The Globodera rostochiensis Gr29D09 Effector with a Role in Defense Suppression Targets the Potato Hexokinase 1 Protein.

The potato cyst nematode (Globodera rostochiensis) is an obligate root pathogen of potatoes. G. rostochiensis encodes several highly expanded effector gene families, including the Gr4D06 family; however, little is known about the function of this effector family. We cloned four 29D09 genes from G. rostochiensis (named Gr29D09v1/v2/v3/v4) that share high sequence similarity and are homologous to the Hg29D09 and Hg4D06 effector genes from the soybean cyst nematode (Heterodera glycines). Phylogenetic analysis revealed that Gr29D09 genes belong to a subgroup of the Gr4D06 family. We showed that Gr29D09 genes are expressed exclusively within the nematode's dorsal gland cell and are dramatically upregulated in parasitic stages, indicating involvement of Gr29D09 effectors in nematode parasitism. Transgenic potato lines overexpressing Gr29D09 variants showed increased susceptibility to G. rostochiensis. Transient expression assays in Nicotiana benthamiana demonstrated that Gr29D09v3 could suppress reactive oxygen species (ROS) production and defense gene expression induced by flg22 and cell death mediated by immune receptors. These results suggest a critical role of Gr29D09 effectors in defense suppression. The use of affinity purification coupled with nanoliquid chromatography-tandem mass spectrometry identified potato hexokinase 1 (StHXK1) as a candidate target of Gr29D09. The Gr29D09-StHXK1 interaction was further confirmed using in planta protein-protein interaction assays. Plant HXKs have been implicated in defense regulation against pathogen infection. Interestingly, we found that StHXK1 could enhance flg22-induced ROS production, consistent with a positive role of plant HXKs in defense. Altogether, our results suggest that targeting StHXK1 by Gr29D09 effectors may impair the positive function of StHXK1 in plant immunity, thereby aiding nematode parasitism. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Identification of genes governing resistance to PCN (Globodera rostochiensis) through transcriptome analysis in Solanum tuberosum.

Potato cyst nematodes (PCNs) are major pests worldwide that affect potato production. The molecular changes happening in the roots upon PCN infection are still unknown. Identification of transcripts and genes governing PCN resistance will help in the development of resistant varieties. Hence, differential gene expression of compatible (Kufri Jyoti) and incompatible (JEX/A-267) potato genotypes was studied before (0 DAI) and after (10 DAI) inoculation of Globodera rostochiensis J2s through RNA sequencing (RNA-Seq). Total sequencing reads generated ranged between 33 and 37 million per sample, with a read mapping of 48-84% to the potato reference genome. In the infected roots of the resistant genotype JEX/A-267, 516 genes were downregulated, and 566 were upregulated. In comparison, in the susceptible genotype Kufri Jyoti, 316 and 554 genes were downregulated and upregulated, respectively. Genes encoding cell wall proteins, zinc finger protein, WRKY transcription factors, MYB transcription factors, disease resistance proteins, and pathogenesis-related proteins were found to be majorly involved in the incompatible reaction after PCN infection in the resistant genotype, JEX/A-267. Furthermore, RNA-Seq results were validated through quantitative real-time PCR (qRT-PCR), and it was observed that ATP, FLAVO, CYTO, and GP genes were upregulated at 5 DAI, which was subsequently downregulated at 10 DAI. The genes encoding ATP, FLAVO, LBR, and GP were present in > 1.5 fold before infection in JEX-A/267 and upregulated 7.9- to 27.6-fold after 5 DAI; subsequently, most of these genes were downregulated to 0.9- to 2.8-fold, except LBR, which was again upregulated to 44.4-fold at 10 DAI.

Studies of potato resistance to Globodera rostochiensis revealed novel alleles for 57R marker.

