Systematics of Cyst Nematodes (Nematoda: Heteroderinae)

<i>Heterodera glycines</i>

Cyst nematodes are one of the most important pathogens worldwide. Most cyst nematode species have been reported recently in China. From 2016 to 2020, an extensive survey of cyst nematodes was conducted in the Bashang region of Hebei Province. A total of 158 soil samples were collected, and cyst-forming nematodes were isolated from five soil samples. Morphological and molecular characterization showed that four of the cyst-forming nematode populations were Heterodera glycines (SCN), named populations of ZM, KM, CB and FN, respectively. These SCN populations were collected from Zhangbei County, Kangbao County, Chabei Management Area of Zhangjiakou and Fengning Manchu Autonomous County of Chengde, respectively, where the corresponding cyst densities were 57, 41, 103 and 31 cysts/200 cc soil. Furthermore, the populations of ZM, KM and CB were identified as race 4, whereas the FN population was identified as race 3. The cyst-forming nematode population was collected from Zhangbei County of Zhangjiakou, which was confirmed to be Heterodera schachtii (SBCN), named population ZZ, and the cyst density was 94 cysts/200 cc soil. It is a new disease of Chinese cabbage caused by SBCN based on Koch’s postulates. Fourteen cultivars from five plant families were evaluated as hosts for SBCN. Chinese cabbage (cv. Linglonghuang012) and cabbage (cv. Chunwang) were suitable hosts, while celery (cv. Yuhuang), potato (cv. Helan 15) and eggplant (cv. Junlang) were nonhosts. The obtained results regarding the occurrence, distribution, races of SCN and hosts of SBCN in the Bashang region in this study provide a reference for SCN and SBCN management.

A unique cyst nematode population (Heterodera spp.) was collected from rice roots in Luoding County, Guangdong Province, China. Morphological and molecular analyses revealed that it is significantly different from all previously described cyst nematode species. It is described as Heterodera luodingensis n. sp. and classified in the Cyperi group. H. luodingensis n. sp. is characterized by its lemon-shaped cyst with a prominent terminal vulval cone that is ambifenestrate with abundant bullae and a relatively short vulval slit, measuring 31.3 (24.4 to 38.7) μm in length. The second-stage juveniles (J2s) are characterized by dumbbell-shaped labials, three lip annules, and a lateral field with three incisures. The J2 stylet is 18.7 (16.9 to 19.8) μm long with anterior concave or spherical knobs. The tail is elongate conoid, tapering to a rounded terminus or zig-tapering to a rounded terminus that is 54.9 (43.9 to 64.3) μm long with a hyaline region comprising 40.3 to 52.5% of the tail. Phylogenetic tree analysis based on rDNA 28S D2D3 and ITS fragments showed that H. luodingensis n. sp. is unique and clearly separated it from other cyst nematodes. It is most closely related to H. oryzicola, H. fengi, H. elachista, H. oryzae, and H. guangdongensis. H. luodingensis n. sp. can be distinguished from H. oryzicola by its shorter vulval slit and underbridge, from H. elachista by abundant bullae, shorter vulval slit, and fenestrate width, from H. oryzae by a shorter vulval slit and underbridge, from H. fengi by a shorter vulval slit, and from H. guangdongensis by a longer cyst length and abundant bullae. Based on PCR-RFLP of rDNA-ITS, H. luodingensis n. sp. can be clearly distinguished from H. oryzicola, H. mothi, H. elachista, H. guangdongensis, and H. cyperi. A parasitism test from a pure culture derived from a single cyst in a greenhouse showed that H. luodingensis n. sp. can successfully complete its life cycle on rice, and rice is its type host.

