Species Identification Of Soybean Pratylenchus In Huanghuai Region

Pratylenchus Filipjev(1936),also known as root-rot nematode,is a migratory plant parasitic nematode that occurs widely around the world and can infect most plants.Pratylenchus is one of the most serious plant parasitic nematodes on crops,which seriously threatens the safety of world agricultural production and causes huge economic losses to agricultural production.As an important source of oil and protein in China,soybean has been widely used in food and feed production.In addition,soybean is also one of the main crops damaged by Pratylenchus.At present,there are few reports on Pratylenchus spp.on soybean in Huanghuai,Ningxia and Xinjiang areas.In order to understand the occurrence and harm of Pratylenchus on soybean in these areas more systematically and accurately,and to better control Pratylenchus,this study investigated the species of Pratylenchus on soybean in Huanghuai Ningxia and Xinjiang areas.Firstly,Pratylenchus.were isolated and collected from soybean soil samples by Bellman funnel method,and then the isolated Pratylenchus were identified by morphological and molecular biology techniques.Finally,the pathogenicity of the purified Pratylenchus populations were determined and analyzed to further verify the harm of Pratylenchus to soybean.In this study,315 soybean rhizosphere soil samples collected from Henan,Shandong,Anhui,Jiangsu,Hebei,Xinjiang and Ningxia provinces were isolated by the Berman funnel method.A total of 182 populations of Pratylenchus were isolated,accounting for 58 %.The species of Pratylenchus in the samples were identified by morphological and molecular biological methods.Through single female culture of carrot callus and cultured in dark at 25 °C for 3-5 months,41 Pratylenchus populations were purified in this study.The pathogenicity of different populations of P.coffeae,P.thornei,P.zeae and P.scribnei was determined by indoor pot inoculation.The results are as follows :1.A total of 182 populations of Pratylenchus were isolated from soybeans,P.coffeae was distributed in four provinces of Henan,Anhui,Ningxia and Xinjiang.,P.skrebneri was also distributed in five provinces of Anhui,Jiangsu,Henan,Ningxia and Xinjiang.P.thornei was distributed in three provinces of Henan,Ningxia and Xinjiang.P.zeae was distributed in three provinces of Henan,Ningxia and Xinjiang.P.neglectus was distributed in three provinces of Henan,Hebei and Jiangsu.P.penetrans was distributed in Henan,Hebei and Shandong.A total of 41 different populations of Pratylenchus were cultured on carrots using a single female.They belong to four species of the Pratylenchus genus,namely,P.coffeae,P.scribneri,P.zeae and P.thornei.2.In this study,112 r DNA 28 S D2-D3 region sequences and 91 r DNA-ITS sequences of Pratylenchus were obtained by molecular biology techniques.After NCBI BLAST alignment,50 28 S sequences and 30 ITS sequences were obtained from P.coffeae,and the sequence lengths were 770-792 bp and 1245-1254 bp,respectively.A total of 43 28 S sequences and 48 ITS sequences were obtained from P.scribneri,and the sequence lengths were 785-786 bp and1099-1106 bp,respectively.A total of 13 28 S sequences and 4 ITS sequences were obtained from P.thornei,with sequence lengths of 770-780 bp and 870-897 bp,respectively.Three 28 S sequences and three ITS sequences were obtained from P.zeae,and the sequence lengths were776-783 bp and 678-685 bp,respectively.A total of 3 28 S sequences and 3 ITS sequences were obtained,and the sequence lengths were 776-782 bp and 687-693 bp,respectively.A total of3 28 S sequences and 3 ITS sequences were obtained,and the sequence lengths were 776-782 bp and 687-693 bp,respectively.Based on the r DNA 28 S D2-D3 region sequence and r DNAITS sequence,the phylogenetic analysis of the purified Pratylenchus was carried out by MEGA software.The results showed that the sequences of 41 Pratylenchus spp.populations obtained by purification culture could be clustered in a branch with the reported Pratylenchus species sequences.3.The soybean plants inoculated with different populations of Pratylenchus 60 d under the same grew conditions smaller than the control group,and the stems were slender compared with the control group,and brown lesions appeared in the roots.At the same time,the roots of the diseased plants were selected and photographed after staining.It was found that there were a large number of Pratylenchus inside the roots,which confirmed that P.coffeae,P.scribneri,P.zeae and P.thornei all could infect and harm soybeans.4.30 females of P.thornei were inoculated on the carrot callus and cultured at 25 °C,27.5 °C and 30 °C in the dark,for 90 days.The results showed that t the fecundity of P.thornei was the strongest at 27.5 °C,which was the optimum temperature for the fecundity of P.thornei.