ANTAGONISTIC POTENTIAL OF BACILLUS SUBTILIS AND PSEUDOMONAS FLUORESCENS ON MELOIDOGYNE INCOGNITA OF GREEN ONION (ALLIUM FISTULOSUM)

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1 International Journal of Botany and Research (IJBR) ISSN Vol. 3, Issue 3, Aug 2013, TJPRC Pvt. Ltd. ANTAGONISTIC POTENTIAL OF BACILLUS SUBTILIS AND PSEUDOMONAS FLUORESCENS ON MELOIDOGYNE INCOGNITA OF GREEN ONION (ALLIUM FISTULOSUM) HAYDER MUNSHID, SOBITA SIMON & A. A. LAL Department of Plant Protection, SHIATS, Allahabad, Uttar Pradesh, India ABSTRACT Biological control is considered as new efficient method that becomes widely used for controlling plant parasitic nematodes, as aim to decrease the extent of environment degradation and the effect of the excessive toxic nematicides. Vitro experiments conducted for the evaluation of bio-control agents viz., Pseudomonas fluorescensand Bacillus subtilis, against root-knot nematode, Meloidogyne incognita in onion (Allium fistulosum) revealed that soil application of both P. fluorescens and B. subtilis alone or in combination was able to reduce the nematode population and improve the onion growth parameters in terms of shoot length, root length, shoot fresh and dry weight, root fresh weight. KEYWORDS: Onion, Meloidogyne incognita, Bacillus subtilis, Pseudomonas fluorescens, Carbofuran 3G INTRODUCTION Onion is one of the most popular vegetable that form of daily diet. It is a common vegetable that is cultivated in more than 175 countries of the world. The U.S.A. is a leader in onion production and produces over 7% of the world onion crop [1]. The area under onion cultivation in India was thousand ha. And its production was 82 lacks MT in The major onion producing stats are Maharashtra, Gujarat, Uttar Pradesh, Rajasthan, Orissa, Karnataka, Tamil Nadu, Madhya Pradesh and Bihar. Maharashtra ranks first in onion production with a share of (18%) in terms of productivity. Indian onions are famous for their pungency and are available round the year. There are estimated 6.7 million acres of onion is an indispensable item in every kitchen as condiment and vegetable. It is used either in raw and dehydrated form to add favor and taste to Indian cousins. Since onion has medicinal value, it is used in some pharmaceutical preparation also. It has many uses as, folk remedies and recent report suggests that onion play a part in preventing heart diseases and other ailments. Onion bulb is rich in minerals like phosphorous, calcium and carbohydrate. It also contains proteins and vitamin C. [2]. The root-knot nematodes Meloidogyne spp.are serious pathogens of many economic crops and causing approximately 5% of global crop loss [3]. Root-knot nematodes, Meloidogyne species, are parasitic on a wide variety of plant hosts and are especially common in warmer sandy soils of the Punjab. The nematode infection induces extensive galling and root damage. Vegetable crops usually are among the most susceptible and worst affected by these nematodes [4-6].Meloidogyne incognita, M. javanica, and M. arenaria are present in some of the major onion-producing states in the U.S. such as California, Texas, and Georgia, and there is the potential that onions could be planted in fields with high population densities of these nematodes [7]. Onion is documented as a host for Meloidogyne incognita, M. javanica, and M. arenaria but their relative reproductive ability on onion has not been reported[8-10]. The rhizosphere is a dynamic environment where mutual relationships among nematodes, plant and environment are often of chemical nature. Rhizopbcteriacolonie plant roots and promote plan growth and for reduce damage caused by the disease. Phosphate solubilizing bacteria supply p to plant [11]. Bacteria of the groups Bacillus and Pseudomonas have proven to be the most powerful phosphate solubilizing bacteria[12]. Bio-control appears to offer an environmentally safe and ecologically

