Bulletin OEPP/EPPO Bulletin (2018) 48 (1), 64–77 ISSN 0250-8052. DOI: 10.1111/epp.12453 European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/89 (2) Diagnostics Diagnostic PM 7/89 (2) Heterodera glycines Specific scope Specific approval and amendment This Standard describes a diagnostic protocol for Approved in 2008–09. Heterodera glycines.1 Revision approved in 2017–11. This Standard should be used in conjunction with PM 7/ 76 Use of EPPO diagnostic protocols. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology.2 (Niblack et al., 2002). Further information can be found in 1. Introduction the EPPO data sheet on H. glycines (EPPO/CABI, 1997). Heterodera glycines or ‘soybean cyst nematode’ is of major A flow diagram describing the diagnostic procedure for economic importance on Glycine max L. ‘soybean’. H. glycines is presented in Fig. 1. Heterodera glycines occurs in most countries of the world where soybean is produced. It is widely distributed in coun- 2. Identity tries with large areas cropped with soybean: the USA, Bra- zil, Argentina, the Republic of Korea, Iran, Canada and Name: Heterodera glycines Ichinohe, 1952 Russia. It has been also reported from Colombia, Indonesia, Synonyms: none North Korea, Bolivia, India, Italy, Iran, Paraguay and Thai- Taxonomic position: Nematoda: Tylenchina3 Heteroderidae land (Baldwin & Mundo-Ocampo, 1991; Manachini, 2000; EPPO Code: HETDGL Riggs, 2004). Heterodera glycines occurs in 93.5% of the Phytosanitary categorization: EPPO A2 List no. 167 area where G. max L. is grown. Heterodera glycines has a broad host range, particularly within the Leguminosae, and 3. Detection has been detected on crops such as Glycine, Vicia, Trifolium, Phaseolus, Lespedeza and Pisum. However, dam- 3.1 Symptoms age of economic importance is mainly observed on soy- bean. It is also able to attack a number of non-legume The symptoms of H. glycines are not specific. General crops including some ornamentals such as Geranium and symptoms are, for example, patches of poor growth in a soy- Papaver, but also many weeds of at least 23 families bean crop. Sometimes the plants in these patches show yel- (Moore, 1984). Field populations of H. glycines exhibit lowing, wilting or loss of leaves with reduced seed diversity in their ability to develop on resistant G. max L. production. There is usually a sharp dividing line between cultivars. Therefore, several race tests for H. glycines popu- affected and non-affected areas of the field. In the affected lations have been proposed (Golden et al., 1970 & Riggs areas, rows are slow to close and may remain so throughout et al., 1981). As these tests proved not always to be reliable the season. The most severe damage is often in the centre of for race characterization, a revised classification scheme for the affected area. Damaged areas are frequently located near genetically diverse H. glycines populations was proposed the field entrance where machinery moves into the field, or in areas where soil from another field is deposited by wind or water. Reduction in seed yield is usually the first sign that 1The use of names of chemicals, equipment or commercial kits in these EPPO Standards implies no approval of them to the exclusion of others that may also be suitable. 3Recent development combining a classification based on morphologi- 2http://www.eppo.int/QUARANTINE/diag_activities/EPPO_TD_1056_ cal data and molecular analysis refer to ‘Tylenchomorpha’ (De Ley & Glossary.pdf Blaxter, 2004) 64 ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 64–77 PM 7/89 (2) Heterodera glycines 65 Soil Roots No cysts, second-stage juveniles and males Yes Yes Morphology Molecular tests Stages with typical PCR tests can be performed on: Uncertain No the Heterodera the Heterodera et al., i group 2000 PCR-ITS-RFLP, Appendix 1) b) Cysts, second-stage juveniles or eggs Yes Appendix 2). c) Cysts or cyst mixtures (according to Uncertain Morphological 3). No H. glycines Yes H. glycines H. glycines not Fig. 1 Diagnostic procedure for H. glycines. [Colour figure can be viewed at wileyonlinelibrary.com] an infestation is present. Usually the combination of reduced 4. Identification growth and yellowing is named ‘yellow dwarf disease’ for soybean infested with H. glycines. Root infestation increases Identification is based on cysts and second-stage juveniles. the number of lateral roots and reduces the number of The presence of males allows the distinction between Rhizobium nodules and nitrogen fixation. Young females and H. glycines and Heterodera lespedezae. Heterodera cysts appear white, yellow or brown, about the size of a pin- glycines can be difficult to distinguish from other species head just visible with the naked eye on the root-surface. in the schachtii group using morphological and morpho- They may be confused with soybean Rhizobium nodules. metrical features. In case of doubts with morphological identification, molecular tests can be used. Molecular tests can be used directly on cysts or juveniles (morphological 3.2 Sampling identification to genus level may still be required; see sec- Guidance on sampling is provided in PM 9/5(1) Heterodera tion 4.2). glycines: procedures for official control. 