Australasian Plant Pathol. (2011) 40:277–285 DOI 10.1007/s13313-011-0043-0
Molecular characterisation of cereal cyst nematodes in winter wheat on the Huang-Huai floodplain of China using RFLP and rDNA-ITS sequence analyses Bo Fu & Hong-xia Yuan & Yu Zhang & Xing-song Hou & Gao-lei Nian & Peng Zhang & Xiao-ping Xing & Bing-jian Sun & Ian T. Riley & Hong-lian Li
Received: 24 November 2010 / Accepted: 8 February 2011 / Published online: 22 February 2011 # Australasian Plant Pathology Society Inc. 2011
Abstract In response to the recent discovery of Heterodera filipjevi central Henan and the uncertain taxonomic status of Heterodera avenae more widely in China, heteroderid specimens from winter wheat at 21 locations in Henan and adjacent provinces were subjected to RFLP and rDNAITS sequence analysis. H. filipjevi was found in six locations in Henan, including two mixed with H. avenae; H. avenae being found at all other locations. A new RFLP profile type was found for H. filipjevi, three new types for H. avenae and the Australian type (Heterodera australis) were found for the first time in China (at two locations). Otherwise, H. avenae and H. filipjevi were of RFLP types previously reported in China. Phylogenic analysis of the rDNA sequences showed H. filipjevi in China was less diverse than H. avenae, with greatest similarity to specimens from Italy and the USA, which is consistent with a more recent introduction. In contrast, H. avenae in China was clearly distinct from H. avenae found elsewhere, except for the discovery of the Australian types. Although the Australian types clustered together, this fell within the variation found for the remainder of the specimens from China, which may represent a single species. These data reveal additional genetic diversity within the two cereal cyst nematode species in China, which is likely to have implications for the development of their control by host resistance. B. Fu : H.-x. Yuan : Y. Zhang : X.-s. Hou : G.-l. Nian : P. Zhang : X.-p. Xing : B.-j. Sun : H.-l. Li (*) College of Plant Protection, Henan Agricultural University, Zhengzhou 450002 Henan, China e-mail:
[email protected] I. T. Riley School of Agriculture, Food and Wine, University of Adelaide, Adelaide, SA 5005, Australia
Keywords Cereal cyst nematode . Heterodera avenae . Heterodera filipjevi . Heterodera australis . Henan . China . Winter wheat . Wheat . Nematode . Plant parasitic nematode . Distribution . Occurrence . Molecular analysis . RFLP . rDNA-ITS . Sequence . Phylogenic analysis . Huanghuai floodpain . Geographic distribution . Occurrence . Taxonomic status . New records
Introduction The cereal cyst nematodes (CCN) are widespread and damaging root parasites of wheat and related cereals in the subfamily Pooideae. Heterodera avenae is the most widespread CCN species, followed by H. filipjevi, H. latipons and several less prevalent species (reviewed by Nicol and Rivoal 2008). H. filipjevi has recently been found in central Henan, China (Li et al. 2010; Peng et al. 2010) and Oregon, USA (Smiley et al. 2008) extending its known range into regions where only H. avenae was previously known. With the increasing recognition of the diversity of H. avenae in China, both in pathotypes (Yuan et al. 2010) and genotypes (Zheng et al. 2000, Rivoal et al. 2003, Subbotin et al. 2003), the recent discovery of H. filipjevi demonstrates that further investigation of CCN diversity in China is warranted. Subbotin and Sturhan (2003) and Rivoal et al. (2003), in the broadest investigations of diversity within CCN (H. avenae and related species) to date, selected RFLP, rDNA-ITS sequence and RAPD analyses from a range of biochemical and molecular methods used in earlier investigations of this group. These methods have likewise been adopted in subsequent more geographically-focused studies (Maafi et al. 2003; Madani et al. 2004; Yan and Smiley
