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55Russian Journal of Nematology, 2015, 23 (2), 145 - 152
Heterodera sturhani sp. n. from China, a new species of the Heterodera avenae species complex (Tylenchida: Heteroderidae)
Sergei A. Subbotin1' 2
'Plant Pest Diagnostics Centre, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA
95832-1448, USA
2Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect
33, 119071, Moscow, Russia e-mail: [email protected]
Accepted for publication 25 November 2015
Summary. Heterodera sturhani sp. n. is described as a new species from the Heterodera avenae complex. The new species is morphologically similar to H. pratensis and H. riparia in many characteristics of cysts and second-stage juveniles. It can be differentiated from H. pratensis by smaller cyst sizes and from H. riparia by longer body and tail lengths of the second-stage juveniles. The sequence and phylogenetic analysis of the coxI mtDNA gene sequences and the PCR-coxI-RFLP separate H. sturhani sp. n. from all other species of the H. avenae complex. Phylogenetic analysis of the coxI gene sequences enables discrimination between A (France) and B (Syria) types of H. avenae. The independent species status of H. australis is confirmed. The multiple species concept of the cereal cyst nematodes is proposed and discussed.
Key words: coxI, Heterodera australis, PCR-RFLP, phylogeny.
At present, the Heterodera avenae complex includes the following nine species: H. arenaria Cooper, 1955; H. avenae Wollenweber, 1924; H. aucklandica Wouts & Sturhan, 1995; H. australis Subbotin, Sturhan, Rumpenhorst & Moens, 2002; H. filipjevi (Madzhidov, 1981) Stelter, 1984; H. mani Mathews, 1971; H. pratensis Gabler, Sturhan, Subbotin & Rumpenhorst, 2000; H. riparia (Kazachenko, 1993) Subbotin, Sturhan, Rumpenhorst & Moens, 2003 and H. ustinovi Kirjanova, 1969. Three species, H. avenae, H. filipjevi and H. australis, from this complex are major nematode pests in cereal growing areas, whereas the other six species only parasitise various grasses. The European cereal cyst nematode, Heterodera avenae, and the Filipjev cereal cyst nematode, H. filipjevi, were found in many countries in Europe, Asia and North America, where they have often overlapping distributions, whereas the Australian cereal cyst nematode H. australis has limited distribution and was reported in Australia and recently in China (Subbotin et al., 2010).
During last few years, special attention was devoted to studies of the cereal cyst nematode in China. In 1989, the cereal cyst nematode was first
reported in Hubei Province, China (Chen et al., 1992). Subsequent surveys revealed that this nematode is widely distributed and has now been found in more than 16 provinces (autonomous regions or cities) in China, including Hubei, Shanxi, Henan, Hebei, Beijing, Inner Mongolia, Qinghai, Anhui, Shandong, Shaanxi, Gansu, Jiangsu, Tianjin, Tibet and Xinjiang. More than 4 million ha of the major wheat-producing regions considered be infected by the cereal cyst nematode with annual reduction of wheat yield valued at 1.9 billion Chinese Yuan (Peng et al., 2009; Cui et al., 2015). Molecular and morphological analysis of samples of the cereal cyst nematode revealed the presence of three species: H. avenae, H. filipjevi (Li et al., 2010; Peng et al., 2010; Fu et al., 2011) and H. australis (Fu et al., 2011) in China. However, Subbotin et al. (2003, 2010) noticed that that nematode samples identified as H. 'avenae' from China appeared to form a distinct group within the H. avenae complex. These populations are close morphologically and genetically to H. pratensis and are not similar to H. avenae and might represent a separate species. Sturhan & Rumpenhorst (1996) also showed that Chinese cereal cyst nematode populations had a
different IEF protein profile from those of H. avenae and other species.
In this paper, the analysis of coxI gene sequences provided evidence of the separate status of Chinese
H. 'avenae' from other species of the H. avenae complex. The samples from China previously identified as H. 'avenae' (type C) are formally described here as a new species under the common name the Sturhan cereal cyst nematode.
MATERIALS AND METHODS
DNA extraction, PCR, RFLP and sequencing.
DNA was extracted from single cysts. Protocols of DNA extraction with proteinase K, PCR and sequencing were described by Tanha Maafi et al. (2003). The primer set with the forward Het-coxiF (5' -TAGTTGATCGTAATTTTAATGG-3') and the reverse Het-coxiR (5' -CCTAAAACATAATGAAA ATGWGC-3') was used for amplification of the partial mitochondrial cytochrome oxidase I (coxI mtDNA) gene with the following thermal profile: 4 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 45°C and 1 min 30 s at 72°C, with a final extension at 72°C for 10 min. A few microliters of purified PCR product were digested by restriction enzyme BcuI (Thermo Fisher Scientific, USA) in the buffer stipulated by the manufacturer. The digested DNA was run on a 1.4% TAE buffered agarose gel, stained with ethidium bromide, visualised on UV transilluminator and photographed. Sequencing was made in Quintara Biosciences (San Francisco, USA). New sequences were deposited in the GenBank under accession numbers: KU147188-KU147203.
