CONCLUSIONS: The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

Globally, phytonematodes cause significant crop losses. Understanding the functions played by the plant rhizosphere soil microbiome during phytonematodes infection is crucial. This study examined the distribution of phytonematodes in the paddy fields of five provinces in Thailand, as well as determining the keystone microbial taxa in response to environmental factors that could be considered in the development of efficient biocontrol tactics in agriculture. The results demonstrated that Meloidogyne graminicola and Hirschmanniella spp. were the major and dominant phytonematodes distributed across the paddy fields of Thailand. Soil parameters (total P, Cu, Mg, and Zn) were the important factors affecting the abundance of both nematodes. Illumina next-generation sequencing demonstrated that the levels of bacterial diversity among all locations were not significantly different. The Acidobacteriota, Proteobacteria, Firmicutes, Actinobacteriota, Myxococcota, Chloroflexi, Verrucomicrobiota, Bacteroidota, Gemmatimonadota, and Desulfobacterota were the most abundant bacterial phyla observed at all sites. The number of classes of the Acidobacteriae, Clostridia, Bacilli, and Bacteroidia influenced the proportions of Hirschmanniella spp., Tylenchorhynchus spp., and free-living nematodes in the sampling dirt, whereas the number of classes of the Polyangia and Actinobacteria affected the amounts of Pratylenchus spp. in both roots and soils. Soil organic matter, N, and Mn were the main factors that influenced the structure of the bacterial community. Correlations among rhizosphere microbiota, soil nematodes, and soil properties will be informative data in considering phytonematode management in a rice production system.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Plant-parasitic nematodes have enormous economic and social impacts. The majority of plant-parasitic nematodes are soil dwelling and feed on plant roots. Exudates from actively growing roots initiate hatch of some nematode species, thus ensuring infective juveniles emerge in close proximity to host plant roots. Several gradients of volatile and non-volatile compounds are established around plant roots, at least some of which are used by nematodes to orientate toward the roots. Plant-parasitic nematodes are microscopic in size (less than 1 mm in length and between 15 and 20 μm in diameter), so investigations into behavior are challenging. Various in vitro techniques have been used to evaluate the effects of root exudates. The techniques can also be used to evaluate the comparative attractiveness of different plants or cultivars of the same plant species. This chapter describes some examples of different types of basic in vitro assays.

The endoparasitic fungus Hirsutella rhossiliensis is an important biocontrol agent of cyst nematodes in nature. To determine the potential parasitism of the fungus on a non-natural host, the pinewood nematode (Bursaphelenchus xylophilus) living in pine trees and the endophytic ability of the fungus on plants, in this paper, we first constructed and utilized a green fluorescent protein (GFP)-tagged H. rhossiliensis HR02 transformant to observe the fungal infection process on B. xylophilus and its colonization on Arabidopsis roots. Then, we compared the fungal parasitism on three species of nematodes with different lifestyles, and we found that the fungal parasitism is correlated with nematode species and stages. The parasitic effect of H. rhossiliensis on adults of B. xylophilus is similar to that on second-stage juveniles (J2) of the root-knot nematode Meloidogyne incognita after 24 h of inoculation, although the virulence of the fungus to second-stage juveniles of M. incognita is stronger than that to those of B. xylophilus and Caenorhabditis elegans. Moreover, the endophytism of H. rhossiliensis was confirmed. By applying an appropriate concentration of H. rhossiliensis conidial suspension (5 × 106 spores/mL) in rhizosphere soil, it was found that the endophytic fungus can promote A. thaliana growth and reproduction, as well as improve host resistance against M. incognita. Our results provide a deeper understanding of the fungus H. rhossiliensis as a promising biocontrol agent against plant-parasitic nematodes.

The current investigation aimed to isolate and identify predatory fungal strains and evaluate their efficacy in mitigating the effects of plant-parasitic nematodes. We successfully isolated three distinct nematophagous fungal strains from soil samples, identified as Arthrobotrys megalosporus, A. oligospora, and A. sinensis, using conventional and molecular identification methodologies. In vitro trials illustrated the high capture efficiency of these fungi against plant-parasitic nematodes. Over an exposure period of 48 h to Aphelenchoides besseyi, Bursaphelenchus xylophilus, and Ditylenchus destructor, A. megalosporus (GUCC220044) displayed predation rates of 99.7%, 83.0%, and 21.1%, respectively. A. oligospora (GUCC220045) demonstrated predation rates of 97.3%, 97.3%, and 54.6%, and A. sinensis (GUCC220046) showed rates of 85.1%, 68.3%, and 19.0% against the same cohort of nematodes. The experimental outcomes substantiate that all three identified fungal strains demonstrate predatory activity against the tested nematodes, albeit with varying efficiencies.

BACKGROUND: Plant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant.
RESULTS: We present the Sperling prep - a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode, Heterodera schachtii, multiplexed in the same nematode.
CONCLUSIONS: We were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes.

Plant-parasitic nematodes conduct a series of sophisticated behaviors to complete their life cycles. Among these, locomotion behaviors, including finding the host and migrating to the feeding site, directly affect the success of parasitism. Thus, disrupting locomotion behaviors has the potential to control these parasites. γ-Aminobutyric acid (GABA) is the prominent inhibitory neurotransmitter in nematodes. GABA-immunoreactive neurons are mostly found in motor neurons, where they regulate behaviors in the model nematode C. elegans. However, the GABA system in most stylet-bearing nematodes has received little attention. Using immunohistochemistry, we found variation in the pattern of GABA-immunoreactivity among two major plant-parasites and a fungal feeder. Some of these GABA-immunoreactive neurons lack clear homologs to C. elegans. Pharmaceutical assays showed that applying GABA, its agonist, and its antagonist, can disrupt the locomotion behaviors of these nematodes, although sensitivity to a given compound varied between species. Our data suggest that the GABA system is a potential target for the control of plant-parasitic nematodes.

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Since the discovery of Wolbachia in plant-parasitic nematodes (PPNs), there has been increased interest in this earliest branching clade that may hold important clues to early transitions in Wolbachia function in the Ecdysozoa. However, due to the specialized skills and equipment of nematology and the difficulty in culturing most PPNs, these PPN-type Wolbachia remain undersampled and poorly understood. To date, there are few established laboratory methods for working with PPN-type Wolbachia strains, and most research has relied on chance discovery and comparative genomics. Here, we address this challenge by providing detailed methods to assist researchers with more efficiently collecting PPNs and screen these communities, populations, or single nematodes with a newly developed PPN-type Wolbachia-specific PCR assay. We provide an overview of the typical yields and outcomes of these methods, to facilitate further targeted cultivation or experimental methods, and finally we provide a short introduction to some of the specific challenges and solutions in following through with comparative or population genomics on PPN-type Wolbachia strains.