Current status and progree of the cereal cyst nematodes Heterodera avenae and H. filipjevi on wheat in China“

Peng, D,, Nicol, J.M., Huang, W, Chen, S Zheng, J. Li, H,

Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China E-mail:dlpeng@ippcaas.cn

4th Asian Conference on Plant Pathology， Darwin ，April 26-29

CCN is a Sedentary nematode forming white adult females on the root system which later become hard brown CYSTS which survive in soil.

• Below ground: bushy-knotted’ appearance usually with several females visible at each root, which later become cysts.

• Above ground: pale green patches of poor growth with yellow lowered leaves and generally plants with few tillers.

• Generally one generation per cropping season• Only cereal host (H. avenae)• Yield losses worldwide between 5-50%.• The damage is greater when the crop is stressed.

Cereal Cyst Nematodes caused seriouseffect on what yield and

The ‘knotted’ root system and white females which turn into

cysts

Symptoms：Yellow, darf

2005年菏泽

April,1992, dingzhou， Hebei Province

December，2007，Xuchang Henan

Confusion withnot speciality, confused with lack water

fertilizer Lack

2010/11/12Hufeng county，Sha’an Province,

Wheat variety Xiaoyan 22

CCN Symptoms: 2010/4/22, Xingyan, and XuchangHena

Quick spread by havesting tractor

Frequency 43%

Hubei 1989

The Cereal cyst nematode - CCN (Heterodera spp.) wasfirst found and reported from Hubei province in the centreof China in 1989 ( Peng, Chen, Wang, 1989)

CCN Distribution

Hubei, Tianmen, 1989 Peng Chen & WangHenan: Anyang, 1989 Liu, et al.

Zhenzhou 2005 Li et al.Hebei, Dingzhou， 1990 Peng et al.Beijing, Chanping 1991 Peng et al.Shanxi, Taigu 1992 Peng et al.Inner Mongolia, 1992 Peng et al.Qinghai 1992 Peng et al. Hou, 2006Anhui, 1998 Zheng, et al.,

2005, Huiping，Ian et al.,Shangdong 2005 Julie Deliang et al.Gansu 2007 Ian，Huixia et al,Shan’xi 2007 Peng et al.,Jiangsu 2010 Li, HM, et al.,Ningxia 2011.1 Huang, Peng

13 provinces

More than 300 counties

Province Year Scientist

CCN has been found in 13 provinces

CCN caused seriuos problem on wheat production, estimated occurrence area 40 Mha

5 province of large wheat produce in China

Henan： yield loss general 17-42%，serious 80%，17 MhaHebei： yield loss general 15-25%，serious 40%，7MhaShangdong：17MhaJiangsu： 3MhaAnhui： 5Mha

Hubei Sha′anxi Gansu Qinghai Ningxia Shanxi

Frequency of isolation and population density of CCN

Survey results from 1998-2008Global reports indicate a damaging threshold for CCN is 5 eggs/g soil

IMPACTS on wheat production in China

Cereal cyst nematodes: complex avenae filipjevi H filipjevi exist in China wheat field Population is higher than avenae population wheat production get more challenge and threaten

Heterodera filipjeviXuchang, Linying, WeihYanjing、Tangying

Species: H avenae H filipjevi

菲利普孢囊线虫H filipjeviNingxia Qinghai

Heterodera filipjevi

HinfI TaqI

M:100bp;: 1 positive control, HN-34 Henan xuchang，HN-29 Henan Xuchang， H glycines；HN-25zhenzhou，HB-22 Hebei， HB-23 Hebei， Shijiazhuang； QM-1； QH-1 ； SD-7 ; SD-8 ；

H. filipjevi: Linying, Xuchang, Weihui and Yanjing, Also found in Ningxia, Qinghai

H avenaeH filipjevi

Heterodera.filipjevi ITS-RFLP

Plant Disease ，2010.10：1262First Report of the Cyst Nematode (Heterodera filipjevi) on Wheat in Henan Province, China

Banpoupu

Xuchang

Henan

Henan privinceLinying (Peng Dl et.al., 2009)Xuchang (Peng Dl et.al., 2009)Weihui (Peng Dl et.al., 2009)Yanjing (Peng Dl et.al., 2009)Tanying，Henan province (Peng Dl et.al., 2010)Huaiyang (Li HL, 2010)Jiaozuo Boai (Li HL, 2010)Shangshui (Li HL, 2010)Lulong, luoyang (Li HL, 2010)

NX31 Ningxia (Peng Dl et.al., 2010)QH19 Qinghai (Peng Dl et.al., 2010)

Heterodera filipjevi found in 9 county, Henan

Sequences registration in GenBank

ITS sequences

Linying：GU083595,Xuchang：GU083596,Weihui：HM147944,Yanjing：HM147945

D2D3 region

Linying：GU083592Xuchang：GU083593Weihui： GU083594Yanjing： GU83597

http://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-04-10-0309

Molecular characteristics analysis of Heterodera avenae, H filipjevi

Stucture of ribosomal DNA clusters

TW 81 - 5’ GTTTCCGTAGGTGAACCTGC-3’AB 28 - 5’ ATATGCTTAAGTTCAGCGGGT-3’

18 S 5.8 S 28 SITS1 ITS2

AB28TW81rDNA1 rDNA2

rDNA1.58S

IGS 5’-ETS 3’-ETS

The amplification of the rDNA ITS regions of each population yielded one fragment of approximately 1045(Heterodera avenae), 1054 bp ( H filipjevi ). No PCR products were obtained in the control lacking the DNA template

M：DL2000 marker；1-17：HY127B， HY65A，ZHZ162A，HY5B，CE18，DT142A，YL4B，GH132A，YBT11B，HY126B，HZXZA，DT2A，XS3，HG24，SF2，HA11，HA10；18： negative control.