Climate change is expected to cause a mean annual temperature increase in Germany of 2 °C by 2050 and up to 4 °C by 2100. This is likely to have effects on crop development and pathogen development as well. For this Ph.D. thesis, experiments were conducted to investigate the effect of increasing temperatures on the beet cyst nematodes Heterodera schachtii and Heterodera betae and thus study the changes of their interaction with their host plant the sugar beet (Beta vulgaris subsp.). Differences in hatch between the beet cyst nematode species were assessed at constant temperatures in incubators as well as simulated temperature conditions set to be 4 °C higher than the standard temperature regime. The optimal temperatures for hatch were found to be different for both cyst nematode species. The optimal temperature range for hatching of H. schachtii was found to be between 15 and 30 °C and for H. betae between 20 and 30 °C. Emergence of juveniles of both beet cyst nematode species began at 5 °C, however in very low percentages. For both beet cyst nematode species no significant differences were found in the final cumulative hatch percentages when comparing the standard temperature regime with the by 4°C increased temperature regime. In the climate chambers, the influence of temperature on the interspecific competition between both beet cyst nematode species was studied. Both beet cyst nematode species performed better at higher temperatures. But there were no clear indications that under competition one of the two species will profit more from higher temperatures under the predicted climate change. In conclusion, the damage done by cyst nematodes in sugar beet is expected to increase with global warming. H. schachtii and H. betae are likely to continue to cause damages. But in competition, neither of the two species will profit more from rising temperatures compared to the other. The effect of experimental soil warming on H. schachtii population development and sugar beet performance was assessed for sugar beet cultivars that were susceptible, tolerant or resistant to H. schachtii. In this study, soil heating lead to a significant increase in the final number of recovered cysts on the tolerant cultivar and susceptible cultivar. The resistant cultivar did not allow nematode reproduction at all. Therefore no effect of soil heating could be detected. Plant-nematode interaction varies greatly depending on the cultivar. Thus cultivar choice is an important element when trying to prevent nematode infestation or controlling nematode populations in the field. In case of high nematode population densities and with the expected increasing soil temperatures, planting nematode-resistant cultivars will become even more important in the future, as an effective tool to reduce nematode populations and prevent damages.

Cyst-forming nematodes have been known since the second half of the 19th century as one of the causes of "soil fatigue" and highly harmful agricultural pests. In Ukraine, the sugarbeet cyst nematode Heterodera schachtii Schmidt, 1871; cereal cyst nematode H. avenae Wollenweber, 1924; golden potato cyst nematode Globodera rostochiensis Wollenweber, 1923; clover cyst nematode H. trifolii Goffart, 1932; alfalfa cyst nematode H. medicaginis Kirjanova &amp; Krall, 1971; hop cyst nematode H. humuli Filipev, 1934 are currently economically significant species. Their distribution mainly coincides with the traditional cultivation of sugar beets, grain cereals, potatoes, clover, alfalfa and hops. Long-term studies of the impact of abiotic, biotic, and anthropogenic factors on their populations made it possible to identify the dominant species and improve the set of protective measures. Anti-nematode crop rotations are the main limiting factor in preventing mass reproduction of cyst-forming nematodes, provided there is scientifically justified crop rotation. To prevent the mass accumulation of alfalfa and clover cyst-forming nematodes, the maximum share of perennial grasses in crop rotations should not exceed 30% (crops under cover of grain cereals + perennial legumes – one-two-year use); oat nematode – 40% of grain cereals; beet nematode – 20% of Chenopodioideae and cruciferous crops; golden potato nematode – 20% of potatoes (10% susceptible + 10% resistant varieties). The rational saturation of modern crop rotations with intermediate crops makes it possible to reduce the level of crop weediness caused by potential host plants of cyst-forming nematodes and to increase their anti-nematode effectiveness in general. The developed ecologically oriented system of phytosanitary control provides mandatory measures, which must be applied regardless of the level of initial population density, and additional measures specific to each culture and species of cyst-forming nematodes. Considering the current high specialization of crop production, the use of biological preparations in modern systems of integrated plant protection will contribute to the greening of agricultural output. Pre-planting treatment of tubers with metabolic biological preparations (Streptomyces avermitilis) of multifunctional action ensured the achievement of a higher (79.2–91.7%) efficiency of biological cleaning of the soil from the golden potato nematode, compared to the use of only potato-resistant varieties. A logical combination of various anti-nematode measures makes it possible to effectively keep the density of populations at an economically insignificant level and prevent crop yield losses.

