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Molecular diagnosis and direct quantification of cereal cyst nematode(Heterodera filipjevi) from field soil using TaqMan real-time PCR

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Journal of Integrative Agriculture 2023年第8期22卷 2591-2601页

作者： JIAN Jin-zhuo huanG Wen-kun KONG Ling-an JIAN Heng Sulaiman ABDULSALAM peng De-liang peng huan State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193P.R.China Key Laboratory of Plant Pathology of Ministry of Agriculture and Rural Affairs College of Plant ProtectionChina Agricultural UniversityBeijing100193P.R.China Division of Agricultural Colleges/Department of Crop Protection Ahmadu Bello UniversityZaria 810107Nigeria

Heterodera filipjevi continues to be a major threat to wheat production *** detection and quantification of cyst nematodes are essential for more effective control against this nematode *** the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying *** from DNA extracts of *** primers and probe designed from the obtained RAPD-SCAR marker fragments of *** showed high specificity to *** using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera *** 3 Pratylenchus *** qPCR assay is highly sensitive and provides improved *** detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify *** in naturally infested field *** was a high correlation between the *** numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR *** potentially provides a useful platform for the efficient detection and quantification of *** directly from field soils and to quantify this species directly from DNA extracts of field soils.

关键词： cereal cyst nematode Heterodera filipjevi molecular diagnosis quantification TaqMan real-time PCR

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我国北方地区大豆孢囊线虫遗传多样性及环境因子相关性分析

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吉林农业大学学报 2024年第3期46卷 379-390页

作者：杨飞燕史树森欧师琪徐伟刘淑艳彭焕彭德良吉林农业大学植物保护学院长春130118 中国农业科学院植保研究所植物病虫害生物学国家重点实验室北京100193

大豆孢囊线虫(Heterodera glycines)是一种严重为害我国乃至世界大豆产量和品质的病害。为阐明H. glycines地理种群的遗传结构,利用微卫星分子标记技术(Inter-simple sequence repeats,ISSR)对我国北方地区5省13个种群130个孢囊进行分... 详细信息

大豆孢囊线虫(Heterodera glycines)是一种严重为害我国乃至世界大豆产量和品质的病害。为阐明H. glycines地理种群的遗传结构,利用微卫星分子标记技术(Inter-simple sequence repeats,ISSR)对我国北方地区5省13个种群130个孢囊进行分析。结果表明:13个种群内多态性位点百分比9.52%~33.33%;平均等位基因数(Na) 1.095 2~1.333 3;有效等位基因数(Ne) 1.053 3~1.217 7;平均期望杂合度(He) 0.033 0~0.126 8;Shannon指数0.050 3~0.188 1,表明在各个H. glycines种群内存在较高的遗传一致性;3条ISSR引物扩增出片段的多态性位点百分比80.95%(30/42片段),种群间遗传分化系数(Gst) 0.732 5,基因流(Nm)0.182 6,表明在种群间存在极低的基因流和高水平遗传分化。成对种群间Nei's遗传一致性值是0.630 5(HLJM和JLCC之间)到0.960 6(HLDQ和HLAD之间),遗传距离的范围是0.040 2(HLDQ和HLAD之间)到0.461 3(HLJM和JLCC之间),聚类分析结果表明,13个种群在遗传相似系数为0.65时分成2个类群。遗传多样性指数与生态因子相关性分析表明,大豆种植期月平均最高温度与遗传多样性指数呈中等及极强以上水平相关,表明在一定温度范围内,温度越高,大豆孢囊线虫的遗传多样性水平越高,其他因子之间无显著相关性。两种群间的遗传一致性和遗传距离分别与地理距离间存在微弱相关性(R2=0.005 8,R2=0.009 6),结果揭示了所选的北方地区的13个H. glycines种群间存在明显分化,具有高水平遗传多样性的结构特征。

关键词：北方地区大豆孢囊线虫遗传多样性遗传分化 ISSR-PCR

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Molecular Characterization and Functional Analysis of a New Acid Phosphatase Gene(Ha-acp1) from Heterodera avenae

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Journal of Integrative Agriculture 2014年第6期13卷 1303-1310页

作者： LIU Yan-ke huanG Wen-kun LONG Hai-bo peng huan HE Wen-ting peng De-liang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences

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 speciifcally 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.

关键词： Heterodera avenae acid phosphatase RNA interference

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Assaying the potential of twenty-one legume plants in Medicago truncatula and M. sativa for candidate model plants for investigation the interactions with Heterodera glycines

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Journal of Integrative Agriculture 2016年第3期15卷 702-704页

作者： KONG Ling-an WU Du-qing huanG Wen-kun peng huan HE Wen-ting peng De-liang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences

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.

关键词： soybean cyst nematode alfalfa life cycle female index pathosystem

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Molecular characterization and functional analysis of two new lysozyme genes from soybean cyst nematode(Heterodera glycines)

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Journal of Integrative Agriculture 2019年第12期18卷 2806-2813页

作者： WANG Ning peng huan LIU Shi-ming huanG Wen-kun Ricardo Holgado Jihong Liu-Clarke peng De-liang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193P.R.China NIBIO Norwegian Institute of Bioeconomy ResearchPb 115N0-1431 AsNorway

Soybean cyst nematode(SCN, Heterodera glycines(I.)) is one of the most important soil-borne pathogens for soybeans. In plant parasitic nematodes, including SCN, lysozyme plays important roles in the innate defense system. In this study, two new lysozyme genes(Hg-lys1 and Hg-lys2) from SCN were cloned and characterized. The in situ hybridization analyses indicated that the transcripts of both Hg-lys1 and Hg-lys2 accumulated in the intestine of SCN. The q RT-PCR analyses showed that both Hg-lys1 and Hg-lys2 were upregulated after SCN second stage juveniles(J2 s) were exposed to the Grampositive bacteria Bacillus thuringiensis, Bacillus subtilis or Staphylococcus aureus. Knockdown of the identified lysozyme genes by in vitro RNA interference caused a significant decrease in the survival rate of SCN. All of the obtained results indicate that lysozyme is very important in the defense system and survival of SCN.

