Meloidogyne Research Articles

A Comparison of Three Automated Root-Knot Nematode Egg Counting Approaches Using Machine Learning, Image Analysis, and a Hybrid Model.

Meloidogyne spp. (root-knot nematodes [RKNs]) are a major threat to a wide range of agricultural crops worldwide. Breeding crops for RKN resistance is an effective management strategy, yet assaying large numbers of breeding lines requires laborious bioassays that are time-consuming and require experienced researchers. In these bioassays, quantifying nematode eggs through manual counting is considered the current standard for quantifying establishing resistance in plant genotypes. Counting RKN eggs is highly laborious, and even experienced researchers are subject to fatigue or misclassification, leading to potential errors in phenotyping. Here, we present three automated egg counting models that rely on machine learning and image analysis to quantify RKN eggs extracted from tobacco and sweet potato plants. The first method relied on convolutional neural networks trained using annotated images to identify eggs (M. enterolobii R2 = 0.899, M. incognita R2 = 0.927, M. javanica R2 = 0.886), whereas a second contour-based approach used image analysis to identify eggs from their morphological characteristics and did not rely on neural networks (M. enterolobii R2 = 0.977, M. incognita R2 = 0.990, M. javanica R2 = 0.924). A third hybrid model combined these approaches and was able to detect and count eggs nearly as well as human raters (M. enterolobii R2 = 0.985, M. incognita R2 = 0.992, M. javanica R2 = 0.983). These automated counting protocols have the potential to provide significant time and resource savings annually for breeders and nematologists and may be broadly applicable to other nematode species.

Wild mungbean resistance to the nematode Meloidogyne enterolobii involves the induction of phenylpropanoid metabolism and lignification.

Root-knot nematodes (Meloidogyne spp.) are plant parasites causing annual economic losses amounting to several billion US dollars worldwide. One of the most aggressive species is M. enterolobii, a growing threat to agriculture due to its broad host range and ability to overcome many known resistance genes. Mungbean, a nutritionally and economically valuable crop, is particularly vulnerable to nematodes and pathogens. However, research focusing on mungbean resistance to M. enterolobii is scarce, and the corresponding defense mechanisms are poorly understood. Here, we screened mungbean accessions and identified an accession strongly resistant to M. enterolobii. Transcriptome analysis revealed 2730 differentially expressed genes (DEGs) in this resistant accession (CPI106939) compared to 1777 in the susceptible accession (Crystal) 7 days after nematode inoculation. The gene ontology (GO) upregulated in CPI106939 with functions related to plant-pathogen interactions, plant hormone signaling, oxidative stress, and plant immunity. Plant defense-related genes (WRKY, PAL, MAPK, POD and PR) were also significantly induced in CPI106939. Metabolome analysis showed that four secondary metabolites related to phenylpropanoid metabolism and lignification were significantly enriched in CPI106939. The induced immune response and secondary metabolites may underpin the enhanced resistance to M. enterolobii, providing insight into the resistance mechanisms in accession CPI106939 as well as candidate genes controlling the interaction between mungbean and its nematode parasite. Our study therefore provides foundations for the breeding of new varieties with intrinsic M. enterolobii resistance.

Dual nematode infection in Brassica nigra affects shoot metabolome and aphid survival in distinct contrast to single-species infection.

Previous studies showed that aphid performance was compromised on Brassica nigra infected by root-lesion nematodes (Pratylenchus penetrans, Pp), but less, or positively influenced by root-knot nematode (Meloidogyne spp., Mi) infection. These experiments were on single-nematode infections, while naturally, roots are infected with several nematode species simultaneously. We performed greenhouse assays to assess the effects of single (Mi, Pp) and concurrent (MP)-nematode infections on aphid performance. Using targeted and untargeted profiling of leaf and phloem metabolomes, we examined how single- and concurrent-nematode infections affect shoot metabolomes, and elucidated the possible consequences on aphid performance. We found that the metabolic response towards double-infection is different from single-species infections. Moreover, Mi- and Pp-infections triggered discrete changes in B. nigra leaf and phloem metabolic profiles. Both Pp and MP-infections reduced aphid survival, suggesting that the biological effect could primarily be dominated by Pp-induced changes. This concurred with increased indole glucosinolates and hydroxycinnamic acid levels in the leaves, in particular the putative involvement of salicylic acid-2-O-β-D-glucoside. This study provides evidence that concurrent infection by different nematode species, as is common in natural environments, is associated with distinct changes in aboveground plant metabolomes, which are linked to differences in the survival of an aboveground herbivore.