Phylogenetic analysis. The newly obtained coxI sequences were aligned with corresponding published gene sequences of other species from the Avenae group (Toumi et al., 2013) using ClustalX
I.83 with default parameters. Heterodera hordecalis and H. latipons were chosen as outgroups. The sequence dataset was analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) under the HKY+G model. BI analysis was initiated with a random starting tree and was run with four chains for 1.0 * 106 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities are given on appropriate clades. The coxI sequence alignment was also used to construct phylogenetic network estimation using statistical parsimony with the TCS software (Clement et al., 2000).
RESULTS AND DISCUSSION
Heterodera sturhani sp. n.
(Fig. 1, Table 1)
Descriptions and measurements for specimens designated as the paratypes (China, Beijing, Fangshan district, sample 1) are given according to Subbotin et al. (2003). Descriptions and measurements for other populations were published by Liu W.Z. et al. (2005), Liu J. et al. (2009), Liu K. et al. (2012) and Wang et al. (2013).
Holotype cyst. L (excluding neck) = 595 ^m; W (slightly deformed) = 330 ^m.
Paratype cysts (n = 9). L = 597 ± 25 (504-696) ^m; W = 437 ± 13 (384-504) ^m; L/W = 1.4 ± 0.05 (1.2-1.5); fenestral length = 46 ± 0.8 (43-50) ^m; mean semifenestral width = 23 ± 1.3 (19-31) ^m; vulval bridge width = 10 ± 0.9 (6.6-16) ^m; vulval slit length = 8.6 ± 0.4 (6.6-12) ^m; vulva-anus distance = 57 ± 1.5 (50-62) ^m.
Paratype second-stage juveniles (n = 20). L = 513 ± 3.5 (480-537) ^m; a = 25 ± 0.2 (23-27); b = 4.5 ± 0.1 (4.0-4.8); c = 8.1 ± 0.1 (7.5-9.1); stylet length = 24 ± 0.1 (24-25) ^m; lip region height = 4.1 ± 0.06 (3.4-4.4) ^m; lip region width = 9.3 ± 0.09 (8.8-9.9) ^m; DGO = 6.4 ± 0.1 (5.4-6.9) ^m; anterior end to valve of median bulb = 72 ± 1.0 (6787) ^m; anterior end to excretory pore = 103 ± 1.0 (98-112) ^m; pharynx length = 116 ± 1.2 (107-126) ^m; body diam. at mid-body = 21 ± 0.1 (20-22) ^m; body diam. at level of anus = 15 ± 0.1 (15-16) ^m; tail length = 63 ± 0.9 (55-72) ^m; hyaline part of tail length = 40 ± 0.6 (34-45) ^m.
Cyst. Lemon-shaped with low vulval cone. Subcrystalline layer distinct; egg sac not observed. Cuticle with irregular zig-zag pattern; colour varying from pale to medium brown. Vulval cone bifenestrate, vulval slit short. Bullae numerous, distinct.
Second-stage juvenile. Body of heat-killed specimens slightly curved ventrally. Labial region slightly offset, flatly rounded, with two indistinct annuli. Labial framework strongly sclerotised. Stylet strong, cone slightly shorter than shaft. Stylet knobs slightly concave, sloping slightly posteriorly. Lateral field with four lines, but outer lines mostly indistinct and outer bands completely areolated. Pharyngeal glands well developed. Tail gradually tapering to a narrow, rounded terminus.
Males. Not found.
Etymology. The species was named in honour of Dr Dieter Sturhan for his great contributions in systematics of cyst forming and other nematodes.
Fig. 1. Heterodera sturhani sp. n. A: white females (China, Shanxi province); B, C: vulval plate (Paratype; China, Beijing); D, E: anterior and posterior ends of the second-stage juveniles (Paratype; China, Beijing) (after Subbotin et al, 2003). Scale bars: A = 120 ^m, B, C = 10 ^m; D, E = 9 ^m.
