| The plant parasitic nematodes(PPNs)are important pathogens causing severe yield losses for global agricultural production.However,the knowledge to many PPN species remains in limitation due to their tiny body,abundance species,wide host range as well as complicate feeding habits.Identification of PPN species and understanding their biological characteristics and genetic variations is critical for making control strategies.In this study,a Pratylenchoides population isolated from the roots and rhizosphere soil of weakened tomato plants in Qinghai Province was identified,the characteristics in fungal feeding and plant parasitism of Aphelenchoides smolae intercepted from the Netherlands was investigated,and the polymorphisms of r RNA 28 S D2 domain in seven PPN populations were analyzed.The main results are as follows:1.The Qinghai population was identified as P.leiocauda based on morphological and molecular characterizations.It is characterized by lateral field with six incisures,labial region with 3-4 annuli and slightly offset from body;stylet stout,knobs broadly rounded and sloping posteriorly;pharyngeal glands overlapping intestine dorsally,the dorsal and one subventral nuclei located mostly anterior to pharyngo-intestinal valve,and another subventral nucleus located posterior to valve;female tail conical,terminus without annulus or having 2-3 annuli;male spicules long,large bursa extending continually to tail terminus.Phylogenetic analyses on basis of r RNA 18 S,28S D2-D3 and ITS sequences revealed that the Qinghai population grouped with P.crenicauda,P.erzurumensis and P.laticauda in an independent branch.2.The inoculation tests of A.smolae were carried out on four fungal species including Alternaria sp.,Botrytis cinerea,Fusarium sp.and Pestalotiopsis sp.,other four nematode populations including A.medicagus,two populations of A.besseyi and Ditylenchus destructor were used as the control.Meanwhile,four Aphelenchoides populations were inoculated on five varieties of three plant species including Chrysanthemum morifolium,Glycine max and Medicago sativa.The results showed that A.smolae and A.medicagus could feed on mycelia of four fungal species,but their reproduction rates(Rf)were significantly different.The Rf value of A.smolae on B.cinerea was the highest,while on Fusarium sp.was the lowest.The alfalfa population of A.besseyi reproduced very poorly on Fusarium sp.mycelia.The Rf values of D.destructor on Fusarium sp.were appearantly higher than that of four Aphelenchoides populations.After inoculation with A.smolae,the symptom on young leaves of chrysanthemum is showed as brown and necrosis,on alfalfa as malformation and shrink,and on soybean as brown lesion spot,which indicating A.smolae has weak plant parasitism.While,after inocluation with A.medicagus,only mild symptom were produced on alfalfa,and with A.besseyi popultions,apparent symptom were produced on all tested plants.All Rf values of four Aphelenchoides populations on chrysanthemum,soybean and alfalfa were less than l,indicating none of them were the suitable host.3.The polymorphisms of DNA and c DNA sequences in r RNA 28 S D2 domain from seven PPN populations were analyzed using monoclonal and high throughput sequencing methods.These PPN populations are D.destructor,Helicotylenchus microlobus,Meloidogyne incognita,Pratylenchus coffeae,P.neglectus,P.thornei and Rotylenchulus reniformis,respectively.The results indicated that r RNA polymorphisms were widely presented in these species.The level of polymorphisms were apparently different,and most of the mutations were conserved and did not affect ribosomal function.The sequence diversity obtained by high-throughput sequencing was significantly higher than that obtained by monoclonal sequencing.Meanwhile,the vast majority of single nucleotide polymorphism(SNP)sites in monoclonal sequencing could be found in high-throughput sequencing,indicating it can be an effective tool for analyzing r RNA polymorphism.Polymorphisms in DNA sequences were retained during the transcription process of r RNA.However,there are a large number of short sequences of different genotypes in c DNA sequences,suggesting the splicing of r RNA maybe presented during the transcription process.In conclusion,the results have enriched the taxonomic information of PPN in China,and provided a theoretical basis for parasitism study of Aphelenchoides species and for making plant quarantine measures at Chinese ports.Meanwhile,it also provided useful information for the upcoming research on effects of r RNA polymorphism level on individual organisms’ survival and evolution. |