Globodera rostochiensis resistance has been an important trait in potato (Solanum tuberosum) breeding for decades. Our aim was to complement phenotypic testing with genetic marker analysis. We analysed the results of G. rostochiensis resistance greenhouse testing in 4601 tubers of 2918 breeding clones from 11 years. Applicability of H1 gene markers TG689 and 57R was compared. We implemented the latter with the positive predictive value of 99.1% and negative predictive value of 60.0% into the breeding scheme. The 57R marker alleles of 22 Estonian cultivars and 470 breeding clones were determined. Two unique 57R alleles, 57R-887 and 57R-1155, were found in Estonian cultivar 'Anti'. The 887 bp allele has two deletions (14 bp and 490 bp) accompanied by several other indels and SNPs within the 57R marker region. The 1155 bp allele has three deletions (7 bp, 20 bp and 210 bp) accompanied by several other indels and SNPs within the same region. Partial resistance to G. rostochiensis in 'Anti' suggests that the newly described alleles could affect the H1-mediated resistance directly or indirectly.

First detection of the potato cyst nematode (Globodera rostochiensis) in a major potato production region of China.

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are the very important quarantine nematode pests of potato [Stone 1973]. Both species cause serious potato tuber yield losses. These species are subjected to strict quarantine regulations in many countries (EPPO 2017). G. rostochiensis was detected in Sichuan and Yunnan province, China in 2022 (Jiang et al. 2022). A survey for cyst nematodes in potato fields was conducted in Guizhou Province from 2018 to 2020. A total of 200 samples, including roots and soil, were collected from 40 potato fields in Hezhang (N27 06.145, E104 39.153) and Weining (N26 50.541, E104 09.885) counties in Guizhou Province, China. The Cobb decanting and sieving method was used to isolate cysts and J2s from the soil samples (Southey 1986). The potato roots were stained with acid fusion to observe cyst development. Morphological and molecular analyses indicated that 27 (13.5%) of the samples contained G. rostochiensis. The cyst density ranged between 1-85 cysts per 100 cm3 of soil and a mean density was 15 cysts per 100 cm3 soil. The smoothly rounded cysts were in brown and golden color, and the terminal cone was absent and circumfenestrate. The key morphometrics of cysts (n=20) were 695 ± 26 (685-757) µm in length excluding neck and 690 ± 30 (668 to769) µm in width; the number of cuticular ridges between anus and vulval fenestra was 16.3 ±2.1 (14 to 18); fenestral length was 15.1 ± 2.1 µm (13.18 to 19.27); distance from anus to the edge of fenestra was 61.12 ± 8.9 (49.22 to76.27) µm; and Granek's ratio was 4.54 ± 0.8 (3.97-5.26). The key morphometrics of J2 (n = 20): 468.0 ± 20.1 (427 to - 521) µm in body length, 20.58 ± 0.7 (20.2 to 21.8) µm in stylet length, 43.9 ± 5.6 (40.3 to 53.9) µm in tail length, and 23.1 ± 1.8 (21.77 to 25.32) µm in hyaline region length. The cyst and J2 morphologies were consistent with those of G. rostochiensis (Subbotin et al. 2010, EPPO 2017). Genomic DNA was isolated from cysts (n=20). DNA extraction was performed in a volume of 20 µl containing 3 µl 10× PCR buffer, 3 µl Proteinase K (600 µg µl-1), 14 µl distilled water and a single cyst was added and ground in an ice bath as described by Ou et al. (2008). The internal transcribed spacer (ITS) regions were amplified using the universal primers: rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA2 (5'-TTTCACTCGCCGTTACTAAGG-3') (Fleming 1998 ), and the 28S rDNA-D2/D3 regions were amplified using the primers: D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Subbotin et al. 2006). After the brackets at the beginning and end of the sequences were closed-up, the ITS rDNA sequences (GenBank Accession No. MZ042367 and MZ042368) showed 99.66% - 99.92% identity to G. rostochiensis sequences available in GenBank (FJ212166.1, GQ294513, FJ212164.1 and KJ409617.1). Sequences from the 28S region (GenBank Accession No. MZ057597 and MZ057598) were 99.23% - 99.74% similar to those of G. rostochiensis isolate from Slovakia (KJ409625.1), Italy (KJ409631.1) and United Kingdom (KJ409633.1). We used species specific primers ITS5（5'-GGAAGTAAAAGTCGTAACAAGG-3'）and PITSr3 (5'AGCGCAGACATGCCGCAA-3') to amplify the product (Bulman & Marshall 1997; EPPO 2017). A single 434bp fragment was obtained from Hezhang and Weining populations. A host test for the Hezhang and Weining populations were performed by inoculating 1,000 eggs per plant of varieties Qingshu 9, Huize 2 and Hezuo 88 grown in the pots containing 800 cm3 of sterilized soil (soil: sand ratio was 3:1), and four replications were tested in greenhouse under 16 h light, 22°C in the day and 8 h dark in the night. At 90 days post inoculation, 32.6 ± 7, 31.2 ± 8, and 29.5 ± 8 females and cysts were extracted from the infected roots and soils of the varieties Qingshu 9, Huize 2 and Hezuo 88, respectively. No females and cysts were observed on the control plants. The trial indicated that potato cultivars Qingshu 9, Huize 2 and Hezuo 88 are hosts for the Hezhang and Weining populations of Globodera rostochiensis. To the best of our knowledge, this is the first detection of potato cyst nematode Globodera rostochiensis in Guizhou Province, China.

