【目的】菲利普孢囊线虫（Heterodera filipjevi）是全球小麦生产上的一种重要病原物，早期快速准确的检测对于小麦孢囊线虫病的防控至关重要。本研究基于TaqMan探针法的实时荧光定量PCR（real-time fluorescent quantitative PCR with TaqMan probes, TaqMan-qPCR）技术建立了一种直接从田间土壤中快速检测和定量分析H. filipjevi的方法。【方法】根据 H. filipjevi的RAPD-SCAR 序列（KC529338）设计TaqMan-qPCR特异性的引物和探针，利用已鉴定的27个线虫群体的DNA进行引物特异性检测；利用单条 J2 DNA稀释液（4^-0，4^-1，4^-2，4^-3，4^-4，4^-5，4^-6 和 4^-7）和单个雌虫DNA稀释液（10^-0，10^-1，10^-2，10^-3，10^-4，10^-5，10^-6和10^-7），以及基因组 DNA 的稀释液（100 μg.μL^-1，10 μg.μL^-1，1 μg.μL^-1，0.1 μg.μL^-1，0.01 μg.μL^-1，0.001 μg.μL^-1，100 pg.μL^-1 和 10 pg.μL^-1）对引物的灵敏度进行检测。通过人工将不同数量（0.1, 1, 10, 100和1000条）的 H. filipjevi添加到灭菌土中，建立了线虫数量的对数与CT值的标准曲线和回归方程，对32份田间土壤样品中H. filipjevi数量进行了评估，并对实时荧光定量PCR检测方法与传统定量方法进行了相关性分析。【结果】本研究建立的菲利普孢囊线虫TaqMan-qPCR检测技术具有极高的特异性和灵敏度，能够从13 种27 个线虫种群中特异的检测出菲利普孢囊线虫，检测阈值低至4^-3个单条J2 DNA、10^-3个单条雌虫DNA和0.01μg. μL^-1基因组DNA。田间土壤样品检测发现，实时荧光定量PCR定量检测方法与传统定量检测法呈现出较好的线性关系（R²= 0.8259）。【结论】本研究建立的TaqMan-qPCR检测H. filipjevi技术特异性强、灵敏度高，能广泛适用于田间土壤开展H. filipjevi快速检测和定量分析。

为明确我国主要甜菜产区线虫的种类和危害程度，2015-2018年我们对我国甜菜主要产区进行了系统的调查和检测。在新疆伊犁州新源县的甜菜地调查发现，部分区域甜菜长势弱，植株黄化、矮化明显，受害植株根系有明显的须根团，根上有大量的白色雌虫。采用形态学和形态测量学的方法对孢囊线虫的雌虫、孢囊和二龄幼虫进行显微观察和测量，结果表明，在我国新疆新源县甜菜上发现的孢囊群体的2龄幼虫、孢囊及阴门锥的特征和关键形态测量值与国外已报道的甜菜孢囊线虫基本一致。采用分子生物学的方法对孢囊线虫新疆新源群体的核糖体DNA-ITS、28S-D2/D3和线粒体DNA COI基因进行克隆、测序和分析发现，新源群体的核糖体DNA-ITS, 28S-D2/D3 和线粒体DNA COI基因序列和国外已报道的甜菜孢囊线虫的一致性为99.81-100%，进化分析显示新疆新源群体与国外甜菜孢囊线虫群体聚类为一个进化分支，同时采用甜菜孢囊线虫特异性引物SHF6和rDNA2，从新疆新源群体中扩增出长度为255 bp的特异性条带，分子生物学检测结果表明：我国新疆甜菜上发现的孢囊线虫为甜菜孢囊线虫。采用柯赫氏法则，将500头二龄幼虫分别接种到甜菜（SD21816）和油菜（德油6号）根系中，15天后在甜菜和油菜根系上分别发现了137和157个孢囊和雌虫，表明新疆新源群体能够在甜菜和油菜上完成生活史，且形态学和分子生物学鉴定结果和田间样品完全一致。综上所述，经形态学鉴定和分子特征分析确认，在我国新疆新源县甜菜上发现的孢囊线虫为我国检疫性有害生物——甜菜孢囊线虫，这也是甜菜孢囊线虫在我国的首次明确报道。

