International Journal of Microbiology & Parasitology. Research Article

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1 Open Access Scientific Publisher Research Article IN VITRO EVALUATION OF NEMATICIDAL ACTION OF NEEM AND BT FORMULATIONS ON MELOIDOGYNE INCOGNITA Harjinder Kaur 1, Harpreet Kaur 1, Praveen Rishi 2 1 Department of Zoology and Environmental Sciences, Punjabi University, Patiala, Punjab , India 2 Department of Microbiology, Panjab University, Chandigarh, Punjab , India ABSTRACT Correspondence should be addressed to Harjinder Kaur Received April 23, 2015; Accepted May 10, 2015; Published June 25, 2015; Copyright: 2015 Harjinder Kaur et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cite This Article: Kaur, H., Kaur, H., Rishi, P.(2015). In vitro evaluation of nematicidal action of neem and bt formulations on meloidogyne incognita. International Journal of Microbiology & Parasitology, 1(1).1-10 Background & Objectives: Meloidogyne incognita is a one of the major pathogens of tomato in India and cause severe crop damage. M. incognita can be managed effectively by chemical treatments but many of the nematicides are expensive, pose human and environmental risk. Management of M. incognita with biological control agents has been receiving growing consideration. Therefore, in the present study neem and Bt were used to control M. incognita through egg hatching experiment in laboratory conditions. Methods: Nematicidal activities of neem and Bacillus thuringiensis have been investigated against root-knot nematode (M. incognita) in laboratory conditions. For this purpose, the effects of aqueous extract of the neem leaf and seed alone, Bt alone (whole cell suspension) and combination with each other on the hatch of eggs of Meloidogyne incognita were evaluation for different time intervals (1 h to 72 h). Results: The NSE inhibit the egg hatching at first hour was 50% which increased to 100% at 72 h. NLE was also effective and egg hatch inhibition was 94.3% and 96.8% at 48h and 72h respectively. Egg hatch inhibition in standard concentrations (100% w/v) of neem, increased with increase in incubation time from 1 h to 72 h. Effect of whole cell suspension (WCS) (1.1X 10 9 CFU/ml) + neem seed extract on egg hatching after 12 h exposure was the most effective treatment and caused significantly least number of egg hatching (0.2%) as compared to the water control. The present study indicated that WCS formulation of Bacillus thuringiensis strain MTCC CODE 1953 was effective and caused significantly 100% egg hatch inhibition after 72 h exposure as compared to the water control. KEYWORDS: M. incognita, WCS(whole cell suspension), NLE(neem leaf extract), NSE(neem seed extract), CFU(colony forming unit) INTRODUCTION Tomato (Lycopersicon esculentum, Mill) is the second most cultivated vegetable crop in the world, after potato, with an annual production of nearly 100 million tons of fresh tomatoes in 3.7 million ha world wide. Tomato is short duration crop, high yielding, economically attractive and its area of cultivation is increasing daily [16]The fruit contain antioxidants, vitamins and minerals and can be processed into juices, ketchup, puree, eaten raw in salads or cook in to stew [5]. Root knot nematodes (Meloidogyne incognita) are one of the major pathogen of tomato world wide and limit fruit production, estimated yield losses ranging from 28-68% [16]. M. incognita infestations on tomato (Lycopersicon esculentum Mill) are common in 1