4.1 Morphology 3.3 Extraction For morphological identification, it is recommended to In order to identify H. glycines all stages should be examine specimens mounted in fixative on microscope obtained. Extraction methods from soil are presented in PM slides under a light microscope. Interference phase micro- 7/119 Nematode extraction. scopy is recommended for observing juveniles. ª 2018 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 48, 64–77 66 Diagnostics (32 the fenestral width,bridge bears respectively the 50semifenestrae vulval slit. (45 (ambifenestrate Thethe type) vulval slit (Fig vulval is 3).of wider bridge The the than cone vulval young and is cysts; lost dividing inzag leaving older pattern the an specimens, of openwidth the fenestra ridges. fenestra thin from The crossed in walled vulval 320cone. by cuticle region Cysts two to usually range 610 Cysts intact in in are length from lemon-shaped 340 with to 920 awalled protruding cyst. neckfemale and body wallareas, changes the upon so-called semifenestrae. deathtransverse slit, The to is mature, a surrounded palesent by brown yellow thin on tough- walled crescent aopen shaped posteriorly terminal through cone-shaped theies vulva. projection. are Vulva and paired The anus andorly vulva, are coiled, projecting pre- nearly a (sloping) filling knobs.crystalline the In layer entire young body prominent. femalesegg and the Stylet sac ovar- is are slenderally present with and covered posteri- contain withbody, up lemon reticulate to shaped 200 ridges. withSedentary eggs. projecting Gelatinous females neck. The The matrix are sub- body white or is to usu- pale yellow, withand swollen body length shouldlet be length, determined. stylet knoband shape bullae and should tail be andtra noted. hyaline shape, For tail vulva second-stage length width juveniles and sty- thejuveniles presence can of be an obtainedmally underbridge directly from viable present cysts. mined for in aand infested reliable the identification. presence soilThe of These characteristics males stages samples; of in both areMorphological the cysts characteristics second-stage nor- and sample of second-stage H. should juveniles glycines be deter- are presented in Fig. 2. teristics of their second-stagewhich juvenile characters. also providesSome the important most members important ofschachtii this diagnostic group charac- are listedwhich in Table includes 1 more than 10Appendix 1). species. Characterstowards of species the levelinclude both within cyst thetified and genus with juveniles.forming keys This genera from have key beenera Wouts is described within and & also these thekey can useful Baldwin for be subfamily the (1998) iden- identification Heteroderinae. of which So cyst- and far non-cyst-forming gen- six cyst- Cysts Sedentary females For cysts, the vulval cone should be examined and fenes- Drawings of male, female, cyst and second-stage juvenile Heterodera glycines Baldwin & Mundo-Ocampo (1991) produced a useful – 50) l -group are given below in the section ‘Cysts’. m. Bullae are prominent and elongated, is a member of the l m. Cuticle surface has a zig- – Heterodera 60) schachtii l l mand40 mandin group, (see Table 1. Several diagnostic characters for differentiation of the second-stage juvenile of Heterodera glycines from some closely related species of the schachtii-group ª 2018 OEPP/EPPO, Species Body length (lm) Stylet length (lm) Stylet knobs Tail length (lm) Hyaline part of tail length (lm) References H. glycines 375–540 (470) 22.0–26.0 (24.0) Concave anteriorly 40.0–61.0 (47.0) 21.0–33.0 (27.0) Graney & Miller (1982); Burrows & Stone (1985) H. daverti 400–480 (457) 24.0–26.0 (25.0) Slightly concave 49.0–60.0 (55.0) 28.0–39.0 (33.3) Wouts & Sturhan (1977) H. betae 550–660 (595) 29.5–33 (31.0) Deeply concave 65.0–84.0 (71.0) 32.0–50.0 (39.0) Wouts et al. (2001) Bulletin OEPP/EPPO Bulletin H. ciceri 440–585 (525) 27.0–30.0 (28.6) Concave anteriorly 53.0–72.0 (60.0) 31.0–42.0 (36.0) Vovlas et al. (1985) H. galeopsidis 495–620 (553) 25.9–28.2 (27.1) Concave anteriorly 60.5–75.1 (68.1) 33.2–44.7 (40.3) Hirschmann & Triantaphyllou (1979) H. lespedezae 401–531 (481) 23.4–25.8 (24.3) Concave anteriorly 45.7–60.7 (53.5) 20.4–37.8 (26.3) Hirschmann & Triantaphyllou (1979) H. medicaginis 420–510 (460) 24.0–26.0 (25,0) Concave anteriorly 41–60 (52.0) 22.0–33.0 (28.5) Gerber & Maas (1982) H. rosii 430–661 (549) 26.6–33.8 (31.3) Concave anteriorly 58.4–76.9 (65.9) 36.9–44.6 (40.4) Duggan & Brennan (1966) H.