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Phytonematode infections are a major constraint to agriculture and can cause pre-harvest losses of up to one-third of the crop in affected fields. With increasing restrictions on the use of chemical pesticides due to environmental and human hazards, and increasing nematode pressure due to climate change, soil degradation and agricultural intensification, more sustainable ways to manage such plant pests are needed to meet the growing demand for food. Therefore, the reproduction of thermophilic Meloidogyne spp. was evaluated in comparison with that of the northern root-knot nematode Meloidogyne hapla on the wild strawberry Fragaria vesca vs. semperflorens cv. Alexandria, a potential source of resistance to nematode infection. M. hapla showed a high reproductive rate in F. vesca, while the thermophilic Meloidogyne species tested showed significant lower reproductive rates. This suggests that F. vesca vs. semperflorens cv. Alexandria, although not resistant to the nematode species tested, could be used in a management system to down-regulate nematode pressure. In addition, this study helps to reinforce the importance of crop wild relatives in the search for resistance traits to support a more sustainable agriculture.

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification approach that has been used to detect a variety of animal and plant pathogens. However, the RPA assay is rarely used for the molecular diagnosis of plant parasitic nematodes. In this study, we developed RPA assays for the detection of four Meloidogyne spp.; Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The RPA amplification step could be completed at 38 °C in 20 min without a thermal cycling instrument. The RPA assays were able to distinguish these four Meloidogyne spp. from closely related Meloidogyne species and other plant parasitic nematodes. The detection limits of the RPA assays were 10−2, 10−2, 10−1, and 10−1 dilutions of DNA from a single J2 nematode of M. incognita, M. javanica, M. arenaria, and M. enterolobii, which were less sensitive than polymerase chain reaction (PCR) detection methods. In addition, the RPA assays could detect these four Meloidogyne spp. directly from infested tomato roots. The simplicity, rapidity and practicability all indicated that the RPA assay will be an effective tool for molecular diagnosis of plant parasitic nematodes.

Molecular taxonomy and evolution of nematodes have been recently the focus of several studies. Mitochondrial sequences were proposed as an alternative for precise identification of Meloidogyne species, to study intraspecific variability and to follow maternal lineages. We characterized the mitochondrial genomes (mtDNAs) of the root knot nematodes M. floridensis, M. hapla and M. incognita. These were AT rich (81-83%) and highly compact, encoding 12 proteins, 2 rRNAs, and 22 tRNAs. Comparisons with published mtDNAs of M. chitwoodi, M. incognita (another strain) and M. graminicola revealed that they share protein and rRNA gene order but differ in the order of tRNAs. The mtDNAs of M. floridensis and M. incognita were strikingly similar (97-100% identity for all coding regions). In contrast, M. floridensis, M. chitwoodi, M. hapla and M. graminicola showed 65-84% nucleotide identity for coding regions. Variable mitochondrial sequences are potentially useful for evolutionary and taxonomic studies. We developed a molecular taxonomic marker by sequencing a highly-variable ~2 kb mitochondrial region, nad5-cox1, from 36 populations of root-knot nematodes to elucidate relationships within the genus Meloidogyne. Isolates of five species formed monophyletic groups and showed little intraspecific variability. We also present a thorough analysis of the mitochondrial region cox2-rrnS. Phylogenies based on either mitochondrial region had good discrimination power but could not discriminate between M. arenaria, M. incognita and M. floridensis.

Root-knot nematodes (RKN) of the tropical group reproducing as a rule with mitotic parthenogenesis are highly polyphagous and damaging pests causing great losses in crop production. Morphometrical and molecular characters of three closely related tropical RKN species have been analysed in order to identify species-specific diagnostic parameters and examine the relationship between species. Morphometrical characters of M. ethiopica, M. luci and M. inornata isolates were similar and overlapped when compared between species and isolates of the same species indicating a very close relationship between the three species. Additionally, structure of map-1 genes was analysed, which provided the evidence of identical structure of map-1 family genes in M. ethiopica, M. inornata and M. luci. The unique structure of map-1 genes when compared to the other tropical RKN species further supports close relationship of the three species and therefore we have classified them together as M. ethiopica group. In addition, our results showed that the emergence of a virulent M. luci population does not correlate with the loss of map-1 genes as it was previously indicated for M. incognita (Castagnone-Sereno et al. 2009). Further, we have developed novel molecular methods to aid in identification of these nematodes: a PCR based method with primers specific for the M. ethiopica group comprising M. ethiopica M. luci and M. inornata, and a PCR based method with primers specific for the tropical RKN group species. Our results show that species of Meloidogyne ethiopica group share high similarity at morphological and genetic level.