2 India and cause severe crop damage [1]. In view of tomato importance, there is great need to control M. incognita. Over the last years, agricultural chemicals have been the main control method for M. incognita in intensive agriculture. The pesticides introduced new environmental conditions to which plant pathogens had to adopt, frequently by becoming resistant. Due to the adverse effects of pesticides on the environment and human health, there is a need for alternative control methods to manage M. incognita. As modern agriculture moves to adopt more environment friendly practices there is increasing interest in the use of plants and microorganisms as source of biocontrol agents [18] The present study was carried out to determine the impact of neem alone, Bt alone and neem+ Bt combinations on the egg hatching of M. incognita in laboratory. MATERIALS AND METHODS was centrifuged at 5000rpm for 15 minutes. Supernatant was discarded and pellete was dissolved in 1ml of PBS. Serial dilutions in duplicate were plated i.e. 10-2, 10-4, 10-6, 10-8, and [17]. Agar plates were incubated at 30 C for 24 h. Small Bt colony groups on nutrient agar plates were counted and CFU/ml was calculated. Preparation of Bt (whole cell suspension) formulation: Whole Cell Suspension 20ml of nutrient broth was taken and inoculated with one colony of Bt. Kept overnight at 30 C (flask-1). Flask-2 (20 ml fresh nutrient broth) was inoculated with 0.2ml of Bt from flask-1. Flask-2 was incubated for 6 h. Flask-2 containing Bt cell suspension ( CFU/ml) was obtained [11] Preparation of aqueous neem leaf extract and neem seed extract : 2 Preparation of bt and neem formulations Accession of Bacillus thuringiensis : Bacillus thuringiensis strain MTCC CODE 1953 was accessed from Institute of Microbial Technology IMTECH, Sector-39 (A), Chandigarh , India. Collection of neem leaves and neem seed powder During the period of four years i.e , mature leaves and seeds of neem (Azadirachta indica) were collected from Punjabi University campus. Leaves were shade dried and were grinded in electric grinder. Oil was extracted from the neem seeds and rest of neem khali was grinded to obtain the powder form. Accession of Carbofuradan 3-G : Carbofuradan 3-G was accessed from Bharat Seeds, Luv Kush market, Patiala and was used as chemical check. Revival and maintainance of Bt culture : Flask-1 containing 20ml nutrient broth was inoculated with lyophilized culture of Bt and incubated at 30 C for 24 h. Bt stock formation was done by pouring 0.1ml of nutrient broth and Bt culture from flask-1 into eppendorf having 0.4ml sterilized glycerol stock and was preserved at -20 C for further use in experimentation. Bt culture was maintained on agar plates, for this 20 ml of nutrient broth inoculated with a loopful of Bt was thoroughly mixed and incubated at 30 C for 24 h followed by streaking on agar plates in quadrant manner with the help of inoculating needle. Streaked plates were kept in inverted position at 30 C for 24 h to obtain Bt colonies. Standardization of CFU/ml of Bt : 20ml nutrient broth was taken in 250 ml conical flask-1 and inoculated with one colony of Bt, kept overnight for incubation at 30 C. 3ml nutrient broth from flask-1 was transferred into the eppendorf to observe the optical density on spectrophotometer and 0.2ml was inoculated into fresh nutrient broth in flask-2. Flask-2 was incubated for 6-8 h. After incubation (Flask-2) 20 ml nutrient broth 25g of neem leaf powder and neem seed powder was blended in electric blender in 250ml distilled water for 15 minutes, kept in water bath for 8h at 60 C, autoclaved at 15Ib pressure at 121 C, allowed to cool and filtered through the muslin cloth. Filtrate was considered as standard solution (100%)[3] xperimental design for egg hatching of M. incognita Egg hatching experiment:- The egg masses shining white in appearance and of same size adhering to the galled roots were handpicked with the help of fine forceps under a stereomicroscope. Egg masses were treated with 1% sodium hypochlorite for 2 minutes to sterilize and were shaken for 5 minutes in electric shaker. Each counting dish contained 2ml of egg suspension (approximately 500 eggs) + 2ml neem extracts alone (100% concentration)/ Bt formulations alone (1.1 X 10 9 CFU/ml) / combination of neem + Bt (1:1). Egg masses incubated in distilled water served as water control [9]Following treatment combinations were evaluated. Neem leaf extract Neem seed extract Whole cell suspension Cell free supernatant Cell pelleted residues Spore/crystal proteins Whole cell suspension + neem leaf extract Whole cell suspension + neem seed extract Cell free supernatant + neem leaf extract Cell free supernatant + neem seed extract Cell pelleted residues + neem leaf extract Cell pelleted residues + neem seed extract Spore/crystal proteins + neem leaf extract Spore/crystal proteins + neem seed extract Carbofuradan 3-G (chemical check) Water control

