In vitro antagonistic activity of Pseudomonas spp . against Rhizoctonia soloni

The present research work deals with the in-vitro study of antagonistic activity of Pseudomonas spp. against plantpathogenic fungi Rhizoctonia soloni. The bacterial strains were isolated from rhizospheric soil of Korea district of Chhattisgarh. A total of 28 bacterial cultures were isolated from 25 representative soil samples collected from five blocks of Korea district of Chhattisgarh, out of which four were identified as Pseudomonas spp. (PKS10Pseudomonas syringae, PKM11Pseudomonas syringae, PKJ25Pseudomonas alcaligenes and PKB27Pseudomonas alcaligenes) from Plant Pathology Division, IARI Delhi. These four Pseudomonas spp. were examined for their ability to antagonize R. soloni in in vitro plate assay by dual culture inoculation along with the standard check (PmtccPseudomonas isolate from IMTECH Chandigarh). The antagonistic activity was interpreted by restricted growth zone of the fungal pathogen in dual culture. The diameter of hyphal growth of the fungi in dual culture with isolates PKS10, PKM11, PKJ25, PKB27, Pmtcc and control were 3.82, 3.68, 2.73, 3.41, 3.25 and 7.3 cm respectively. All the four Pseudomonas isolates PKS10, PKM11, PKJ25, PKB27 and Pmtcc (standard check) inhibited the hyphal growth of Rhizoctonia spp. by 47.67, 49.58, 62.60, 53.28 and 55.47% respectively. One among four Pseudomonas isolates, (PKJ25) P. alcaligenes was found to suppress the growth of fungal pathogen significantly in dual culture by 62.60% and was more effective than other isolates. This study suggests that P. alcaligenes isolates might be used as potential biological control agents against plant pathogenic fungi Rhizoctonia soloni.


INTRODUCTION
Rhizoctonia solani has been encountered as one of the potent soil-borne fungal pathogens, which develops in both cultured and non-cultured soils.R. solani are highly destructive phytopathogens (Curtis et al., 2010), known to cause symptoms of damping-off and root rot diseases to wide range of vegetable and crop plants including tomato (Abu-Taleb et al., 2011;Karima et al., 2012).Various methods have been reported for controlling of damping-off disease but biological control is an efficient and ecofriendly methods.Many microbial species are involved in the biocontrol of phytopathogens such as Trichoderma viride (Hafez et al., 2013) fluorescens and Bacillus subtilis (Sivasakthi et al., 2014).Adhikari et al. (2013) reported the antagonistic nature of rhizospheric bacteria against R. solani.
Pseudomonas spp. is one of the most promising groups of rhizospheric bacterial inhabitants that are extensively investigated to be used as biocontrol of pathogens in agriculture (O'Sullivan and O'Gara, 1992;Ganeshan and Kumar, 2006;Maurya et al., 2014).They show antagonistic activity against diverse phytopathogens such as Pythium spp.(Leoper, 1988) and Rhizoctonia spp.(Howell and Stipanovic, 1979).Various species of Pseudomonas are thought to play an important role in plant growth promotion and disease suppression (Kloepper et al., 1980;Jayaswal et al., 1990).In particular P. fluorescence (Howell and Stipanovic, 1979;Weller and Cook, 1983) and P.cepacia (Hebbar et al., 1992;Jayaswal et al., 1990) have attracted a considerable attention on account of their potential for biological control.Shalini and Srivastava (2008) screened out antifungal activities of P. fluorescence against phytopathogenic fungi.Antifungal activity of fluorescent Pseudomonads against R. solani is correlated to the production of secondary metabolites (Mina et al., 2013;Sharma et al., 2014;Mezeal, 2014).Pseudomonas aeruginosa rhizobacterial isolates PTR-3 exhibited antagonism of over 68.9% by restricting in vitro mycelial growth of R. solani up to 1.9 cm (Kamei et al., 2014).
In the present investigation the in vitro biocontrol efficacy of Pseudomonas spp.isolated from Korea district of Chhattisgarh is reported against R. soloni; they are able to antagonize plant pathogenic fungi in in-vitro condition, hence can be used as potential biocontrol agent.