The Impact of Management Strategies on the Development and Status of Potato Cyst Nematode Populations in Switzerland: An Overview from 1958 to Present.

Globodera rostochiensis and G. pallida are some of the most successful and highly specialized plant parasitic nematodes and among the most regulated quarantine pests globally. In Switzerland, they have been monitored by annual surveys since their first detection in Swiss soil in 1958. The dataset created was reviewed to produce an overview of the development and actual status of potato cyst nematodes (PCNs) in Switzerland. Positive fields represent 0.2% of all the samples analyzed, and their distribution is limited to central-west and western Switzerland, suggesting that new introduction of PCNs and the spread of the initial introduced PCN populations did not occur. In this way, the integrated management used in Switzerland appears to be effective. However, the increasing availability of potato varieties with resistance to G. rostochiensis and the limited availability of varieties with resistance to G. pallida, together with other biotic and abiotic factors, have promoted changes in the dominance of either species. Consequently, an extended monitoring program is of interest to Swiss farmers, to avoid favoring virulent traits that could be present in Swiss Globodera populations.

Genome Resources of Two Pathotypes of the Potato Cyst Nematode Globodera rostochiensis from New York.

The potato cyst nematode Globodera rostochiensis is a regulated pest posing a serious threat to potato production worldwide. Although the endemic pathotype (Ro1) of G. rostochiensis has been confined to New York State for several decades as a result of quarantine regulations and management with resistant potato cultivars, a virulent pathotype, Ro2, has emerged, for which control measures are scarce. The ability to detect Ro2 early in fields is necessary to sustain the success of G. rostochiensis quarantine in the United States. Here, we report the comparative analysis of whole-genome sequences of multiple single-cyst-derived Ro1 and Ro2 lines, propagated from original field populations. The identified discriminant variants are good targets for developing molecular diagnostic tools for differentiating G. rostochiensis pathotypes in New York.

The relations of broad nematode resistance to quality characteristics as a consequence of marker-assisted selection in potato breeding programs.

Cultivating resistant varieties of potato is the most effective and environmentally sound method of protecting potato crops against pests and diseases. Potato cyst nematodes (PCN) are major nematode pests causing severe constraints in potato production worldwide. There are five pathotypes of Globodrea rostochiensis (Ro1-Ro5) and three of G. pallida Pa1-Pa3. Cultivation of potato varieties with broad nematode resistance may influence the growth of the wide spectrum of PCN pathotypes, but there is limited availability of such varieties on the market. The use of molecular markers allows for the effective selection of resistant genotypes at early stages of breeding. However, the impact of early selection for nematode resistance on the agronomic value of the final selected clones is a cause of concern for potato breeders. This study investigates the relationships between the presence of the combined resistance genes H1, Gro1-4 and GpaVvrn , which confer resistance to the nematodes, and certain agricultural traits. Clones with broad nematode resistance conferred by the genes H1, Gro1-4 and GpaVvrn presented yields and tuber morphology traits similar to those of the clones without identified resistance genes.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100-200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Biology, pathotype, and virulence of Globodera rostochiensis populations from Kenya.