3 International Journal of Microbiology & Parasitology Egg hatch inhibition:- Egg hatch inhibition was recorded after different time intervals i.e. 1h, 6h, 12h, 24h. 48h and 72h. Eggs were washed several times with distilled water and number of hatched larvae were recorded. After completion of each treatment, eggs were resuspended in 5ml distilled water for 24 h to check whether the egg masses kept in the treatment had been temporarily or permanently inactivated. The emergence of juveniles was again recorded after 24 h [2] Statistical analysis Mean values for each experiment were calculated. Data recorded was analyzed statistically by using analysis of variance (ANOVA), Pearson correlation and means were compared with the Tuckey s Multiple Range test. RESULTS Observations indicated that egg hatch inhibition in NSE at first hour was 50% which increased to 100% at 72 h (Table 1 and 6). NLE was also effective and egg hatch inhibition was 94.3% and 96.8% at 48h and 72h respectively (Table 5 and Table 6). In vitro incubation (6h) of eggs of M. incognita in aqueous neem leaf extract resulted in 27.5% egg hatch inhibition as compared to the water control (Figure 2). In the present study, egg hatch inhibition in standard concentrations (100% w/v) of neem, increased with increase in incubation time from 1 h to 72 h. In the present study 6 h treated eggs with standard concentration of aqueous neem leaf extract and whole cell suspension + neem leaf extract showed no egg hatching in distilled water after 24 h incubation as compared to the water control (Table 2). In vitro effect of whole cell suspension (1.1X 10 9 CFU/ml) + neem seed extract on egg hatching after 12 h exposure was the most effective treatment and caused significantly least number of egg hatching (0.2%) as compared to the water control. Whole cell suspension alone was also effective and resulted in 86.7% egg hatch inhibition after 24h incubation (Table 3and Figure 4). After 12 h incubation whole cell suspension + neem seed extract and whole cell suspension + neem leaf extract were also very effective causing 99.3% and 98.7% egg hatch inhibition respectively Table 3 The present study indicated that WCS formulation of Bacillus thuringiensis strain MTCC CODE 1953 was effective and caused significantly 100% egg hatch inhibition after 72 h exposure as compared to the water control. Egg hatching inhibition with whole cell suspension + neem leaf extract and neem leaf extract was at par and were equally effective (Figure 6). DISCUSSION This study was conducted to evaluate the nematicidal affect of aqueous neem extracts and Bt formulations on egg hatching of M. incognita in laboratory conditions. Azadirachta indica with 20% concentration caused increase in egg hatch inhibition till 48 h [6] The rate of hatching with petroleum ether leaf extract of chinaberry (Melia azedarach) was maximum at 48 h and minimum at 12 h which showed that hatching rate was directly proportional to the exposure period [7] The different concentrations of BioNem (commercial formulation of Bacillus firmus) significantly caused inhibited egg hatching [19] Secondary metabolites produced by the rice endophytic bacterium, Bacillus megaterium have shown similar effect and reduced hatching in Meloidogyne graminicola [13] Isolates B5, B4, B11 were more effective and inhibited hatching of M. incognita to large extent than other isolates tested [17]. The culture filterate of the strain CHAO of Pseudomonas fluorescens (p<0.05) caused 40% egg hatch inhibition [15] The hatching of eggs was completely inhibited when egg masses of M. incognita were exposed to the higher concentration i.e cells/ml of Bt strain (J107), while only six eggs hatched when exposed to Bt strain (J115) for 6 days [4] Two commercial Bt insecticides, SAN 415 and Dipel, inhibited the percentage of egg hatching of M. javanica by 37% and 34% respectively [12] The complete culture and cell suspension of two yeast species, Candida incommunis and Wicherhamiella domercqiae and recorded 100% and 89.9% inhibition respectively. On the other hand, Bacillus amyloliquefaciens suspension of microbial cells caused the lowest inhibition percentage (72.97%) [10]. The bioagent candidates like, Bacillus subtilis, Saccharomyces avarum and Saccharomyces ludwiggi proved harmful to M. javanica egg masses [14] The bacterial volatiles of Bacillus megaterium YFM3.25 resulted in strong inhibition of egg hatching and complete egg hatch inhibition was recorded at six th day [8] 3