关键词： soybean cyst nematode lysozyme Hg-lys1 Hg-lys2 innate defense against bacteria

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A new pathotype characterization of Daxing and huangyuan populations of cereal cyst nematode(Heterodera avenae) in China

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Journal of Integrative Agriculture 2015年第4期14卷 724-731页

作者： CUI Jiang-kuan huanG Wen-kun peng huan LIU Shi-ming WANG Gao-feng KONG Lin-an peng De-liang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural Sciences

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.

关键词： cereal cyst nematode Heterodera avenae Daxing population huangyuan population pathotype

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Occurrence,identification and phylogenetic analyses of cereal cyst nematodes(Heterodera spp.)in Turkey

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Journal of Integrative Agriculture 2017年第8期16卷 1767-1776页

作者： CUI Jiang-kuan peng huan LIU Shi-ming Gul Erginbas Orakci huanG Wen-kun MustafaImren Abdelfattah Amer Dababat peng De-liang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193P.R.China CIMMYT(International Maize and Wheat Improvement Centre) Ankara 06511Turkey Department of Plant Protection Faculty of Agriculture and Natural ScienceAbant lzzet Baysal UniversityBolu 14280Turkey

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 （CentralAnatolia, Marmara, Aegean, SoutheastAnatolia, 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 patho- genic 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 6r 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 DiyarbakJr 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.

关键词： species specific PCR cereal cyst nematode molecular identification ITS-rDNA wheat pathogen

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A fragment of a 70-kDa Heterodera glycines heat shock protein(HgHSP70)interacts with soybean cyst nematode-resistant GmSHMT08

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Journal of Integrative Agriculture 2022年第10期21卷 2973-2983页

作者： LIU Zhi ZHANG Liu-ping ZHAO Jie JIAN Jin-zhuo peng huan huanG Wen-kun KONG Ling-an peng De-liang LIU Shi-ming State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193P.R.China MARA–CABI Joint Laboratory for Bio-safety Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193P.R.China

Soybean cyst nematode(SCN)Heterodera glycines is considered as the major constraint to soybean *** SHMT08 at Rhg4 locus on chromosome 08,encoding a serine hydroxylmethyltransferase,is a major gene underlying resistance against *** in Peking-type ***,the molecular mechanism underpinning this resistance is less well characterized,and whether Gm SHMT08 could interact with proteins in *** remains *** this study,yeast two-hybrid screening was conducted using Gm SHMT08 as a bait protein,and a fragment of a 70-kDa heat shock protein(Hg HSP70)was screened from *** that exhibited interaction with Gm *** interaction was verified by both GST pull-down and bimolecular fluorescence complementation *** finding reveals Hg HSP70 could be applied as a potential candidate gene for further exploring the mechanism on Gm SHMT08-mediated resistance against SCN ***.

关键词： soybean cyst nematode Heterodera glycines GmSHMT08 HgHSP70 interaction resistance

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Golden Promise barley(Hordeum vulgare) is a suitable candidate model host for investigation interaction with Heterodera avenae

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Journal of Integrative Agriculture 2017年第7期16卷 1537-1546页

作者： LUO Shu-jie KONG Ling-an peng huan huanG Wen-kun CUI Jiang-kuan LIU Jing QIAO Fen JIAN Heng peng De-liang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Key Laboratory of Plant Pathology Ministry of Agriculture/College of Plant Protection China Agricultural University

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 inter- action 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 ger^es 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 no.t only has published genome context but also be compatible to BSMV VIGS.

关键词： Golden Promise barley Heterodera avenae candidate model host interaction

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Evaluation of the biocontrol potential of Aspergillus welwitschiae against the root-knot nematode Meloidogyne graminicola in rice(Oryza sativa L.)

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Journal of Integrative Agriculture 2019年第11期18卷 2561-2570页

作者： LIU Ying DING Zhong peng De-liang LIU Shi-ming KONG Ling-an peng huan XIANG Chao LI Zhong-cai huanG Wen-kun State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection Chinese Academy of Agricultural SciencesBeijing 100193P.R.China College of Plant Protection Hunan Agricultural UniversityChangsha 410128P.R.China Agriculture Bureau of Hanshou County Hunan ProvinceChangde 415900P.R.China

The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas,and nematicides are neither ecofriendly nor cost *** acceptable biological agents are required for controlling this destructive *** this study,the biocontrol potential of Aspergillus welwitschiae AW2017 was investigated in laboratory and greenhouse *** in vitro ovicidal and larvicidal activities of *** metabolites were tested on *** in laboratory *** effect of *** on the attraction of *** to rice and the infection of rice by *** was evaluated in a *** bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal *** best larvicidal activity was observed at a concentration of 5×AW2017,which caused an 86.2%mortality rate at 48 h post *** highest ovicidal activity was recorded at a concentration of 5×AW2017,which resulted in an approximately 47.3%reduction in egg hatching after 8 d compared to the *** greenhouse conditions,the application of *** significantly reduced the root galls and nematodes in rice roots compared to the *** a concentration of 5×AW2017,juveniles and root galls in rice roots at 14 d post inoculation(dpi)were reduced by 24.5 and 40.5%,*** addition,the attraction of *** to rice roots was significantly decreased in the AW2017 treatment,and the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control *** results indicate that *** is a potential biological control agent against *** in rice.

关键词： Meloidogyne graminicola Aspergillus welwitschiae biological fungus nematode development nematicidal potential

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