Isolation and characterization of Pseudomonas
In the present investigation soil samples were collected from randomly selected locations in the field region from Korea district of Chhattisgarh by composite sampling method (Walworth, 2004).
Korea District is North-Eastern District of Chhattisgarh State of India.Geographically, state lies in Latitude between 23 02 ' 42" to 23 deg. 44' 46" North and Longitude between 81 46' 42" to 82 deg. 33' 43" East.Height from Sea Level is 700 Meters.The District is bound on the North by Shidhi District of Madhya Pradesh, on the South by Bilaspur Districts, on the East by its parent District Surguja and on the West by Shahdol District of Madhya Pradesh (Figure 1).Temparature (Average) is 32C (Max) and 17C (Min); land area covers 5978 Sq.Km and forest area is 59.03%.The Climate is ideal with a beautiful monsoon, a mild summer and a bearable winter.Average Rainfall is 1410.9mm, Soil type red-yellow and major crop-paddy (http://korea.gov.in/glance.htm(National Informatics centre Korea Chhattisgarh).Korea district is divided into 5 blocks-Baikunthpur, Manendragarh, Khadgawan, Sonhat and Janakpur.
All total of 25 soil samples were collected from 5 blocks of agro based areas of Korea district of Chhattisgarh, five representative soil samples from each block (Baikunthpur, Manendragarh, Khadgawan, Sonhat, Janakpur), during May-June 2009, all these were drawn from post harvested fields (Venkateswarlu et al., 1984).Total of 28 bacterial cultures were isolated from 25 soil samples of 5 blocks of Korea district of Chhattisgarh.
Isolation of rhizospheric bacteria was carried out by serial 10-fold dilutions technique (Pandey et al., 2006) on Nutrient agar and Pseudomonas agar base (all from Hi Media).Four out of 28 bacterial cultures were identified as Pseudomonas spp.by Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, Pusa campus, New Delhi, India -12, for there confirmation through molecular marker (16s rRNA and 16s rDNA sequencing) (Kanimozhi and Panneerselvam, 2010).Isolated and identified bacterial cultures were characterized by studying cultural characteristics of individual isolates on nutrient agar, nutrient broth and Pseudomonas agar base medium.Morphological characteristics of bacterial cultures were studied microscopically.The bacteria were also tested for their biochemical reaction, antibiotic sensitivity and tolerance of pH (from 3 to 11 at intervals of 2 pH units), temperature (from 4.0 to 40°C) and salt concentration (from 0.5-20% NaCl) by growing them on Nutrient Agar Medium and broth.

Isolation of fungal pathogen
Fungal pathogen R. soloni was isolated from stem of infected tomato plantlet with dampingoff disease from local field.The fungal parthogen was characterized by microscopic examination (Ganesan and Gnanamanickam, 1987) and its pure cultures were maintained on PDA for further use (Devi et al., 1989).

Assay for in vitro antagonism
A loopful of bacterial culture was placed (5mm in diameter) at one edge on the periphery of PDA plate and mycelial discs (5mm in diameter) were cut from actively growing fungal culture and placed opposite to the bacterial inoculation on PDA plate (Picture 1) (Ganesan and Gnanamanickam, 1987;Podile et al., 1988;Babu et al., 2000).All four isolates of Pseudomonas (PKS10, PKM11, PKJ25 and PKB27) were tested in vitro for their antagonistic activity against test plant pathogens, Rhizoctonia spp.(causal agent of dampingoff of tomato) along with standard check Pmtcc (Pseudomonas isolate from MTCC Chandigarh used as standard) in comparison to control (pure cultures of fungal pathogen taken as control for in vitro assay).

RESULTS AND DISCUSSION
Two among four Pseudomonas isolates belong to spp.P. syringae (PKS10 and PKM11) and P. alcaligenes (PKJ25 and PKB27).These four isolates of Pseudomonas were from rhizosphere of Oryza sativa (Kailashpur, Sonhat Block), Abelmoschus esculentus (Barbaspur, Manendragarh Block), Oryza sativa (Umarwah, Janakpur Block) and Lycopersicum esculentum (Dumaria, Baikunthpur Block) respectively.characterization of Pseudomonas spp.are given in Table 2. Pseudomonas isolates PKS10 and PKM11 formed creamy colonies on Nutrient agar Medium and yellow colonies on Pseudomonas agar medium whereas isolates PKJ25 and PKB27 formed white colonies on NAM and PAB, PKS10 and PKM11 showed surface growth while PKJ25 and PKB27 showed turbidity in liquid medium.These isolates had the morphological features like singly arranged, flagellated, non-endospore forming, non -capsulated, motile, Gram negative rods.The isolates PKS10 and PKM11 showed growth from 25--11 (optimum pH for PKS10-7 and PKM11-9) and salt concentration up to 5%.Isolates PKJ25 and PKB27 were a concentration of NaCl upto 5% and a wide range of initial pH from 5 to 11 (optimum pH for PKJ25-5 and PKM11-5).Similar characteristics were described for bacteria belonging to genus Pseudomonas and species fluorescens (Sharma et al., 2007;Malviya and Singh, 2012) and putida (Pandey et al., 2006;Malboobi et al., 2009b) respectively.The result interpreted by Malboobi et al. (2009a) in their investigation on PSB tolerance to extreme climates supported our findings.They intensively examined the isolates for tolerance toward high temperature, high concentration of NaCl and wide range of pH and found that all PSB strains survived at high temperature and could tolerate concentration of 2.5% NaCl and alkaline pH.All four isolates were sensitive to gentamicin (<10 µg ml -1
Result of in vitro antagonistic effect of different Pseudomonas isolates against plant pathogenic fungi under dual culture technique is depicted in Table 3.Among 4 of the Pseudomonas isolates, isolate PKJ25 inhibited the mycelial growth and was inhibitory to the Rhizoctonia soloni as compared to the other three and Pmtcc.The diameter of hyphal growth of the fungi with isolates PKS10, PKM11, PKJ25, PKB27, Pmtcc and control were 3.82, 3.68, 2.73, 3.41, 3.25 and 7.3 cm respectively.All the four Pseudomonas isolates PKS10, PKM11, PKJ25, PKB27 and Pmtcc inhibited the hyphal growth of Rhizoctoniaby 47.67, 49.58, 62.60, 53.28 and 55.47% respectively.