The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are important pests of potato globally. Due to their extensive damage potential and the challenge of managing them, these nematodes are under strict regulations in many countries; however, despite these regulations, PCN continue to spread into new areas and countries. In Kenya, G. rostochiensis was first reported in 2015 and G. pallida was reported three years later, both in Nyandarua County. Research was conducted to characterize the biology, pathotype, and virulence of G. rostochiensis populations from Kenya in glasshouse and laboratory studies. The development of G. rostochiensis was assessed in roots of susceptible potato 'Désirée' and resistant 'Laura' carrying the H1 resistance gene. The 'HAR1' population from Kenya and 'Ecosse' from Germany were not able to produce females in the roots of the resistant potato 'Laura'. The rate of root penetration by G. rostochiensis juveniles did not differ (p > 0.05) between populations and cultivars. However, in the resistant cultivar, juveniles developed into males only. A total of 736 cumulative degree-days at 6°C base temperature (DD6) were required by 'HAR1' to complete the life cycle on 'Désirée', whereas 'Ecosse' completed the life cycle within 645 DD6. The Kenyan populations lacked obligatory diapause and high numbers of juveniles hatched immediately after maturity. Consequently, the Kenyan populations had the potential to complete up to three reproduction cycles in less than a year. On selected potato cultivars, the populations from Kenya failed to reproduce on 10 out of 13 commercial cultivars tested. The 10 cultivars carried the H1 resistance gene, which suggests that the G. rostochiensis populations tested belong to the Ro1/4 pathotype group. The virulence of the G. rostochiensis populations from Kenya did not differ from that of the standard reference population 'Ecosse' and therefore can be effectively managed with the commercially available potato cultivars carrying the H1 resistance gene.

First report of morphological and molecular characterization of Moroccan populations of Globodera pallida.

Potato cyst nematodes (PCNs) are the most important potato pest causing major crop losses across the world with a quarantine status in many countries. In Morocco, several potato crops are infected with PCNs and the monitoring of potato production as well as the control of import and export of potato seeds are currently carried out by morphological methods. The present work was aimed to use molecular and morphometric methods for identifying and differentiating PCN species in Morocco for the first time. The morphological identification of PCN species from collected soil samples were carried out using the shape of the cysts, the length of the stylet, the number of cuticular ridges, and the Granek's ratio. The J2 had a slightly shorter body length, the number of cuticular ridges was 9 and the Granek's ratio averaged 2.2. The morphobiometric analysis revealed proximity of the Moroccan population to G. pallida species. PCNs sampled from contaminated fields were analyzed molecularly using PCR. DNA amplification was performed using the multiplex PCR method and PCR-RFLP from the ITS region of the total genomic DNA compared to multiplex PCR-specific DNA sequences. All confirmed the presence G. pallida in all samples of the Moroccan PCN populations.

The mechanism of potato resistance to Globodera rostochiensis: comparison of root transcriptomes of resistant and susceptible Solanum phureja genotypes.

BACKGROUND: Globodera rostochiensis belongs to major potato pathogens with a sophisticated mechanism of interaction with roots of the host plants. Resistance of commercial varieties is commonly based on specific R genes introgressed from natural populations of related wild species and from native potato varieties grown in the Andean highlands. Investigation of molecular resistance mechanisms and screening the natural populations for novel R genes are important for both fundamental knowledge on plant pathogen interactions and breeding for durable resistance. Here we exploited the Solanum phureja accessions collected in South America with contrasting resistance to G. rostochiensis. RESULTS: The infestation of S. phureja with G. rostochiensis juveniles resulted in wounding stress followed by activation of cell division and tissue regeneration processes. Unlike the susceptible S. phureja genotype, the resistant accession reacted by rapid induction of variety of stress response related genes. This chain of molecular events accompanies the hypersensitive response at the juveniles' invasion sites and provides high-level resistance. Transcriptomic analysis also revealed considerable differences between the analyzed S. phureja genotypes and the reference genome. CONCLUSION: The molecular processes in plant roots associated with changes in gene expression patterns in response to G. rostochiensis infestation and establishment of either resistant or susceptible phenotypes are discussed. De novo transcriptome assembling is considered as an important tool for discovery of novel resistance traits in S. phureja accessions.