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2010), and further investigation of CCN in China using such approaches would build on these data. The molecular investigations of H. avenae in China (Zheng et al. 2000; Rivoal et al. 2003, Subbotin and Sturhan 2003; Ou et al. 2008) have noted differences from the H. avenae found in Europe and adjacent parts of Asia and Africa. Although morphologically indistinguishable, Subbotin and Sturhan (2003) concluded that the Chinese populations are distinct from H. avenae on molecular grounds and suggested further work is needed to determine their taxonomic status. This parallels the proposal (Subbotin et al. 2002) for the Australian populations of H. avenae to be classified as a distinct taxon, H. australis, on molecular grounds alone. However, the lack of biological and morphological differentiation of H. australis from H. avenae has meant the proposed new species has not been universally adopted (Vanstone et al. 2008; Riley and McKay 2009). The status of H. filipjevi in China has not presented similar challenges. The five populations currently characterised (Li et al. 2010; Peng et al. 2010) are reported to have a high degree of similarity with those found Europe, North America and West Asia. However, given that the populations in China represent an outlier from the main geographic range of the species, closer examination may reveal consistent differences or even provide evidence of the source of its introduction to China. To assist in the further clarification of the status of H. avenae in China and determine the distribution and diversity of H. filipjevi in Henan, CCN specimens were collected for molecular characterisation from winter wheat from 21 locations on the Huang-Huai floodplain of Northern China. The focus was on central to northern Henan where H. filipjevi had been found, supplemented with a smaller number of specimens collected in adjacent provinces.
Methods Nematode specimens Heteroderid specimens were collected from winter wheat from 21 locations on the Huang-huai floodplain of China (Fig. 1). Mature females were collected from roots from 19 locations in May 2008 and cysts from stored soil from two locations (BDNS and XFNS, Table 1). Across Henan, the locations were selected to represent the major wheatgrowing regions known to be infested with CCN (Henan Ministry of Agriculture, unpublished report). Researchers in the adjacent provinces provided advice on representative infested locations (or provided cysts in the case of BDNS and XFNS). The specimens were stored at −20 C until further processed. The species present were examined morphologically (Handoo 2002) and for possible heterogeneity by ISSR-
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PCR (Godwin et al. 1997) on 5–10 individuals (unpublished data). On the basis of these results, two specimens from each location were selected for this study. Where ISSR-PCR indicated mixed species (LYLU and XCYZ) one specimen of each species was included. DNA extraction, PCR, RFLP and sequencing Total DNA from single females was extracted as described by Subbotin and Waeyenberge (2000). The resulting DNA suspension (1 μl) was added to a PCR mixture containing 25 μl of 2× Taq PCR master mix 1 μl each of forward and reverse primers (1 μM), and double-distilled water to make a final volume of 25 μl. The primers were TW81 5’GTTTCCGTAGGTGAACCTGC-3’ and AB28 5’ATATGCTTAAGTTCAGCGGGT-3’ (Subbotin et al. 1999). For RFLP analysis, 7 μl of each PCR product were digested separately with the following restriction enzymes; Taq I, Alu I, Hae III, Hinf I, Rsa I, Pst I, Cfo I, Tru9 I (Promega, USA) in a buffer stipulated by the manufacturer. The digested PCR product was loaded on a 2% agarose gel, separated by electrophoresis (100 V, 50 min), stained with ethidium bromide and recorded with a gel imaging system (Ultra-Violet Products Ltd, Cambridge, UK). The PCR and RFLP procedures were repeated three times to verify the results. PCR products of these specimens (and a second specimen from 13 of the 21 locations, see Table 1) were cloned and sequenced (Genomics Company, Wuhan, Hubei, China). To validate and further explore the RFLP profiles, virtual digestion using sequences was performed with Vector NTI (a bioinformatics software package, Invitrogen Corporation, USA). Phylogenic analysis Thirty four new sequences (this study), 22 reference sequences (details in Fig. 4) from the H. avenae group and one for Globodera pallida (as an out group) were edited with Genetyx-Win (Ver 5.0, Genetyx Co., Tokyo, Japan) and aligned with ClustalX (Ver 1.83, www.clustal.org). Only sequences of ITS1, 5.8 S gene and ITS2 were used for further analyses. All alignments were analysed with maximum-parsimony (MP) algorithms (MEGA 4: Molecular Evolutionary Genetics Analysis, www.megasoftware.net) to examine the phylogenic relationships within the H. avenae group.
