Molecular characterisation of cereal cyst nematodes in winter wheat on the Huang-Huai floodplain of China

using RFLP and rDNA-ITS sequence analyses

Hong-lian LiA , Bo FuA and Ian T. RileyB

ACollege of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan, China (HAU)

B South Australia Research and Development Institute (SARDI)

Outline

1. Background

2. Methods

3. Results

4. Summary and discussion

1. Background

China is the biggest wheat producing country with a large area ( about 23 million ha), accounting for about 20% of world’s annual wheat production in recently years.

Huang-Huaifloodplain winterwheat area

Zoning plan of wheat in China

Huang-Huai floodplain is largest wheat producing area in China, with about 50% total wheat production.

The cereal cyst nematodes (CCN) are widespread and damaging root pathogen of wheat in China, speciallyin Huang-Huai floodplain winter wheat area.

• There are 12 accepted CCN species and some unadmitted species in the world.

• Heterodera avenae is the most widespread CCN species, followed by H. filipjevi, H. latipons and several less prevalent species.

H. avenae H. filipjevi H. latipons

In China, the pathogen of CCN in most of provinces is H. avenae , only find H. filipjevi in Henan, the centre of Huang-Huai floodplain.(Li et al. 2010, Peng et al. 2010)

The distribution of CCN in China

Based on the increasing recognition of the diversity of H. avenae in China, both in pathotypes and genotypes, and the recent discovery of H. filipjeviindicated that further investigation of CCN diversity in China is needed.

• So, we collected CCN specimens from 21 locations on the Huang-Huai floodplain for analysing the molecular characterisation

• To clarify the status of H. avenae group in China and determine the distribution and diversity of H. filipjeviin Henan.

2.1 Nematode specimens

Specimens were collected from 21 locations on the Huang-huai area

2. Materials and Methods

• Mature females were collected from roots from 19 locations and cysts from stored soil from 2 locations (BDNS and XFNS).

2.2 DNA extraction, PCR, RFLP and sequencing

Total DNA from single females was extracted as described by Subbotin and Waeyenberge (2000).

The primers were TW81 5’-GTTTCCGTAGGTGAACCTGC-3’ and AB28 5’-ATATGCTTAAGTTCAGCGGGT-3’ (Subbtoin 1999).

8 restriction enzymes; TaqⅠ, AluⅠ, HaeIII, HinfⅠ, RsaⅠ,

PstⅠ, CfoⅠ, Tru9Ⅰ used for RFLP analysis

2.3 Phylogenic analysis

24 new ITS sequences (in this study) 22 reference sequences from the Heterodera

avenae group and one for Globodera pallida (as an out group)

Four distinct RFLP profiles were obtained , with two consistent with H. avenae (A,B) and two with H. filipjevi (C,D).

3. Results

3.1 RFLP profiles

A B C D

Profile of fifteen specimens consistent with populations of H. avenae in China—type “C” (A).

Profile of two populations same as H. avenae in Australia (B)

H. avenae in ChinaType C

H. avenae in AustraliaAustralis Type

•3 specimens had profiles consistent with H. filipjevi (C),•but one specimen matched H. filipjevi except for a different

pattern for Hae III (D).

The specimen from JZBA had a 550 bp bands，compared withthe H. filipjevi （420, 350 and 180 bp）.

550 bp

The dendogram obtained by phylogenic analysis (Fig. 4.) places the specimens from the present study into one of two main clades

3.2 Phylogenic analysis

and BClade A

25 sequences obtained in this study along with reference sequences for H. avenae of the australisand ‘China’ types in a second sub-clade

Within this second sub-clade there is a cluster of the four australis type sequences from China (locations ZZXS and ZZXY) with the three reference sequences of this type from Australia and EU106165 from China

Clade A includes reference sequences for H. avenae (‘a’ and ‘b’ types), H. aucklandica and H. mani in one sub-clade

Clade B includes 8 sequences in this study and 5 reference sequences for H. filipjevi, indicate that H. filipjevi occurs in at least 6 locations in China.

The sequence from China cluster together with reference sequence from Italy and the USA, separating to a small degree from all other reference sequences.

4. Summary and discussion

4.1 Occurrence of Heterodera avenae and H. filipjevi on Huang-huai floodplain of China

H. avenae was confirmed to be widespread across Henan and was the only species found in the adjacent provinces

H. filipjevi was found at 6 locations, also widely dispersed in central Henan and at two location it mixed with H. avenae

4.2 The RFLP and phylogenic analyses

To provide some indication of the genetic diversity of H. filipjevi in China，it means more complex in control this disease with resistant cultivars

The finding of the H. avenae‘australis’ type (H. australis) at two locations.

The clustering of H. avenae(types ‘a’ and ‘b’) with H. aucklandica, H. mani in the phylogenic analysis of rDNA-ITS sequences

This result indicates the limitations of ITS sequence analysis for identifying H. avenae and closely related species.

4.3 More work need to do:

Pathotypes diversity of different CCN populations Screening of resistant cultivars to different CCN

populations

• Pathogenicity of H. filipjevi in China• Distribution of H. filipjevi in other areas of China

• Molecular diagnosis

Thanks to :

NSFC （National Natural Science Foundation of China）

NSFC –CIMMYT Major International cooperation projects

China- Australia Special Fund，MOST

The Special R & D Fund for Public Benefit Agricultural，

China( No.200903040), MOA

Thanks to

For their many suggestion and helps

Prof. Tang Wenhua Dr. Julie Nicol Dr. Ian Riley

Thanks