Root-knot nematodes (RKNs) of the genus Meloidogyne spp., are among the most economically important groups of plant-parasitic nematodes worldwide, causing significant economic losses through yield reduction across a wide range of crops. In Croatia, although the presence of Meloidogyne spp. has been documented for decades, data at the species level was limited. As accurate identification is crucial for implementation of effective management strategies, we attempted to fill this gap. This study presents the results of a national survey of RKNs affecting potato crops as well as an early warning programme targeting vegetable crops, conducted across Croatia between 2022 and 2024. Nematodes were identified using morphological analyses (female perineal patterns and second-stage juveniles) and molecular methods (PCR with group-specific and species-specific primers, as well as DNA sequencing). Meloidogyne spp. were detected in 61 out of 210 samples, corresponding to an infestation rate of 29%. Four species were identified: M. incognita, M. hapla, M. arenaria, and M. javanica. Notably, M. incognita and M. javanica are reported here for the first time in Croatia. These results provide updated insights into the distribution and identity of RKNs in Croatia, thereby establishing a foundation for the implementation of sustainable management strategies.

Secreted parasitism proteins encoded by parasitism genes expressed in esophageal gland cells mediate infection and parasitism of plants by root-knot nematodes (RKN). Parasitism gene 16D10 encodes a conserved RKN secretory peptide that stimulates root growth and functions as a ligand for a putative plant transcription factor. We used in vitro and in vivo RNA interference approaches to silence this parasitism gene in RKN and validate that the parasitism gene has an essential function in RKN parasitism of plants. Ingestion of 16D10 dsRNA in vitro silenced the target parasitism gene in RKN and resulted in reduced nematode infectivity. In vivo expression of 16D10 dsRNA in Arabidopsis resulted in resistance effective against the four major RKN species. Because no known natural resistance gene has this wide effective range of RKN resistance, bioengineering crops expressing dsRNA that silence target RKN parasitism genes to disrupt the parasitic process represents a viable and flexible means of developing novel durable RKN-resistant crops and could provide crops with unprecedented broad resistance to RKN.

Rice (Oryza sativa) production is seriously impacted by root-knot nematodes (RKN), including Meloidogyne graminicola, Meloidogyne incognita, and Meloidogyne javanica, in upland and irrigated culture systems. Successful plant infection by RKN is likely achieved by releasing into the host cells some effector proteins to suppress the activation of immune responses. Here, we conducted a series of functional analyses to assess the role of the Meloidogyne-secreted protein (MSP) 18 from M. incognita (Mi-MSP18) during rice infection by RKN. Developmental expression profiles of M. javanica and M. graminicola showed that the MSP18 gene is up-regulated throughout nematode parasitic stages in rice. Reproduction of M. javanica and M. graminicola is enhanced in rice plants overexpressing Mi-MSP18, indicating that the Mi-MSP18 protein facilitates RKN parasitism. Transient expression assays in onion cells suggested that Mi-MSP18 is localized to the cytoplasm of the host cells. In tobacco, Mi-MSP18 suppressed the cell death induced by the INF1 elicitin, suggesting that Mi-MSP18 can interfere with the plant defense pathways. The data obtained in this study highlight Mi-MSP18 as a novel RKN effector able to enhance plant susceptibility and modulate host immunity.