Results RFLP analysis Genomic DNA was successfully extracted from the specimens and a single fragment of about 1100 bp amplified by
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Fig. 1 Locations sampled and species identities for Heterodera associated with winter wheat on the Huang-huai floodplain of China. Sampling focused on central and northern Henan with one or two samples collected in adjacent provinces
PCR. No PCR products were amplified in the controls without nematode DNA template. Four distinct RFLP profiles were obtained (Fig. 2), with two consistent with H. avenae and two with H. filipjevi. Fifteen specimens had profiles consistent with populations of H. avenae in China (Fig. 2a) and two matched populations from Australia (Fig. 2b). Three specimens had profiles consistent with H. filipjevi (Fig. 2c), but one specimen matched H. filipjevi except for a different pattern for Hae III (Fig. 2d). The specimen from JZBA had bands at about 550, 420, 350 and 180 bp (Fig. 2d Lane 4) compared the expected bands for H. filipjevi at about 420, 350 and 180 bp. Application of virtual digestion to the sequences of the specimens included in the RFLP assays (and additional sequences from many of the same locations) provided
addition detail and showed further heterogeneity within these two CCN species for Hae III, Taq I and Tru9 I (Table 2). Virtual digestion provides precise fragment lengths, including small fragments that cannot be seen on a gel, and allow location of the restriction site (Fig. 3). The upper part of Table 2 provides six profile types that have been reported for H. avenae and H. filipjevi for these three restriction enzymes. Of these types, four were found among the specimens under study. Nineteen sequences were the same as those already reported for H. avenae China (Table 2 profile type 4) and eight were the same as for H. filipjevi as recently reported in China (Table 2 profile type 6). The profiles for the remaining seven sequences differed from any already reported in China. These new profiles for China were from two locations with profiles that matched H. avenae from Australia (Table 2
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Table 1 Details of locations sampled in Heterodera from winter wheat on the Huang-huai floodplain of China Codea
City
District/County
Province
Lat. (ON)
Long. (OE)
Genbank codeb Specimen 1
Specimen 2
AYAY BDNS FYYS HDYN HZHL
Anyang Baoding Fuyang Handan Heze
Anyang Nanshi Yingshang Yongnian Helou
Henan Hebei Anhui Hebei Shandong
36.0674 38.8619 32.7813 36.8063 35.1667
114.3464 115.5569 116.2024 114.4953 115.4915
HM027886 HQ380868 HQ380870 HQ380872 HQ380876
– HQ380869 HQ380871 HQ380873 –
JZBA KFQX LFXF LYLU LYYI PYQF SQSY XAYL XCXC XCYZ XFNS XXYJ ZKHY ZKSS ZZXS ZZXY
Jiaozuo Kaifeng Linfen Luoyang Luoyang Puyang Shangqiu Xi'an Xuchang Xuchang Xiangfan Xinxiang Zhoukou Zhoukou Zhengzhou Zhengzhou
Boai Qixian Xiangfen Luolong Yiyang Qingfeng Suiyang Yanliang Xuchang Yuzhou Xiangyang Yanjin Huaiyang Shangshui Xushui Xingyang
Henan Henan Shanxi Henan Henan Henan Henan Shaanxi Henan Henan Hubei Henan Henan Henan Henan Henan
35.1487 34.7147 35.9072 34.7125 34.5311 35.9360 34.3808 34.6490 34.0488 34.1203 32.1082 35.3987 33.8174 33.4821 34.7867 34.7390
113.0817 114.7368 111.4088 112.5351 112.1414 115.0262 115.6818 109.2329 113.7425 113.5507 112.0073 114.3764 114.8058 114.6416 113.4829 113.4114
HM027888 HQ380882 HQ380877 HQ380880 HM027890 HM027891 HQ380881 HQ380892 HM027892 HM027893 HQ380884 HQ380890 HQ380874 HM027896 HQ380886 HQ380888
HM027889 – HQ380878 HQ380879 – – HQ380883 – – – HQ380885 HQ380891 HQ380875 HM027897 HQ380887 HQ380889
