Evaluation of the Level of Defense Enzymes Induced by Antagonistic Fungi against Root Knot Nematode, Meloidogyne javanica in Pistachio Seedlings

Introduction: Pistachio is one of the most important export products of the country and root knot nematode is one of the threatening factors in its production. The most prevalent species in pistachio orchards are those of Meloidogyne javanica and M. incognita. Plants have a wide range of defense mechanisms effective against the invasion of various pathogens and pests. These mechanisms include pre-existing physical barriers, chemical barriers, and induced defense responses. Beneficial soil microorganisms such as Trichoderma spp. and arbuscular mycorrhizal fungi (AMF) can protect plants from infection by direct mechanisms such as production of toxins, enzymes, and other metabolites or by inducing systemic resistance. Induce resistance to the root knot nematode occurs following an increased accumulation of different antagonistic compounds such as peroxidase and polyphenol oxidase. Increased levels of these enzymes has been observed in different plants that are responsible for the induced protection against Meloidogyne. Materials and Methods: In this study potential of antagonistic fungi, vesicular-arbuscular mycorrhiza (Funneliformis mosseae, Rhizophagus intraradices, Funneliformis caledonius) and Trichoderma (Tricoderma aureoviride,Tricoderma harzianum) and mixed of both fungi against Meloidogyne javanica under greenhouse conditions was investigated. In order to investigate the effect of antagonistic fungi on the induction of defense enzymes in pistachio seedlings of Badami Riz Zarand cultivar, the study was performed as a factorial experiment based on completely randomized design with twelve treatments in three replications in the greenhouse. Pistachio seedlings were inoculated at the stage of 8-10 leaf with 60 g of mycorrhizal mixture and three months later, these plants were sampled for examination. In order to ensure the purity of the fungi and the colonization of the roots, staining of the plant roots was performed and microscopic examination was performed to observe different organs of the fungus. After confirming the mycorrhiza treatment, Trichoderma treatment was applied. For Trichoderma treatment, 10 g of mixture of isolates T1, T2, T3, T4 and T5 per kg of soil was used. Pure nematode populations were prepared from a single egg mass on Early Urbana tomatoes. Pistachioseedlings were inoculated with 5000 second-stage juveniles of nematode 21 days after Trichoderma treatment.Nematode indices including the number of galls, egg masses and second juveniles per gram of root, the number of eggs in each egg mass and reproductive factor after 75 days for each treatment were measured.The second experiment was performed to investigate the effect of biological treatments on nematode indices in a completely randomized design with six treatments and three replications. Defense enzymes level of Peroxidase (POX), Polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) determined at four times of one and a half, three, five and a half and twelve days after nematode inoculation. Results and Discussion: Disease indices showed a significant decrease in fungi treatments compared to the nematode control. Trichoderma treatment showed a significant difference in all indicators compared to the other treatments. Reproductive index in Trichoderma treatment and its combination with mycorrhizal treatment decreased by 72 and 33.3 and nematode population decreased by 73.4% and 36.2% compared to the control, respectively. Extracellular hydrolytic enzymes play important role in the infection process of Tichoderma species against plant-parasitic nematodes. In mycorrhiza treatment, although there was no significant difference in gall index and egg mass with the control, but the number of second juveniles and nematode reproductive factor showed a significant decrease compared to the control. This may be due to the small size of the egg mass and the effect on the number of eggs. Previous results on mycorrhizal species of Rizophagus irregularis in tomato plant showed that egg masses and number of eggs in treated roots have a significant reduction. The maximum enzymatic activity for POX, and PPO was obtained in different treatments on the twelfth day. The maximum amount for PAL activity in seedlings treated with mycorrhizal and Tricoderma fungi was observed after five and a half days. The level of different enzymes increases after treatment with mycorrhiza and Trichoderma in plants, each of which plays a role in limiting the development of nematodes. Conclusion:Due to the high proliferation of nematodes in pistachio orchards and due to the environmental hazards of nematicides, the use of biocontrol agents is recommended. The studied fungal strains in the present study could be used as components in an integrated approach to manage M. incognita on pistachio plants. The use of plant growth promoting fungi such as mycorrhiza and Trichoderma while improving plant growth, can reduce the use of fertilizers and chemical pesticides and are effective for better plant growth and development and disease control. However, further studies on the use of combinations of these fungi with other bacterial antagonists and even animal manures as well as garden studies are necessary.