RESUMEN
BACKGROUND: Plant-parasitic nematodes are economically important pests responsible for substantial losses in agriculture. Researchers focusing on plant-parasitic nematodes, especially on finding new ways of their control, often need to assess basic parameters such as their motility, viability, and reproduction. Traditionally, these assays involve visually counting juveniles and eggs under a dissecting microscope, making this investigation time-consuming and laborious. RESULTS: In this study, we established a procedure to efficiently determine the motility of two plant-parasitic nematode species, Heterodera schachtii and Ditylenchus destructor, using the WMicrotracker ONE platform. Additionally, we demonstrated that hatching of the cyst nematode H. schachtii can be evaluated using both the WMicrotracker ONE and by assessing the enzymatic activity of chitinase produced during hatching. CONCLUSIONS: We present fast and straightforward protocols for studying nematode motility and hatching that allow us to draw conclusions about viability and survival. Thus, these methods are useful tools for facilitating fast and efficient evaluation in various fields of research focused on plant-parasitic nematodes.
RESUMEN
Viticulture is a traditional branch of agriculture in the Czech Republic. Grapevines (Vitis vinifera L.) are cultivated on more than 18,000 hectares in the wine-growing regions of Bohemia and South Moravia. South Moravia alone accounts for more than 90 % of the total wine-growing area in the country. Grapevine yellows are a complex of diseases associated with the phytoplasma presence. Phytoplasmas of at least five different groups can cause similar symptoms in grapevines, and they can be distinguished only on a molecular basis (EPPO 2016). One of them, the grapevine Flavescence dorée phytoplasma (GFDP), which belongs to the 16SrV group, is listed in Annex II, Part B, of the Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 as a Union quarantine pest known to occur in the Union territory. Official surveys for GFDP in the Czech Republic have been carried out since 2007. In 2016, the first occurrence of Scaphoideus titanus Ball, 1932, the main vector of GFDP, was reported in the South Moravian Region (EPPO Reporting Service 2016). This is a matter of concern because it indicates that there is a risk of disease dissemination to other geographical locations. In September 2021, a total of 250 samples of V. vinifera (preferentially focused on symptomatic plants) and four samples of the wild plant host Clematis vitalba L. were collected from 50 vineyards in South Moravia. Total DNA was extracted using High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). For phytoplasma screening, a real-time PCR test for generic detection of phytoplasmas was used (Christensen et al. 2004). Samples evaluated as positive were further tested by PCR using phytoplasma universal P1 and P7 primers (Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR using the 16SrV group-specific primers fB1 and rULWS1 (Smart et al. 1996). For identification of 16SrV phytoplasma, sequence analysis of the secY-map genetic locus was performed. Two sets of primers were used: FD9f5/MAPr1 primers for the first PCR and FD9f6/MAPr2 for the nested PCR (Arnaud et al. 2007) with PCRBIO TaqMix (PCR Biosystems, London, UK). The nested PCR products were purified and sequenced (Eurofins Genomics, Ebersberg, Germany). The sequences were compared with sequences from the GenBank database. Phytoplasma of the 16SrV group was detected in three samples: V. vinifera cv. Gewürztraminer with symptoms of leaf reddening with no rolling and no other typical symptoms; C. vitalba L. with leaf curling (Fig. 1A); symptomless C. vitalba. The obtained sequences of the secY-map locus of all three 16SrV-positive samples were identical to the sequence of GFDP, isolate Vv-SI257 (Acc. No. FN811141), detected in grapevine in Tuscany (Italy), which belongs to 16SrV group. The sequence of the V. vinifera cv. Gewürztraminer isolate was submitted to GenBank under Acc. No. OQ185203. This isolate belongs to the Map-FD3 cluster (Fig. 1B), and the genotype identified is M51 (corresponding to FD-C), which has already been found in C. vitalba and outbreaks of Flavescence dorée in grapevines in some other European countries (Malembic-Maher et al. 2020). Based on the abovementioned results, this is the first report of the GFDP in the Czech Republic.
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Nepenthespudica, a new species from North Kalimantan, Indonesia, is described and illustrated. The species belongs to the N.hirsuta group (sensu Cheek and Jebb 1999) but exhibits some characters that are unique within the group or even within the genus. Above all, it produces underground, achlorophyllous shoots with well-developed, ventricose lower pitchers that form in soil cavities or directly in the soil. No lower pitchers are formed above ground. The main part of its prey are ants, besides other litter- and soil-inhabiting species of invertebrates. A number of infaunal species were found in both aerial and underground pitchers, mainly Diptera and nematodes. Nepenthespudica is known only from a few neighbouring localities in the Mentarang Hulu district of North Kalimantan, where it grows on ridgetops at an elevation of 1100-1300 m. Its discovery underlines the natural richness of Borneo's rainforest and the necessity to preserve this important ecosystem with its enormous and still undiscovered biodiversity.
RESUMEN
A new clitellate species of Pristininae (Naididae), Pristina armata n. sp., found in the pitcher of the carnivorous plant Nepenthes sp., is reported from East Kalimantan, Indonesia. P. armata n. sp. is a very small clitellate, less than 1 mm long in fixed state, and without proboscis on the prostomium. Signs of reproduction by paratomy were observed, but the generic placement remains preliminary because sexually mature individuals were not found. P. armata n. sp. is characterized by giant hook-like dorsal chaetae at IV. The description of P. armata n. sp. was based on six fixed specimens of different size and stage of development. Noteworthy is the habitat of P. armata n. sp. in Nepenthes pitchers, this being the first clitellate species described from such a habitat. P. armata n. sp. may be a member of the nepenthebionts' community, realizing its life cycle inside the digestive fluid of the Nepenthes pitcher, or it belongs to nepenthephiles, species that commonly occur in this habitat but do not specialize on it.