2 16 Hayder Munshid, Sobita Simon & A. A. Lal feasible option for plant protection with great potential for promoting sustainable agriculture. Bacteria, fungi, protozoans, viruses, nematodes and plants have been reported as antagonists to plant parasitic nematodes [13]. Plant-associated microorganisms have important roles in natural and induced suppressiveness of soil-borne diseases. Several culture ablerhizobacteria have been tested for their biocontrol potential against plant parasitic nematodes [14-21]. The efficiency of carbofuran has been reported by various authors such as Steele (1977) that stated that carbofuran causes the dysfunction of the nematode as component of complex diseases and also inhibits development of female and egg production. Plantparasitic nematodes were also prevented from entry into tomato roots after application of carbofuran[22]. MATERIALS AND METHODS Nematodes Inoculum Meloidogyne incognita was collected from the 10 different localities of the farmer's field. At each location, root of 20 composite plants were collected showing stunted and yellowing, tomato plant from each filed. Roots were carefully washed under tap water in the bucket to remove adhering particular. Egg masses of M. incognita were picked with forceps and rinsed with sterile water then placed in 0.5% sodium hypochlorite (NaOcl) solution, agitated for 4 minutes and rinsed with sterile water on a 26 mesh Sieve [23]. The eggs were incubated for 3-5 days using a modified baermann funnel method [24].To obtain second stage Juvenile. M. incognita J 2 were extracted from gall of fresh root also after grounded the tissue in grinder. Population densities of J 2 were determined from 1ml aliquots before inoculation of the onion plant. Preparation of Onion Nursery The seed beds of onion plant were prepared in the pots. Onion (A. fistulosum) seeds were sown in 2-cm deep in earthen pots filled with 5kg sterilized soil and FYM@50 g/pot. Thirty days seedlings were used for experiment. Source of Pseudomonas fluorescence and Bacillus subtilis Pseudomonas fluorescne and Bacillus subitilis were isolated from tomato rhizosphere in king b media and nutrient ager, identified on this basis of morphological physiological and biochemical characteristics [25 and 26]. The isolates were stored under appropriate conditions in the incubator at the C. The individual cultvre of Pseudomonas fluorescne in kings B media and Bacillus subitilisnutrient agar media was centrifuged at 10,000g for 10 mints to separate bacterial from culture media. After centrifugation supernatants was discarded and pellets (bacterial cells) was washed by centrifugation three times with sterilized distilled water and for that suspended in sterilized distilled water. The centrifugation of bacterial suspension used for experiment was 10 7 CFU/ml. Inoculation of M. incognita Twenty eight days old to onion seedlings were transplanted into 25 cmdiameter clay pots containing 2 kg steamsterilized sandy loam soil (bacterial treated) pots. Pots were divided into sex groups, each contained four replicate and were arranged in a complete randomized block design, watered and received the normal agricultural practices. One week after transplant of onion except control (Plant alone) all the treatments of seedling were inoculated with 1000 J 2 larvae of M. incognita per pot. 90 days after nematodes inoculation, the plants were uprooted from the pots and the roots were gently separated from soil, washed with tab water and dried by pressing lightly between blotting paper. Observations were made on plant growth parameters viz., shoot length, root length, fresh plant weight, dry plant weight, root weight, number of larvae per gram root,number of root-knot per plant. Root gall index was recorded on 1-5 scale as given by [27], 1 = no galling. 2 = 1-10 galls. 3 = galls. 4 = galls. 5 = more than 100 galls/root system.

3 Antagonistic Potential of Bacillus subtilis and Pseudomonas fluorescens 17 on Meloidogyne incognita of Green Onion (Allium fistulosum) RESULTS & DISCUSSIONS The bacterial bio-agent Pseudomonas fluorescens and Bacillus subtilis were evaluated alone and in combination for their predacity against Melodiogyne incognita results of the present investigation are presented as under. Shoot Length(cm) of Onion Plants were inoculated with Meloidogyne incognita significant reduction in shoot length were observed in untreated plants with bio-agents and carbofuran 3G. The shoot length (cm) of onion at 60 and 90 d.a.i after inoculation with Meloidogyne incognita of the treatment T 4 (Pseudomonas fluorescens) in combination with Bacillus subtilis significantly increased as compared with T 2 (P. fluorescence) alone, T 5 (Carbofuran 3G) and T 3 (B. subtilis). T 0 (onion alone) and T 1 (M. incognita alone).whereas the treatments T 2, T 5, T 3, T 0 and T 1 were found non-significant from each other.[28]also reported the use of Bacillus subtilis may also improve plant growth by suppressing non parasitic root pathogen or by the production of biologically active substances or by unavailable mineral and organic compounds in to forms available to plants. Result of table (1) indicates that the fresh shoot weight (g) at 90 d.a.i of M. incognita were significantly lower in nematode inoculated plants from Pseudomonas fluorescens and Bacillus subtilis and carbofuran 3G treated plants non-significantly different was found in T 2, T 3, T 4 and T 5 treatments. Table 1: Effect of Bio-Control Agents on Plant Growth Characters 90 d.a.i after Inoculation of Meloidogyne incognita on Onion Treatments Shoot Length Shoot Length Freshweight Dry weight/g Rootlength/ Rootweight/ (cm) 60 d.a.i (cm) 90 d.a.i /g 90 d.a.i 90 d.a.i (cm) 90 d.a.i (cm) 90 d.a.i Onion alone M. incognita P. flourescens(10ml) B. subtilis(10ml) P.flourescens(5ml) + B.subtilus(5ml) T5 - carbofuran 3G C. D. (P = 0.05) T 0 - Onion T 1 - Meloidogyneincognita T 2 - Meloidogyne incognita + Pseudomonas fluorescens T 3 - Meloidogyne incognita + Bacillus subtilis T 4 - Meloidogyne incognita + Bacillus subtilis + Pseudomonas fluorescens T 5 - Meloidogyne incognita + Carbofuran 3G Figure 1