Plant-parasitic nematodes are important pathogens of agricultural crops. Of particular importance are cyst nematode species of Globodera and Heterodera, which remain a major constraint to global production of crops such as potato, soybean, and wheat. Recent restrictions on the use of some synthetic nematicides has created an urgent need for alternative methods of controlling cyst nematodes. This project aimed to (i) document fungi found associated with cyst nematodes, (ii) determine if the fungi associated with cyst nematodes are dependent on growth conditions or nematode species, (iii) identify core mycobiomes for each of the eight cyst nematode species, (iv) identify the core mycobiome across all cyst nematode species studied, and (v) determine if the fungi associated with Globodera species are structured geographically. Sixty cyst nematode populations from eight species— Globodera sp., G. ellingtonae, G. pallida, G. rostochiensis, H. avenae, H. carotae, H. glycines, and H. schachtii—were collected from several geographic locations globally and screened for fungi using both culture-dependent and -independent methods. The core mycobiome was determined for each species. In total, 744 species of fungi were found to be associated with cyst nematodes. Fungal species shared across all cyst nematode species included Pochonia sp., Sporothrix sp., Neocosmospora solani, Exophiala equina, Mortierella sp., and Sarocladium kiliense. The core mycobiomes were conserved between cyst nematode species irrespective of their geographic origin. Cyst nematodes collected from the field or from greenhouse cultures also recruited similar core mycobiomes. These findings could be relevant when choosing fungi to evaluate as biocontrol agents against cyst nematodes.

Identification of Heterodera schachtii on sugar beet in Xinjiang Uygur Autonomous Region of China

| DET EC T ION | SymptomsAbove ground symptoms due to PCNs are not specific and often go undetected.General symptoms include patches of poor growth in the crop, with plants some times showing yellowing, wilting or death of foliage; tuber size is reduced and roots are extensively branched with soil stuck to them.However, there are many other causes of these symptoms.Plants should therefore be lifted for a visual check for the presence of cysts and young females on the roots, or a soil sample should be taken for testing.Young females and cysts are just visible 1 Use of brand names of chemicals or equipment in these EPPO Standards implies no approval of them to the exclusion of others that may also be suitable. 2http://www.eppo.int/QUARANTINE/ diag_activ ities/ EPPO_TD_1056_Gloss ary.pdf.

