated using its extinction coeffi- cient of 9630 m”1 29. Enzyme activity was expressed as nmol trans-cinnamic acid min”1 mg”1protein.
Evaluation of bio-control agents under glass house condition
Efficacy of those bacterial and fungal isolates (Pseudomonas spp. and Trichoderma spp.) causing enhanced seedling growth and inhibition to F. udum in vitro were selected and tested for their ability to reduce pigeonpea wilt under glass house conditions with different treatment combinations by using root-dip-inoculation technique. The disease incidence is calculated using the following formula.

Wilt incidence (%) = Number of diseased seedlings/ Total number of seedlings ×100

Evaluation of bio-control agents under field condition
The isolates of Trichoderma and Pseuomonasa having maximum inhibition of growth of F. udum under laboratory conditions were chosen for field evaluation and consortium of Trichoderma(T. viride + T. harzianum) and Pseudomonas (P. fluorescence + P. putida ) preparations were used for pigeonpea seed treatment(Cv. BSMR- 736) @ 4 g/kg seed. The bio-control agents were multiplied on talc based formulations then enriched in farm yard manure and applied to the soil along with the seed treatments. The seeds were placed in a clean container, sprinkled with water until they were wet, adequate quantity of the bio-control agent available in a talc based dry powder form30 was added and the container was agitated thoroughly until the seeds were uniformly coated. Farm yard manure (FYM) to be used for soil application was enriched with Trichoderma(T. viride + T. harzianum) or Pseudomonas (P. fluorescence + P. putida ) consortium. The enrichment process involved adding of 2 kg of the respective bio-control agent to 1 ton of FYM and incubating the mixture at ambient temperature under shade for a period of 15 days prior to planting pigeonpea. Optimum moisture content (15%) was maintained by adding water as needed during this period. The bio-control agent enriched FYM was added at 15 tons/ha after enrichment.
Field trials were conducted in Fusarium udum wilt sick plot at the farm of Agricultural Research Station (ARS), Kalaburgi (Karnataka) for two growing seasons in 2013/14 and 2014/15. The experiment was set up in a randomized block design (RBD) with three replications of seven treatments each with a plot size of 2.5 ×1.8 m. The treatments used included Trichoderma consortium and Pseudomonas consortium treated seeds sown after soil application of Trichoderma consortium and Pseudomonas consortium enriched FYM applied at 15 tons/ha. Carbendazim @ 0.3 per cent was used for soil drenching. A non-treated control was also included in the trials. All recommended agronomic practices for the region were followed to raise a good crop. The germination percentage was noted 15 days after planting and the incidence of wilt was monitored periodically and the terminal incidence was recorded 120 days after planting. The wilt incidence was calculated by counting the total number of diseased plants in 1m2 which was then divided by the total number of plants in the area and expressed as percentage.
Statistical analysis
Data were statistically analyzed using the standard procedures for completely randomize design, complete randomized block and split designs 31. The averages were compared at 1% and 5% level using least significant differences (L.S.D) 32.

Results and discussion

Evaluation of bio-control agents under laboratory conditions
Four isolates of Trichoderma spp and two isolates of Pseudomonas spp were evaluated for their efficacy as antagonists against F. udum (FU- 37), the cause of vascular wilt of pigeonpea using dual culture technique. All isolates were found to cause significant reduction in fungal growth as compared to the control.
The per cent inhibition of F. udum ranged from 46.52 to 70.84 per cent. Among tested fungal antagonists, the maximum inhibition of F. udum growth was observed in T. harzianum (Th-R) bioagents as compared to other bio-control agents and inhibited maximum fungal growth (74.52 %) of F. udum followed by Trichoderma spp (ICRISAT- T) (72.23 %). T. viride (TV-R) and Trichoderma spp (GLB) with 70.84% and 67.91% respectively. In bacterial bioagents P. fluorescens (RP- 46) inhibited to the extent of 50.28 per cent. Least inhibition was recorded with 46.52 per cent in P. putida (RP- 56)[Table. 1]. In- vitro evaluation of antagonistic microorganisms against F. udum recorded maximum inhibition of F. udum against T. viride (87.03 %) and T. harzianum (85.40 %), P. fluorescens (81.87 %) 33. Evaluation of six bioagents against F. udum though dual culture technique and recorded highest inhibition from G. virens (Pantnagar) and T. viride (Coimbatore) 34.