马铃薯孢囊线虫Globodera rostochiensis是国际公认的重要检疫性有害线虫，严重危害马铃薯。2018-2020年，在全国农业技术推广服务中心组织的全国马铃薯检疫性线虫调查中，从云南省昭通市鲁甸县和四川省越西县和昭觉县马铃薯根系发现金色孢囊线虫，经形态学观察鉴定、分子生物学rDNA-ITS及28S的D2-D3区域特征分析比对及种特异性引物ITS5和PITSr3检测确定为马铃薯金线虫Globodera rostochiensis（Wollenweber）Skarbilovich, 1959. 在隔离温室内采用盆栽人工接种的致病性研究结果表明，该三个金线虫种群都能侵染马铃薯（品种青薯9号）并繁殖，接种12周后，马铃薯上形成成熟雌虫，完成生活史。这是马铃薯金线虫在我国云南和四川省首次记录。

Stunt nematodes (Tylenchorhynchus spp.) are obligate migratory root ecto-parasitic nematodes found in the fields of many cultivated crops.  These nematodes, with phyto-sanitary potential, are frequently ignored or misdiagnosed as pests, and this may pose a threat to food security.  The accuracy of its identification based on a morphological approach has been challenged recently, due to the overlapping of the morphological and morphometric characters of the species.  Consequently, the objective of this study is to identify and characterize stunt nematodes present in 54 fields cultivated with cereal crops (wheat, maize and rice) in the savannahs of northern Nigeria, using integrative taxonomy and molecular approaches.  The molecular and morphological studies identified and confirmed the presence of T. annulatus as the occurring specie in the savannahs of northern Nigeria.  The phylogenetic analysis was carried out using the internal transcribed spacer (ITS) and 28S genes of ribosomal DNA further confirmed the presence of T. annulatus.  The first molecular characterization and sequences of the ITS and 28S rDNA gene for T. annulatus from Nigeria were provided by this research.  Also, according to our literature search, this is the first report on T. annulatus in wheat, maize and rice in the savannahs of northern Nigeria.  Further study to test the pathogenicity of the parasitic nematode species found in this survey is recommended for the prioritization and development of efficient management strategies.

葡萄根结线虫（Meloidogyne vitis）是在云南省葡萄根部发现的一种新的根结线虫种类，该线虫在侵染地高密度存在，已对葡萄造成严重损害。葡萄根结线虫病的发生可能对我国葡萄产业发展构成威胁，为了建立一种快速、可靠的葡萄根结线虫特异性分子检测方法，本研究以葡萄根结线虫核糖体DNA内转录间隔区(ribosomal DNA-internal transcribed spacer，rDNA-ITS)基因片段为靶标，设计筛选葡萄根结线虫种特异性检测引物，通过优化反应体系，并对所设计引物的可靠性、特异性及灵敏性进行检测验证，建立了葡萄根结线虫PCR快速分子检测技术体系。结果表明，优化后的引物最佳退火温度为53℃，该引物能够对不同龄期葡萄根结线虫进行检测；特异性检测结果表明，本研究建立的PCR分子检测技术能够从葡萄根结线虫中扩增获得长度为174 bp的特异性片段，选用的5种非靶标根结线虫则无任何扩增条带，从而将葡萄根结线虫和其他5种非靶标根结线虫有效区分开；灵敏度检测结果表明，该PCR分子检测技术能够有效的检测单头2龄幼虫和10^-4头雌虫的DNA；此外，该PCR分子检测技术能够从混合线虫种群中特异性地检测出葡萄根结线虫，并能够有效检测出土壤中的葡萄根结线虫，检测灵敏度为0.5 g土壤中可检测出2头2龄幼虫或一头雄虫。本研究建立的快速、灵敏、特异的PCR分子检测技术可用于葡萄根结线虫单头2龄幼虫的直接鉴定、混合线虫群体中葡萄根结线虫的检测及0.5 g土壤中2头2龄幼虫或一头雄虫的直接检测。本研究建立的PCR分子检测技术能够准确、快速地检测出葡萄根结线虫，将为葡萄根结线虫的发生危害调查和高效绿色防控策略的制定提供技术支撑。

