Morphological And Molecular Phylogeny On Some Trichodoridae Species And Populations

Stubby root nematodes(Trichodorus spp. and Paratrichodorus spp.) are a group of ectoparasitic nematodes that not only affect root growth but also lead to root stagnation of an extensive range of crops by their feeding on plant root cells, and the important is that some of which are virus vectors and can transmit viruses to many economically important crops such as flowers, vegetables, crops, fruit trees, etc. Many countries and organizations in the world have listed them as quarantine pests. Due to the morphological similarity of the key identification characters among the species, it is very time-consuming and laborious only based on the morphological characters to identify the species. In this study, Trichodorus and Paratrichodorus populations from 13 proviences of China and Belgium were systematical studied based on the morphological and rDNA characters. The major results obtained are as follows:1. Among 1,546 soil samples collected from 13 provinces of China, including Beijing, Shandong, Shanxi, Henan, Jiangsu, Anhui, Hunan, Chongqing, Zhejiang, Fujian, Yunnan, Hainan and Guangdong, or intercepted in azalea imported from Belgium,6.3% of which were detected the presence of Trichodorus and Paratrichodorus populations, and 23,15,31,10 and 8 soil samples contain T. nanjingensis, T. pakistanensis, P. porosus, P. renifer and P. minor, respectively. The five species account for 23.7%,15.5%,40.0%,10.3% and 8.2% of the total Trichodorus and Paratrichodorus populations detected and this shows T. nanjingensis, T. pakistanensis and P. porosus are predominant species in the genus Trichodorus and Paratrichodorus occurring in China, respectively. This is the first detailed report of P. renifer and P. minor in Mainland China.Several populations of Trichodorus nanjingensis were collected from the rhizosphere of Chinese mulberry, ornamental flowers in Zhejiang province, and orchards in Beijing, respectively. The Beijing populations of T. nanjingensis account for 39.1% of the total nematode populations detected in Beijing, which indicates T. nanjingensis is dominant species of perennial plant parasitic nematodes in Beijing. Through the morphological and morphometrical comparisons, it revealed slight variation between intra-species populations of T. nanjingensis from different geographic regions of China (Beijing, Zhejiang). But 2 males of population 129 and 1 male of population 088 have one cervical papillae instead of two, respectively. According to comparisons of 18S regions of rDNA, it is found the similarity of populations used in this study and those (AJ439579 and AJ439580) in GenBank is in the range of 99.2-100.0%. Sequence alignments of ITS1, ITS2 region of rDNA and D2D3 region of 28S RNA gene revealed the sequence consistency between these populations.Among soil samples collected from Hainan, Chongqing, Yunnan and Fujian of China, fifteen Trichodorus pakistanensis populations from the rhizosphere of sugar cane, longan, litchi and other economic crops were got. The Fuzhou, Xiamen, Zhangzhou populations of T. pakistanensis account for 20.6% of the total nematode populations detected in Fujian, and 80% of total T. pakistanensis populations in this study, which means T. pakistanensis is dominant trichodorid species of Fujian orchard. This is the first report of distribution of T. pakistanensis in Hainan and Chongqing of China. The males of T. pakistanensis usually have three cervical papillae with two anterior and one posterior to the S-E pore, but in this study it was found that there are 11.1%, 15.4%,14.3%,15.4%,15.4%,23.1% males in population 108,109,114,119,121,122 with one cervical papillae anterior to the S-E pore, respectively. Sequence comparisons of 18S region of rDNA revealed high similarity between intra-species populations of T. pakistanensis from different geographical regions of China, and the similarity to those in GenBank (AJ439581 and AJ439582) is 98.5-99.3%. Sequence alignments and RFLP analysis of ITS1, ITS2 region of rDNA and D2D3 region of 28S RNA gene revealed the sequence consistency between these populations.Morphometrical comparisons of 17 populations of T. cedarus from China (Ningbo and Hangzhou, Zhejiang), Japan and Korea revealed great variation of onchiostyle between intra-species populations. According to the length of onchiostyle, all the 17 populations can be divided into two groups including long onchiostyle group and short onchiostyle group. This phenomenon was found in the three coutries, China, Japan and Korea, populations. PCR-RFLP and sequence alignments of 18S, ITS1, ITS2 region of rDNA and D2D3 region of 28S RNA gene revealed the sequence consistency between these populations.Based on both morphological and molecular characters, one new Trichodorus species was described and reported. Trichodorus n. sp. is from the rhizosphere of loquat in Hangzhou, Zhejiang. The identification code in the polytomous key of Trichodoridae (Decraemer& Baujard, 1998) for this species are D1-C1-L2-K2-A1(2)-B2-E3-F2(1)-G?-H3-I2/3-J1-M1-N1-O1-P1-Q?-R?-S5 and F3-D3-P2-A1(2)-B2-C1-E0-GO-H3-I2-J2-K3-L1-M2/7-N1-05 for females and males, respectively. The morphological characteristic has a relatively high similarity to T. tricaulatus but differs from it in shape of vagina, shape and length of spicule.The two species can easily differentiate in the polytomous key of Trichodoridae [H3 and H1 for female of Trichodorus n. sp. and T. tricaulatus, respectively; C1-M2/7 and C1/2-M2/7/6 for male of Trichodorus n. sp. and T. tricaulatus, respectively].2.31 populations of Paratrichodorus porosus were collected from Hunan, Zhejiang, Fujian, Yunnan and Guangdong of China. Morphological characters of several populations with high nematode density were compared. Not only no male was found within all the samples, but no sperm in uteri of females was detected. By comparisons of 18S regions of rDNA, it is found the similarity of Chinese populations and those (DQ345524 and AJ438059) in GenBank is in the range of 99.7-100.0%,95.5-96.1%, respectively.Morphological and molecular information of Paratrichodorus renifer parasiting on roots of waxberry, maple trees and Chinese rose in Zhejiang were reported, and this is the first report about the distribution of P. renifer in Mainland China. The Chinese populations and that population intercepted in azalea imported from Belgian have high similarity on morphological, morphometrical and molecular datas.Eight populations of Paratrichodorus minor collected from the rhizosphere of vegetabales and orchard in Hainan, Yunnan, Fujian provinces in China were described. The sequence similarity of 18S RNA region of populations used in this study, including 8 Chinese populations and 3 American populations, and three sequences from GenBank, AJ438053 (Portugal), AJ438054 (Brazil) and AJ438056 (USA), is in the range of 99.1 - 100.0%. This is first detailed description of morphological characteristics and the distribution of P. minor in Mainland China.3. Phylogenetic trees of species and populations of Trichodorus and Paratrichodorus used in this study were constructed based on sequences of the 18S, ITS1, ITS2 region of rDNA and D2D3 region of 28S gene. This is first report of the rDNA data of T. cedarus, a new Trichodorus species and Heterodera sinensis.4.18S region of rDNA of 94 plant parasitic nematode species and populations, belonging to Tylenchida, Aphelenchida, Dorylaimida and Triplonchida, were amplified with primer sets NEMF1/18S and A/18S, and were used to construct the phylogenetic trees by Mega 4 software, respectively. The analysis of the two phylogenetic trees indicates that the sequences amplified by the primer set, A/18S, contain more information, which is good for molecular phylogeny of plant parasitic nematodes.