Etiology Of A New Stem-node Rot Disease Of Rice

Since2009, the rice has been appeared on a special kind of stem-node rot symptoms in a number of counties of Hunan Province.The highest incidence of the disease was up to80%, and resulted in serious losses. In order to identify the pathogen of the disease,we conducted a study of the etiology.1Pathogen IdentificationThe symptoms of infected rice that came from Fenghuang, Xiangxiang and Pingjiang were dead heart or dead spike, irregular-shaped brown strip in sheath, and the range of1cm above stem-nodes were black and rotten,easily broken, after peeling the sheath. The stem-node rot disease of rice should be a new disease. The symptoms of infected rice of Lingling did not stem-node rot but only stem rot; By separating microscopic examination found that there are9strains isolated from infected rice that came from Fenghuang,Xiangxiang and Pingjiang (respectively numbered:HNFH01～HNFH03, HNXX01～HNXX03and HNPJ01～HNPJ03),and their morphology are similar. In addition, there have3stains isolated from infected rice that came from Lingling (respectively numbered:HNLL01～HNLL03),and compared with the other stains found fewer whorled conidiophores and conidia more neatly arranged on phialide; The spore suspension was dropt into the heart leaves, sprayed on to the flowers and injected into the stem-nodes. Then the rice of been inoculated shown that the sheath has strip and dark brown spots, the spikes were a lot of empty,white and black, and the stem-nodes were rotten and black. These symptoms are consistent with the wild rice stem-nodes rot disease; PCR amplification of HNFHOK HNXXO1、HNPJ01and HNLL01were carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5. Sequencing of PCR products of HNFHO1、HNXXO1、HNPJ01showed100%identity to Sarocladium attenuatum CBS414.81(GenBank Accession No.AY566997) and HNPJ01was100%identitied to Sarocladium oryzae CBS361.75(GenBank Accession No. AY566993), after BLAST search. Therefore, identification of the pathogen of the stem-node rot disease of rice was Sarocladium.2Comparison of the Biological Characteristics of two Types of Sarocladium Comparison of conidium and conidiospore morphology, cultivation characters and temperature, illumination conditions etc. impact on hypha growth and sporulation with3strains of S.attenuatum and2strains of S. oryzae. The results show that there are some differences, but overall, S.attenuatum and S. oryzae were significantly different.3Classification status of two types of SarocladiumSince the taxonomic status of S.attenuatum and S. oryzae is in dispute, use of the differences of rDNA sequence to determine the status. The results show the design of specific primers and national patent primers can identified S.attenuatum and S. oryzae. The phylogenetic tree display S.attenuatum and S. oryzae gather into two groups. Therefore, the two types of Sarocladium should be different species or varieties.4Transformation of red fluorescent protein gene in Sarocladium attenuatumIn order to research colonization and infection process systems of pathogen of the rice stem-node rot disease, transformation of red fluorescent protein gene in S.attenuatum mediated by Agrobacterium tumefaciens.The15randomly picked transformants were selected of hygromycin-resistance, they were continuously cultured of5generations in the PDA plates with or without hygromycin, finally, there are eight transformants still showed hygromycin-resistant; rDNA of the transformants were extracted, primers which designed by the sequence of DsRed gene and hygromycin-resistance gene, used for PCR amplification of the transformants, the result showed that the DsRed gene was integrated into the genomes of tested transformants; The transformants could generate red fluorescence when they were excited by558nm laser. These results indicated that the DsRed gene had been successfully transformed into S.attenuatum-HNXXO1and expressed in the tested transformants.