Differential expression of NBS-LRR-encoding genes in the root transcriptomes of two Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis.

BACKGROUND: The characterization of major resistance genes (R genes) in the potato remains an important task for molecular breeding. However, R genes are rapidly evolving and frequently occur in genomes as clusters with complex structures, and their precise mapping and identification are complicated and time consuming. RESULTS: Comparative analysis of root transcriptomes of Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis revealed a number of differentially expressed genes. However, compiling a list of candidate R genes for further segregation analysis was hampered by their scarce annotation. Nevertheless, combination of transcriptomic analysis with data on predicted potato NBS-LRR-encoding genes considerably improved the quality of the results and provided a reasonable number of candidate genes that provide S. phureja with strong resistance to the potato golden cyst nematode. CONCLUSION: Combination of comparative analyses of tissue-specific transcriptomes in resistant and susceptible genotypes may be used as an approach for the rapid identification of candidate potato R genes for co-segregation analysis and may be used in parallel with more sophisticated studies based on genome resequencing.

Climate change is predicted to alter the current pest status of Globodera pallida and G. rostochiensis in the United Kingdom.

The potato cyst nematodes Globodera pallida and G. rostochiensis are economically important plant pathogens causing losses to UK potato harvests estimated at £50 m/ year. Implications of climate change on their future pest status have not been fully considered. Here, we report growth of female G. pallida and G. rostochiensis over the range 15 to 25°C. Females per plant and their fecundity declined progressively with temperatures above 17.5°C for G. pallida, whilst females per plant were optimal between 17.5 and 22.5°C for G. rostochiensis. Relative reproductive success with temperature was confirmed on two potato cultivars infected with either species at 15, 22.5 and 25°C. The reduced reproductive success of G. pallida at 22.5°C relative to 15°C was also recorded for a further seven host cultivars studied. The differences in optimal temperatures for reproductive success may relate to known differences in the altitude of their regions of origin in the Andes. Exposure of G. pallida to a diurnal temperature stress for one week during female growth significantly suppressed subsequent growth for one week at 17.5°C but had no effect on G. rostochiensis. However, after two weeks of recovery, female size was not significantly different from that for the control treatment. Future soil temperatures were simulated for medium- and high-emission scenarios and combined with nematode growth data to project future implications of climate change for the two species. Increased soil temperatures associated with climate change may reduce the pest status of G. pallida but benefit G. rostochiensis especially in the southern United Kingdom. We conclude that plant breeders may be able to exploit the thermal limits of G. pallida by developing potato cultivars able to grow under future warm summer conditions. Existing widely deployed resistance to G. rostochiensis is an important characteristic to retain for new potato cultivars.

Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec, Canada.

The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world.

Sustainable management of the potato cyst nematode, Globodera rostochiensis, with two microbial fermentation products.

