Isolation, Identification And Molecular Analysis Of Neovossia Horrida

Smut is a kind of pathogenic fungi which is harmful to crops, especially to gramineous plants. The characteristic of smut is that it produces a large number of black powdery teleutospores in injury site of host plant. The smut has a wide variety of subspecies and wide ranges of host. According to difference of host plant or different injury site of the host, these plant diseases can be named wheat loose smut, wheat dwarf bunt, wheat flag smut, corn smut, sphacelotheca reiliana, rice kernel smut, reilianum of sorghum, stinking smut of barley, Sugarcane whip smut, and so on.Rice kernel smut is a fungal disease which can cause pathological changes of rice grains by Tilletia barclayana. When rice are harmed by these pathogenic bacteria, the disease seeds are more than ten to dozens of seeds which becoming black powder teleutospores. This disease has a wide distribution range in rice producing countries of Asia, America, Africa and most of the rice-growing area of China. After the introduce of hybrid rice in the 1970s, this disease further increased in farmland of the hybrid rice which influenced yield of rice and seed quality seriously.In this study, twelve samples of rice kernel smut were collected from Sichuan, Anhui, and Jiangxi province. The pathogenic bacteria was isolated from these samples by spore suspension culturing method. Eleven pathogenic strains were isolated and their morphology were observed by optical microscope and scanning electron microscopy. The result indicates that the mature chlamydospore of these strains is deep brown to black and opaque, and the thorn wart which is distributed in surface of chlamydospore is lightish and has top tip. Under certain temperature and light conditions, chlamydospore can germinate in water and produce microspores without other special nutrients. In process of chlamydospore germination, it grows a non-septatehypha which has some varying length of branches somewhile. The top of the non-septatehypha presents verticillated digitations. The microspore grows in these verticillated digitations of the non-septatehypha and is linear, slightly curved, colorless transparent and no separation. It can bud secondary microspore whose shape is acicular or banana-shaped, or sprout hypha directly.Because of high similar morphology of these isolated pathogenic bacteria, the QB-1 strain was selected to observed its morphology and molecular biological identification based on its ITS sequence. The result shows that morphology of this strain were exactly consistent with the morphology of Tilletia barclayana. In addation, phylogenetic analysis of its ITS sequence is the closest to ITS sequences of known Tilletia horrida in NCBI database. Therefore, the QB-1 strain can be considered to a Tilletia barclayana strain. Under different temperature, light and pH conditions, the growing states of QB-1 strain was observed. The result indicates that the optimum growth temperature of QB-1 strain is 28 ℃; the optimum growth pH value is 5-7; the light has no effect on the growth of QB-1 strain.High quality genome DNA of QB-1 strain was extracted by different method. The genome DNA was random interrupted and was used to pUC18 plasmid construction with inserted sequence as 180 bp. The genome sequencing of QB-1 strain was performed using Illumina Solexa GA II sequencing technology. The sequence assembly was achieved using the SOAPdenovo alignment tool. The result shows the 19 Mb draft genome of QB-1 strain is composed of 792 Scaffold, and contains 9,366 predicted genes whose total length is 16,309,943 bp, and average length is 1,741 bp, which account for 81.12% proportion of the draft genome of QB-1 strain. The G+C content of the draft genome is 35.41%. the protein encoded by predicted genes of QB-1 was compared with proteins of Pathogen Host Interactions database (PHI). The result shows that 4,653 proteins of QB-1 strain are similar to proteins of PHI database, which account for 49.7% proportion of all predicted proteins in QB-1 strain. The predicted proteins which its similarity is greater than or equal to 50% is 130, accounted for 2.7% of the total predicted proteins which have the similarity with proteins of PHI database.