4 18 Hayder Munshid, Sobita Simon & A. A. Lal Dry Shoots Weight (g) at 90 Days The plant shoot was dried at 60 C for four d.a.i and the dry weight was recorded. Significantly increased in all the plant treated with P. flourescens, B. subtilustogether as compared to other treatments including control. Treatments (T 2, T 3, and T 5 ) were found significantly as compared to treatment (T 1 ) M. incognita. Whereas the treatments (T 2, T 3 and T 5 ) (T 0 and T 1 ) were found Non-significantly different from each other. (Table. 1) Table 1. Indicated root length of onion plants treated with P. fluorescensand B. subtilis and weight (g) over the plants inoculated with M. incognita alone and carbofuran 3G treatments. Root length (cm) of nematode alone treated plants and control (plant alone) were found non-significant from each other. Moreover, plants treated with bio-agents together and alone resulted in increased root length from nematode inoculated plants. Among the treatments carbofuran 3G nonsignificant result was found. [29]ReportedBacillus subtilis improved plant growth system of chickpea. [30]ObservedPseudomonas fluorescens alone or in combination with 10g/ plant of each bio-agent applied in the soil was effective to improve growth of mulberry with increased leaf yield. Table 2: Effect of Bio-Control Agents on Nematodes Population 90 d.a.i after Inoculation of Meloidogyne incognita on Onion Treatments Number of Number of Gall galls/plant Larvae/g Index Onionalone M. incognita P. flourescens(10ml) B. subtilis(10ml) P. flourescens(5ml) + B. subtilus(5ml) T5 - carbofuran 3G C. D. (P = 0.05) Number of Root-Knots and Larvae of M.incognita in Onion Root Result of table 2; from pot experiment indicated that addition of treatments with bacterial suspensions and carbofuran 3G into soil suppressed root galling and final population of M. incognita. Results of table 2. shows the highest reduction in Meloidogyne incognita galling at 90 d.a.i was found when Pseudomonas fluorescens and Bacillus subtilis treated together in the soil before transplanted onion as compared with Pseudomonas fluorescens and Bacillus subtilis alone treatment. However, both the bio-agent significantly reduced root-knot nematode as compared with control (nematode alone). Number of Larvae/g of Root Table no 2. Indicate that number of M. incognita larvae in 1g of onion root found significantly reduced in the treatment of carbofuran 3G as compared with other bio-agent treatments. But the bio-agent are significantly reused number of larvae as compared with control (nematode alone). Among the bacterial bio-agents non-significant result was found in T 3, T 1 and T 4. [31]found that Bacillus subtilis, Pseudomonas aeruginosa decrease nematode population densities, suppressed nematode, infection and galls on lettuce plants roots. [32]Who recorded that Bacillus subtilis significant reduced eggs hatching of M. javanica in vitro. [33]Observed the bacterial treatment shows the most effective to reduced nematode populations next to nematicidevydate 10 Gas compared with fresh leaf extract of neem, garlic and marigold. Bacterial antibiotics and other toxic compound present in metabolites as well as direct interaction might be responsible for the J 2 immobility, production of metabolites by rehizosphere bacteria causes lysis of nematode eggs and affects vitally of root-knot nematode J 2 stage. [14, 34-36].[37]Reported that production of metabolites, including 2,4diacetylphol-

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