a
In the text, this code is appended with 1 or 2 for locations where two cysts were analysed. b One female (Specimen 1) from each location was used for RFLP analysis and sequencing. An second female (Specimen 2) was sequenced for 13 of the 21 locations
profile types 2 and 3), and two locations with three previously unreported profiles (Table 2 profile types 7, 8 and 9). Type 7 has an additional restriction site in the 412 bp Hae III fragment found commonly in H. avenae (Fig. 3) dividing it into 356 and 60 bp fragments. Type 8 has an additional
restriction site in the 307 bp Tru9 I fragment found commonly in H. avenae in China (Fig. 3, Table 2 profile types 4) dividing it into 243 and 64 bp fragments. Type 9 has Taq 1 and Tru9 I fragments as previously reported for profile types 2 and 1, respectively, but not in this combination. In three
Fig. 2 Four RFLP-rDNA-ITS profiles found for Heterodera associated with winter wheat on the Huang-huai floodplain of China. Lanes: M, 100 bp ladder; 1, unrestricted fragment; 2, Taq I; 3, Alu I; 4, Hae III; 5, Hinf I; 6, Rsa I; 7, Pst I; 8, Cfo I; 9, Tru9 I; Profile: a AYAY,
BDNS, FYYS, HDYN, HZHL, KFQX, LFXF, LYLU, LYYI, PYQF, SQSY, XAYL, XCYZ, XFNS and XXYJ; b ZZXS and ZZXY; c XCXC, ZKHY and ZKSS; d JZBA. Profiles A and B were consistent with Heterodera avenae, and C and D with H. filipjevi
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Table 2 rDNA-ITS sequence fragment length profiles obtained by virtual digestion by three restriction enzymes (Hae III, Taq I and Tru 9) using Vector NTI software (Invitrogen Corporation) of sequences Heterodera species
Sequence source
Reference sequence restriction profiles H. avenae Type A, Morocco, Subbotin et al. 2003, Genbank AY148367 Type B, India, Subbotin et al. 2003, Genbank AF274397 Australia, Subbotin et al. 2003, as H. australis, Genbank AY148395 Australia, Subbotin et al. 2003, as H. australis, Genbank AY148396 China, Subbotin et al. 2003, Genbank AY148382 China, Ou et al. 2008, same location as ZZXS in this study, Genbank EU106165 H. filipjevi Iran, Subbotin et al. 2003, Genbank AF498380 Sample sequence restriction profiles H. avenae AYAY, BDNS, BDNS2, FYYS, FYYS2, HDYN, HDYN2, HZHL, KFQX, LFXF, LFXF2, LYLU, LYYI, PYQF, SQSY, XAYL, XCYZ, XXYJ, XXYJ2 XFNS XFNS2 ZZXS ZZXS2, ZZXY ZZXY2 H. filipjevi
JZBA, LYLU2, SQSY2, XCXC, ZKHY, ZKHY2, ZKSS, ZKSS2 JZBA2
from Heterodera avenae and H. filipjevi. Both reference sequences and those obtained from samples collected on the Huang-huai area of China are listed. Previously unreported profiles are set in bold
Restriction enzyme
Profile type
Hae III
Taq I
Tru9 I
416·353·176·52·24·24
385·274·150·129·65·43
549·486·9
1
416·353·176·52·24·24
384·274·150·129·65·43
549·486·9
1
416·353·176·52·24·24
532·275·129·65·43
542·486·9·7
2
416·353·176·52·24·24
384·274·148·129·65·43a
542·486·9·7
3
416·353·176·52·24·24
384·275·148·129·65·43a
542·307·179·9·7
4
416·249·176·106·52·24·24
386·276·148·129·65·43
542·489·9·7
5
424·378·173·52·24
339·316·134·118·79·65
551·493·7
6
416·353·176·52·24·24
384·275·148·129·65·43
542·307·179·9·7
4
356·353·181·60·52·24·24b 416·353·176·52·24·24 416·353·176·52·24·24 416·353·176·52·24·24 416·353·176·52·24·24
384·275·148·129·65·44 384·275·148·129·65·43 532·275·129·65·43 532·275·129·65·43 385·275·148·129·65·43
542·307·179·9·8 542·243·179·64·9·7b 549·486·9 542·486·9·7 542·487·9·7
7 8 9 2 3
424·378·173·52·24
339·316·134·118·79·65
551·493·7
6
551·424·52·24b
339·316·134·118·79·65
551·493·7
10
a
These two profiles were combined in error in Table 3 of Subbotin et al. 2003
b
Previously unreported RFLP profile
