Screening for Biocontrol Agent against Sclerotium rolfsii Sacc, Stem Rot Pathogen of Groundnut

R. R. RAKH & L. S. RAUT* Department of Microbiology, Shri Guru Buddhiswami Mahavidyalaya, Purna (Jn.) *Department of Microbiology, Sant Tukaram College, Parbhani E mail: drrrrakh@rediffmail.com; drrrrakh@gmail.com

Abstract: Stem rot pathogen of groundnut, Sclerotium rolfsii, was isolated from the infected groundnut plant part. Later on screened for effective biocontrol agent against the stem rot pathogen, by using dual culture method, by using bacteria isolated from various sources such rhizospheric soil, extreme environment. 137 bacteria were tested against pathogen and among them two bacteria found effective against the stem rot pathogen, Sclerotium rolfsii. These two bacteria belongs to Pseudomonas genus, which was later on identified by 16 S rRNA sequencing, and found to as Pseudomonas cf. monteilii 9, Pseudomonas aeruginosa AL 98. Key words: Sclerotium rolfsii, rhizospheric soil, Pseudomonas, Pseudomonas cf. monteilii 9, Pseudomonas aeruginosa AL 98 Introduction: Biological control is an environment-friendly strategy to reduce crop damage caused by plant pathogens. Biological control of soil-borne pathogens with antagonistic bacteria and fungi has been intensively investigated (Paulitz, et al., 1996). Antagonistic microorganisms from rhizosphere are ideal biocontrol agents, as the rhizosphere provides the frontline defense for root against infection by the pathogens (Lumsden, et al., 1995). Biocontrol of phytopathogen using antagonistic microorganism offer a highly economical and ecofriendly alternative to the use of synthetic pesticides. Rhizospheric bacteria serve as excellent agents to control soil-borne plant pathogens. Bacterial species like Bacillus, Pseudomonas, Serratia and Arthrobacter have been proved in controlling the fungal diseases. Bacteria identified as plant growth promoting rhizobacteria and biocontrol strains often belong to the following genera. (i) Bacillus (Nair et al., 2002), (ii) Streptomyces (Adb-Allah, 2001) and (iii) Pseudomonas (Mark et al., 2006). The mode of action of biocontrol agents against various soil borne plant pathogens, include biosynthesis of various secondary metabolites such as antibiotics, hydrolytic enzymes, siderophores, and competition for nutrients. Successful bacterial antagonists often show a synergistic combination of mechanisms responsible for a successful antifungal interaction (Reddy, et al., 2008). Sclerotium rolfsii Sacc is a soil-borne plant pathogen of worldwide importance with a very extensive host range including more than 500 plants species. Most S. rolfsii diseases have been reported on dicotyledonous hosts, but with several monocotyledonous species also being infected. Sclerotium rolfsii is especially severe on legumes, Solanaceous crops, cucurbits and other vegetables grown in rotation with beans (Tu, 1978; Wydra 1996). Among the soil-borne fungal diseases of groundnut, stem rot caused by S. rolfsii is a potential threat to production and is of considerable economic significance for groundnut grown under irrigated conditions. Stem-rot caused by S. rolfsii is sporadic in most of the