Evaluation of fungicides under laboratory conditions
In contact fungicides, Mancozeb and capton recorded maximum inhibition (> 75%) of mycelial growth at 0.20 and 0.30 per cent and chlorothalonil showed 62.50 per cent inhibition at 0.10 per cent concentration, more than 65 per cent inhibition at 0.2 and 0.3 per cent concentrations. In systemic fungicides, carbendazim 25 per cent + mancozeb 50 per cent showed 100 per cent inhibition at all concentrations (0.05, 0.10 and 0.20 %). Benomyl, carbendazim, thiophanate methyl showed 100 per cent inhibition at 0.2 per cent concentration and more than 90 per cent inhibition was recorded in 0.05 and 0.1 per cent concentrations of benomyl and carbendazim (Table. 2). The fungicides suppressed Fusarium by altering and inhibiting cell metabolism and these biochemical alterations may lead to inhibition in fungal growth35. Carbendazim may directly inhibit conidial germination and sporulation of F. oxysporum36 as well as colonization 37. In- vitro evaluation of different fungicides against F. udum and reported carbendazim and thiram fungicides were quite effective in inhibiting the growth of the fungus at 1000 and 2000ppm, which gave 93.8 and 91.3 per cent inhibition, respectively38. Similarly, efficacy of different fungicides tested against F. udum by using poisoned food technique in vitro revealed that carbendazim inhibited the growth of pathogen at all concentrations(100, 250 and 500 ppm)39.
Seedling vigour
In the moderately resistant cultivar (CV. BSMR- 736), P. fluorescens (RP- 46) + P. putida (RP- 56) treated seeds showed highest germination (95.34 %), mean root length (20.63 cm), shoot length (7.56 cm) and vigour index of 2688.40, which differed significantly from all other isolates. Whereas in susceptible cultivar (CV. ICP 2376) also the same combined isolates P. fluorescens (RP-46) + P. putida (RP-56) treated seeds showed highest germination (93.67 %), mean root length (16.36 cm), shoot length (7.1 cm) and vigour index (2193.67) which differed significantly from all other isolates (Table. 3). Highest vigour index was shown by the combined isolates of P. fluorescens (RP- 46) + P. putida (RP- 56) and as far as germination and vigour index is concerned all the isolates differed significantly in both the cultivars (BSMR- 736 and ICP 2376). These findings are in confirmation with the earlier workers 24 concluded that germination and vigour index were considered as indices of systemic induction of resistance and observed that the indigenous isolate of P. fluorescens (RP- 56) showed highest induction of resistance resulting in highest seed germination and vigour indices in chilli seeds against F. solani. Similarly, other researchers also observed the increased ISR, vigour index, germination by plant growth promoting rhizobacteria (PGPR) and Trichoderma spp40.
Induction of defense mechanisms
Major defense related enzymes focussed in the present study were peroxidase (PO), polyphenol oxidase (PPO), phenyalanine ammonia lyase (PAL). The increased activity of all these enzymes is possible when any biocontrol agent having the capacity to suppress the disease is applied through a reliable established method, so that it has consistent performance for a longer time period. Peroxidases are used primarily for the synthesis of secondary metabolites and are known to be induced by various types of stresses including pathogen infection 41. Peroxidases have been implicated in a number of physiological functions that may contribute to resistance phenol oxidation, lignification and in the deposition of phenolic material into plant cell walls during resistant interaction 42. Both PAL and PPO play important roles in biosynthesis of phenolics, phytoalexins and lignin, the three key factors responsible for disease resistance 43. Phenylalanine ammonialyase catalyzes the conversion of phenylalanine to trans cinnamic acid, a key intermediate in the synthesis of salicylic acid. Enhanced PAL and PPO activity was reported in tomato infected by Fusarium oxysporum 40. Present study revealed that higher accumulation of PO was observed in moderately resistant cultivar (BSMR- 736) than susceptible cultivar (ICP 2376). The treatment P. fluorescens (RP- 46) + F. udum (FU-37) showed maximum PO activity (0.96 change in absorbance at 470 nm/ min/mg protein) followed by P. fluorescens (RP-46) + P. putida (RP- 56) + F. udum (FU-37) which were significantly different from all other treatments. Accumulation of PO started three day after challenge inoculation. The maximum accumulation was observed on 6th day after challenge inoculation and the level of expression of the enzyme declined in subsequent days. Plants inoculated with pathogen alone had comparatively less PO activity but compared to control, the activity was higher (Figure 1). Increased activity of cell wall bound peroxidases has been elicited in different plants such as cucumber 44, rice 45, tomato 46 and tobacco 47 due to pathogen infection. PO1 isoform was prominently expressed in P. ûuorescens isolate Pf1-treated root tissues against F. oxysporum f. sp. lycopersici 40.
