Antagonistic Activities of Streptomyces against Root Knot Nematode of Kiwifruit

Introduction: Iran is among the world leading kiwifruit producers with 2.816 ha cultivated and 31.567 tones production. Plant parasitic nematodes cause damages to a variety of agricultural crops throughout the world. Interest in biological control of nematodes has increased because of the need for alternative methods to fumigant and non-fumigant nematicides and overall improvement of IPM programs. Bacterial species with nematicidal activity have also been used with some success for controlling root-knot diseases, including Streptomyces spp., Serratia spp., Bacillus spp. and Pseudomonas spp. The goal of the current study was to isolate, identify and investigate the potential of local Streptomyces bacteria for controlling and reducing root-knot nematode population in the north of Iran. Materials and Methods: In order to evaluate the effect of antagonistic bacteria on control of root-knot nematode of Kiwifruit, 100 isolates of bacteria were collected from Kiwifruit rhizosphere in the north of Iran and screened for pigmented microorganisms especially Streptomyces by applying standard serial dilution plate technique, using starch casein nitrate agar and glycerol asparagine agar. Morphological characterizations were achieved by the microscopic method. The microscopic characterization was done by cover slip culture method. The mycelium structure, color and arrangement of conidiospore and arthrospore on the mycelium were observed through the oil immersion (100X). The observed structure was compared with Bergey’s Manual of Determinative Bacteriology and the organism was identified. Various biochemical tests performed for the identification of the potent isolates are as follows: casein hydrolysis, starch hydrolysis, urea hydrolysis, esculin hydrolysis, acid production from sugar, NaCl resistance, temperature tolerance. Soil samples (100g) were collected, and then processed for nematode egg and larvae extraction Hussey method. The suspension was pipetted into counting Petri plate and examined under a stereomicroscope. Nematode larvae were identified to generic level and were counted. Evaluation of Actinomycetes isolates against root-knot nematodes in vitro performed according to Sun et al. Seedlings (Six-month-old) of kiwifruit (Actinidia deliciosa) were sown in 30 cm3 pots containing autoclaved sandy loam soil (1:1). Pots were divided into three groups by three replicates. Bioagents were individually incorporated into the soil at a dose rate of 10 cm3 (Heavy cell suspension of all isolates was prepared at rate 105 spores ml-1) were added to the soil. After seven days, (when bacterial cells reach its maximum growth peak) plants were inoculated with 2000 freshly hatched second stage juveniles(J2) of Meloidogyne spp. Pots were fertilized with recommended dose and kept at 25ºC ± 3ºC in complete randomized design. After two months plants were uprooted then galls and egg masses were counted and their indices were recorded according to Sharma et al. Fresh weight of roots was also registered. Treatments means were compared by the Duncan Multiple Range Test at 0.05 level of probability. The growth responses of kiwifruit (roots weight and number of galls and egg mass) were also recorded. Statistical analyses were achieved using SAS. Results and Discussion: Among 25 isolates identified as Streptomyces genus, 9 Actinomycetes isolates showed the antagonistic potential in vitro and reduced the rate of egg hatching in seven days and larval mortality in four days. Streptomyces sp3. Streptomyces sp4., Streptomyces sp5., Streptomyces sp9. And Streptomyces sp12. were able to reduce egg hatching 16.29%, 19.99%, 27.11%, 20.22% and 18.41% and increased the percentage of larval mortality 45%, 33.3%, 37.53%,35.01% and 37.50%, respectively. They showed the greatest effect and selected for evaluating in greenhouse condition. In addition, Streptomyces sp9.and Streptomyces sp4. reduced galls by 65.35% and 64.56% compared with the phenamiphus 57.47% had good performance. Results showed Streptomyces can be considered as an alternative for control of root-knot nematode. This is the first report of biocontrol of root-knot nematode in Kiwifruit by Streptomyces. The production of most antibiotics is species specific, and these secondary metabolites are important so the Streptomyces spp. can compete with other microorganisms that may come in contact, or even within the same genus. Another important process involving the production of antibiotics is the symbiosis between Streptomyces and plants, as the antibiotic protects the plant against pathogens and plant exudates allows the development of Streptomyces. Almost 80% of the world’s antibiotics are known to come from Actinomycetes, mostly from the genus Streptomyces. Conclusion: The present study clearly indicates that the use of Streptomyces sp9. And Streptomyces sp4. that significantly enhanced kiwifruit growth and reduced root-knot nematode populations and that it could be proposed for eco-friendly bionematicide use. Strains belonging to genus Streptomyces can, therefore, act as biocontrol agent with plant growth promoting ability. Furthermore, their potential metabolic diversity, mycelia growth habit, rapid growth rate, colonization of semi-selective substrates and ability to be genetically manipulated make them well-suited for soil inoculation. Additionally, ability to form desiccation-resistant spores which assists their spread, persistence and formulation make them preferred biocontrol agents. Biological control agents offer one of the best alternatives to reduce the use of pesticides.