1000bp→

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Molecular characteristics of Heterodera avenae and H filipjevi

H filipjevi HN-32,HN-34 codes:M-100bp, N-unrestricted PCR product , 1/2-AluI, 3/4-CfoI, 5/6-HifI, 7/8-SatI, 9/10-PstI, 11/12-RsaI, 13/14-TaqI, 15/16-Tru9I.

H avenae HN-16,HN-30 codes:M-100bp, N-unrestricted product , 1/2-AluI, 3/4-CfoI, 5/6-HifI, 7/8-SatI, 9/10-PstI, 11/12-RsaI, 13/14-TaqI, 15/16-Tru9I.

AluI CfoI HifI SatI PstI RsaI TaqI Tru9I AluI CfoI HifI SatI PstI RsaI TaqI Tru9I

Henan、Hebei, Shandong，Jiangsu，Gansu， Qinghai，

76 populations ITS

Henan、Hebei, Shandong，Jiangsu，Gansu，Qinghai，

76 populations D2D3

Heterodera avenae complex species

Europe

H. avenaeH. mani H. aucklandica

Asia

France

H. “avenae”

H. pratensis

H. ustinovi

H. filipjevi

H. australis

H. arenaria

with underbridge

without underbridge

rDNA-ITS and D2/D3 OF Heterodera avenae from Gansu province

rDNA-ITS of Heterodera avenae

A: AluⅠ；B: CfoⅠ；C: Bsh1236Ⅰ；D: AvaⅠ；E: RsaⅠ；F: BsuRⅠ；G: HinfⅠ；H: MvaⅠ；I: TaqⅠ). M: DL 100 bp marker）; U: uedigestion; 1-7: GsWx, GsGg, GsWw, GsMq, GsTs1, GsTs2, GsYz

rDNA-ITS-RFLP analysis of Heterodera avenae from Gansu province

Species specific primer for diagnosis Heterodera filipjevi

Forward: TW81 Reversed HfR1

cDNA library of H avenae J2, 6000 EST7 genes cloned already

species genes Accessionnumber

Year

Heterodera avenae Ha-eng-1 FJ839965 2009.3.19

Heterodera avenae Ha-CBP1 GQ178086 2009.05.21

Heterodera avenae Ha-PEL1 GQ998895 2009.09.25

Heterodera avenae Ha-PEL2 GU138156 2009.10.29

Heterodera avenae Ha-dsl-1 HM147942 201-04-27

Heterodera avenae Ha-dsl-2 HM147943 201-04-27

Heterodera avenae Ha-expb1 HQ386233 2010-10-12

PARASITISM GENES

A new endoglucanase-like gene (Ha-eng-1a GenBank ID: FJ839965) was cloned from H. avenae J2 by RACE

5’race 3’race

The full-length cDNA designated asHa-eng-1a is 1565 bp long

Conserved domain search shows the deduced protein Ha-eng-1a contains a catalytic domain and a carbohydrate-binding module of family 2 (CBM II)

Ha-eng-1a

Gts-eng-1

Gr-eng-1

Hs-eng-1

Hg-eng-1

cyst nema

Rs-eng-1a

Da-eng-1

Rs-eng-1b

Dd-eng-1

migratory nema

Mi-eng-1

GHF5

GHF45 Bx-eng-1

100

100

47

100

80

69

51

93

0.00.20.40.60.8

Phylogenetic tree of the new endogulcanase gene Ha-eng-1a.

Southern blot analysis shows Hg-eng-1a is a member of a multigene family

Ha-eng-1a clustered in a group of cyst nematode endogulcanases and classified into the GHF5.

Exon / intron organization of Ha-eng-1a gDNA

Ha-eng-1a gDNA clone is 2129 bp long containg eight introns

Ha-eng-1a specifically expressed in the two subventral gland cells of H. avenae

Recombinant protein Ha-eng-1a showed a strong cellulase activity in vitro

Cellulase activity assay on carboxymethylcellulose (CMC) agar plate test

Tissue localizaiton of Ha-eng-1a mRNA

A new cellulose binding protein gene (Ha-cbp-1， GQ178086 ) was cloned from H. avenae J2 by RACE

3’RACE 5’RACE

The full-length cDNA designated as Ha-CBP-1 is 964 bp long composed of a 393-bp open reading frame, a 55-bp 5’-untranslated region (5’-UTR) before the ATG initiation codon and a 513-bp 3’-UTR containing a polyadenlylation signal (AATAAA)

The predicted HA-CBP-1 shared similarities of 76% and 75%with the HG-CBP-1 and HS-CBP-1, respectively.

