Irrigation and Rootstocks to Manage Northern Root-Knot Nematode During Wine Grape Vineyard Establishment

Vineyard replanting in Washington state can be negatively impacted by the plant-parasitic nematode Meloidogyne hapla. Chemically-focused nematode management programs do not offer long-term suppression, however, this may be achieved through the adoption of cultural approaches such as rootstocks and irrigation. Nematode-resistant rootstocks are used extensively in other regions, but many have not been tested against M. hapla. Vineyards in eastern Washington are irrigated, so manipulating available soil water may also impact nematode development. In 2017, two field trials were established in eastern Washington to evaluate the effects of: 1) late-summer water limitation on M. hapla population development, and 2) host status of 1103 Paulsen, 3309 Couderc and Matador rootstocks for M. hapla. The efficacy of these cultural management approaches was evaluated under three initial M. hapla densities (0, 50, and 250 M. hapla J2 per 250 g soil) in both trials. Reducing irrigation to manage M. hapla infestation of grape roots was ineffective and may cause harm to the vines by inducing too much water stress. Conversely, rootstocks effectively reduced population densities of M. hapla. Overall, rootstocks show the most promise as a cultural tool to manage M. hapla during the establishment phase in Washington vineyards.

Occurrence and seasonal changes in the population of root-knot nematodes on honeybush (Cyclopia sp.)

Summary Root-knot nematodes in the genus Meloidogyne are an important group of plant-parasitic nematodes causing severe damage on agricultural crops worldwide. A study was conducted to identify the species of root-knot nematodes causing damage on honeybush monocultures and to assess the seasonal variations in the nematode population. Soil samples were collected from six experimental sites in Genadendal, Western Cape province of South Africa from 2016 to 2017. DNA was extracted from single-second stage juveniles and species identifi cation was done using species-specifi c sequence-characterised amplifi ed regions (SCAR) primers. Meloidogyne hapla and M. javanica were identifi ed from the sites. Mean population density of the nematodes varied significantly (p < 0.05) in the six sites, with the peak population being recorded in summer of 2017. The study suggests that seasonal variation in temperature and moisture could contribute to changes in the population density of root-knot nematodes in the soil.

Early detection of the Root-knot Nematode Meloidogyne hapla through developing a robust qPCR approach compliant with the MIQE guidelines

Root-knot nematodes (RKNs) are major threats to crops through attacking the roots, which induces an abnormal development of the plant. Meloidogyne hapla Chitwood is of particular concern as it is currently expanding its distribution area and displays a wide host range. Effective plant protection against this RKN requires early detection as even a single individual can cause severe economic loses on susceptible crops. Molecular tools are of particular value for this purpose and among them, qPCR presents all the advantages, i.e. sensitivity, specificity, rapidity of diagnosis at a reduced cost. Although few studies already proposed detecting M. hapla through this technique, they lack experimental details and performance testing, and suffered from low taxonomic resolution and/or required expensive hydrolysis probes. Here we propose a qPCR detection method that uses SybrGreen with developed primers amplifying a fragment of COI mitochondrial region. The method is developed and evaluated following the MIQE guidelines to ensure its quality, i.e. sensitivity, specificity, repeatability, reproducibility, robustness. The results demonstrate that the newly developed method fulfills its goals as it proved specific to M. hapla and allowed for a reproductible detection level as low as 1.25 equivalent of a juvenile individual. All criteria associated with the MIQE guidelines were also met, what makes it of general use for the reliable early detection of M. hapla.