Fig. 2. Phylogenetic relationships between cyst nematode species of the Avenae group as inferred from the analysis of the coxl mtDNA gene sequences. A. Statistical parsimony network showing the phylogenetic relationships between haplotypes of H. sturhani sp. n. and H. pratensis. Small black cycles represent missing haplotypes. Pie chart sizes are proportional to the number of samples with a particular haplotype. B: Bayesian majority rule consensus tree inferred from the analysis of the coxl gene sequence alignment under the HKY + G model. Newly obtained sequences are indicated by bold letters.
Table 1. Species and populations of the Heterodera avenae complex used in the present study
Species Location Host Sample code Accession number for coxI sequence Source or reference
H. australis Australia, Yorke Peninsula Cereals CD1485 KU147202 Subbotin et al. (2003)
H. avenae Syria, Deir Al-Zor Wheat - KC172908 Toumi et al. (2013)
H. avenae Syria, Al-Hasakah Wheat - KC172909 Toumi et al. (2013)
H. avenae France, St Georges du Bois Cereals CD1461 KU147188 R. Rivoal; Subbotin et al. (2003)
H. filipjevi Iran, Aligoudarz Wheat - KC172910 Toumi et al. (2013)
H. filipjevi Syria, Al-Hasakah Wheat - KC172911 Toumi et al. (2013)
H. hordecalis Tunisia Wheat - KC172912 Toumi et al. (2013)
H. sturhani sp. n. China, Beijing, Tongzhou district Cereals 514a KU147195 D. Peng; Subbotin et al. (2003)
H. sturhani sp. n. China, Beijing, Pinggu district Cereals 515a KU147199 D. Peng; Subbotin et al. (2003)
H. sturhani sp. n. China, Beijing, Fangshan district Cereals 513b KU147196 D. Peng; Subbotin et al. (2003)
H. sturhani sp. n. China, Shanxi province, Wenxi Cereals CD1555 KU147197, KU147198 J. Zheng
H. latipons Syria, Deir Al-Zor Wheat - KC172913 Toumi et al. (2013)
H. mani USA, California Grasses CD1252 KU147203 S.A. Subbotin
H. pratensis Russia, Leningrad region, Putilovo Grasses 565, 595 KU147200, KU147201 Subbotin et al. (2003)
H. pratensis The Netherlands, near Rotterdam Grasses 559 KU147189 Subbotin et al. (2003)
H. pratensis Germany, Missunde, near Schleswig Grasses 537a KU147194 D. Sturhan; Subbotin et al. (2003)
H. pratensis Germany Grasses 555 KU147192 D. Sturhan; Subbotin et al. (2003)
H. pratensis Belgium, Zwin Grasses Zwi78 KU147191 S.A. Subbotin
H. pratensis Germany Grasses 601 KU147193 D. Sturhan
H. pratensis Germany Grasses 507a KU147190 D. Sturhan
Fig. 3. PCR-coxI-RFLP profiles generated by BcuI for several Heterodera species. Lanes - M: 100 bp DNA marker (Promega, USA); 1: H. sturhani sp. n. (China, Shanxi province, CD1555a); 2: H. sturhani sp. n. (China, Beijing, CD515a); 3: H. pratensis (Germany, 537); 4: H. pratensis (Germany, 555); 5: H. mani (USA, California, CD1252); 6: H. australis (Australia, CD1485); 7: H. avenae (France, CD1461).
Type locality and host. Fangshan district, Beijing, China; cereals.
Type materials. Holotype and paratypes are deposited at the Nematode collection of Julius Kühn-Institut, Bundesforschungsinstitut für Kulturpflanzen Institut für Epidemiologie und Pathogendiagnostik, Münster, Germany.
Differential diagnosis. Heterodera sturhani sp. n. is morphologically similar with H. pratensis and H. riparia and overlaps with these species in many characteristics of cysts and second-stage juveniles. Heterodera sturhani sp. n. can be differentiated from H. pratensis by smaller average cyst size (L = 540-641 ^m; W = 324-480 ^m vs L = 650-760 ^m; W = 490-570 ^m) and from H. riparia by longer average body length (488-570 vs 446-486 ^m) and longer average tail length (62-67 vs 57-61 ^m) of the second-stage juveniles. The new species differs from H. avenae by smaller average cyst size (L = 540-641 ^m; W = 324-480 ^m vs L = 600-808 ^m; W = 465-507 ^m) and smaller average fenestral length (40-46 vs 43-55 ^m). Heterodera sturhani sp. n. can be distinguished from H. mani by the structure of stylet knobs, which are flat or slightly concave vs strongly developed and deeply concave in second stage juveniles.