4 Table 1: Showing in vitro effect of various formulations of neem, Bt and combination of neem + Bt on egg hatch inhibition of M. Incognita after 1 h exposure as compared to the water control Treatments No. of eggs hatched after 1 No. of 1 h treated eggs hatched Hatch inhibition h of treatment (mean±sd) in distilled water after 24 h after 1 h (%) Neem leaf extract 10.4 ± 1.5 0± Neem seed extract 8.4 ± 1.1 0± Whole cell suspension 8.2 ± 0.8 0± Whole cell suspension +neem 3.6 ± ± leaf extract Whole cell suspension + 3 ± ± neem seed exract Carbofuradan 3-G 3.4± ± Water control 17± ±0.5 - Table 2:Showing in vitro effect of various formulations of neem, Bt and combination of neem + Bt on egg hatch inhibition of M. Incognita after 6 h exposure as compared to the water control Treatments No. of eggs hatched after 6 h of treatment (mean±sd) No. of 6 h treated eggs hatched in distilled water after 24 h Hatch inhibition after 6 h (%) Neem leaf extract 14.2±1.09 0± Neem seed extract 16±1.4 3± Whole cell suspension 12.4± ± Whole cell suspension 4.6±1.1 0± neem leaf extract Whole cell suspension + 5± ± neem seed exract Carbofuradan 3-G 15.6± ± Water control 19.6± ±0.8 4

5 Table 3:Showing in vitro effect of various formulations of neem alone, Bt alone and combination of neem + Bt on egg hatch inhibition of M. Incognita after 12 h exposure as compared to the water control Treatments No. of eggs hatched after 12 h of treatment No. of 12 h treated eggs hatched in distilled water after 24 h Hatch inhibition after 12 h (%) Neem leaf extract 15.2±1.3 0± Neem seed extract 10.4±1.5 0± Whole cell suspension 0.6±0.54 0± Whole cell suspension +neem 0.4±0.5 0± leaf extract Whole cell suspension + neem 0.2±0.4 0± seed exract Carbofuradan 3-G 5.8±1.3 0± Water control 33.2± ± Table 4:Showing in vitro effect of various formulations of neem, Bt and combination of neem + Bt on egg hatch inhibition of M. Incognita after 24 h exposure as compared to the water control Treatments No. of eggs hatched after 24 h of treatment No. of 24 h treated eggs hatched in distilled water after 24 h Hatch inhibition after 24 h (%) Neem leaf extract 15.4±0.5 2± Neem seed extract 9.6±1.5 3± Whole cell suspension 5.4± ± Whole cell suspension +neem leaf extract Whole cell suspension + neem seed exract 6.6± ± ± ± Carbofuradan 3-G 40± ± Water control 40.8± ±0.8-5