The response of the pepper with and without Me1 gene to Mi-1.2-virulent Meloidogyne incognita (Kofoid & White,1919) (Tylenchida: Meloidogynidae) isolates

Root-knot nematodes are important organisms that infect vegetables. Due to the intense use of Mi-1.2, virulent populations that break resistance have become widespread and have become an important factor limiting the use of this gene. Me1 resistance gene on pepper provides resistance against Meloidogyne arenaria (Neal, 1889) Chitwood, 1949 Meloidogyne incognita (Kofoid & White,1919) Chitwood, 1949 and Meloidogyne javanica (Treub, 1885) Chitwood, 1949 (Tylenchida: Meloidogynidae) species. However, there is limited information on the effectiveness of the Me1 gene against Mi-1.2 virulent populations. Therefore, it is important to know the reaction of pepper cultivars carrying the Me1 resistance gene against Mi-1.2 virulent populations. In this study, the response of resistant pepper cultivar MT-01 F1 (bearing Me1) and susceptible pepper cultivar Safran F1 against both Mi-1.2 natural virulent M. incognita isolates and Mi-1.2 selected virulent M. incognita isolate was investigated under controlled conditions. This study was conducted in Akdeniz University Faculty of Agriculture Department of Plant Protection Nematology Laboratory in 2021. All isolates caused many egg masses and galls on the resistant tomato cultivar Seval F1 as expected, and the susceptible pepper cultivar Safran F1. Five isolates were found to cause egg masses and gall formation, while the V3 isolate did not multiply on the resistant pepper cultivar MT-01 F1. The results showed that pepper cultivars carrying the Me1 gene exhibited different responses against Mi-1.2 virulent isolates.

Nematode Diversity and Root-Knot Nematode Management Approaches for Sustainable Mulberry Production

Mulberry ecosystems are beset by the clandestine menace of nematodes, precipitating devastating consequences on plant productivity. This review highlights the importance of nematode diversity in mulberry ecosystems and explores various eco-friendly and sustainable approaches for managing the root-knot nematode, Meloidogyne incognita involving cultural, biological and host resistance methods. Various eco-friendly management strategies viz., cultural approaches like summer deep ploughing, mulching and trap crops; biological methods like bio-agents and plant extracts; and resistant varieties against the root-knot nematode infecting mulberry are reviewed and discussed providing an impetus for integrating multiple approaches for effective and sustainable root-knot nematode management.

Constitutive Level of Specialized Secondary Metabolites Affects Plant Phytohormone Response to Above- and Belowground Herbivores

Plants defend themselves chemically against herbivory through secondary metabolites and phytohormones. Few studies have investigated how constitutive variation in secondary metabolites contributes to systemic herbivory response. We hypothesized that plants with lower constitutive defenses would induce a stronger phytohormone response to spatially separated herbivory than plants with high constitutive defense. We used growth chamber bioassays to investigate how aboveground herbivory by Colorado potato beetle (Leptinotarsa decemlineata, CPB) and belowground herbivory by northern root-knot nematode (Meloidogyne hapla, RKN) altered phytohormones and glycoalkaloids in roots and shoots of two lines of wild potato (Solanum chacoense). These lines had different constitutive levels of chemical defense, particularly leptine glycoalkaloids, which are only present in aboveground tissues. We also determined how these differences influenced the preference and performance of CPB. The susceptible wild potato line responded to aboveground damage by CPB through induction of jasmonic acid (JA) and OPDA. However, when challenged by both RKN and CPB, the susceptible line retained high levels of JA, but not OPDA. Beetles gained more mass after feeding on the susceptible line compared to the resistant line, but were not affected by nematode presence. Belowground, JA, JA-Isoleucine, and OPDA were higher in the resistant line compared to the susceptible line, and some compounds demonstrated response to local herbivory. In contrast, the susceptible line did not induce phytohormone defenses belowground. These findings allow us to predict that constitutive level of defense may influence the threshold of herbivory that may lead to plant-mediated effects on spatially separated herbivores.