Molecular characterisation of Heterodera sturhani sp. n. and its relationships with other species. The coxI mtDNA gene alignment included 22 sequences and was 425 bp in length. Intraspecific variation for H. sturhani sp. n. was 0-3 bp (0.7%) and for H. pratensis was 0-3 bp (0.7%). Two coxI haplotypes (Hstl and Hst2) were found for H. sturhani sp. n. and two haplotypes (Hprl and Hpr2) were revealed for H. pratensis. The coxI gene sequences of H. sturhani sp. n. differ from those of H. pratensis by up to 5 bp (1.2%). Differences between H. avenae type A (France) and type B (Syria) were 21-26 bp (6.4-7.0%) and between H. australis and H. avenae types were 36-46 bp (9.112.0%). Phylogenetic relationships of H. sturhani sp. n. with other species are given in Figure 2. Heterodera sturhani sp. n. showed close relationships with H. pratensis (Fig. 2A & B) and, perhaps, originated from the later species. Heterodera australis was presented as a separate lineage from H. avenae (types A and B) and other species in the phylogenetic tree. Thus, the coxI mtDNA sequence analysis confirms the validity of H. australis as was previously proposed based on other biochemical and molecular data (Subbotin et al., 2002).
The PCR-coxI-RFLP profiles generated by Bcul for H. sturhani sp. n. and several other species from the H. avenae complex are given in Figure 3. This
restriction enzyme cut only PCR products obtained from H. sturhani sp. n. samples.
The results of previous published analyses of the ITS rRNA gene sequences (Subbotin et al., 2003; Fu et al., 2011) and the present analysis of the coxI mtDNA gene sequences showed that the cereal cyst nematode from China is different from H. avenae and represents a separate evolutionary lineage. Moreover, several studies (Peng & Cook, 1996; Zheng et al., 1997; Yuan et al., 2010; Cui et al., 2015) demonstrated that these populations belong to new pathotypes, which are distinct from European ones according to the pathotype scheme proposed by Andersen & Andersen (1982). Thus, molecular, biological and morphological results justify the erection of a new species H. sturhani sp. n. and its separation from H. avenae. Although molecular results show close relationships of H. sturhani sp. n. and H. pratensis, they are different in plant-host range. Heterodera sturhani sp. n. parasitises cereals, whereas H. pratensis is presently known only as a parasite of grasses (Subbotin et al., 2010).
The ability of nematodes to parasitise cereals might have appeared independently in several evolutionary lineages of the H. avenae species complex and different geographical regions. Nematodes belonging to these lineages also showed differences in their virulence and pathogenicity for cereal crops. Thus, under the common name of "the cereal cyst nematode" there presently exists several species, which could be named as the European cereal cyst nematode, H. avenae, the Filipjev cereal cyst nematode, H. filipjevi, the Australian cereal cyst nematode, H. australis and now the Sturhan cereal cyst nematode, H. sturhani sp. n. Although, morphological characters can fail to differentiate species of the cereal cyst nematodes from each other or from sibling species parasitising grasses, the molecular markers (ITS rRNA and coxl) presently provide reliable differentiation of this complex at a species level. Acceptance of the multiple species concept of the cereal cyst nematodes raises an important question about reconsidering the present quarantine regulations and other measures, which prevent dispersal of some nematode species that are present only in geographically isolated regions. Special concern should be directed to H. sturhani sp. n., which could be considered as a potential invasive species for Europe, America and Australia.
ACKNOWLEDGEMENTS
The author thanks Drs D. Sturhan (Germany), R. Rivoal (France), D. Peng (China) and J. Zheng (China) for providing nematode materials. The
author acknowledges support from the Russian
Foundation of Basic Research (project number 14-
04-00953-a).
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S.A. Subbotin. Heterodera sturhani sp. n. из Китая, новый вид из группы видов 'Heterodera avenae' (Tylenchida: Heteroderidae).
Резюме. Heterodera sturhani sp. n. описан как новый вид из группы видов 'Heterodera avenae'. Новый вид по многим морфологическим признакам цисты и личинки второго возраста схож с H. pratensis и H. riparia. Он может быть дифференцирован от H. pratensis меньшими размерами цист и от H. riparia большей длиной тела и длиной хвоста личинки. Филогенетический анализ нуклеотидных последовательностей гена цитохром с-оксидазы I и рестрикционный анализ ПЦР продуктов этого гена позволяют четко отличать H. sturhani sp. n. от всех близких видов. Филогенетический анализ нуклеотидных последовательностей гена цитохром с-оксидазы I также позволил разделить два типа (А и В) Heterodera avenae друг от друга и подтвердить видовой статус H. australis. Таким образом, под названием "овсяная цистообразующая нематода" следует рассматривать не один, а несколько видов.
