The Morplogical Characteristics, Genetic Diversity And Sensitive Detection Of The Tylenchulus Semipenetrans, Collected From Different Hosts And Regions In China

The citrus nematode, Tylenchulus semipenetrans (TS) Cobb, 1913, causing agent of "slow decline" of citrus is considered one of major nematode pests of citrus. TS widely distributes in China, causes significant economic losses in all citrus-growing regions of China. In this study, TS from different geographies in China were compared and analyzed based on morphology, cross-inoculation parasitism and molecular biology, to develop a diagnostic protocol with a single step PCR using the primers to detect low number of TS in soil nematode community extracted by the Modified Baermann funnel method.5 populations of TS were collected from root and soil samples in different citrus orchards in 4 provinces and Chinese-fir(Cunninghamia lanceolata) plantations in Fujian province in China. And then, the samples were observed and measured with the microscope. The results showed that the measurement of TS was consistent with previous study. The morphologic of TS is conservative. In all populations, there is another type of tail. The near terminal of tail is obtuse circle, like stick, constriction reduced. The results enriched morphometrics of TS.Several cross-inoculations through indoor, the field or the forest land showed that TS can infect Chinese fir, but failed to infect citrus seedling roots when Chinese fir and citrus were all planted in Chinese-fir plantations which were infected with TS. However, TS can infect citrus, but failed to infect Chinese fir seedling roots when Chinese fir and citrus were all planted in the citrus orchards. The result is inconsistent with the predecessor's results.The 27 rDNA-ITS sequences of TS from 4 provinces were sequenced, submitted to the GenBank, and obtained the accession numbers, so the results enriched nucleic acid database of TS. rDNA-ITS sequences of TS were compared and analyzed. The results indicated that the rDNA-ITS sequences of TS are conservative with minimum differences. The sequence similarity between the TS populations was above 98.4% and the sequence variation rate is between 0 and 1.6%. Phylogenetic analysis of sequences indicated that there was differene in sequence characteristics and rich multiplicity. According to the sequence similarity and the system cladogram, it was demonstrated that TS's ITS sequence was unrelated with geographical position and the host. It proved that it could not distinguish the different physiologic race of TS.Species-specific primers were designed based on the sequences of rDNA ITS regions of 14 populationa of TS collected in different citrus groves or Chinese fir plantations in China. The primers were sufficiently sensitive to yield an expected size band for individual second-stage juvenile (J2) or different life stages of TS. Specificity and reliability of species-specific primers were validated by further 16 additional populations of TS collected from 4 provinces in China. Non-target nematode specificity of species-specific primers was demonstrated by testing 14 non-target nematode species respectively which were commonly found in citrus groves. The sensitivity and reliability of primers were also confirmed to detect low numbers of J2 of TS in nematode soil communities containing large numbers of non-target nematode extracted by the Baermann funnel method. It appears that the diagnostic primers will be useful for accurate identification and also as a decision management tool for growers in China.