A similar pattern of increased activity of PPO was observed in moderately resistant cultivar rather than susceptible cultivar. Here also the PPO activity was maximum on 6th day after challenge inoculation and expression level was reduced afterwards. The same P. fluorescens (RP- 46) + F. udum (FU-37) treatment recorded 1.21 and 0.98 change in absorbance at 420 nm/ min/mg protein) which significantly differed from all other treatments in BSMR- 736 and ICP- 2376 respectively. In moderately resistant cultivar (BSMR 736) the lower activity of PPO was recorded in T. harzianum (Th-R) + F. udum (FU-37) as compared to F. udum alone treated plants. Where as in susceptible cultivar the lower PPO activity was recorded in T. viride (Tv-R) + T. harzianum (Th- R) + F. udum (FU-37) interactions as compared to F. udum alone treated plants (Figure1).
PAL is the key enzyme in inducing synthesis of Salicylic Acid (SA) which induces systemic resistance in many plants. PAL plays an important role in the biosynthesis of phenolics and phytoalexins 48. PAL activity showed increased trend 6th day after challenge inoculation. Similar to PO and PPO, the PAL activity also maximum in P. fluorescens (RP- 46) + F. udum (FU-37) treatment ((31.26 nmol transcinnamic acid/hr/mg protein) but in T. harzianum (Th-R) + F. udum (FU-37), the PAL activity was lower compared to F. udum alone treated plants and in healthy control the activity was recorded up to 19.91 transcinnamic acid/hr/mg protein(Figure 1).
Similarly, roots collected from P. fluorescens treated seedlings induced early and enhanced level of PAL, PO and PPO in tomato plants challenged with F. o f. sp. lycopersici 40. Induction of high PO, PPO and phenolic activity was noticed in tomato against Fusarium wilt pathogen, F. o f.sp. lycopersici when treated with T. harzianum 49. Increased level of defense related enzymes, viz., PAL, PO and PPO was found in co-inoculation of plant growth promoting rhizobacteria, Rhizobium and challenge inoculation with F. udum of pigeonpea 50. Increased activity of defense related enzymes mainly PO, PAL, total phenol and â 1,3 glucanase was noticed due to application of P. fluorescens isolates in chilli plants challenge inoculated with F. solani causing wilt of chilli 51.
Evaluation of bio-control agents under glass house condition
In moderately resistant cultivar (CV. BSMR- 736), least wilt incidence (8.34 %) was recorded in P. fluorescens (RP- 46) treatment followed by P. fluorescens (RP- 46) + P. putida (RP- 56) with mean incidence (13.89 %). Whereas in susceptible cultivar (Cv. ICP 2376) also least wilt incidence (29.17 %) was recorded in P. fluorescens (RP- 46) treatment followed by P. fluorescens (RP- 46) + P. putida (RP- 56) with mean incidence of 42.06 per cent (Table. 4). Similar study conducted by other workers also by biological control of pigeonpea wilt under glasshouse condition and found T. viride and T. harzianum isolate- C as effective bicontrol agents52. Evaluation of 20 isolates of fluorescent pseudomonads and Bacillus spp. in the laboratory and glasshouse condition and six isolates were considered as potential for the biocontrol of the disease on the basis of antibiotic sensitivity, antifungal activity 53. Efficacy of T. viride, carbendazim, Rhizobium, T. viride + carbendazim, T. viride + Rhizobium, carbendazim + Rhizobium and T. viride + Rhizobium + carbendazim against F. udum in a pot experiment and observed that all treatments significantly reduced the wilt incidence over the control (73.30 %) except Rhizobium alone (64.40 %) 54. In- vitro efficacy of eleven P. fluorescens strains (I1, I2, I3, I4, I5, I6, I7, I8, I9, I10 and I11) in controlling wilt of pigeonpea and recorded seed treatment with P. flurescens strains I10 from pigeonpea plants resulted in the lowest (16.66 %) incidence of the disease 55.