groundnut growing areas like Tamil Nadu, Andhra Pradesh, Karnataka (Pande, et al., 2000). The traditional agricultural practice to control the phytopathogen is by using variety of fungicides e.g. Bavistin, Captan etc. but a sever disadvantage of the traditional method, that it is not effective to check the Sclerotium during the cropping duration (90- 100 days) and is not eco-friendly. Hence, as an alternative attempt has been made to give an eco-friendly strategy for the control of Sclerotium during this work. The objective of the current investigation was to screen for an efficient biocontrol agent, Pseudomonas spp. Bacillus spp., from rhizospheric soil of healthy plants such as Neem and evaluate its potential in controlling the soil-borne pathogen, Sclerotium rolfsii, causing stem rot of groundnut. Materials and Methods: Collection of diseased groundnut plants and seeds: Diseased groundnut plants were collected from different locations such as, field at village Therla, Dist. Beed, from farm of Marathwada Agricultural University, Parbhani, from field at village Shirasgav, near Parbhani, and various fields from different district of Marathwada region, and brought to laboratory, Department of Microbiology, Shri Guru Buddhiswami Mahavidyalaya, Purna (Jn.) Parbhani in poly-ethylene bags. Infected groundnut seeds (Photo Plate 1) were procured from farmers as well as from market for isolation of seed borne pathogens. Isolation of Phytopathogens: Diseased samples showing typical symptoms of stem rot i.e. wilting of total plants, white mycelial growth at collar region of plant (Photo Plate 1) were selected and used as sample source for the isolation of causative agent. Infected portion of stem was cut into small pieces with sterilized scalpel, cleaned with distilled water, then surface sterilized with 0.1% HgCl2 solution for 30 second and again washed thrice with sterile distilled water. Small 1 to 2 pieces were transferred aseptically on Potato Dextrose Agar (PDA) plates containing Chloramphenicol (30 mg/100 ml) with the help of sterilized forceps under aseptic condition (Rakh, 2010). Inoculated Petri plates were incubated at 25C for 5-7 days for growth of the pathogen. Isolation of Rhizobacteria: Rhizospheric soil from different healthy plants such as soybean, neem, groundnut, tur etc. were collected in poly-ethylene bags and brought to the research laboratory. 1 gm of soil sample was inoculated into 100 ml nutrient broth and kept for incubation at room temperature for 24 h. For isolation of Azotobacter and Pseudomonas spp, 1ml of this nutrient broth was transferred to selective enrichment media such as Nitrogen Free Mannitol Broth for Azotobacter spp., Cetrimide Broth for Pseudomonas spp. and kept for incubation at room temperature for 24 h. From enriched Nitrogen Free Mannitol Broth and Cetrimide Broth, a loopful of culture was streaked on Nitrogen Free Mannitol Agar (Ashby (1907) and Cetrimide Agar (Brown and Lowbury, 1965), respectively and the plates were incubated at room temperature till colonies were observed (24 48 h). The isolated colonies developed were then purified on Nutrient Agar slants and used for screening against the phytopathogen for biocontrol ability. For isolation of Bacillus spp., a modified method of Kim et al., (1997) was employed. A 1ml of enriched Nutrient Broth was added to 10 ml sterile distilled water and kept at 80C for 20 min. later on a loopful of culture was streaked on nutrient agar plates. Plates were incubated at room temperature for 48 h. Typical white colonies were picked up individually and purified on nutrient agar slants. All the isolates were tentatively named during this research to avoid confusion.

Screening for Potential Biocontrol agents: All the isolates were screened for potential antagonistic activity against S. rolfsii, on Kings B agar (Ran, et al., 2003) using dual culture technique. (Rangeshwaran and Prasad, 2000) An agar disc (5 mm) was cut from an actively growing (96 h) S. rolfsii, and placed on the surface of fresh Kings B Agar medium at the one side of the Petri plates. A loopful of actively growing bacterial isolates (each) was placed opposite to the fungal disc. Plates inoculated with phytopathogen and without bacteria were used as control. Each experiment was carried out in triplicates. Plates were incubated at room temperature for 7 days. Degree of antagonism was determined by measuring the radial growth of pathogen with bacterial culture and control and Percent inhibition was calculated by the following equation (Riungu et al., 2008). Colony diameter of pathogen Colony diameter of pathogen + alone (Control) Antagonist Infection = Colony diameter of pathogen alone An efficient biocontrol agent obtained from screening was identified according to Bergeys Manual of Systematic Bacteriology (1984) by using cultural, biochemical characteristics as well as 16s rRNA sequencing. 16s rRNA sequencing of culture was carried out at Agharkar Research Institute (ARI) Pune, Maharashtra. Result and Discussion: Isolation of Sclerotium rolfsii: After 7 days incubation on PDA plates, the fungus produced abundant white septate mycelia, 1.53.0 m diameter with clamp connections at each septation, aerial hyphae and also numerous spherical, or ellipsoidal, white sclerotia, 0.5 2.0 mm diameter, which turned brown on maturation (Photo Plate 1). Based on morphological and culture characteristic, the disease causing organism was identified as Sclerotium rolfsii (Mesquita et al., 2007). Isolation of Rhizobacteria: During this research work, 137 rhizospheric bacteria were isolated from soil of different healthy plants such as Soybean, Neem, Groundnut, Tur etc. Amongst 137 rhizospheric isolates, 67 were Pseudomonas spp., 9 were Azotobacter spp. and 61 were Bacillus spp. All the rhizospheric isolates were tentatively named as DSM 1 to DSM 61, SGBM 1 to SGBM 67, AZO 1 to 9. Screening for Biocontrol agents against Phytopathogens: For screening of potential Biocontrol agent all 137 isolated strains were tested for their antagonistic activity against isolated pathogens of groundnut viz. S. rolfsii, by dual culture method. It was observed that two isolates namely SGBM 1 and SGBM 7 were found as efficient antagonists against S. rolfsii (Photo Plate 2 & 3). The results of antagonistic activity in vitro for both the Pseudomonas strains were observed by dual culture technique. It was revealed that both culture i.e. SGBM 1 and SGBM 7 were able to inhibit Sclerotium rolfsii (94 %) as shown in table 1. Our results when compared with the results earlier reported by Kishore et al., (2005), for control of Sclerotium rolfsii with Pseudomonas aeruginosa in dual culture. It was found that our results with Pseudomonas are far better than the above mentioned results because of the fact that there was only 32-74 % inhibition recorded where as 94 % inhibition was recorded for S. rolfsii. Further when the inhibitory activity of both strain was confirmed by microscopic observation of the clear zone, it revealed lysed mycelium of Sclerotium rolfsii. Similar results observed by Kishore et al., 2005b, where they found that seed endophytes, P. aeruginosa GSE 18 and GSE 19 effectively reduced stem rot severity in the greenhouse. These two strains were x 100

antagonistic to eight fungal pathogens of groundnut and induced hyphal deformations in S. rolfsii. 16S rRNA sequencing of SGBM 1 showed 98 % homology with Pseudomonas cf. monteilii 9 while 16S rRNA sequencing of SGBM 7 showed 98 % homology with Pseudomonas aeruginosa AL98.