in-vitro antagonism Pseudomonas isolate against R. soloni
Our result suggests Pseudomonas isolate PKJ25 (P.alcaligenes) was efficient and significantly suppressed the vegetative growth of the test fungi Rhizoctonia by   Khan and Zaidi (2002) for R. soloni and Fusarium oxysporium.Akhtar and Siddiqui (2009) suggested the use of plant growth promoting rhizobacteria for the biocontrol of root-rot disease complex of chickpea and their studies showed that the three Pseudomonas spp.had inhibitory effect on Macrophomina phaseolina; P. alcaligenes was one of the biocontrol agent.Jayaraj et al., (2007) tested 08 fluorescent Pseudomonads isolated from tomato rhizosphere and observed highest growth inhibition (15.5 mm) of Pythium aphanidermatum and controlled damping off of tomato by 68.5%.The antagonistic nature of P. aeruginosa rhizobacterial isolates PTR-3 and PCF-3 against R. solani was also reported by Kamei et al. (2014).Their finding suggest that rhizobacterial isolates PTR-3 restricted mycelial growth of R. solani up to 1.9 (cm) and were found to exhibit antagonism of over 68.9%.Sharma et al. (2014) reported that Pseudomonas spp.isolates showed antifungal activity against Rhizoctonia spp. in the range of 7.27-53.84%inhibition.Also, P. fluorescens isolate restricted the linear growth of R. solani by 81.3% as reported by Mezeal (2014).
Thus, present study receives strong support from the above observations and the information generated through this study will help for future studies on the antagonistic affect of native microorganisms on soil in Chhattisgarh (India) and consequently for the maintenance of native microorganisms as microbial antagonists for enhancement of crop production.

Figure 1 .
Figure 1.Political map of Korea district of Chhattisgarh, India.

Picture 1 .
Pure culture plate of Pseudomonas spp.(plate 1) and Rhizoctonia soloni (plate 2).Dual culture plate of antagonism by Pseudomonas against Rhizoctonia soloni on PDA (plate 3).(a) Rhizoctonia soloni (b) Pseudomonas isolate PKJ25.Zone of inhibition was recorded after 1 week of incubation, by measuring the restricted growth zone between the edges of fungal and bacterial colonies (Picture 2).Plate with pure Pseudomonas inoculum corresponding to pure fungal inoculum was taken as control.Inoculated Petri plates were incubated at 25±1C for 07 days (Picture 3).The assays of dual culture interaction were conducted in triplicates in Completely Randomized Design and repeated twice.The per cent inhibition of mycelial growth of the pathogens was calculated using the following formula(Perveen and Bokhari, 2012): I = (C -T/C) × 100 Where, I = Inhibition (%) or antagonistic effect, C = colony diameter of test fungus in control plate and T = colony diameter of the same test fungus in dual culture against Pseudomonas as antagonist.

Table 1 .
Occurrence, identities, sources and abbreviation of Pseudomonas in Rhizospheric soil of Korea district of Chhattisgarh.

Table 2 .
Characterization of different Pseudomonas isolates.

Table 3 .
In vitro antagonistic effect of different Pseudomonas isolates against plant pathogenic Fungi under dual culture technique.
Devi et al. (1989)fluorecens on plant pathogenic fungi R. solani, Sclerotium rolfsii.Devi et al. (1989)suggested that antagonistic bacteria Pseudomonas fluorescent isolates (Pfr1-14) obtained from rice rhizosphere suppressed the rice ShB pathogen, R. solonii in vitro by inhibiting mycelial growth and sclerotial germination.Similar report of antagonistic effect of Fluorescent Pseudomonas was reported by