Evaluation of bio-control agents under field condition
Trials to evaluate the ability of the bio-control agents to control vascular wilt under Fusarium wilt sick plot were set up using moderately resistant cultivar( Cv. BSMR- 736) during 2013/14 & 2014/15 using Trichoderma and Pseudomonas consortium applied as seed treatments alone and in combination with a soil treatment. Chemical soil drenching with Carbendazim, commonly used by growers in the region for control of F. udum were also included in the trial. In 2013/14 Kharif percent wilt incidence was found to be significantly reduced, showing a nearly 35.18- 80.16 % reduction in wilt incidence in the treatments overall when compared to the non-treated control (Table 5). Whereas in 2014/15 Kharif percent wilt incidence was found to be significantly reduced from 38.09- 83.13 per cent when compared to the non-treated control thereby emphasizing the contribution of seed and soil treatments towards yield enhancement.
In Kharif season 2013/14 soil drenching with 0.2% carbendazim fungicide recorded significantly lowest mean wilt incidence (7.06 %) with highest yield (1723.96 kg / ha). Among the bio-control agents seed treatment + soil application of PGPR consortium, recorded wilt incidence of 10.31% and yield of 1594.79 kg per ha (Table. 5). Whereas in Kharif season 2014/15 with same treatments combinations, observed that again in the same treatment viz., soil drenching with 0.2% carbendazim fungicide recorded significantly lowest mean wilt incidence (5.30 %) with highest yield (1653.13 kg/ ha). Effective control of wilt of pigeonpea by soil drenching with 0.1% carbendazim but their approach was preventive not curative and they applied pre decided soil drenching at 30 days after sowing without considering wilt appearance56. Plant growth promotion activity of Trchoderma is well established 57- 59 and researchers have reported significant yield enhancement 60. Root colonization by Trchoderma strains frequently enhances root growth, development, and crop productivity, resistance to abiotic stresses and the uptake and use of nutrients 61. Seed treatment @ 4g per kg seeds + soil application of PGPR (P. fluorescens and P. putida) consortium @ 25 kg per ha in FYM @ 50 kg per ha, recorded the wilt incidence (7.28%) and yield (1540.63 kg/ ha) among bio-control agents. Soil application of P. fluorescens formulation was effective against the wilt 62. Numerous strains of P. fluorescens and B. subtilis have been found suppressive against soil borne fungal pathogens. In the present study used strains of PGPR P. flourescens and P. putida consortium found to be an efficient plant growth promoter. Its application resulted to significantly greater production of dry matter and yield of pigeonpea. The possible mechanism involved in the suppression may be the competition and rhizosphere colonization63. Antibiosis is the other mechanism by which the biocontrol bacteria would have suppressed F. udum. Antibiotics such as pholoroglucinols64 and pyrolnintrin65 produced by P. fluorescens and agrocin-84 66, bulbiformin 67 etc. by B. subtilis have been reported to be fungicidal in nature. In present study Trichoderma spp consortium was found less effective in controlling wilt of pigeonpea as compared to Pseudomonas spp consortium. Similar relative effectiveness of the biocontrol agents have been reported against wilt of chickpea and pigeonpea 68- 69.
