Co-Silencing of the Voltage-Gated Calcium Channel β Subunit and High-Voltage Activated α1 Subunit by dsRNA Soaking Resulted in Enhanced Defects in Locomotion, Stylet Thrusting, Chemotaxis, Protein Secretion, and Reproduction in Ditylenchus destructor

The voltage-gated calcium channel (VGCC) β subunit (Cavβ) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Cavβ subunit gene of D. destructor, namely, DdCavβ, was subjected to molecular characterization. In situ hybridization assays showed that DdCavβ was expressed in all nematode tissues. Transcriptional analyses showed that DdCavβ was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCavβ was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCavβ or HVA Cavα1 alone and co-silencing of the DdCavβ and HVA Cavα1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Cavα1 and Cavβ subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

First Detection of Ditylenchus destructor Parasitizing Maize in Northeast China

Maize is one of the most important crops in the world. Heilongjiang province has the largest maize area in China. Plant-parasitic nematodes are important agricultural pests, which cause huge economic losses every year and have attracted global attention. Potato rot nematode Ditylenchus destructor is a plant-parasitic nematode with a wide range of hosts and strong survival ability in different environments, which brings risks to agricultural production. In 2020, D. destructor was detected in seven maize fields in Heilongjiang province. Morphological identification and molecular approach were used to characterize the isolated D. destructor. The observed morphological and morphometric characteristics were highly similar and consistent with the existing description. The DNA sequencing on the D2/D3 region of the ribosomal DNA 28S and the phylogenetic analysis showed that D. destructor population obtained from maize and other isolates infesting carrot, sweet potato, and potato were in subclade I supported by a 96% bootstrap value. Additionally, the phylogenetic analysis of the ITS rRNA gene sequence further indicated that this D. destructor population from maize clustered in a clade I group and belonged to ITS rRNA haplotype C. An inoculation experiment revealed that D. destructor was pathogenic on the maize seedlings in pots and caused the disease symptoms in the stem base of maize seedlings. This is the first report of D. destructor causing stem rot of maize in Heilongjiang province, and contributes additional information on disease control and safe production of maize in the region.

Nematodofauna of potato tubers in the Republic of Moldova

According to the multiannual researches, it was observed that, in the conditions of the Republic of Moldova, the Nematodofauna of potato tubers (Solanum tuberosum) of different varieties, collected from deposits, is specific, with the dominance of saprophytes due to the spread of bacteriosis and fungal infections. In the potato tubers, sick with dithylenchosis (phases 4, 5), the presence of 30 spec8es of nematoodes was identified, belonging to 24 genera, 14 families, 5 orders, 2 classes: 1. Class Secernentea, Orders Aphelenchida, Rhabditida and Tylenchida; 2. Class Adenophorea, Orders Dorylaimida and Plectida. Most species (19 species) are saprophytes of the Class Secernentea, Order Rhabditida with the dominance of the Superfamilies Cephaloboidea and Diplogasteroidea. According to the trophic-ecological group, the species detected in the potatoes tubers are included in all 5 groups: plant feeding, hiphal feeding, bacterial feeding, animal predation, omnivorous. In all researched varieties, both in frequency (100%) and density (thousands of individuals/gram of infested tissue) distinguished species Ditylenchus destructor Thorne 1945, Order Tylenchida, Family Anguinidae, which is also the main parasite in the culture of Solanum tuberosum in the Republic of Moldova, capable of causing dithylenhosis, as well as epiphytotics – mass infestation of potatoes in deposits

Parasitic nematodes in potatoes of different varieties and their interrelations with some arthropods

Introduction. Potato (Solanum tuberosum L.) is susceptible to infestation with an associated complex of different species of parasitic and saprophytic nematodes, bacteria, fungi, and arthropods, which diminish the quality of the product. Material and methods. 10 varieties of potatoes were investigated (Agata, Albastriu-mov, Bella rosa, Concorde, Desiree, Irga, Iagodca, Roko, Romano, Sprinter), cultivated on the territory of the Republic of Moldova. The extraction of nematodes and mites was performed using the Baermann funnels, modified by Nesterov.Results. Multiannual researches on the degree of potato infestation have shown that tubers are preferred by various arthropods (Acaridae), Agriothes spp. (Elateridae), Gryllotalpa gryllotalpa, (Grillotalpidae) and Sciaridae spp . (Sciaridae), which form different interactions with the parasitic nematodes of tubers (Ditylenchus destructor, D. dipsaci).Conclusions. Solanum tuberosum infested by parasitic nematodes D. destructor in association with saprophytic nematodes and dry rot are colonized by nematophag ous (mites – 80%) and phytophagous arthropods (wireworms – 40%; mole cricket – 50%; flies – 40%). Among the researched arthropods, Rhizoglyphus echinopus were more frequently found, which together with other species actively consume the primary and secondary parasitic nematodes, their mortality constituting up to 90%. In the traumatized by some arthropods potatoes, with the soil, secondary parasitic nematodes, also penetrate, carrying bacterial and fungal infections, subsequently stimulating the total rot of potato tubers.

