Identification of Fungal Endophytes in Aerobic Rice Cultivation of Golestan Province

Rice is one of the most important grains in the world and large part of its consumption is related to Middle East. Iran ranks 23rd in production, 26th in cultivation, and 13th in rice consumption with nearly 560,000-hectare rice lands and an annual production of nearly 2.5 million tons. In natural and agricultural ecosystems, plants are associated with large populations of microorganisms in the rhizosphere, endosphere (inside the root), leaves (phyllosphere), as well as pollen and seeds. These microorganisms spend all or part of their life cycle inside the healthy tissues of host plant without any obvious sign of disease. The stable coexistence between endophytes and plants is due to production of biological secondary metabolites with a unique structure, which positively affects the survival of plant growth in adverse conditions. Studies have shown that endophytic fungi increase resistance to drought, insects, diseases and host tolerance level, especially in stress conditions. Considering the increase in the risk of dehydration and the lack of sufficient water resources and the development and adoption of aerobic rice cultivation, it is important to identify endophytic fungi that increase tolerance to drought stress and improve the growth factors of the host plant. There are relatively few studies on endophytic fungi that colonize healthy tissues of rice plants under aerobic conditions. Therefore, the aim of this study is to evaluate endophytic fungal collections in different parts of aerobically cultivated rice plants.

In this study, in order to identify endophytic fungi in aerobic cultivation of rice in Golestan province, sampling were carried out from several aerobic rice fields in Golestan province, including Gorgan, Kordkuy, Bandar Gaz, Aliabad and Kalaleh cities during the months of May to September in 2019-2020. Plant tissues were transferred to the laboratory (Department of Plant Protection) in separate paper bags along with specifications. Purification was performed on a 2% water-agar medium using Hyphal tip method. Based on investigations and morphological data, a number of isolates were selected as representatives for molecular identification. Genomic DNA was extracted from mycelia grown in PDB (Potato Dextrose Broth) culture medium according to the CTAB method with a slight modification, and the polymerase chain reaction (PCR) was performed according to Nemati and Abdulhazadeh method. The ITS region (ITS4-5.8S-ITS5) of the selected isolates was amplified during the PCR reaction using ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') primers and The β-tubulin region was amplified by T1 (5'-AACATGCGTGAGATTGTAAGT-3') and β Sandy-R (5'-GCRCGNGGVACRTACTTGTT-3') primers. Purification and sequencing of PCR products was done by Pishgam Biotechnology Company. The sequences of ITS and β-tubulin regions were modified and extracted using Chromas 2.6.6 software. Nucleotide sequences of the selected isolates were compared with the available sequences in the Gene bank by Blast algorithm and the sequences data were submitted in the NCBI database website. Phylogenetic trees were drawn using the Maximum likelihood method and MEGA 6.06 software.

Based on preliminary morphological studies, 20 isolates were selected as representatives of purified isolates and were identified by molecular analysis. In this study, a total of 39 fungal isolates were obtained, that the highest number of the isolates belonged to the pods of rice plants with the frequency of 17 isolates and the lowest number of isolates belonged to the roots of rice plants with 10 isolates. Among the 6 genera identified in this research, the largest number of isolates was belonged to Alternaria and Fusarium genera, with 18 and 6 isolates, respectively, and the highest number of species was belonged to Fusarium genus with 6 species. Fungal species include: F. chlamydosporum, F. proliferatum, F. incarnatum, F. solani, F. fujikuroi, F. verticillioides, S. bactrocephalum, N. sphaerica, N. oryzae, A. alternata, E. nigrum, C. cladosporioides. Among these species, Nigrospora sphaerica, E. nigrum and S. bactrocephalum are reported for the first time in this study as rice plant endophytes in Iran.

Investigation of endophytic fungi in the present study indicates that some of these isolates, including F. chlamydosporum, F. proliferatum, F. fujikuroi are pathogenic fungi. It means that range of the endophytic life and pathogenic life cannot be separated and determined and environmental conditions and nutritional status determine the type of interaction. F. chlamydosporum causes Fusarium root and crown rot, is reported in some sources as an endophytic fungus in various plants.