钾（K）是一种重要的营养元素，可以提高作物的抗逆性/耐受性。K在抗植物寄生线虫中的应用表明，K处理可以减少线虫病的发生，提高作物产量。然而，K在水稻抗拟禾谷根结线虫（Meloidogyne graminicola）中的研究仍然缺乏。本研究首先用K₂SO₄直接处理线虫，发现K₂SO₄对线虫的死亡率、侵染率以及发育水平无显著影响；接着通过温室盆栽接种，发现0.5 mM K₂SO₄处理水稻后，根中的根结和线虫数量分别下降了57.2±4.4% 和59.2±6.6%，成年雌虫比例（70.9±5.6%）显著低于对照（90.7±5.1%），同时幼虫比例（27.0±6.3%）显著高于对照（6.0±3.2%），而水稻的生长不受影响；统计Pluronic明胶中水稻根尖吸引的线虫数量，发现接种后6小时K₂SO₄处理与清水处理之间并无显著差异；对接种后7天根结中巨细胞的形态、大小和数量进行显微观察，发现两个处理间也不存在显著差异；接着检测根结中胼胝质沉积，发现K₂SO₄处理后其沉积面积增加了67.9%，同时其合成基因OsGSL1和降解基因OsGNS5分别显著上调和下调；另外检测H₂O₂累积发现，接种后8和24 小时K₂SO₄处理的根中H₂O₂含量分别增加了78.2% 和118.7%，同时其合成基因OsRbohB也显著上调；再对水杨酸、茉莉酸、乙烯以及油菜素内酯等信号通路相关基因和病程相关蛋白基因的表达进行定量分析，发现在线虫侵染初期K₂SO₄处理显著上调了某些抗病相关基因的表达；最后对K通道基因OsAKT1和转运蛋白基因OsHAK5缺陷型植株进行接种，发现根结和线虫数量显著增加并且线虫的发育加快，同时K₂SO₄的作用降低。这些说明K₂SO₄通过激发基础防御反应提高了水稻对线虫的抗性，并且K通道和转运蛋白积极参与了寄主抗性。K及其通道和转运蛋白在寄主抗性中的应用，为进一步研究水稻抗线虫机制以及钾在植物抗生物胁迫中的功能奠定了基础。低钾能诱导水稻对拟禾谷根结线虫的抗性，为田间有效利用钾肥防控线虫病害提供了理论依据。

大豆孢囊线虫（SCN, Heterodera glycines）严重制约大豆生产。大豆抗线虫数量性状遗传位点Rhg4上的丝氨酸羟甲基转移酶编码基因（GmSHMT08）对大豆孢囊线虫有显著的抗性，但该基因如何介导了对大豆孢囊线虫的抗性机制仍不明晰，GmSHMT08能否与大豆孢囊线虫产生的蛋白发生互作仍不明确。本研究以GmSHMT08作为诱饵，通过酵母双杂交体系在线虫中筛选出了与GmSHMT08互作的一个热休克蛋白70片段（HgHSP70p）。通过GST pull-down和荧光双分子互补，进一步验证了HgHSP70p与GmSHMT08之间存在互作关系。本研究发现的HgHSP70基因可以作为关键候选基因，用于进一步探究GmSHMT08介导的对大豆孢囊线虫的抗性机制。