Design, Synthesis, and Nematocidal Evaluation of Waltherione A Derivatives: Leveraging a Structural Simplification Strategy.

Southern root-knot nematodes are among the most pernicious phytoparasites; they are responsible for substantial yield losses in agricultural crops worldwide. The limited availability of nematicides for the prevention and control of plant-parasitic nematodes necessitates the urgent development of novel nematicides. Natural products have always been a key source for the discovery of pesticides. Waltherione A, an alkaloid, exhibits potent nematocidal activity. In this study, we designed and synthesized a series of quinoline and quinolone derivatives from Waltherione A, leveraging a strategy of structural simplification. Bioassays have revealed that the quinoline derivatives exhibit better activity than quinolone derivatives in terms of both nematocidal and fungicidal activities. Notably, compound D1 demonstrated strong nematocidal activity, with a 72 h LC50 of 23.06 μg/mL, and it effectively controlled the infection of root-knot nematodes on cucumbers. The structure-activity relationship suggests that the quinoline moiety is essential for the nematocidal efficacy of Waltherione A. Additionally, compound D1 exhibited broad-spectrum fungicidal activity, with an EC50 of 2.98 μg/mL against Botrytis cinerea. At a concentration of 200 μg/mL, it significantly inhibited the occurrence of B. cinerea on tomato fruits, with an inhibitory effect of 96.65%, which is slightly better than the positive control (90.30%).

Phylogenetic position of Meloidogyne javanica associated with tomato, based on ITS rDNA partial sequences Limpopo Province, South Africa

The decline in the yield of tomato (Solanum lycopersicum L.) is largely attributed to the prevalence of nematodes belonging to the Meloidogyne species, commonly known as root-knot nematodes. The Meloidogyne species of nematodes are regarded as the most significant contributing factor to the reduction in tomato yield. Therefore, the present investigation was conducted in 2023 at the University of Limpopo to reveal that Meloidogyne javanica (Treub) Chitwood was the identified cause of the nematode. The ITS rDNA was sequenced and amplified from female nematodes, following which the blast result showed that the South African population exhibited a high similarity (99% identity) to those populations studied in the United States (AF387094 and U96305) and China (AY438555). A comparison to the USA population (AF387094), revealed four nucleotide differences. Utilizing the maximum likelihood method for phylogenetic analysis, it was demonstrated that our population and the other populations of M. javanica form a robust clade (91% bootstrap values).

Genetic variations underlying root-knot nematode resistance in sweetpotato

Root-knot nematode (Meloidogyne incognita) causes severe crop damage and large economic losses worldwide. Several cultivars of sweetpotato [Ipomoea batatas (L.) Lam)] have been developed with root-knot nematode-resistant traits; however, many of these cultivars do not have favorable agronomic characteristics. To understand the genetic traits underlying M. incognita resistance in sweetpotato, whole genome resequencing was conducted on three RKN-susceptible (Dahomi, Shinhwangmi, and Yulmi) and three RKN-resistant (Danjami, Pungwonmi, and Juhwangmi) sweetpotato cultivars. Three SNPs (single nucleotide polymorphisms) in promotor sequences were shared in RKN-resistant cultivars and were correlated with disease resistance. One of these SNPs was located in G6617|TU10904, which encoded a homolog of RIBOSOMAL PROTEIN EL15Z, and was associated with reduced expression in RKN-resistant cultivars only. Alongside SNP analysis, mRNA-seq data were analyzed for the same cultivars with and without nematode infection, and 18 nematode-sensitive genes were identified that responded in a cultivar-specific manner. Of these genes, expression of G8735|TU14367 was lower in sensitive cultivars than in RKN-resistant cultivars. Overall, this study identified two genes that potentially have key roles in the regulation of nematode resistance and will be useful targets for nematode resistance breeding programs.

Impact of genetically modified bacteria and natural rocks on root-knot nematode bio-control, improving productivity and chemical properties of grapes

Impact of genetically modified bacteria and natural rocks on root-knot nematode bio-control, improving productivity and chemical properties of grapes

Nematode-trapping fungus Arthrobotrys oligospora recruited rhizosphere microorganisms to cooperate in controlling root-knot nematodes in tomato.

