Unnati Sonawala

Plant-parasitic nematodes are a diverse, polyphyletic group of plant pathogens which can infect most plant tissues and all major crops. Amongst the most damaging clades are the cyst nematodes, which can remain dormant in the soil for decades as infection-competent, developmentally arrested, second-stage juveniles in eggs. Hatching is stimulated by a variety of factors. However, the impact of hatching factor responsiveness on nematode morphology, physiology, gene expression, and infection biology has not been explored. We examined the impact of hatching time on the beet cyst nematode, Heterodera schachtii . We found that late hatchers invaded host roots and established feeding sites in greater numbers than early hatchers. We demonstrate variation in baseline parasitism gene expression and in responsiveness of genes to effectostimulins, small, plant-derived molecules which upregulate parasitism genes. Three quarters of effectostimulin-induced transcriptional changes were also modulated, either positively or negatively, by hatching time. While there were no observable morphological differences between early and late hatching nematodes on the day of their emergence from the egg, the late hatchers displayed signs of faster utilisation of internal energy reserves after 7 days at 4°C, as evidenced by less body area attributed to fat, than early hatchers. Finally, we found no evidence of substantive genetic differences between early and late hatchers, they were representative of a single population, despite the observed differences in infection, gene expression, and physiology. Taken together, non-genetic differences likely drive late hatchers to more rapidly utilise their internal energy reserves, to be more responsive to host-derived signals, and to be ultimately more infective than their early hatching counterparts.

Plant-parasitic nematodes are a diverse group collectively capable of infecting most major crops. Among the most damaging are the cyst nematodes, which can remain dormant in the soil for decades as infection-competent, developmentally arrested, second-stage juveniles in eggs. Hatching is stimulated by a variety of factors. However, the impact of hatching factor responsiveness on nematode morphology, physiology, gene expression, and infection biology has not been explored. We examined the impact of hatching time on the beet cyst nematode, Heterodera schachtii . We found that late hatchers invaded host roots and established feeding sites in greater numbers than early hatchers. We demonstrate variation in baseline parasitism gene expression and in responsiveness of genes to effectostimulins—small, plant-derived molecules that upregulate parasitism genes. Three quarters of effectostimulin-induced transcriptional changes were also modulated, either positively or negatively, by hatching time. Although there were no observable morphological differences between early- and late-hatching nematodes on their day of hatching, the late hatchers displayed signs of faster utilization of internal energy reserves after 7 days at 4°C, as evidenced by less body area attributed to fat, than early hatchers. Finally, we found no evidence of substantive genetic differences between early and late hatchers—they were representative of a single population, despite the observed differences in infection, gene expression, and physiology. Taken together, nongenetic differences likely drive late hatchers to more rapidly utilize their internal energy reserves, to be differentially responsive to host-derived signals, and to be ultimately more infective than their early-hatching counterparts. Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Roots of quince (Cydonia oblonga Mill.) exhibiting lesions/darkening were collected from private property in Sheffield, England (53°23'23.1"N 1°30'53.4"W), along with rhizospheric soil. Nematode extraction revealed the presence of Pratylenchus specimens (60 nematodes per g of root; 100 nematodes/cm3 of soil). No above-ground symptoms were evident. Females were slender with the labial region slightly offset from the body and comprising three annuli (Figure 1A and 1B), measuring 505.6 µm (427.0 - 580.5), a = 22.5 (19.1 - 25.1), b= 6.85 (5.3 - 10.0), c= 17.3 (9.4 - 27.0), and V% = 81.6 (78.9 - 83.0) (n = 10). Basal knobs were rounded but frequently found as slightly indented/cupped in some individuals (1B and 1C). The tail was rounded and smooth (Figure 1E). Males were common and slightly smaller than females (Figure 1D), measuring 482 µm (477.81 - 489.22), a = 25.9 (24.6 - 27.3), b= 7.3 (6.2 - 9.5), c= 15.2 (14.0 - 15.0) (n = 4). These characteristics are consistent with Pratylenchus penetrans (Loof, 1991; Castillo and Vovlas, 2007). To validate morphological observations, molecular identification was carried out using 28S rDNA D2A/D3B primers (Gamboa-Cortés et al., 2023). DNA was extracted from hand-picked individuals according to Sonawala et al. (2024). PCR was carried out using 5 µL of the digested sample with KOD Xtreme polymerase (Sigma Aldrich) using an initial denaturation at 94˚C for 2 min; 40 cycles of 98˚C for 10 s, 55˚C for 30 s and 68˚C for 1 min). Purified PCR products (Monarch Gel Extraction Kit) were sequenced, compared to those publicly available for Pratylenchus spp. and Xiphinema index, and aligned and trimmed using Clustal Omega (Madeira et al., 2024) on Geneious (2025.0.3) using default settings. Model selection (TPM3+G4) and phylogenetic tree inference (SH-aLRT, 1000 bootstraps (Minh et al., 2020)) were performed on the aligned sequences using IQ-TREE 2 version 2.2.0 (Figure 2A). Phylogenetic analysis corroborated the morphological identification of P. penetrans (Figure 2A). Nematodes were multiplied and maintained on maize (Zea mays L.) 'B73' and tomato (Solanum lycopersicum L.) 'Moneymaker' in growth chambers (light/dark cycle of 16h/8h at 30°C/21°C, respectively). To assess the host status of quince, five plants of C. oblonga (acquired commercially), and an equal number of Maize 'B73', were inoculated with 1,200 individuals. The inoculum was administered into two holes (2 cm depth) close to the stem and partially filled with vermiculite to avoid heat stress. Plants were assessed 90 days post-inoculation, as previously reported for sufficient reproduction of Pratylenchus brachyurus on Eucalyptus spp. (Souza and Inomoto, 2021). For quince and maize, similar reproductive values (2.3 and 2.2) and nematodes per gram of root (410 and 426) were found, respectively. Quince-infested roots were lesioned and decaying (Figure 2B), and reduced in weight by 31% compared to non-inoculated controls (7.8 g and 11.3 g, respectively, p<0.05, Student's t-test). Taken together, these data demonstrate that P. penetrans successfully reproduces on quince, and induces significant damage to host roots, indicating both host status and potential economic impact. The presence of P. penetrans should be considered when establishing quince orchards, as trees can become a source of inoculum over-season and/or to fast-growing crops (e.g., wheat or maize) grown near them. To our knowledge, this is the first report of P. penetrans on quince in England.

Pathogens are engaged in a fierce evolutionary arms race with their host. The genes at the forefront of the engagement between kingdoms, including effectors and immune receptors, are part of diverse and highly mutable gene families. Even in this context, we discovered unprecedented variation in the HYPer-variable (HYP) effectors of plant-parasitic nematodes. We discovered single effector gene loci that can harbour potentially thousands of allelic variants. These alleles vary in the number, but principally in the organisation, of motifs within a central Hyper Variable Domain (HVD). Using targeted long-read sequencing, we dramatically expand the HYP repertoire of two plant-parasitic nematodes, Globodera pallida and G. rostochiensis, such that we can define distinct sets of species-specific “rules” underlying the apparently flawless genetic rearrangements. Finally, by analysing the HYP complement of 68 individual nematodes we made the unexpected finding that despite the huge number of alleles, most individuals are homozygous. Taken together, these data point to a novel mechanism of programmed genetic variation, termed HVD-editing, where alterations are locus-specific, strictly governed by rules, and can theoretically produce thousands of variants without errors.