Morphological, Molecular And Biological Characteristics Of Rotylenchulus Reniformis Occurring In China

Reniform nematodes(Rotylenchulus Linford & Oliverira,1940) are a group of semiparasitic nematodes which affect the growth of roots especially root fibril by feeding on plant root cells and what is more serious is that they can cause roots necrosis and decayed. Among 10 species of Rotylenchulus genus, Rotylenchulus reniformis is the type species and a plant parasitic nematode with wide distribution and host range. The hosts of R. reniformis refer to above 300 plant species, including crops, fruit trees, vegetables and ornamental plants, etc. Many countries list it as quarantine pest because it accounts for the serious loss of cotton, soursop, pineapple and sweet potato, etc. In this study, reniform nematodes from 5 provinces or cities in China were systematical studies based on the morphological, rDNA and biological characteristics. The major results obtained are as followed:1. Among 1,021 soil samples collected from 5 provinces or cities of China, including Hainan, Guangdong, Fujian, Zhejiang and Chongqing, 33 populations of reniform nematodes were obtained and all the populations were identified as R. reniformis based on the morphological characters and morphometrics. It is indicated that R. reniformis is the dominant species of the genus Rotylenchulus in China. Meanwhile this is the first report about the distribution of R. reniformis in Zhejiang and Chongqing respectively.2. The hosts of R. reniformis in China were confirmed. The hosts of 33 populations of R. reniformis in this study referred to 16 families and 24 genera of plants. Among the hosts longan, Dimocarpus longana, is a new host of R. reniformis.3. All the 33 populations of R. reniformis were amphimictic, and the number of male and immature female was almost equal. The morphology characters of immature female and male among different geographical populations were roughly identical, except for the tail shape variation of some populations. Morphometrical comparisons revealed that most morphometrics of R. reniformis populations in China were confirmed to that of the type species and prior reported populations and some variation were detected, such as b value, c value, DGO, body length and h, which were think to be intra-species variation of different geographical populations. The body surface characters of males and immature females of HN17 population obtained by SEM enriched the morphometrical character.4. rDNA ITS region of the 33 geographic populations of R reniformis were amplified, cloned, and sequenced. Phylogenetic tree were made combined with the amphimictic populations sequrnces (AY335191, AY335192, FJ374686) and parthenogenetic popuplation sequence (AY335190). Amplification of ITS region of the 33 populations yielded an approximately 800bp in length. Sequencing analysis and 3 ITS types, Type A, B, and C, were detected, in which HN8 and HN17 belonging to type B, FJ56 belonging to type C and the other 30 populations from China belonging to type A. Compared with the amphimictic and parthenogenetic popupations, variation of type A is very small among 33 populations and the homology similarity were 98.6-100% and 90.5-91.1%, respectively. Variation in type B and C were relatively large with type A and the homology similarity among 3 types was 94.0-95.0%, and compared with the amphimictic and parthenogenetic popupations the homology similarity were 93.7-94.7% and 89.9-91.0% respecticvely. Meanwhile, the homology similarity betwwen HN8 and HN17 in type B is 99.9% and 94.5-94.7% between type B and C. All of R. reniformis populations constructed one group in phylogenetic tree, in which amphimictic populations divided with parthenogenetic popupation and conformed two main branches. In amphimictic branch, type B and C constructed a group and type A with the amphimictic populations from GenBank formed another. 3 main groups were formed in R. reniformis populations. The results were the same with that of PCR-RFLP of ITS region. Alignment of ITS 1 and ITS2 of the amphimictic populations showed variations, between which ITS1 was larger than that of ITS and ITS2. The homology similarity between amphimictic and parthenogenetic popupations was relatively low.5. D2D3 region of 28S RNA gene of the 33 R. reniformis populations were amplified, cloned, and sequenced. Phylogenetic tree was made combined with the relevant sequences (R. reniformis and R. macrodoratus DQ328713 and DQ328711). It was found that amplification of D2D3 region of 28S RNA gene of 33 R. reniformis populations yielded an approximately 780bp in length. Sequencing analysis and alignment showed that intra-specific variation were very small, the homology similarity compared with DQ328713 was 98.2-99.8%, and among them homology similarity of HN8, HN17 and FJ56 with DQ328713 was 99.1-99.8%. But inter-specific difference between R. reniformis and R. macrodoratus was quite large, the homology similarity was only 77.1-78.6%. The phylogenetic analysis about D2D3 region of 28S RNA gene of different populations were studied and this is the first report about the intra-specific variation in R.reniformis D2D3 region of 28S RNA gene. It enrich the molecular information of R. reniformis, and will be very helpful for molecular identification of the species.6. The life cycle and reproductivity of R. reniformis in China were studied systematically. The whole life cycle of R. reniformis at 27±2℃was obtained by inoculating soybean, and the reproduction factor decreased with the increase inoculum levels.7. Different inoculumn levels of R. reniformis on tobacco, tomato and cucumber showed that below 50 nematodes/pot inoculumn levels no clear symptoms observed on infected plants, whereas the shoots and roots of the plants with 500 nematodes/pot, especially plants that with 5000 nematodes/pot inoculumn levels were heavily dwarfing and the root system were not developed and fibrous root system was fewer. The trend of the reproduction factor on three different crops was same with that of soybean.The main results obtained in this study will be helpful to study the morphology and molecular taxonomy and identification of reniform nematodes, and it will be useful to acquire the informations of damage caused by R. reniformis and will be very helpful for management of the nematode disease.