6 Table 5: Showing in vitro effect of various formulations of neem, Bt and combination of neem + Bt on egg hatch inhibition of M. Incognita after 48 h exposure as compared to the water control Treatments No. of eggs hatched after 48 h of treatment No. of 48 h treated eggs hatched in distilled water after 24 h Hatch inhibition after 48 h (%) Neem leaf extract 4±1.2 0± Neem seed extract 6.2± ± Whole cell suspension 3±1.2 0± Whole cell suspension +neem 6.2±0.8 0± leaf extract Whole cell suspension + 3± ± neem seed exract Carbofuradan 3-G 33±2 0± Water control 70.8± ±1.1 - Table 6:Showing in vitro effect of various formulations of neem, Bt and combination of neem + Bt on egg hatch inhibition of M. Incognita after 72 h exposure as compared to the water control Treatments No. of eggs hatched after 72 h of treatment No. of 72 h treated eggs hatched in distilled water after 24 h Hatch inhibition after 72 h (%) Neem leaf extract 2.6±0.8 0± Neem seed extract 0±0 0±0 100 Whole cell suspension 0±0 0±0 100 Whole cell suspension +neem 3±0.7 0± leaf extract Whole cell suspension + neem 1.4±0.5 0± seed exract Carbofuradan 3-G 0±0 0±0 100 Water control 83.4±2.07 1±0.5 6

7 Figure 1: Showing the effect of various formulations of neem, Bt and combination of neem + Bt on egg hatching of M. Incognita (in vitro) after 1 h exposure and number of treated eggs hatched in distilled water after 24 h incubation Values are mean of five replicates. Mean in each column followed by the same letter do not differ at p<0.05 according to Figure 2:Showing the effect of various formulations of neem, Bt and combination of neem + Bt on egg hatching of M. Incognita (in vitro) after 6 h exposure and number of treated eggs hatched in distilled water after 24 h incubation Values are mean of five replicates. Means in each column followed by the same letter do not differ at p<0.05 according to 7

8 Figure 3:Showing the effect of various formulations of neem, Bt and combination of neem + Bt on egg hatching of M. incognita (in vitro) after 12 h exposure and number of treated eggs hatched in distilled water after 24 h incubation Values are mean of five replicates. Means in each column followed by the same letter do not differ at p<0.05 according to Figure 4: Showing the effect of various formulations of neem, Bt and combination of neem + Bt on egg hatching of M. incognita (in vitro) after 24 h exposure and number of treated eggs hatched in distilled water after 24 h incubation Values are mean of five replicates. Means in each column followed by the same letter do not differ at p<0.05 according to 8

9 Figure 5: Showing the effect of various formulations of neem, Bt and combination of neem + Bt on egg hatching of M. Incognita (in vitro) after 48 h exposure and number of treated eggs hatched in distilled water after 24 h incubation Values are mean of five replicates. Means in each column followed by the same letter do not differ at p<0.05 according to Figure 6: Showing the effect of various formulations of neem, Bt and combination of neem + Bt on egg hatching of M. incognita (in vitro) after 72 h exposure and number of treated eggs hatched in distilled water after 24 h incubation Values are mean of five replicates. Means in each column followed by the same letter do not differ at p<0.05 according to REFERENCES [1] Abd-Elgawad MMM, Aboul-Eid HZ. Effects of oxamyl, insect nematodes and serratia marcescens on a polyspecific nematode community and yield of tomato. Egyptian J Agronematol 2001; 5: [2] Adegbite AA, Adesiyan SO. Root extracts of plants to control root knot nematode on edible soybean. World J Agric Sci 2005; 1(1) : [3] Akhtar M, Mahmood I. Nematode populations and short-term tomato growth in response to neem-based products and others soil amendments. Nematropica 1994b; 24: [4] Al-Banna L, Khyami-Horani H. Nematicidal activity of two Jordanian strains of Bacillus thuringiensis on rootknot nematodes. Nematol Medit 2004; 32 : [5] Beutner S, Bloedorn B, Frixet S, Blanco IH, Hoffman T, Martin H. Quantitative assessment of antioxidant properties of natural colarants and phytochemicals: carotenoid, flavonoid, phenol and indigoids. The role of β- carotene in antioxidant functions, J Sci Food Agric 2001; 81: [6] Bharadwaj A, Satyawati S. Effect of some plant extracts on the hatch of Meloidogyne incognita eggs. Int J Bot 2007; 3 (3) : [7] Fourie H, Mc Donald AH. Nematodes. ARCLNR Leaflet. Crop Prot 2000; 18 (4):

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