The success of dual application could be attributed to establishment and rapid build-up of bio-control agents in the soil which would assist in reducing the infectivity of soil borne inoculum or suppression of the pathogen70 enabled by the soil application while the seed treatment would prevent proliferation of the seed borne inoculum. This is particularly relevant for soil borne pathogens such as F. udum where drenching the soil with chemicals is not only deleterious to the environment but is also practically not feasible due to high cost as pigeonpea is generally cultivated under rainfed condition without much investment for crop production as well as protection. Considering the fact that the cost of seed treatment chemicals under Indian conditions is almost three times that of the indigenously produced bio-control agents such as those used in this study, the use of two different methods and added amounts of inoculants will not significantly affect the cost of production. Moreover, prolonged use of the bio-control agents will increase their rhizosphere population 5, 30 and thereby enhancing the disease suppressive characteristics of the soil. Therefore, this study not only reports the availability of isolates of Trichoderma and Pseudomonas that can effectively control vascular wilt of pigeonpea but also promote the plant growth and induces resistance in plants.

References

1. FAO, 2014 http://faostat.fao.org.
2. Nene, Y.L., Sheila, V.K., Sharroa, S.B. A world list of chickpea (Cicer arietinum L) and pigeonpea (Cajanus cajan L. Millsp.) pathogens. Legume Pathology Progress Report- 7, Patancheru, A. P. 502324, India: International Crops Research Institute for the Semi- Arid Tropics.
3. Pande, S., Sharma, M., Guvvala, G., Telangre, R. High throughput phenotyping of pigeonpea disease, stepwise identification of host plant resistance. Information Bulletin No. 93, ICRISAT, India, 2012; pp. 1- 31.
4. Garret, S.D. Inoculum potential. In: Plant Pathology: An Advance Treatise. Ed. J G Horsfall and A E Dimond, Volume 3, pp. 23-56. Academic Press, New York. (1960).
5. Naik, MK., Sen, B. Bio-control of plant disease caused by Fusarium spp. In: Mukherjee KG. (Ed.), Recent Developments in Bio-control of Plant Diseases. Aditya Publishing House, India, 1995; p. 32.
6. Laha, G.S., Verma, V.P. Role of fluorescent pseudomonads in the suppression of root rot and damping-off of cotton. Indian Phytopath, 1998; 51: 275- 278.
7. Rangeshwaran, R., Prasad, RD., Anuroop, C.P. Field evaluation of two bacterial antagonists, Pseudomonas putida (PDBCAB 2) against wilt and root-rot of chickpea. J Boil. Contrl, 2001; 15: 165-170.
8. Singh, H.B. Trichoderma: A boon for biopesticides industry. J. Mycol. Pl. Pathol, 2006; 36: 373-384.
9. Marx, J. The roots of plant-microbe collaborations. Science, 2004; 304: 234-236.
10. Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Marra, R., Woo, S.L. Trichoderma plant-pathogen interactions. Soil Biol. Biochem. 2008; 40: 1-10.
11. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M. Trichoderma species-opportunistic, avirulent plant symbionts. Nature Reviews, 2004; 2: 43-56.
12. Van der, E.S., Van Wees, S.C., Pieterse, C.M.J. Jasmonate signaling in plant interactions with resistance-inducing beneficial microbes. Phytochemistry, 2009; 70: 1581-1588.
13. Salisbury, F.B. The role of plant hormones. In: Wilkinson RE (eds) Plant–Environment Interactions. Marcel Dekker, New York, USA, 1994; 39-81.
14. Barazani, O., Friedman, J. Is IAA the Major Root Growth Factor Secreted from Plant-Growth-Mediating Bacteria?. J. Chem Ecol, 1991; 25: 2397-2406.
15. De Salamone, I.E.G., Hynes, R.K., Nelson, L.M. Cytokinin production by plant growth promoting rhizobacteria and selected mutants Can J Miccrobiol, 2001; 47: 404-411.
16. Pierson, E.A., Weller, D.M. Use of mixture of fluorescent pseudomonads to suppress take-all and improve the growth of wheat. Phytopathology, 1994; 84: 940–947.
17. Datnoff, L.E., Nemec, S., Pernezny, K. Biological control of Fusarium crown and root rot of tomato in Florida using Trichoderma harzianum and Glomus intraradices. Biol. Control, 1995; 5: 427–431.
18. Duffy, B.K., Weller, D.M. Use of Gaeumannomyces graminis var. graminis alone and in combination with fluorescent Pseudomonas spp. to suppress take-all of wheat. Plant Dis, 1995; 79: 907–911.
