中国孢囊线虫新记录种——野生豆孢囊线虫记述及其对豆科植物的寄生性测定

Heterodera sojae, a New Cyst Nematode Record in China and Its Parasitism to Legume Crops

【目的】在江西婺源县孢囊线虫调查中,从大豆根及根际土壤中采集到一个孢囊线虫群体,其形态特征和分子特征与大豆孢囊线虫(Heterodera glycines)存在较大差异,因此对其形态学和分子特征以及寄生性等进行详细研究,以明确其种类及对豆科作物寄生性,为病害防控提供科学依据。【方法】从婺源县采集的大豆根及根际土样,用筛淘法分离孢囊,大豆根系上的孢囊用直接解剖法获取。用浅盘法分离根际土样中的2龄幼虫和雄虫。选取具典型特征的饱满孢囊制作阴门锥切片,浅盘法分离的2龄幼虫和雄虫制作永久玻片,在显微镜下观察记录孢囊、2龄幼虫和雄虫的形态特征,并进行形态特征测量。采用两对植物线虫通用引物AB28和TW81以及D2A和D3B分别扩增内转录间隔区(internal transcribed spacer,ITS)和28S大亚基D2-D3区段,经序列测定,用MEGA软件采用邻接法(neighbor-joining(NJ)method)构建该线虫群体与孢囊线虫属其他种群的ITS和LUS D2-D3系统进化树,分析其系统发育关系。在温室内采用盆栽人工接种的方法,测定江西孢囊线虫群体对11种豆科作物的寄生性,同时测定国内40个大豆栽培品种对该孢囊线虫的抗病性。【结果】形态学观察和特征值测量结果表明,从江西婺源大豆上采集的孢囊线虫群体,其孢囊形态、2龄幼虫以及雄虫的形态特征与野生豆孢囊线虫(Heterodera sojae)的形态特征基本一致;ITS序列(MG859982)比对发现其与野生豆孢囊线虫ITS序列(KU160510和KU160512)的同源性为99%和98%,而与大豆孢囊线虫ITS的同源性仅为81%(KY794762.1)。D2-D3序列(MG859981)与野生豆孢囊线虫(KU160511)相似度最高,为99%。序列系统进化树分析发现以ITS序列与D2-D3序列构建系统发育树的结果相一致,江西孢囊线虫群体与野生豆孢囊线虫分布在同一支,节点支持率为100%,而与大豆孢囊线虫聚在另一个分支中。结合形态学和分子生物学结果,将江西婺源大豆上的孢囊线虫群体鉴定为野生豆孢囊线虫,系中国新记录种。寄生性研究结果表明,在参鉴的11种豆科作物中,野生豆孢囊线虫的2龄幼虫能在大豆和相思豆(红豆)上侵染和繁殖,完成生活史;虽然能侵染豇豆、豌豆、扁豆、绿豆、赤豆、刀豆、菜豆和苜蓿8种作物根系,但不能完成生活史。测定的40个大豆栽培品种中,19个高感、11个中感、5个中抗、5个高抗。【结论】在我国江西婺源新发现的一种寄生于大豆的孢囊线虫为野生豆孢囊线虫,系中国新记录种;人工接种条件下相思豆(红豆)也是野生豆孢囊线虫的寄主,40个大豆栽培品种中30个对该孢囊线虫表现感病。

【Objective】In a survey for cyst nematodes in Jiangxi Province,China,a cyst nematode population was collected and extracted from soybean roots and rhizosphere soil.The morphological characteristics of this population are different from that of the soybean cyst nematode(Heterodera glycines).The objective of this study is to research morphological and molecular characteristics and parasitism test of this population,identify their species and parasitism to legume crops,and to provide scientific basis for disease prevention and control.【Method】Soil samples were collected in Wuyuan County,Shangrao City,Jiangxi Province.The cysts were collected by sieving method or dissected from the roots.The 2nd stage juveniles(J2s)and males in soil were extracted by using the shallow disk method.The cysts with typical characteristics were selected to make a slice of the vulva cone,the J2s and males separated by shallow disk method were made into permanent slides.Morphological characteristics of cysts,J2s and males were observed and measured under microscope.For molecular studies,single cyst from soybean roots was picked and then ground to extract the genomic DNA.The ribosomal RNA-ITS region and the D2-D3 expansion segments of 28S large subunit rRNA gene were amplified with universal primers AB28,TW81 and D2A,D3B,respectively.The sequencing results were deposited in the GenBank database and compared with nematode sequences using the BLAST homology search program.The closest sequences were selected for phylogenetic analyses.The MEGA software was used to construct the ITS and LUS D2-D3 phylogenetic tree of cyst nematodes using neighbor-joining method.The parasitism of this cyst nematode to 11 legume crops and 40 soybean cultivars was tested by artificial inoculation in greenhouse.【Result】Morphological characteristics of cysts,J2s and males of population in this study agreed with Heterodera sojae reported in Korea.Sequence from the ITS-rDNA(MG859982)was 99%and 98%identical to those of H.sojae from Korea(KU160510 and KU160512)and only 81%identical to the soybean cyst nematode(H.glycines)(KY794762.1).The D2-D3(MG859981)sequence was 99%identical to H.sojae from Korea(KU160511).In addition,analysis of phylogenetic trees constructed by the ITS and LUS D2-D3 both showed that the Jiangxi population and H.sojae were grouped in the same clade with a reliability of 100%.The cyst nematode population from Jiangxi was identified as H.sojae-a new record species in China.Inoculation of 11 legume crops showed that J2s of H.sojae could infect 10 crops(soybean,jequirity,cowpea,pea,lentil,mung bean,adzuki bean,sword bean,green beans and alfalfa),but could complete the life cycle only on soybean and jequirity.Soybean cultivars resistant identification test results showed that 19 cultivars of the 40 inoculated soybean cultivars were highly susceptible,11 cultivars were moderately susceptible,5 cultivars were moderate resistant and 5 cultivars were highly resistant.【Conclusion】The population of cyst nematode collected on soybean from Jiangxi,China was identified as H.sojae-a new cyst nematode record in China.Parasitism tests indicated that soybean and jequirity were suitable hosts for H.sojae,and 30 of the 40 soybean cultivars were susceptible to phenotypic disease of cyst nematodes.