The objective of this study was to elucidate the role and mechanism of changes in the rhizosphere microbiome following A. oligospora treatment in the biological control of root-knot nematodes and identify the key fungal and bacterial species that collaborate with A. oligospora to biocontrol root-knot nematodes. We conducted a pot experiment to investigate the impact of A. oligospora treatment on the biocontrol efficiency of A. oligospora against Meloidogyne incognita infecting tomato. We analyzed the rhizosphere bacteria and fungi communities of tomato by high-throughput sequencing of the 16S rRNA gene fragment and the internal transcribed spacer (ITS). The results indicated that the application of A. oligospora resulted in a 53.6% reduction in the disease index of M. incognita infecting tomato plants. The bacterial diversity of rhizosphere soil declined in the A. oligospora-treated group, while fungal diversity increased. The A. oligospora treatment enriched the tomato rhizosphere with Acidobacteriota, Firmicutes, Bradyrhizobium, Sphingomonadales, Glomeromycota and Purpureocillium. These organisms are involved in the utilization of rhizosphere organic matter, nitrogen, and glycerolipids, or play the role of ectomycorrhiza or directly kill nematodes. The networks of bacterial and fungal co-occurrence exhibited a greater degree of stability and complexity in the A. oligospora treatment group. This study demonstrated the key fungal and bacterial species that collaborate with the A. oligospora in controlling the root-knot nematode and elaborated the potential mechanisms involved. The findings offer valuable insights and inspiration for the advancement of bionematicide based on nematode-trapping fungus.

Piriformospora indica modifies cucumber’s tolerance to Meloidogyne incognita by regulating various agro-physiological traits, antioxidant enzymes, and abscisic acid pathway genes

Root knot nematode (RKN), Meloidogyne incognita, is considered a major soil-borne pathogen that can cause severe yield losses for vegetables and diverse crops. Usually, reducing of M. incognita damage is mainly relies on the application of nematicides and good agricultural practices. However, the use of synthetic nematicides is restricted due to concerns about their impact on the environment and human health. As a result, the use of alternative strategies is becoming necessary to combat RKN resistance. This study evaluates the antagonistic impact of the root mutualistic fungus Pirforomospora indica on M. incognita. It also assesses its influence on the nutritional status, photo-synthesis, antioxidant enzyme activity, endogenous abscisic acid (ABA) levels, and selected ABA related-responsive genes in cucumber plants. Roots of cucumber seedlings were inoculated with P. indica and the second-stage juveniles (1000 J2 per plant). The results demonstrated that P. indica significantly reduced M. incognita invasion in roots, resulting in a 24% reduction in root galling and 42.6% decline in final population. Inoculating plants with both P. indica and RKN increased performance of root fresh and dry weight, as well as improved photochemical efficiency of PSII (Fv/Fm), photosystem II efficiency (PSII), catalase (CAT), peroxidase (POD), and superoxide dis-mutase (SOD). Furthermore, P. indica colonization, either alone or in combination with M. incognita, significantly improved number of fruits per plant, average fruit weight, the plant's marketable yield, and leaf nutrient content (N, P, K and Mg), Moreover, there was an increase in IAA content combined with a decrease in ABA content in roots of dual inoculation plants, if compared to M. incognita infested plants. The highest ABA content was recorded in the root of RKN-cucumber plants. The decline in ABA content due to P. indica treatment was consistent with the modulation of ABA pathway genes, specifically PP2C, PYL1, RK2,1, and RK2,2. The mixed of P. indica and M. incognita led to a decrease in the expression of PP2C, PLY1, RK2,1, and RK2,2 in comparison to the control group. These results indicate that P. indica application could help reduce the negative effects of RKN on important crops.

Impact of Bioagents on Growth and Yield Parameters of Root-knot Nematode Infested Mulberry Plants