Moist Chamber Technique for isolation of Sclerotium from Infected Seeds Infected Groundnut Seed

Isolated Sclerotium rolfsii Stem Rot Infection of Groundnut Photo Plate 1: Isolation of Sclerotium rolfsii from Infected Groundnut Stem and Seed

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Photo Plate 2: Dual Culture Technique of SGBM 1 against Sclerotium rolfsii A) Control of Sclerotium rolfsii B) SGBM 1 Culture with Sclerotium rolfsii

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Photo Plate 3: Dual Culture Technique of SGBM 7 against Sclerotium rolfsii A) Control of Sclerotium rolfsii B) SGBM 7 Culture with Sclerotium rolfsii

Table 1: Screening for Biocontrol Agent against Sclerotium rolfsii Sacc

Tentative Name of Bacteria SGBM 1 SGBM 2 SGBM 3 SGBM 4 SGBM 5 SGBM 6 SGBM 7 SGBM 8 SGBM 9 SGBM 10 SGBM 11 SGBM 12 SGBM 13 SGBM 14 SGBM 15 SGBM 16 SGBM 17 SGBM 18 SGBM 19 SGBM 20 SGBM 21 SGBM 22 SGBM 23 SGBM 24 SGBM 25 SGBM 26 SGBM 27 SGBM 28 SGBM 29 SGBM 30 SGBM 31 SGBM 32 SGBM 33 SGBM 34 SGBM 35 SGBM 36 SGBM 37 SGBM 38 SGBM 39 SGBM 40 SGBM 41 SGBM 42 SGBM 43 Inhibition Zone of Sclerotium rolfsii 4 3 2 1 1 1 4 3 2 3 0 3 0 0 0 1 2 3 0 0 2 2 1 0 0 0 1 1 3 1 2 1 0 0 0 1 1 3 2 2 1 0 2 Tentative Name of Bacteria SGBM 46 SGBM 47 SGBM 48 SGBM 49 SGBM 50 SGBM 51 SGBM 52 SGBM 53 SGBM 54 SGBM 55 SGBM 56 SGBM 57 SGBM 58 SGBM 59 SGBM 60 SGBM 61 SGBM 62 SGBM 63 SGBM 64 SGBM 65 SGBM 66 SGBM 67 DSM 1 DSM 2 DSM 3 DSM 4 DSM 5 DSM 6 DSM 7 DSM 8 DSM 9 DSM 10 DSM 11 DSM 12 DSM 13 DSM 14 DSM 15 DSM 16 DSM 17 DSM 18 DSM 19 DSM 20 DSM 21 Inhibition Zone of Sclerotium rolfsii 3 2 3 0 3 0 0 0 1 2 3 0 0 2 2 1 0 0 0 1 1 3 1 2 1 0 0 0 1 1 3 2 2 1 0 2 1 2 1 0 0 0 1 Tentative Name of Bacteria DSM 24 DSM 25 DSM 26 DSM 27 DSM 28 DSM 29 DSM 30 DSM 31 DSM 32 DSM 33 DSM 34 DSM 35 DSM 36 DSM 37 DSM 38 DSM 39 DSM 40 DSM 41 DSM 42 DSM 43 DSM 44 DSM 45 DSM 46 DSM 47 DSM 48 DSM 49 DSM 50 DSM 51 DSM 52 DSM 53 DSM 54 DSM 55 DSM 56 DSM 57 DSM 58 DSM 59 DSM 60 DSM 61 AZO 1 AZO 2 AZO 3 AZO 4 AZO 5 Inhibition Zone of Sclerotium rolfsii 0 0 0 1 2 3 0 0 2 2 1 0 0 0 1 1 3 1 2 1 2 2 1 0 0 0 1 1 3 1 2 2 1 0 0 0 1 1 3 1 0 0 3 Tentative Name of Bacteria AZO 8 AZO 9 SGBM 44 SGBM 45 DSM 22 DSM 23 AZO 6 AZO 7 Inhibition Zone of Sclerotium rolfsii 1 2 3 0 0 2 2 3

Each number is mean of three replicates. 0 - none, 1= inhibition zone 1-25 %, 2= inhibition zone 26-50 %, 3= inhibition zone 51-75 %, 4= inhibition zone 76-100 %.

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