大豆孢囊线虫是世界范围内大豆生产的重要病原之一，生物防治目前已成为大豆孢囊线虫病防治的重要手段。黑曲霉NBC001由本实验室从小麦孢囊线虫群体上分离获得，其发酵液拌种在盆栽中不仅可以有效防治大豆孢囊线虫，而且对大豆具有一定的促生作用。本研究将在田间评价NBC001对大豆孢囊线虫的防治效果及对大豆根际土壤产生的微生态效应。研究结果表明在田间应用黑曲霉NBC001发酵浓缩液拌种可以有效防治大豆孢囊线虫病，防效达31.7%。高通量测序结果显示黑曲霉NBC001对大豆根际土壤微生物多样性和群落结构无显著影响，表明NBC001发酵浓缩液拌种对土壤生态环境安全。在大豆定植10天时，黑曲霉 NBC001促进了大豆根际土壤中放线菌门Actinobacteria，酸杆菌门Acidobacteria，叶瘤菌属Phyllobacterium，雷尔氏菌属Ralstonia和H16的丰度；而降低拟杆菌门Bacteroidetes，芽单胞菌门Gemmatimonadetes，Adhaeribacter，芽单胞菌属Gemmatimonas，鞘氨醇单胞菌属Sphingomonas，Flavisolibacter的丰度。在定植90 d时，影响程度减小，仅增加气微菌属Aeromicrobium和RB41属的丰度，降低H16的丰度，说明其对大豆根际土壤微生物物种丰度的影响是短暂的。同时结果也表明黑曲霉NBC001可以增加大豆根际土壤中有益微生物放线菌门、酸杆菌门、气微菌属和叶瘤菌属的丰度。综上所述生防菌黑曲霉NBC001对大豆根际土壤微生物无显著影响，因此在田间应用黑曲霉NBC001对土壤生态环境安全。研究结果将为黑曲霉NBC001的安全应用奠定理论基础，为大豆孢囊线虫病生物防治提供高效生防菌株。

Received  3 March, 2018    Accepted  2 April, 2018

The root-knot nematode Meloidogyne graminicola, which is distributed worldwide, is considered a major constraint on rice production in Asia.  The present study used the root gall index and number of nematodes inside the roots to evaluate resistance/susceptibility to M. graminicola in different subpopulations of Oryza sativa (aus, hybrid aus, indica, hybrid indica, temperate japonica, tropical japonica).  Nematode development in highly resistant varieties was also evaluated.  Analyses of randomly selected 35 varieties showed the number of M. graminicola nematodes inside the roots correlated very strongly (r=0.87, P≤0.05) with the nematode gall index, and the results from pot and field experiments revealed similar rankings of the varieties for resistance/susceptibility.  Among the 136 tested varieties, temperate japonica displayed the highest gall index, followed by tropical japonica, indica, hybrid indica, aus, and hybrid aus. Zhonghua 11 (aus), Shenliangyou 1 (hybrid aus) and Cliangyou 4418 (hybrid indica) were highly resistant to M. graminicola under both pot and field conditions.  Further examination of nematode development suggested that compared to susceptible rice, M. graminicola penetrated less often into highly resistant varieties and more frequently failed to develop into females.  The promising varieties found in the present research might be useful for the breeding of hybrid rice in China and for the further development of practical nematode management measures.   

Plant-parasitic nematodes are very common on cereal crops and cause economic losses via reduction in grain quality and quantity. During 2014, 83 soil samples were collected from wheat and barley fields in 21 districts of 13 provinces across five regions (Central Anatolia, Marmara, Aegean, Southeast Anatolia, and Black Sea Region) of Turkey. Cyst-forming nematodes were found in 66 samples (80%), and the internal transcribed spacer (ITS) sequencing and species-specific PCR identified the species in 64 samples as Heterodera filipjevi, Heterodera latipons, and Heterodera avenae. The predominant pathogenic cereal cyst nematode was H. filipjevi, which was found in all five regions surveyed. H. avenae was only detected in Southeast Anatolia whereas H. latipons was detected in Southeast Anatolia and Central Anatolia. ITS-rDNA phylogenetic analyses showed that H. avenae isolates from China clustered with H. australis, and Turkish isolates were closely related to European and USA isolates of this species. H. filipjevi from Turkey and China were clustered closely with those from the UK, Germany, Russia, and the USA. The density of many of these populations exceeded or approached the maximum threshold level for economic loss. To our knowledge, this is the first report of H. filipjevi in Diyarbakir, Edirne, and Kutahya provinces, and the first report of H. avenae in Diyarbakir Province. These results exhibit the most rigorous analysis to date on the occurrence and distribution of Heterodera spp. in Turkey’s major wheat-producing areas, thus providing a basis for more specific resistance breeding, as well as other management practices.