Mulberry (Morus alba L.) is a vital crop for sericulture and its productivity is crucial for the industry's sustainability. This study investigated the influence of bioagents viz., Purpureocillium lilacinum, Trichoderma harzianum, Trichoderma viride, Pochonia clamydosporia and Pseudomonas fluorescens, alongside control treatments including carbofuran 3G and neem cake on growth and yield parameters in severely root-knot nematode infested mulberry plants. The treatments involved are generally recommended to manage root-knot nematode infestation. The results revealed that T. viride @ 5 kg/ha significantly improved plant growth, leaf area and leaf yield leading to enhanced productivity. The findings of this study underscore the potential of bioagents in fostering environmentally sustainable and economically viable sericulture. Aims: The study aims to assess the impact of bioagents on growth and yield parameters of root-knot nematode, Meloidogyne incognita infested mulberry plants. Study Design: The data collected from the experimental field were analyzed statistically by using one-way RCBD. Place and Duration of Study: The experiment was carried out in the root-knot nematode infested mulberry garden in Kalyapura Village, Shidlaghatta Taluk, Chikkaballapura District, Eastern Dry Zone (Zone-5) of Karnataka, India between January to September 2022, covering three harvests. Methodology: The treatments were incorporated near the root zone of mulberry plants within a week after pruning as per the recommendations. The observations were recorded once before the imposition of the treatments (initial) and during the first (60 DAT) and second (120 DAT) shoot harvests in mulberry after imposing the treatments. Results: Among the bioagents Trichoderma viride showed better results for shoot height (120.00 cm), number of leaves per plant (511.75), leaf area (102.10 cm2) and leaf yield per plant (528.38 g) during the second harvest i.e., 120 DAT. The minimum leaf yield per plant was recorded in the untreated check (216.43 g). Conclusion: Incorporating T. viride @ 5 kg/ha mixed with 5 tons FYM near the root zone of mulberry infested with RKN showed promising results in promoting plant growth and yield.

Identification of a Promising Novel Genetic Source for Rice Root-Knot Nematode Resistance through Markers Associated with Trait-Specific Quantitative Trait Loci.

Direct-seeded rice (DSR) is gaining popularity among farmers due to its environmentally safe and resource-efficient production system. However, managing the rice root-knot nematode (RRKN), Meloidogyne graminicola, remains a major challenge in DSR cultivation. Developing genetic resistance is a pragmatic and effective approach compared to using hazardous pesticides. Pusa Basmati 1121 (PB1121) is the most popular Basmati rice variety, but it is highly susceptible to RRKN. In contrast, Phule Radha (PR) has shown highly resistant reaction to RRKN, as reported in our earlier study. We generated an F2:3 population from the cross of PB1121/PR and evaluated it for RRKN resistance-related traits under artificial inoculation conditions. The distribution pattern of traits in the F2:3 population indicated that resistance may be governed by a few major-effect genes and many minor-effect genes. The molecular markers reported to be associated with QTLs governing RRKN resistance traits were used to test in the current population. Although the simple linear regression identified significant associations between the markers and RRKN resistance-associated traits, these associations were spurious as the LOD score was below the threshold limit. This indicates that PR possesses novel genomic regions for resistance to RRKN as it does not possess any of the earlier reported QTLs.

Study on the Function of SlWRKY80 in Tomato Defense against Meloidogyne incognita.

WRKY transcription factors (TFs) can participate in plant biological stress responses and play important roles. SlWRKY80 was found to be differentially expressed in the Mi-1- and Mi-3-resistant tomato lines by RNA-seq and may serve as a key node for disease resistance regulation. This study used RNAi to determine whether SlWRKY80 silencing could influence the sensitivity of 'M82' (mi-1/mi-1)-susceptible lines to M. incognita. Further overexpression of this gene revealed a significant increase in tomato disease resistance, ranging from highly susceptible to susceptible, combined with the identification of growth (plant height, stem diameter, and leaf area) and physiological (soluble sugars and proteins; root activity) indicators, clarifying the role of SlWRKY80 as a positive regulatory factor in tomato defense against M. incognita. Based on this phenomenon, a preliminary exploration of its metabolic signals revealed that SlWRKY80 stimulates different degrees of signaling, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH), and may synergistically regulate reactive oxygen species (ROS) accumulation and scavenging enzyme activity, hindering the formation of feeding sites and ultimately leading to the reduction of root gall growth. To our knowledge, SlWRKY80 has an extremely high utilization value for improving tomato resistance to root-knot nematodes and breeding.

Screening, identification of root-knot nematode resistance sources using multivariate analysis and validation of molecular markers linked to Me genes in chilli (Capsicum annuum L.)

Screening, identification of root-knot nematode resistance sources using multivariate analysis and validation of molecular markers linked to Me genes in chilli (Capsicum annuum L.)