Maize is the largest crop planted in China. Nine species of cyst nematodes have been reported to affect maize production. Heterodera zeae, H. avenae and Punctodera chalcoensis can cause significant maize yield losses annually (Luc et al. 2005). In 1971, the maize cyst nematode H. zeae was first detected in Rajasthan, India (Koshy et al. 1971). Subsequently, it has been reported in many other countries such as the United States, Greece, Pakistan, and Egypt. In China, H. zeae was first identified in the maize fields of Laibin City, Guangxi Zhuang Autonomous Region (Wu et al., 2017). Cui et al. (2020) identified H. zeae in a maize field of Yuzhou City, Henan Province of Central China in 2018. From 2018 to 2022, a survey of cyst-forming nematodes was conducted in Southwest China. Fifteen soil samples of about 500 g each were collected from Luding County, Ganzi Prefecture of Sichuan Province. No major aboveground symptoms were shown on maize, but a few females were observed on the roots of maize in one field. The cysts and second-stage juveniles (J2s) were collected from each soil sample using Cobb's screening gravity method. A total of 8.50±2.0 cysts per 100 ml of soil on the average were observed in the field. A thin subcrystalline layer was discernible only in young cysts. Morphological and molecular studies of cysts and J2s indicated that the nematodes were identified to be H. zeae in a maize-field. Morphologically, the cysts were in a lemon shape, light brown or pearly white in color. The vulval cone was prominent. Fenestra ambifenestrate, and semifenestra were separated by a fairly wide vulval bridge, fenestral length and width were variable, and the cyst wall was shown in a zigzag pattern. The J2s' body was in a vermiform, tapering at both ends, with a hyaline tail. Stylet was strongly developed with round or slightly anteriorly directed knobs. Morphological measurements of the cysts (n = 9) determined that the mean body length was 417.2 μm (403.6 to 439.4 μm), body width was 429.7 μm (397.6 to 456.9μm); length-width ratio was 1.4 (0.75 to 3); fenestra length was 525.3 μm (498.5 to 570.7 μm); and the mean semifenestra width was 458.6 μm (403.6 to 546.3 μm). Morphometric measurements of second-stage juveniles (n = 20) showed a body length of 419.7μm (355.8 to 492.5 μm); a stylet length of 20.8 μm (19.51 to 23.3μm); a tail length of 41.5 μm (20 to 49.4 μm); and a hyaline tail length of 20.7 μm (16.6 to 24 μm). The main morphological characteristics and measured values were basically consistent with those described by Cui et al. (2022), and all of which were similar to those of H. zeae. Amplification of DNA from random single cysts (n = 5) was conducted using the protocol described by Cui et al. (2022). The rDNA-internal transcribed spacer (ITS) was amplified and sequenced using a pair of universal primers TW81 (5'-GTTTCCGTAGGTGAA CCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3'). The ITS sequences were deposited at GenBank with the accession number OR811029.1. Alignments of sequences showed an identity of 98% with H. zeae sequences from China (OP692769.2, MW785772.1) and the USA (GU145616.1), which were confirmed using a pair of species-specific primers HzF1 (5'-GGGGAGGTGAATGTGGG-3') and HzR1 (5'-CCTTTGGCAATCGGTGA-3') of H. zeae with a targeted PCR fragment of 393 bp (Cui et al. 2022). Pathogenicity was conducted and confirmed by infection and reproduction on maize. Seeds (cv. Zhengda 619) were sown in three pots that contained 150 ml of a sterile soil mixture (loamy soil: sand=1:1), and 5 cysts (103 eggs/cyst on the average) were inoculated in each pot at 25/30°C, under a 12-h dark/12-h light condition (Cui et al. 2023). Fifteen days after sowing, third- and fourth-stage juveniles were observed in the rootstained with acid fuchsin, and a total of 32 cysts per maize plant on the average were collected at 40 days after sowing. The new cysts' morphological and molecular characteristics were identical to the cysts from the original soil samples. To the best of our knowledge, this is the first report of H. zeae as a pathogen on maize in Sichuan Province, Southwest China. Our findings will be useful for management and further research of maize cyst nematodes.

<i>Meloidogyne chitwoodi</i> and <i>Meloidogyne fallax</i>

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype‐specific host plant resistances are essential for PCN control. However, the poor delineation of G. pallida pathotypes has hampered the efficient use of available host plant resistances. Long‐read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs (venom allergen‐like proteins) diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCNs after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (“DOG boxes”) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland‐produced effectors. Notably, DOG box dosage was only loosely correlated with the expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Resequencing of PCN populations with different virulence characteristics will allow for the linking of these characteristics to the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

In a survey of plant-parasitic nematodes in agricultural fields, cyst-forming nematodes were found in soil planted bamboo in Korea. The aim of this study was to identify the cyst nematodes based on morphological and molecular characteristics. As the results, the morphology and morphometrics of cysts and second-stage juveniles (J2s) were consistent with those of previous descriptions of Heterodera koreana. In phylogenetic analyses based on DNA sequences, these cyst nematodes were clustered together with clade of H. koreana in internal transcribed spacer (ITS) region, and large subunit D2-D3 segments (LSU D2-D3). These nematodes were clustered together with clade of H. koreana in cytochrome c oxidase subunit I (COI) gene, but a haplotype was different when compared with previous reported haplotypes (haplotype A-C) in Japan. This study showed these cyst nematodes were identified as H. koreana, and a new haplotype of H. koreana is distributed in Korea. We suggest that the new haplotype of H. koreana name as haplotype D.

In a survey of plant-parasitic nematodes in agricultural fields, cyst-forming nematodes were found in soil planted bamboo in Korea. The aim of this study was to identify the cyst nematodes based on morphological and molecular characteristics. As the results, the morphology and morphometrics of cysts and second-stage juveniles (J2s) were consistent with those of previous descriptions of Heterodera koreana. In phylogenetic analyses based on DNA sequences, these cyst nematodes were clustered together with clade of H. koreana in internal transcribed spacer (ITS) region, and large subunit D2-D3 segments (LSU D2-D3). These nematodes were clustered together with clade of H. koreana in cytochrome c oxidase subunit I (COI) gene, but a haplotype was different when compared with previous reported haplotypes (haplotype A-C) in Japan. This study showed these cyst nematodes were identified as H. koreana, and a new haplotype of H. koreana is distributed in Korea. We suggest that the new haplotype of H. koreana name as haplotype D.