Potato cyst nematodes (PCN) cause an overall 9% yield loss of total potato production worldwide. Research on sustainable management of PCN is still under progress. Two microbial fermentation products (MFPs) from Alltech, a proprietary blend formulated with a bacterial fermentation media and a Cu component (MFP5075), and a microbial based product (MFP3048), were evaluated against the PCN Globodera rostochiensis. In laboratory tests, effectiveness of the MFPs was recorded in terms of PCN juveniles (J2) hatching from cysts, J2 mortality and their attraction toward potato roots using pluronic gel. Greenhouse trials were conducted to study the effect of the products on PCN infestation in potato plants and a pilot scale experiment was conducted to study the impact of these MFPs on nematode biodiversity in garden soil. All treatments were performed within a concentration range of 0, 0.5, 1, and 2% (v/v) MFP5075 and 2, 6, 10, and 20 g/10 ml (w/v) MFP3048. The attraction assay, juvenile hatching and the PCN infestation in plants results were compared with those in an untreated control and a commercial nematicide (Nemguard™) treatment. After 24 h of treatment with 0.5 and 1% MFP5075, a 13-fold and 43-fold reduction, respectively, relative to J2 survival was recorded compared to that of untreated control. However, no J2 survived at 2% and above concentration of the MFP5075 treatment. Treatment with MFP3048 was effective in causing mortality of J2 only after 48-h. In the attraction assay, a 20-fold and 8-fold reduction in number of J2 attracted toward potato roots was observed, when treated with MFP5075, compared to the untreated and the Nemguard™ treatment, respectively. Subsequently, 30-35 PCN cysts were treated with both products dissolved in potato root diffusate and the results were recorded in terms of number of J2 hatched in each treatment after 10 days. No J2 hatched in the MFP5075 treatment, whereas mean numbers (±SE) of 243 ± 11.5, 30 ± 2.5, and 1.3 ± 0.6 J2 were noted in the untreated control, MFP3048, and the Nemguard™ treatment, respectively. The treatment with the MFPs compromised the integrity of the unhatched J2, which looked granular, whereas the internal organs of the unhatched J2 could be clearly identified in the untreated control. In plant infestation studies, treatment with MFP3048 and MFP5075 caused 90.6 and 84.9 percent reduction in PCN infestation, respectively, in terms of cysts developed on roots compared to untreated control. Overall, results indicate that the MFPs could potentially provide a promising alternative for sustainable PCN management.

A plant cell wall-associated kinase encoding gene is dramatically downregulated during nematode infection of potato.

Plant cell wall associated kinases (WAKs) and WAK-like kinases (WAKLs) have been increasingly recognized as important regulators of plant immunity against various plant pathogens. However, the role of the WAK/WAKL family in plant-nematode interactions remains to be determined. Here, we analyzed a WAK-encoding gene (Soltu.DM.02G029720.1) from potato (Solanum tuberosum). The Soltu.DM.02G029720.1 encoded protein contains domains characteristic of WAK/WAKL proteins and shows the highest similarity to SlWAKL2 from tomato (S. lycopersicum). We thus named the gene as StWAKL2. Phylogenetic analysis of a wide range of plant WAKs/WAKLs further revealed close similarity of StWAKL2 to three WAK/WAKL proteins demonstrated to play a role in disease resistance. To gain insights into the potential regulation and function of StWAKL2, transgenic potato lines containing the StWAKL2 promoter fused to the ß-glucuronidase (GUS) reporter gene were generated and used to investigate StWAKL2 expression during plant development and upon nematode infection. Histochemical analyses revealed that StWAKL2 has specific expression patterns in potato leaf and root tissues. During nematode infection, GUS activity was mostly undetected at nematode infection sites over the course of nematode parasitism, although strong GUS activity was observed in root tissues adjacent to the infection region. Furthermore, mining of the transcriptomic data derived from cyst nematode infection of Arabidopsis roots identified a few WAK/WAKL genes, including a StWAKL2 homologue, found to be significantly down-regulated in nematode-induced feeding sites. These results indicated that specific suppression of WAK/WAKL genes in nematode-induced feeding sites might be crucial for cyst nematodes to achieve successful infection of host plants. Further studies are needed to uncover the role of WAK/WAKL genes in plant defenses against nematode infection.

Characterization and response of two potato receptor-like kinases to cyst nematode infection.