locations (XFNS, ZZXS and ZZXY), these data indicated heterogeneity in the ITS region within the population. A further location had a new profile similar to H. filipjevi, except that it has one less restriction site for Hae III resulting in a 551 bp fragment instead of 378 and 173 bp fragments (Fig. 3, Table 2 profile type 10). At this location (JZBA) the other sequence conformed to the known H. filipjevi profile, indicating heterogeneity in the population. Phylogenic analysis The dendogram obtained by phylogenic analysis (Fig. 4.) places the specimens from the present study into one of two main clades (Clades A and B). Clade A includes reference sequences for H. arenaria, H. aucklandica, H. avenae (‘a’ and ‘b’ types) and H. mani in one sub-clade, and 25
sequences obtained in this study along with reference sequences for H. avenae of the australis and ‘China’ types of Subbotin and Sturhan (2003) in a second sub-clade. The inclusion of the specimens with the new RFLP profile types 7, 8 and 9 (location XFNS and ZZXS, Table 2) is indicative of these populations being H. avenae with similarity to other H. avenae types found in China. Within this second sub-clade there is a cluster of the four australis types from China (locations ZZXS and ZZXY, Table 2) with the three reference sequences of this type from Australia and EU106165 from China. Although, this clustering indicates these eight sequences are closely related, the degree of difference of this group from the whole sub-clade of H. avenae sequences from China is no more than other differences seen throughout this sub-clade. Clade B (Fig. 4) includes five reference sequences for H. filipjevi, the sequence from the population (location
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a
B. Fu et al.
with H. filipjevi. With these frequencies and the fact that none of the seven locations sampled in adjacent provinces were found to have H. filipjevi, this indicates a possible aggregated distribution of this species in China.
Discussion
b
Fig. 3 Restriction sites in rDNA-ITS sequence for enzymes Hae III, Taq 1 and Tru9 I for Heterodera avenae and Tru9 I for H. filipjevi for RFLP profile types (refer to Table 2) for specimens collected from winter wheat on the Huang-huai floodplain of China
XCXC, Table 2)1 reported as the first record of H. filipjevi in China (Li et al. 2010) and eight other sequences, indicating that H. filipjevi occurs in at least six locations in China. This clade includes JZBA2, with the new RFLP profile 10 (Table 2) providing evidence that this population is also H. filipjevi. The sequence from the China cluster together with reference sequence from Italy and the USA, separating to a small degree from the three other H. filipjevi reference sequences from Europe. Occurrence of Heterodera avenae and H. filipjevi on the Huang-huai floodplain of China Fig. 1 maps the occurrence of H. 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 specimens collected in adjacent provinces. H. filipjevi was found at six locations, also widely dispersed in central Henan and at two locations it co-occurred with H. avenae. The relative frequency of the two species in central Henan (that is excluding two locations in northern Henan) was similar; eight of the 12 locations with H. avenae and six 1
Peng et al. (2010) report four rDNA-ITS sequences for H. filipjevi from four locations in China. However, only two of the Genbank codes provided are ITS sequences and both are from the same location as our XCXC. These differ by only two to three base pairs from our sequence, so were not included in the phylogenic analysis.