19. Duffy, B.K., Simon, A., Weller, D.M. Combination of Trichoderma koningii with fluorescent pseudomonads for control of take-all on wheat. Phytopathology, 1996; 86: 188–194.
20. Janisiewicz, W.J. Biocontrol of postharvest diseases of apples with antagonist mixtures. Phytopathology, 1988; 78: 194– 198.
21. Janisiewicz, W.J, Bors, B. Development of a microbial community of bacterial and yeast antagonists to control wound-invading postharvest pathogens of fruits. Appl. Environ. Microbiol, 1995; 61: 3261–3267.
22. Raupach, G.S., Kloepper, J.W. Mixtures of plant growth promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology, 1998; 88: 1158–1164.
23. King, E.O., Ward, M.K., Raney, D.E. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med, 1954; 44: 301-307.
24. Naik, M.K., Madhukar, H.M., Devika Rani, G.S. Evaluation of biocontrol efficacy of Trichoderma isolates and methods of its applications against wilt of chilli caused by Fusarium solani. J. Biol. Control, 2009; 23: 31-36.
25. Dey, R.K., Chaudhary, R.G., Naimuddin. Comparative efficacy of bio-control agents and fungicides for controlling chickpea wilt caused by Fusarium oxysporum f. sp. ciceri. Indian J. Agric. Sci, 1996; 66: 370-373.
26. Abdul-Baki, A.A., Anderson, J.D. Vigour determination in soybean seed by multiple criteria. Crop Sci, 1973; 13: 630-633.
27. Hammerschmidt, R., Nuckles, E.M., Kuc, J. Association of enhanced peroxidase activity with induced systemic resistance of cucumber of Colletotrichum lagenarium. Physiol. Plant Pathol, 1982; 20: 73-82.
28. Mayer, A.M., Harel, E., Shaul, R.B. Assay of catechol oxidase a critical comparison of methods. Phytochemistry, 1965; 5: 783-789.
29. Dickerson, D.P., Pascholati, S.F., Hagerman, A.E., Butler, L.G., Nicholson, R.L. Phenylalanine ammonia-lyase and hydroxy cinnamate: CoA ligase in maize mesocotyls inoculated with Helminthosporium maydis or Helminthosporium carbonum. Physiol. Plant Pathol, 1984; 25: 111–123.
30. Devika Rani, G.S., Naik, M.K., Patil, M.B., Prasad, P.S. Biological control of Fusarium solani causing wilt of chilli. Indian Phytopath, 2009; 62: 190- 198.
31. Snedecor., Cochran. Statistical Methods, Eighth Edition Iowa State University press. 1967 pp. 75-82.
32. Fisher, R.A. Statistical Methods 6th ed. Iowa State Univ. Press, Ames, Iowa, USA, 1948.
33. Goudar, S.B., Kulkarni, S. Bioassay of antagonists against Fusarium udum, the causal agent of pigeonpea wilt. Karnataka J. Agri. Sci, 2000; 13: 64-67.
34. Chaudhary, R.G., Prajapati, R.K. Comparative efficacy of fungal biological agents against Fusarium udum. Ann. Plant Prot. Sci, 2004; 12: 75-79.
35. Ragsdale, N.N., Sisler, H.D. Metabolic effects related to fungitoxicity of carboxin. Phytopathology, 1970; 60: 1422-1427.
36. El-Abyad, M.S., Ismail, I.K., AL-Meshhadani, S.A. Effects of some biocides on Fusarium oxysporium f.sp. lycopersici causing cotton and tomato wilts in Egypt. Trans. Brit. Mycol. Soc, 1983; 80: 283-287.
37. Sarhan, M.M., Ezzat, S.M., Reda, M., Tohamy, A., El-Essawy, A.A., EL-Sayed, A.F. Application of Trichoderma hamatum as a biocontroller against tomato wilt disease caused by Fusarium oxysporum f.sp. lycopersici. Egypt J. Microbiol, 1999; 34: 347-376.
38. Jadav, H.R., Jani, S.M. The potentiality of chemicals against Fusarium udum, the incitant of wilt of pigeonpea. Indian Phytopath, 2003; 56: 314.