The potato cyst nematode species G.rostochiensis and G.pallida are a threat to the cultivation of potatoes. Their presence in the soil embodies a potential financial loss to the farmer either because of harvest reduction, or because of rejection of seed potatoes, and other crops with adhering soil, for certification. Together with crop rotation and the use of nematicides, resistant potato varieties are essential for the control of these parasites. A reliable, simple and quick screening test to characterize and monitor field infestations of potato cyst nematodes according to species should improve possibilities for control, by means of cultivating resistant potato varieties. Both parasites are hard to distinguish, and considerable attention has been paid to the identification of these nematode species by methods other than those based on morphological characteristics. Such methods have mostly been based on biochemical entities, but have not proven suitable for routine applications. In this thesis, the outlines are presented of an immunoassay, which is based on species-differentiating monoclonal antibodies, and which can be used for routine purposes.To raise species-differentiating (monoclonal) antibodies, species-specific antigens must be available. In chapter II the purification of two major groups of heat-stable proteins from homogenates of eggs of G.rostochiensis and G.pallida is described. SDS-polyacrylamide gel electrophoresis revealed two bands specific for G.rostochiensis with apparent molecular weights of 20.7 kD and 18.0 kD, and three bands specific for G.pallida with apparent molecular weights of 21.0 kD, 20.5 kD and 17.0 kD. Two-dimensional gel electrophoresis revealed that actually four thermostable proteins are present in either nematode species, of which two (present in the 17.0 kD and 18.0 kD band respectively) have the same apparent molecular weight but differ in iso-electric point. Conventional antisera made against proteins of either one of the Globodera species were shown to exhibit a strong cross-reactivity with these species-specific proteins.Monoclonal antibodies (McAbs) hold the promise of better differentiating reagents since they recognize a single epitope with a possible reduction in the extent of cross-reactivity. The development of such antibodies using thermostable proteins of either nematode species as an antigen source is described in chapter IV. The antibodies produced by three of the hybridomas (WGP 1, WGP 2 and WGP 3) bind preferentially to thermostable protein antigens of G.pallida while two other hybridomas (WGR 11 and WGR 12) produce antibodies which prefer binding to proteins of G.rostochiensis . Most of the hybridomas which were isolated, however, produce antibodies which bind to thermostable proteins from both potato cyst nematode species. To quantitate the differences in affinity, binding constants were determined (chapter IV) according to the method as described in chapter III. In immunoblotting experiments, it was demonstrated that all G.pallida specific, and some aspecific, McAbs bind to the same two proteins in a mixture of four thermostable proteins from either G.pallida alone, or G.rostochiensis and G.pallida . Besides their very similar physico-chemical characteristics this can be interpreted as another indication that these proteins are homologous. In addition, the reactivity with thermostable proteins from other commonly occurring cyst nematodes was also investigated. Three categories of McAbs could be distinguished: i) WGP 2 and WGP 3, which only bind to thermostable proteins from G.pallida , ii) WGP 1, which binds to proteins of both G.pallida and G.rostochiensis , and iii) all other McAbs, which bind to thermostable proteins of potato cyst, and other cyst nematodes. In chapter V, the topological relationship between the antigenic sites on the G.rostochiensis and G.pallida antigens as defined by six of these McAbs was determined with a competition ELISA.From the McAbs so far characterized, a deliberate choice was made with respect to the development of a routine test for the identification of the potato cyst nematodes G.rostochiensis and G.pallida . In chapter VI, the reactivity of these McAbs in a direct ELISA was predicted and verified with the use of the formerly established binding constants.Thus, the McAbs obtained and characterized in this study establish a basis for the serological identification of the potato cyst nematode species G.rostochiensis and G.pallida by an immunoassay. In chapter VII, the implications of such an assay for the reduction of nematicide applications, and the utilization of resistant potato varieties is discussed.

Records of crop damage by some species of cyst nematodes in eastern England, 1967–77