With the recent detection of H. filipjevi in China (Li et al. 2010; Peng et al. 2010), the most important finding of this study is that H. filipjevi is widespread in central Henan and at a frequency similar to H. avenae (although these values must be treated as indicative only given that the sampling scheme was not designed to estimate prevalence). This is important as it adds significantly to the challenges of managing CCN in wheat in China, especially as Henan is the major wheat-producing province. Already it is clear that pathotype diversity in H. avenae (Yuan et al. 2010) complicates the identification and deployment of host resistance for CCN control in China. With H. filipjevi having different host reactions to H. avenae (Nicol et al. 2009), this complexity is further increased. It is possible that H. filipjevi populations in China will also possess pathotype diversity, although this remains to be investigated. The widespread distribution of H. filipjevi in central Henan indicates it is not a recent introduction. However, based on limited sampling in adjacent provinces, it appears that H. filipjevi populations might be restricted to Henan. Of course, more extensive surveys are needed to confirm this. If an aggregated distribution within the wider wheat growing areas of China is confirmed, it would be reasonable to postulate that H. filipjevi has been established in China for a much shorter period than H. avenae. In which case, it would be also reasonable to predict that the genetic and physiologically diversity of H. filipjevi in China will be less than that found in H. avenae. In the late 1950s, wheat cultivars from Albania were introduced to China and came to Henan via Hubei (Jin and Liu 1964), which could have provided a route of entry for H. filipjevi and allowed enough time for its spread within central Henan. Given this possibility, further sampling in Hubei would be justified. The RFLP and phylogenic analyses undertaken in this study provide some indication of the genetic diversity of H. filipjevi in China. One population had an additional Hae III restriction sites in the rDNA-ITS region compared to those reported elsewhere. The phylogenic analysis groups the H. filipjevi from China separately to several sequences from Europe. H. filipjevi sequences from China (Clade B, Fig. 4) appear to have a lower genetic diversity compared to H. avenae sequences from China (Clade A, Fig. 4), which would be consistent with a later introduction. However, there is evident diversity within this species in China, which could have arisen since its introduction or already
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Fig. 4 Phylogenetic relationships for Heterodera associated with winter wheat on the Huang-huai floodplain of China (for location codes refer to Table 1 and Fig. 1) and related Heterodera spp. with Globodera pallida as an out group based on rDNA-ITS sequence using the maximum parsimony method. A Genbank code is included for each entry. Clade A includes H. avenae specimens from China and reference sequences of H. avenae and closely related species. Clade B includes H. filipjevi specimens from China and representative reference sequence of H. filipjevi
A
HZHL HQ380876 XCYZ HM027893 XXYJ2 HQ380891 H. avenae China AY148382 XFNS2 HQ380887 AYAY HM027886 BDNS2 HQ380869 PYQF HM027891 LFXF HQ380877 HBHD HQ380872 LFXF2 HQ380878 XFNS HQ380884 ZZXS2 HQ380887 ZZXS HQ380886 64 ZZXY2 HQ380889 65 H. avenae China EU106165 H. australis Australia AY148395 ZZXY HQ380888 H. australis Australia AY148393 87 H. australis Australia AY148396 HBHD2 HQ380873 SQSY HQ380881 BDNS HQ380868 XXYJ HQ380890 KFQX HQ380882 52 XAYL HQ380892 LYYI HM027890 LYLU HQ380880 FYYS HQ380870 FYYS2 HQ380371 50 H. mani Germany AY148377 H. arenaria UK AF274396 67 H. avenae 'a' Morocco AY148367 42 H. avenae 'b' India AF274397 H. aucklandica AF274398
H. filipjevi Russia AF274399 H. filipjevi UK AY148403 H. filipjevi Iran AF498380 86 JZBA2 HM027889 XCXC HM027892 B H. filipjevi Italy AY347922 JZBA HM027888 43 LYLU2 HQ380879 SQSY2 HQ380879 52 ZKHY2 HQ380875 ZKHY HQ380874 ZKSS HM027896 H. filipjevi USA GU079654 ZKSS2 HM027897 10 93
A
100
99
98
B
10 100
59 H. hordecalis AF498381 H. latipons AF498382 H. fici AF498385 H. humuli AY347926 100 H. ripae DQ846902 100 H. glycines AF498387 H. schachtii AY166436 100 G. pallida GU084806 10