Precrop Effect of Red Clover (Trifolium pratense L.) and Alfalfa (Medicago sativa L.) on the Population Development of the Northern Root-Knot Nematode Meloidogyne hapla Chitwood, 1949 and on Succeeding Crops—A Pot Study

The northern root-knot nematode, Meloidogyne hapla, is a major pest of many crop species. The objective of the study was to determine how M. hapla population dynamics is affected by two precrops, i.e., Trifolium pratense and Medicago sativa, in three crop durations: one, two and three years of continuous cultivation. Moreover, we set ourselves the task of evaluating the effect of the legume precrop soil on the growth of the succeeding tomato plant (Solanum lycopersicum) and on the nematode population. The experiment was performed outdoors in pots with naturally infected soil. Both precrop species investigated were found to modify the J2 nematode population density in the soil. The galls and nematode females with egg masses were observed on the roots of both studied plant species at the end of each growing season. They appeared to be more abundant on the red clover roots than on those of the alfalfa. The obtained data indicate that the spring soil sampling is more appropriate for the estimation of the M. hapla population density in the red clover precrop soil. The legume precrop soil had a limiting effect on tomato growth and fruit yield. The nematode population negatively influenced tomato growth. The experiment revealed that tomato plants could be planted in alfalfa precrop soil following at least three years of continuous alfalfa cultivation. The same cannot be said of the cultivation of red clover as a precrop for tomatoes.

Occurrence of Meloidogyne hapla relative to nematode abundance and soil food web structure in soil groups of selected Michigan vegetable production fields

Summary Despite considerable knowledge of distribution, biology and parasitic variability (PV) of Meloidogyne hapla in cropping systems, how its PV relates to soil health conditions remains unknown. This study investigated the relationship between the presence or absence of M. hapla with soil food web and the abundance of nematode community in muck (high organic matter) and mineral soils of 15 agricultural fields and adjacent natural vegetation across three vegetable production regions of Michigan, USA. Meloidogyne hapla was present in all regions and all muck soils, but only in some mineral soils. It was present in soils with degraded and disturbed soil food web conditions, but there was no pattern with nematode trophic group abundance. However, principal component analysis showed distinct relationships among M. hapla presence or absence, soils, nematode abundance parameters in agricultural and natural vegetation. The study lays down a foundation for more targeted investigations to understand any links between the PV of M. hapla and its soil environment.

First report of Meloidogyne hapla infecting Yunmuxiang (Aucklandia lappa) in China

Yunmuxiang (Aucklandia lappa) is a tall, perennial herbaceous plant in the compositae family, occurring mainly in Asia and Europe. Yunmuxiang originated in India and was introduced into China in approximately 1940. Since then it has been widely cultivated in the southwest region of China for medicinal uses; it is included in the Chinese Pharmacopoeia. Yunmuxiang is used primarily as a sedative, including for anesthesia (Ting et al. 2012). Severely stunted and withered Yunmuxiang plants with rotted and galled roots were observed in a field in near the city of Lijiang (N 99°46′; E 27°18′) in October 2019. These symptoms were typical of infection by root-knot nematodes.The second-stage juveniles (J2) were collected from the soil in the root zone, and adult females were dissected from roots. Population densities of J2 ranged from 325 to 645 per 100 cm3. Morphological analysis and species-specific PCR were performed on the second stage (J2) and females. Morphological characteristics are as follows: for J2 (n=20) , body length = 360.5 ± 23.4 µm, tail length = 47.2 ± 6.1 µm, and stylet length = 10.4 ± 1.9 µm, distance from dorsal esophageal gland opening to the stylet knot (DGO) = 3.96 ± 0.42 μm; females (n = 20) were pear-shaped, body length = 565.23 ± 86.68 μm, maximum body width = 407.24 ± 60.21 μm, stylet length = 9.93 ± 0.88 μm, DGO = 4.76 ± 0.32 μm, stylet median bulb width (MBW) = 29.67 ± 3.61 μm, perineum morphology is low and low dorsal arch round, with a typical inferior protrusion near the anus. These morphological characteristics are consistent with Meloidogyne hapla as described by Hunt and Handoo (2009). To confirm species identification, DNA was extracted from females (Blok, et al. 1997) and ITS region was amplified using the primers 18S/26S (Vrain et al. 1992). Furthermore, species-specific SCAR primers JMV1/JMV hapla were used as described by Adam et al. (2007). PCR produced 768 bp and 419 bp sequences. Fragments were sequenced (MW512922and MW228371, respectively) and compared with available sequences on NCBI. Sequences were 99.48% identical to the MT249016, KJ572385, and 100% identical to the GQ395574, GQ395569 M. hapla sequences, respectively. Morphological and molecular characterization supports the identification of the isolate found on Aucklandia lappa as M. hapla. Yunmuxiang seed were planted in 20 cm diameter, 10 cm deep plastic pots containing 1000 cm3 sterilized soil. Seedlings were thinned to one per pot. At the 2-3 leaf stage 10 pots were infested with 1500 M. hapla J2 per seedling, using a sterilized micropipette. Plants were maintained at 20-25°C in a greenhouse. Control plants received sterile water, and the pathogenicity test was repeated three times. After 30 days, plants were removed from pots and soil gently removed from the roots. A large number of galls (95.6 ± 2.5) and egg masses (33.5 ± 0.5) were found on each root system. Yunmuxiang was considered a good host for M. hapla in Lijiang. M. hapla is a major plant parasitic nematode with a wide geographic distribution and range of host plants and causes severe yield losses (Azevedo de Oliveira et al. 2018). Through investigation, this is the first report worldwide of M. hapla infecting Aucklandia lappa.

Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode, Meloidogyne hapla, Using Recombinase Polymerase Amplification Assays

Rapid and reliable diagnostics of root-knot nematodes are critical for selections of effective control against these agricultural pests. In this study, recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the northern root-knot nematode, Meloidogyne hapla. The RPA assays using TwistAmp® Basic, TwistAmp® exo and TwistAmp® nfo kits (TwistDx, Cambridge, UK) allowed for the detection of M. hapla from crude extracts of females, eggs and juveniles without a DNA extraction step. The results of the RPA assays using real-time fluorescence detection (real-time RPA) in series of crude nematode extracts showed reliable detection after 13 min with a sensitivity of 1/100 of a second-stage juvenile and up to 1/1000 of a female in reaction tubes. The results of the RPA assays using lateral flow dipsticks (LF-RPA) showed reliable detection within 30 min with a sensitivity of 1/10 of a second-stage juvenile and 1/1000 of a female in reaction tubes. The RPA assay developed here is a successful tool for quick, accurate and sensitive diagnostics of M. hapla. The application of the LF-RPA assay has great potential for diagnosing infestation of this species in the lab, field or in areas with a minimal laboratory infrastructure.

Field Validation of the Effect of Soil Fumigation of Ethanedinitrile (EDN) on the Mortality of Meloidogyne hapla and Carrot Yield Parameters

With the increasing importance of soilborne plant pest nematodes and the relatively recent phase-out of methyl bromide as a key soil fumigant, there is an urgent need for new fumigants with good nematicidal properties. Ethanedinitrile (EDN) is a promising fumigant and preparation because of its physical, agrochemical, and nematicidal properties. However, its efficacy against nematode pests of different crops under field conditions has not been fully validated and understood. Therefore, the aim of our study was to evaluate the effect of two concentrations of EDN on the survival of northern root-knot nematodes (Meloidogynehapla) and on carrot yield and quality under field conditions. The evaluation was performed using naturally infested carrot plots, and EDN application to the soil was followed by covering the treated area with film. A high biological effect was observed for both EDN concentrations tested, with even the lower dose (30 g·m−2) being sufficient for M. hapla suppression. Positive effects of EDN application—likely due to EDN partial conversion into biologically available nitrogen in soil—on carrot root weight, in comparison with the untreated control, were also observed.

The centromere histone is conserved and associated with tandem repeats sharing a conserved 19 bp box in the holocentromere of Meloidogyne nematodes

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 (MIG), 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) copy remained conserved in all extant species, including in distant Meloidogyne hapla, while the other copy evolved rapidly and under positive selection into four different CenH3 variants. This pattern of CenH3 evolution in Meloidogyne species suggests the sub-specialization of CenH3s in ancestral sexual species. Immunofluorescence performed on mitotic Meloidogyne incognita revealed a dominant role of αCenH3 on its centromere, while 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 are also confirmed in the related mitotic MIG 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.