39. Raju, G.P., Ramakrishna Rao, S.V., Gopal, K. In-vitro evaluation of antagonists and fungicides against the redgram wilt pathogen Fusarium oxusporum f.sp. udum (Butler), Synder and Hansen. Legume Res, 2008; 31: 133- 135.
40. Ramamoorthy, V., Raguchander, T., Samiyappan, R. Induction of defense-related proteins in tomato roots treated with Pseudomonas ûuorescens Pf1 and Fusarium oxysporum f. sp. lycopersici. Plant and Soil, 2002; 239: 55- 68.
41. Sasaki-Sekimoto, Y., Taki, N., Obayashi, T., Aono, M., Matsumoto, F., Sakurai, N., Suzuki H., Hirai, M.Y., Noji, M., Saito, K., Masuda, T., Takamiya, K., Shibata, D., Ohta, H. Coordinated Activation of Metabolic Pathways for Antioxidants and Defence Compounds by Jasmonates and Their Roles in Stress Tolerance in Arabidopsis. Plant Journal, 2005; 44: 653- 668.
42. Walter, M.H. Regulation of Lignification in Defense. In: Boller, T. and Meins, F., Eds., Genes Involved in Plant Defenses, Springer-Verlag, Berlin, 1992; 327-352.
43. Daayf, F., Ongena, M., Boulanger, R., El-Hadrami, I., Belanger, R.R. Induction of Phenolic Compounds in Two Cultivars of Cucumber by Treatment of Healthy and Powdery Mildew-Infected Plants with Extracts of Reynoutria sachalinensis. J. Chem Ecol, 2000; 26: 1579-1593.
44. Chen, C., Bélanger, R.R, Benhamou, N., Paulitz, T. Defense enzymes induced in cucumber roots by treatment with plant growth promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol. Mol. Plant Pathol, 56: 13– 23.
45. Reimers, P.J., Guo, A., Leach, J.E. Increased activity of a cationic peroxidase associated with an incompatible interaction between Xanthomonas oryzae pv. oryzae and rice (Oryza sativa). Plant Physiol, 1992; 99: 1044– 1050.
46. Mohan, R., Vijayan, P., Kolattukudy, P.E. Developmental and tissue speciûc expression of a tomato anionic peroxidase (tap 1) gene by a minimal promoter with wound and pathogen induction by an additional 5’-ûanking region. Plant Mol. Biol, 1993; 22: 475– 490.
47. Ahl Goy, P., Felix, G., Metraux, J.P., Meins, Jr. Resistance to disease in the hybrid Nicotiana glutinosa × Nicotiana debneyi is associated with high constitutive levels of â-1,3-glucanase, chitinase, peroxidase and polyphenol oxidase. Physiol. Mol. Plant Pathol, 1992; 41: 11– 21.
48. Daayf, F., Bel–Rhlid, R., Bélanger, R.R. Methyl ester of pcoumaric acid: a phytoalexin-like compound from long English cucumber leaves. J. Chem. Ecol, 1997; 23: 1517– 1526.
49. Ojha, S., Chatterjee, N.C. Induction of resistance in tomato plant against Fusarium oxysporum f. sp. lycopersici mediated through salicylic acid and Trichoderma harzianum. J. Plant Protec. Res, 2011; 52: 220-225.
50. Dutta, S., Mishra, A.K., Dileep Kumar, B.S. Induction of systemic resistance against fusarial wilt in pigeonpea through interaction of PGPR and rhizobia. Soil Biol. Biochem, 2008; 40: 452-461.
51. Anand, M., Naik, M.K., Ramegouda, G., Devika Rani, G.S. Biocontrol and plant growth promotion activity of indigenous isolates of Pseudomonas fluorescens. J. Mycopathol. Res, 2010; 48: 45-50.
52. Pandey, K.K., Upadhyay, J.P. Comparative study of chemical, biological and integrated approach for management of Fusarium wilt of pigeonpea. J. Mycol. Plant Pathol, 1999; 29: 214-216.