Perennial cultivation of green pepper provides pest suppression, ecosystem development and high crop yield

Perennial cultivation (PC) provides economic benefits to farmers and environmental benefits to society such as soil protection. However, little is known about the effects of PC on soil pest occurrence. This 4-year study used overwintered green pepper (GP) and root-knot nematodes (RKN) as a model plant–soil pest system. We compared the levels of RKN, its antagonists, and other organisms in strip-shaped PC microplots to those of annual cultivation (AC) and no-tillage AC (NT) microplots. To promote RKN occurrence, we inoculated the ends of all microplots with RKN in the spring of year 3 of study. During autumn in years 3 and 4, we determined abundance of RKN and antagonistic animals, DNA operational taxonomical units (OTUs) counts of antagonistic microbes. The results showed that the rate of RKN occurrence was lower in PC and NT than in AC due to minimal soil movement, rather than to suppression by antagonists, although some antagonists such as collembolan and nematode-trapping fungi increased in PC. Moreover, plant biomass was higher in PC than in NT and AC during fruiting period due to persistence of the plant body over years.We also found signs of ecosystem development in PC, which is an essential background for pest suppression: the maturity index (a measure of disturbance) of the nematode community, fungal diversity, frequency of plant and animal symbionts/parasites were greater in PC than in NT or AC. These findings suggested that ecosystem development had commenced within the 4-year study period. We conclude that the long-lasting plant body and undisturbed soil environment are specific to PC, and could provide agronomical and ecological benefits.

Field study on the effect of different planting periods on root-knot nematodes, Meloidogyne spp. infecting tomato

Field study on the effect of different planting periods on root-knot nematodes, Meloidogyne spp. infecting tomato

Bio-control efficacy of selected indigenous nematophagous fungi against Meloidogyne enterolobii in vitro and on dry bean (Phaseolus vulgaris L.).

Dry bean (Phaseolus vulgaris L.) is an important commercialized field crop in South Africa for aiding in food security as a cheap protein source. However, it is highly susceptible to root-knot nematodes (RKN), Meloidogyne species. Use of indigenous nematophagous fungi as bio-control agents (BCA) of Meloidogyne nematodes is a promising research focus area. This is because indigenous fungal species are naturally part of the ecosystem and therefore compatible with other biological processes unlike most synthetic chemicals. The objective of the study was to identify indigenous nematophagous fungal BCA and establish their potential efficacy in reducing M. enterolobii population densities on dry bean with and without incorporation of compost. Screened indigenous fungal species included Aspergillus terreus, Talaromyces minioluteus, T. sayulitensis, Trichoderma ghanense, and T. viride. There were observed significant parasitism differences (P ≤ 0.05) among the BCA, with T. ghanense showing the highest egg parasitism (86%), followed by T. minioluteus (72%) and T. sayulitensis (70%). On the other hand, the highest J2 parasitism was observed on T. minioluteus (95%), followed by A. terreus and T. viride (63%). A similar trend was observed under in vivo conditions, with higher efficacy with compost incorporation. This provides a highly encouraging alternative and ecologically complementary Meloidogyne management in dry bean production.

Protective role of native root-associated bacterial consortium against root-knot nematode infection in susceptible plants

Root-knot nematodes (RKNs) are a global menace to agricultural crop production. The role of root-associated microbes (RAMs) in plant protection against RKN infection remains unclear. Here we observe that cucumber (highly susceptible to Meloidogyne incognita) exhibits a consistently lower susceptibility to M. incognita in the presence of native RAMs in three distinct soils. Nematode infection alters the assembly of bacterial RAMs along the life cycle of M. incognita. Particularly, the loss of bacterial diversity of RAMs exacerbates plant susceptibility to M. incognita. A diverse range of native bacterial strains isolated from M. incognita-infected roots has nematode-antagonistic activity. Increasing the number of native bacterial strains causes decreasing nematode infection, which is lowest when six or more bacterial strains are present. Multiple simplified synthetic communities consisting of six bacterial strains show pronounced inhibitory effects on M. incognita infection in plants. These inhibitory effects are underpinned via multiple mechanisms including direct inhibition of infection, secretion of anti-nematode substances, and regulation of plant defense responses. This study highlights the role of native bacterial RAMs in plant resistance against RKNs and provides a useful insight into the development of a sustainable way to protect susceptible plants.