Analysis Of 26S RDNA Sequence Of Nomuraea Rileyi Nr0310 And The Produced Crystal

Nomuraea rileyi is an entomopathogenic fungus that widely distributed in many countries and regions, capable of infecting over 40 insect species. The genetic diversity of the Nomuraea rileyi related to different conditions, including environment, habitats, host species or even individual physiology, which cannot be identified and classified only by the physiological, biochemical and morphological characteristics. In such cases, molecular biology techniques and phylogenetic analysis must be considered especially during classification. The crystal product of Nomuraea rileyi was first observed on the body surface of infected Bombyx mori by Kumar in 1997. But in our laboratory, the crystal product of Nomuraea rileyi was observed both on the body surface and in hemocoel of silkworm, and even on the medium cultured in vitro.Nomuraea rileyi strain Nr0310, the native pathogen of Bombyx mori was identified based on 26S rDNA by using molecular biological methods. The total DNA was extracted from newly harvested hyphae and prepared by using a CTAB protocol. PCR amplification of the 26S rDNA was performed using universal primer pairs of fungi, NL1 and NL4. The 26S rDNA sequence of Nomuraea rileyi strain Nr0310 was compared with available gene sequences in GenBank databases using BLAST search facility at the National Center for Biotechnology Information. Several genus and species such as Cordyceps, Metarhizium, Chamaeleomyces granulomatis, Torrubiella tenuis, N.cylindrosporae and N.atypicola were analyzed. Based on alignment of 26S rDNA gene sequences of the fungi mentioned above, the 26S rDNA sequence of Nomuraea rileyi strain Nr0310 with the GenBank accession number JQ999993 showed the highest similarity (99%) to that of Nomuraea rileyi (AB047211), and they shared the same clade in the phylogenetic tree. Combined with morphological characteristics, this strain was finally identified as Nomuraea rileyi, belongs to family Clavicipitaceae. The strain also showed high similarity of 98 percent to Chamaeleomyces granulomatis (HM635077, HM635078) Cordyceps chlamydospori, but it’s far away from them and the species of N.cylindrosporae and N.atypicola. The crystals were observed both on the body surface and in hemocoel of the silk worm infected by Nomuraea rileyi strain Nr0310. On the solid medium cultured in vilro, a large number of the octahedron diamond or rhombus shaped crystals scatter away from the hyphae. The crystals produced by the Nomuraea rileyi strain Nr0310 would be influenced by the conditions such as pH value, temperature, light and carbon souree and nitrogen source.Under the culture condition of pH 4-5, Nomuraea rileyi strain Nr0310 produced very few or none crystals, and the shape of crystals were changed into rectangular, square hexahedron and irregular shapes; under the culture condition of pH 9-11, the quantity of crystals were reduced but the volume became larger. When the temperature is too high, the crystals were rectangular, square hexahedron and irregular shapes; if the temperature was higher than 47℃, the growth of strain was inhibited, and the process of the crystal production was delayed. Under the dark condition, the germination rate of the conidias were decreased, but once the conidia started to grow, a large number of crystals would be produced, so there is almost no difference in crystal quantity. Limited carbon source and nitrogen source of the culture leaded to the lower amount and rectangular shape of crystals; when using olive oil instead of carbon source and nitrogen source, some hyphae became saccate protrusions and did not produce crystals, but the normal hyphae produced a large number of crystals. These results suggest that the crystal production of Nomuraea rileyi are closely related and shows positive correlation to the growth and development. When the culture condition changes, the growth and development of the strain are affected, so as the crystal production process. Because of the crystals were observed both on the body surface and in hemocoel of infected Bombyx mori, it is might that the crystals secreted by Nomuraea rileyi strain Nr0310 are involved in self-protection, or a toxin beneficial for entomopathogenic fungus during the infection process.