Sequence alignment between deduced amino acid sequence of cellulose binding protein HA-CBP-1 from H.avenae with a cellulose binding protein from other nematodes .

The genomic sequence of Hg-cbp-1 which contained two introns

Phylogenetic trees of six CBPs based on the CBPs amino acid sequence using ME method.

Capacity building to work with CCN in China

2006 – 1st 5.13-18, Cereal Cyst Nematode Workshop held in Hebei province with ATSE Crawford Foundation Australia, and CAAS.

2007 – 2nd 4.12-14, Cereal Cyst Nematode Workshop held in Beijing, IPP-CAAS

2008-3rd 8.3-5, Cereal Cyst Nematode Workshop held in Qinghai, IPP-CAAS

2009-4th 5.9-11, Cereal Cyst Nematode Workshop held in Zhengzhou, IPPCAAS，HNAU

2010-5th 6.13-15,Cereal Cyst Nematode Workshop held in Beijing, IPPCAAS

First China-CIMMYT workshop on CCN

Capacity building to work with CCN in China

CHINA-CIMMYT workshop on CCN, 2010, 6.12-15

10Th Chinese Nematology Symposoms， 2010, 8.13-15

CCN network in China

CIMMYTInstitute of plant Protection, CAASInstitute of plant Protection, HbAAFHenan Agricultural UniversityChina Agricultural UniversityHuazhong Agricultural UniversityHuanan Agricultural UniversityAnhui Agricultural UniversityInner Mongolia Agricultural UniversityQinghai Academy of Agricultural and Forestry SciencesGansu Agricultural UniversityShandong Agricultural UniversityQingdao Agricultural UniversityWest North Agricultural and Forestry UniversitySeveral local plant protection station from Henan, Hebei, Inner Mongolia, Qinghai and Gansu

2010/4/22 henan,

Training

Ningxia, 2010/6/9

Cereal cyst nematodes Projects in China

THREE PROJECTS ：

MOA-China project NO. 200903040NSF-CIMMYT project no. 30921140411MOST-China project NO. 2009DFB30230

National CCN Project in China

MOA-China project（NO. 200903040）Crop Cyst Nematode Managements Research and

Development (2009-2013） Leading Scientist：Deliang PengPartner – CAAS-IPP， 28 University，Institution，

Extension Station, about 50 scientists invovledHAAFS---Dr.Shulong Chen, HAU---Dr.Honglian Li,NAU---Dr.Hongmei Li, IMAU--Dr.Hongyou Zhou

Donor-MOF, MOA 3 M Euros

National CCN Project in China

Cereal cyst nematodes Project in China

NSF-CIMMYT project no. 30921140411:Development of molecular tools for Cereal Cyst Nematode (Heterodera spp.) diagnostics and identification of wheat resistance and nematode parasitsm genes Project Leader: Deliang Peng2010-2012. Partner – CAAS and other Chinese

HAU—Honglian LiHAAFS—Shulong Chen

Donor-NSF China

MOST-China-CIMMYT Project

MOST-China project NO. 2009DFB30230 Project title:

Study on the Pathotypes and and virulence genes of cereal cyst nematode in China 2009-2013

Leading Scientist: Deliang PengPartner – CAAS and associated Chinese NARsDonor-MOST

Ministry of Science and Technology, China

ONGOING RESEARCH

Risk Assessment – yield loss, damage thresholds and population dynamics

Develop a molecular diagnostics Parasitism gene and effector Pathogenecity, pathotype, ecotype, and biology Control of CCN through IPM practices including;Identification of resistance against known pathotype,

followed by subsequent breeding to incorporate resistance.Use of rotation Identification of Biological control agents

Cooperation with other countries ,scientists

ACKNOWLEDGMENT

Nature Sciences Foundation of China

Ministry of Agricultural

Ministry of Sciences and Technology

CIMMYT

SKLBPI

Acknowledgment

Research Team

Deliang PengDongsheng ZhangWenkun Huang Wenting He

Huan PengHaibo LongGaofeng WangXiaochuan GuWenxing Ye

Qiong Wang Binyu WangLingling Dai Xiaoli QiXiaoqiu Zong

The tentative agenda including1. Global status of the distribution of CCN2. The economic importance and population dynamics of CCN on wheat3. Control strategies of CCN in wheat using host resistance4. Control strategies of CCN other than host resistance5. Use of molecular tools for research with CCN (such as pathogen diagnostics, phylogeny studies and host resistance).6. CCN genome and parasitism genes7. Biological control and IPM

ORGANISERS

CIMMYT: Dr Julie NicolIPP-CAAS: Dr Deliang PengChinese Society of Nematologists

Registration and Accommodation

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Proceeding: Official proceedings will be published.

Contact

Prof. Deliang Peng

Institute of Plant Protection, Chinese Academy of

Agricultural Sciences, Beijing 100193, China

E-mail: dlpeng@ippcaas.cn

dlpeng@caas.net.cn

Tel ++ 86 10 62815611Fax + 86 10 62896114

Mobile: 86 13381056763