Root-knot nematodes (Meloidogyne spp.) are among the biotic factors that limit tomato production worldwide. The objectives of this study were to assess the distribution and identify Meloidogyne spp. and associated problems from major tomato growing areas of Ethiopia. A total of 212-rhizosphere soil and 123 root samples were sampled from 40 localities during 2012/13 growing season. A total of 646 respondents participated in the questionnaire to assess knowledge and practice of farmers and factors associated with RKN damage on tomato. Out of the 212 soil samples collected, 47.2% were found infested by various Meloidogyne species 8 weeks after the start of the bioassay test. Out of the 123 root samples collected, 65% had root galls. The highest prevalence (100%) of RKN was found on samples collected from Adami Tullu, Babile, Erer Gota, Hurso, Jittu, Tikur Wuha, Tepo Choronke, Zeway and Koka. The highest incidence (100%) of RKN was found from Adami Tullu, Jara Weyo, Babile, Erer Gota, Hurso, Jittu, Tikur Wuha, Tony farm, Tepo Choronke, Zeway and Koka localities based on direct observation of galls on collected root samples. Out of the 646 respondents, 43.3% reported to have RKN damage symptoms when shown the symptoms while 56.7% of them did not report damage. The highest significant effect on the dependent variable RKN damage on tomato roots comes from the previous crop, soil texture, awareness about RKN and source of irrigation water used. The presence of Meloidogyne incognita, M. javanica, M. arenaria and M. hapla on tomato was confirmed using a combination of molecular and biochemical identification tools. Five isolates of Meloidogyne spp. were not properly identified and could be new species. The two tropical species, M. incognita (48.4%) and M. javanica (41.2%) were the most prevalent species. Both species were also co-infesting tomato plants. Meloidogyne hapla was detected for the first time in an open tomato production farmer’s field at ‘Zeway’ locality with 1620 m.a.s.l. elevation. The occurrence of these Meloidogyne species alone, or in mixed populations from samples collected, clearly shows that RKN are widespread in major tomato growing areas of Ethiopia. In the near future, the economic importance of M. arenaria and M. hapla on tomato production in Ethiopian agriculture should be investigated.

Root knot nematodes (RKN) can infect most of the world's agricultural crop species and are among the most important of all plant pathogens. As yet however we have little understanding of their origins or the genomic basis of their extreme polyphagy. The most damaging pathogens reproduce by obligatory mitotic parthenogenesis and it has been suggested that these species originated from interspecific hybridizations between unknown parental taxa. We have sequenced the genome of the diploid meiotic parthenogen Meloidogyne floridensis, and use a comparative genomic approach to test the hypothesis that this species was involved in the hybrid origin of the tropical mitotic parthenogen Meloidogyne incognita. Phylogenomic analysis of gene families from M. floridensis, M. incognita and an outgroup species Meloidogyne hapla was carried out to trace the evolutionary history of these species' genomes, and we demonstrate that M. floridensis was one of the parental species in the hybrid origins of M. incognita. Analysis of the M. floridensis genome itself revealed many gene loci present in divergent copies, as they are in M. incognita, indicating that it too had a hybrid origin. The triploid M. incognita is shown to be a complex double-hybrid between M. floridensis and a third, unidentified, parent. The agriculturally important RKN have very complex origins involving the mixing of several parental genomes by hybridization and their extreme polyphagy and success in agricultural environments may be related to this hybridization, producing transgressive variation on which natural selection can act. It is now clear that studying RKN variation via individual marker loci may fail due to the species' convoluted origins, and multi-species population genomics is essential to understand the hybrid diversity and adaptive variation of this important species complex. This comparative genomic analysis provides a compelling example of the importance and complexity of hybridization in generating animal species diversity more generally.

Although centromeres have conserved function, centromere-specific histone H3 (CenH3) and centromeric DNA evolve rapidly. The centromere drive model explains this phenomenon as a consequence of the conflict between fast-evolving DNA and CenH3, suggesting asymmetry in female meiosis as a crucial factor. We characterized evolution of the CenH3 protein in three closely related, polyploid mitotic parthenogenetic species of the Meloidogyne incognita group, and in the distantly related meiotic parthenogen Meloidogyne hapla. We identified duplication of the CenH3 gene in a putative sexual ancestral Meloidogyne. We found that one CenH3 (αCenH3) remained conserved in all extant species, including in distant Meloidogyne hapla, whereas the other evolved rapidly and under positive selection into four different CenH3 variants. This pattern of CenH3 evolution in Meloidogyne species suggests the subspecialization of CenH3s in ancestral sexual species. Immunofluorescence performed on mitotic Meloidogyne incognita revealed a dominant role of αCenH3 on its centromere, whereas the other CenH3s have lost their function in mitosis. The observed αCenH3 chromosome distribution disclosed cluster-like centromeric organization. The ChIP-Seq analysis revealed that in M. incognita αCenH3-associated DNA dominantly comprises tandem repeats, composed of divergent monomers which share a completely conserved 19-bp long box. Conserved αCenH3-associated DNA is also confirmed in the related mitotic Meloidogyne incognita group species suggesting preservation of both centromere protein and DNA constituents. We hypothesize that the absence of centromere drive in mitosis might allow for CenH3 and its associated DNA to achieve an equilibrium in which they can persist for long periods of time.