Identification of specific odour compounds from garlic cloves infected with the potato tuber nematode, Ditylenchus destructor, using gas chromatography-olfactometry

Summary The potato tuber or potato rot nematode, Ditylenchus destructor, causes severe damage to garlic (Allium sativum) produced in Japan. Although consumption of a nematode-infected garlic bulb is not harmful to human health, it causes consumer dissatisfaction because the infected bulb deteriorates quickly. In addition, nematode-contaminated garlic cloves are inadvertently used as seed garlic for next season, which then leads to nematode-infested fields and increases yield loss. However, infected garlic bulbs look the same as healthy ones, and hence have been unknowingly distributed and sold. A method is needed to discriminate infected garlic bulbs from non-infected garlic bulbs before distribution in the market, but to date no suitable technique has been developed. The objective of this study was to identify specific odour-active compounds associated with nematode-infected garlic through the use of analytical chemistry. Garlic cloves were infected with cultured nematodes under controlled conditions, and the volatile odour-active compounds were analysed by an ‘odour identification system’ which included gas chromatography-olfactometry (GC-O), GC-fractionation (GC-F), GC-mass spectrometry (GC-MS), and odour/aroma-specific database. Two specific odour-active compounds associated with nematode-infected garlic cloves were identified as allyl methyl disulfide and eugenol, and two others were tentatively identified as (E)-1-allyl-2-(prop-1-en-1-yl)disulfane and allyl methyl trisulfide. These specific odour-active compounds may be useful as indicators to detect nematode-infected garlic bulbs in a non-destructive manner.

METHODOLOGICAL INSTRUCTIONS FOR ASSESSING POTATO VARIETIES FOR RESISTANCE TO STRAWBERRY NEMATODE UNDER LABORATORY CONDITIONS

The tuber nematode Ditylenchus destructor is the causative agent of ditylenchiasis, or dry rot of potatoes, and causes significant damage to the crop both during the growing season and during storage. Methods for evaluating potato varieties for resistance to tuberous nematode in laboratory conditions have been developed to reduce the search term for a source or donor of nematode resistance. Methods for obtaining a suspension of a tuber nematode, methods of inoculation of tubers, a method for assessing potato varieties for susceptibility and resistance to the tuberous nematode Ditylenchus destructor are presented. Infection of tubers is carried out by introducing a suspension of nematodes into the wounds inflicted on the potato tubers. 6 methods were tested. The best infection and reproduction of nematodes occurred when nematodes were introduced into the “lattice”, “holes”, “pyramids”. Depending on the value of the average score of the lesion by ditylenchiasis and reproduction by the tuber nematode, the varieties are divided into 5 groups: highly resistant (average score 0), resistant (0.1–1), moderately resistant (1.1–3), susceptible (3.1–4), highly susceptible (4.1–5).

METHODOLOGICAL INSTRUCTIONS “METHODOLOGY FOR IN VITRO SCREENING OF STRAINS, ISOLATES OF BACTERIA WITH PARASITIC AND NONMATICIDAL PROPERTIES”

The aim of this work was to develop a sequence for in vitro screening of isolates and strains of bacteria with similar activity on nematodes of various ecological groups, respectively, differing in the degree of cuticle permeability and having a different structure of the stoma, which is fundamental for the penetration of bacterial spores and toxins. The objects of screening were the saprobic nematode Caenorhabditis elegans, vinegar eel (Turbatrix aceti), mycohelminths Aphelenchus avenae, Aphelenchoides saprophilus, Paraphelenchus, migrating phytohelminths Ditylenchus destructor, larvae of gall nematodes of the genus Meloidyne. The proposed screening scheme makes it possible to comprehensively assess the effect of various strains and isolates on nematodes of various ecological groups. Strains, bacterial isolates that cause immobilization or death of nematodes, are reevaluated in vitro for other nematode species. In the case when the maximum mortality is observed in the variant with the undiluted isolate suspension, and when the suspension is diluted from minimum to maximum, there is a decrease in mortality to a minimum or does not differ statistically from the control, then a nematicidal effect of the metabolite products of the tested bacteria can be assumed. With a well-organized microbiological part, this algorithm can provide significant material for identifying bacteria with the desired properties, which in the future could serve as the basis for creating new drugs for protecting plants from nematodes and other pathogenic organisms.