53. Siddiqui, S., Siddiqui, Z.A., Iqbal Ahmad. Evaluation of fluorescent Pseudomonads and Bacillus isolates for the biocontrol of a wilt disease complex of pigeonpea. World J. Microbiol. Biotech, 2005; 21: 729-732.
54. Raju, G.P., Rao S.V.R., Gopal, K. Integrated management of pigeonpea wilt caused by Fusarium udum. Indian J. Plant Prot, 2005; 33: 246-248.
55. Gholve, V.M., Kurundkar, B.P. Efficacy of Pseudomonas fluorescens isolates against wilt of pigeonpea. J. Maharashtra Agri. Univ, 2003; 27: 327-328.
56. Vidysekaran, P., Selhuraman, K., Rajappan, K., Vasumathi, K. Powder formulations of Pseudomonas fluorescens to control pigeonpea wilt. Biol control, 1997; 8: 166-171.
57. Harman, G.E., Bjorkman, T. Potential and existing use of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: G.E. Harman and C.P. Kubicek (Eds.), Trichoderma and Gliocladium. Enzymes, Biological control and Commercial Applications, vol.2. taylor & Francis Ltd, London, United Kingdom, 1998; pp.229-265.
58. Benhamou, N., Picard, K. Induced resistance: a new strategy of plant defence against pathogenic agents. Phytoprotection, 1999; 80: 137-168.
59. Whipps, J.M., Lumsden, R.D. Commercial use of fungi as plant disease biological control agents: status and prospects. In: T. Butt, C. Jackson and N. Magan (Eds.), Fungal Biocontrol Agents: Progress, Problems and potential, CABI Publishgn, Wallingford, 2001; pp. 9-22.
60. Khan, M.R., Gupta, J. Antagonistic effects of Trichoderma species against Macrophomina phaseolina on eggplant. J. Plant Dis Prot, 1998; 105: 387-393.
61. Arora, D.K., Alander, R.P., Mukerji, K.G. Handbook of applied mycology. Fungal Biotechnology, 1992; Vol. 4, Marcel Dekker, New York.
62. Bull, C.T., Weller, D.M., Thomasow, L.S. Relationship between root colonization and suppression of Gaemannomyus graminis var tritici by Pseudomonas fluorescens strain2-79. Phytopathology, 1991; 81: 954-959.
63. Berger, F., Hong, Li., White, D., Frazer, R., Leifert, C. Effect of pathogen inoculum anotagonist density and plant species on biological control of Phytopthora and Pythium damping off by Bacillus subtilis cot 1 in high humidity fogging glasshouses. Phytopathology, 1996; 86: 428-433.
64. Mazzola, M., Van Veen, J.H., Laaanbroek, H.J., do Vos, W.M. Mechanism of natural soil suppressiveness to soil borne disesase. Proceedings of 9th International of microbial ecology, Emsterdum, Netherland August 2001. Antonie-van- Leeuwenhoek. 2002; 81: 557-564.
65. Burkhead, K.D., Schisler, D.A., Slininger. Pyrolnintrin production by biological control agent Pseudomonas cepacia B37 W in culture and in colonized wounds of potatoes. Appl. Environ Microbiol, 1994; 60: 2031-2039.
66. Kim, D.S., Cook, R.J., Weller, D.M. Bacillus sp.L324-92 for biological control of three root diseases of wheat grown with reduced tillage. Phytopathology, 1997; 87: 551-558.
67. Brannen, R. Production of antibiotics by Bacillus subtilis and their effect on fungal colonists of various crops. Trans. Br. Mycol. Soc, 1995; 65: 203.
68. Khan, M.R. Preface of Biological control of Fusarial wilt and root knot of legumes. Government of India Publication, Department of Biotechnology, Ministry of Science at technology, New Delhi, India, 2005.
69. Dubey, S.C, Suresh, M., Singh, B.C. Evaluation of Trichederma: species agents first edy. Feplium for integrates management of clicepe a wilt. Biol control 2007; 40: 118-127.
70. Rini, C.R., Sulochana, K.K. Usefulness of Trichoderma and Pseudomonas against Rhizoctonia solani and Fusarium oxysporum infecting tomato. J. Trop. Agric, 2007; 45, 21-28.