existed within the original founder population. The grouping of these with the sequence from Italy is consistent with a possible Albanian connection, but clearly these data are insufficient to adequately address that question. Another key finding is the detection of the H. avenae ‘australis’ type (H. australis of Subbotin and Sturhan 2002) at two locations. This is the first published record of this type outside Australia. Although most H. avenae found were of the type previously reported for China (Subbotin et al. 2003), the detection of the australis type and three new
H. avenae RFLP profile type (types 7, 8 and 9, locations XFNS and ZZXS, Table 2), shows that H. avenae in China is more genetically diverse than previously known and that this diversity can occur within a relatively restricted geographic range. Within a moderately close radius of Zhengzhou, H. avenae of five RFLP types have been found; profile types 2, 3, 4, 5 and 9 in Table 2 (including one new type). The other new RFLP profile types 7 and 8 in Table 2 were from Hubei, the most southerly specimen examined and towards the southern margin of China’s
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wheat growing areas, further indicating the merits of additional sampling in Hubei. It is considered unlikely that H. avenae is endemic to Australia (Riley and McKay 2009) and the detection of the australis type in China adds weight to this position. However, postulation that H. avenae in Australia has a Chinese origin is possibly premature. Riley and McKay (2009) consider the genetic diversity of H. avenae in its native range has not been adequately explored, and imply this is needed to answer such questions. In the phylogenic analysis (Fig. 4), the australis type sequences cluster with other H. avenae sequences from China, so a Chinese origin for this type is plausible. The clustering of all H. avenae sequences along with the sequence from Australia (also noted by Ou et al. 2008), does not provide strong support for H. australis as currently proposed (Subbotin and Sturhan 2002; 2003), especially in the absence of morphological and biological differences. Alternatively, the clustering could be used to suggest that if H. australis is indeed a cryptic species, distinct from H. avenae of Europe, North Africa and West Asia, then its definition might need to be widened to include the other H. avenae types from China. The clustering of H. avenae (types ‘a’ and ‘b’) with H. arenaria, H. aucklandica, H. mani in the phylogenic analysis of rDNA-ITS sequences (Fig. 4), and also with H. pratensis in Subbotin and Sturhan (2003), indicates the limitations of ITS sequence analysis for discriminating H. avenae and closely related species. H. arenaria, H. aucklandica, H. mani and H. pratensis are species with morphometric and biological differences from H. avenae, whereas, H. avenae types ‘a’ and ‘b’ cannot be distinguished in this way from the types found in China (Subbotin and Sturhan 2003). Therefore, it is recommended that until diagnostic morphology or biology is found for these types, or another species is described, the two CCN species in China be regarded as H. avenae and H. filipjevi. The widespread occurrence of both H. avenae and H. filipjevi, along with the recognition of additional intra-specific genetic diversity, in Henan (China’s major wheat-producing province) highlights the need to take this diversity into account when developing control of CCN in China. Already significant pathotype diversity is reported for the common H. avenae type in China (Yuan et al. 2010), so the discovery of the Australian type will add to this. It is also possible that the Chinese populations of the H. avenae ‘australis’ type will have different pathogenicity that of Australian populations. Additionally the pathogenicity of H. filipjevi in China needs to be determined. Acknowledgements Funding was provided by The Special Research and Development Fund for Public Benefit Agriculture (No.200903040) and NSFC-CIMMYT Major International Cooperation Project (No.30921140411). Colleagues from adjacent provinces are thanked for
B. Fu et al. the provision of samples and advice on infested locations, including Chen Shulong (Institute for Plant Protection, Hebei Academy of Agriculture and Forestry Sciences), Wu Huiping (College of Plant Protection, Anhui Agricultural University) and Xiao Yannong (College of Plant Science, Huazhong Agricultural University).
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