Plant-parasitic cyst nematodes (Heterodera and Globodera spp.) secrete CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) effector proteins, which act as ligand mimics of plant CLE peptides to promote successful nematode infection. Previous studies of the Arabidopsis-beet cyst nematode (BCN; H. schachtii) pathosystem showed that Arabidopsis CLE receptors including CLAVATA1 (CLV1), CLV2, and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) are required for BCN CLE signaling. Studies further revealed that nematode CLE signaling through GmCLV2 and StCLV2, an Arabidopsis CLV2 orthologue from soybean (Glycines max) and potato (Solanum tuberosum), respectively, is required for the soybean cyst nematode (SCN; H. glycines) and the potato cyst nematode (PCN; G. rostochiensis) to induce disease in their respective host plant. In this study, we identified and characterized two additional potato receptors, StRPK2 and StCLV1, homologues of Arabidopsis RPK2 and CLV1, for a role in PCN parasitism. Using promoter-reporter lines we showed that both StRPK2 and StCLV1 are expressed in the potato root but vary in their spatial expression patterns. Interestingly, StRPK2 but not StCLV1 was found to be expressed and upregulated at PCN infection sites. Nematode infection assays on StRPK2-knockdown lines revealed a decrease in nematode infection. Collectively, our results suggest that parallel CLE signaling pathways involving StCLV2 and StRPK2 are important for PCN parasitism and that manipulation of nematode CLE signaling may represent a viable means to engineer nematode resistance in crop plants including potato.

The effect of linalool on second-stage juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida.

Linalool is either a toxic compound to a few species of plant parasitic nematodes or attractive to entomopathogenic nematodes. This compound is produced and emitted by several host plants of Globodera rostochiensis and G. pallida, the potato cyst nematodes (PCN). With the aim to reveal the effect of linalool on PCN, laboratory assays were carried out. Survival of PCN second-stage juveniles (J2s) in water + linalool control did not differ; thus, proving linalool to be nontoxic to PCN. Behavioral assays carried out in Petri dishes revealed attractiveness in the form of positive response of J2s of both PCN species towards linalool. Based on these behavioral assays, sensitivity to linalool of G. rostochiensis J2s was higher compared with that of G. pallida J2s. Linalool is the first compound of plant origin to elicit positive response in both PCN species.

Rapid and sensitive detection of potato cyst nematode Globodera rostochiensis by loop-mediated isothermal amplification assay.

Cyst nematodes of the species Globodera rostochiensis and G. pallida are devastating parasites of the potato crop. Early detection of cyst nematodes in the field is critical for adopting an appropriate management strategy. A specific and sensitive loop-mediated isothermal amplification (LAMP) assay using four oligonucleotide primers has been developed to amplify the internal transcribed spacer region (ITS) of ribosomal DNA of potato cyst nematode G. rostochiensis. The PCN-LAMP reaction could be completed within 75 min at 68 °C followed by termination at 85 °C for 7 min. The primers exhibited specificity for G. rostochiensis and did not detect any other tested genera of plant parasitic or entomopathogenic nematodes. LAMP reaction was highly sensitive, suitable for crude genomic DNA and could successfully detect G. rostochiensis DNA up to femtogram quantity. This assay is rapid, cost effective and requires minimal instrumentation. It will facilitate the detection of G. rostochiensis at field and point-of-care labs and help in the interception of infested plant material/soil samples at quarantine stations independent of a professional nematologist. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02830-8.

Potato Cyst Nematodes: Geographical Distribution, Phylogenetic Relationships and Integrated Pest Management Outcomes in Portugal.

The identification and phylogenetic relationships of potato cyst nematodes (PCN) were studied to assess the potential value of geographical distribution information for integrated pest management of potato production in Portugal. This research focused on PCN species, Globodera pallida and Globodera rostochiensis. From 2013 until 2019, 748 soil samples from the rhizosphere of different potato cultivars were surveyed in the Portuguese mainland to detect and identify both species and track their location. PCN are widespread invasive species throughout Portugal. In fact, during the survey period an incidence of 22.5% was estimated for the tested samples. The patterns of infestation vary among regions, increasing from south to north, where PCN were first detected. Currently, both species are present in all potato producing regions of the country, with a greater incidence of G. pallida. Phytosanitary control measures are influencing to the observed results. The use of potato cultivars resistant to G. rostochiensis led to a decrease of this species but had no influence on G. pallida detections, which continues its reproduction freely since there are no effective resistant cultivars for this species. The relationship between the presence, infestation rate, spread and geographical distribution of PCN is discussed in terms of behavioral responses of the potato cultivars and the implications for developing new integrated crop protection measures.