    Heterodera avenae (cereal cyst nematode, CCN) infects many cereal crops and causes serious yield losses worldwide. Interaction studies investigating H. avenae and its hosts are still in their infancy. In this study, a barley model plant, the Hordeum vulgare cultivar Golden Promise, was investigated for its potential as a candidate model host to study its interaction with H. avenae. CCN-infective juveniles were attracted by the root tips and gathered around the root elongation zones of Golden Promise on 0.7% water agar plates. The juveniles invaded the roots and developed successfully until maturation at 40 days after inoculation in sterile sand soil. The cryotomy and syncytium measurements indicated that the syncytia enlarged gradually throughout the development of the nematodes and caused the corresponding root regions to swell obviously. Quantitative real-time PCR analysis showed that the down-regulation of defence-related barley genes and up-regulation of development-related barley genes contribute to the understanding of compatible interaction between H. avenae and Golden Promise. Barley stripe mosaic virus (BSMV) virus-induced gene silencing (VIGS) can be used in the roots of Golden Promise. In conclusion, the Hordeum vulgare cultivar Golden Promise is a suitable candidate model host for interaction studies with Heterodera avenae. The studies presented above document the first CCN host that not only has published genome context but also be compatible to BSMV VIGS.  

Soybean cyst nematode Heterodera glycines is one of the most serious soil-borne pathogens in soybean production. However, the researches were limited in China due to lack of an effective pathosystem. In this study, we screened 21 legume Medicago plants in both Medicago truncatula and Medicago sativa to obtain candidate model plants for establishing a new pathosystem for legume-H. glycines interactions. The nematode infection of tested plants was assayed with Race 3 and 4 respectively, which were two dominant H. glycines inbred races in China soybean producing areas. The results showed that the model legume plant M. truncatula A17 failed to allow Race 3 of H. glycines to complete its life cycle, in contrast, it provided the Race 4 population to form several cyst nematodes, however, the female index (FI) value was approximately 1.6. Three M. sativa cultivars, including Xunlu, Aergangjin and Junren, provided either Race 3 or 4 of H. glycines to develop into mature cysts with their FI value below 5 as well. Our results demonstrated that legume plants in both M. truncatula and M. sativa were not likely to be a model plant for H. glycines because of an extreme high resistance.

The cereal cyst nematode (CCN, Heteroder aavenae) causes serious yield loss on cereal crops, especially wheat, worldwide. Daxing population in Beijing City and Huangyuan population in Qinghai Province, China, are two CCN populations. In this study, the CCN pathotypes of Daxing and Huangyuan populations were characterized by tests on 23 standard “International Test Assortment” with the local species Wenmai 19 as the susceptible control. Tested materials were grouped by three nematode populations’ virulence on resistant genes (Rha1, Rha2, Rha3, Cre1) and nonresistant genes, varieties and lines. Both Daxing and Huangyuan populations were avirulent to Ortolan (Ha1). Barley cvs. Ortolan, Siri, Morocco, Bajo Aragon 1-1, and Martin 403-2 were all resistant to both populations. Cultivars Herta, Harlan 43 and wheat Iskamish-K-2-light were all susceptible to Huangyuan population, all of them, however, were resistant to Daxing population. The other five oats were all resistant to the two tested CCN populations. Except Iskamisch K-2-light, all the other wheat cultivars (Capa, Loros×Koga, AUS 10894, and Psathias) were susceptible to Daxing population. Because the pathotypes of the two tested CCN populations in Beijing and Qinghai were not identical to any of the 13 pathotypes previously characterized by the test assortment, we classified Daxing and Huangyuan populations as the new pathotypes, named Ha91.

For sedentary endo-parasitic nematodes, parasitism genes encoding secretory protein expressed in the subventral glands cells always play an important role during the early parasitic process. A new acid phosphatase gene (Ha-acp1) expressed in the subventral glands of the cereal cyst nematode (Heterodera avenae) was cloned and the characteristics of the gene were analyzed. Results showed that the gene had a putative signal peptide for secretion and in situ hybridization showed that the transcripts of Ha-acp1 accumulated specifically in the subventral gland cells of H. avenae. Southern blot analysis suggested that Ha-acp1 belonged to a multigene family. RT-PCR analysis indicated that this transcription was strong at the pre-parasitic juveniles. Knocking down Ha-acp1 using RNA interference technology could reduce nematode infectivity by 50%, and suppress the development of cyst. Results indicated that Ha-acp1 could play an important role in destroying the defense system of host plants.