Summary Root-knot nematodes Meloidogyne chitwoodi and M. fallax , major soil-borne pathogens of potato, are listed as quarantine pests in the European Union. This classification requires the use of rapid and reliable tools for monitoring such organisms and thus preventing their potential introduction and spread. In this study, we developed and validated two specific and sensitive real-time polymerase chain reaction (PCR) assays in order to identify these two species from isolated individuals and to detect them in soil extracts. The two primer pairs designed, McN and MfN, targeting species-specific regions of the intergenic spacer (IGS) rDNA locus, enabled the amplification of DNA of all the target populations tested and no cross-reaction was observed when tested against a large number of non-target phytoparasitic nematodes. The identification assay was highly sensitive, being effective at a threshold of one female and one second-stage juvenile (J2) and was not impacted by the presence of other nematodes in the samples. The detection assay was able to reliably detect five J2 of M. chitwoodi or M. fallax in a soil extract and is usable in duplex format, thus allowing the simultaneous detection of both species. Our detection assay was also validated with the incorporation of a heterologous internal amplification control (IAC), thus improving our assay for routine diagnostics. The real-time PCR assays also demonstrated high levels of repeatability and reproducibility and was tested with success on routine samples. These assays have also been the subject of interlaboratory testing, confirming their robustness. These new PCR assays provide rapid and accurate identification and detection tools for use in diagnostic laboratories.
Summary Root-knot nematodes (RKN; Meloidogyne spp.) are among the most damaging pathogens of vegetable crops worldwide. They damage roots and reduce yield, with their management becoming challenging because of their wide host range. This study evaluated three microbial bioproducts, Mytech ® ( Purpureocillium lilacinum ), Trichotech ® ( Trichoderma asperellum ) and Regain ® ( Bacillus subtilis ) and a seaweed-based biostimulant (Maximizer ® ) for growth promotion and RKN management in capsicum and spinach. The fungal and bacterial bioproducts were cultured to provide fresh inocula for each experimental trial. In RKN-free pot experiments, Mytech ® and Trichotech ® consistently improved plant height, shoot and root fresh weight and total plant weight in capsicum, whereas Regain ® and Mytech ® enhanced plant height, shoot fresh weight and total plant weight in spinach compared with the non-treated control. In RKN-infected pot experiments, where microbial bioproducts or the seaweed-based biostimulant were applied before nematode inoculation, all treatments using bioproducts and biostimulant improved growth parameters, although responses varied between crops and repeated experiments. All bioproducts and biostimulant treatments significantly reduced nematode infection in both crops. However, significant reductions in nematode density, number of galls and reproduction factor were observed only with Regain ® and Mytech ® in spinach. These findings indicate that microbial bioproducts and seaweed-based biostimulants can promote vegetable growth and reduce RKN infection, although their effects on nematode density are treatment- and crop-dependent. Further field validation of these products is needed to establish their effects on marketable yield and economic returns under farmer conditions.
The awl nematode Dolichodorus heterocephalus was first described by Nathan Augustus Cobb in 1914, based on a female specimen from Silver Springs in Florida and a male from Douglas Lake in Michigan. Later, Golden et al. (1986) designated a female and male from Silver Springs as lectotypes, providing limited morphometric data while incorporating more detailed morphological observations from additional populations collected in Florida and Massachusetts. Their study highlighted considerable intraspecific variability and noted discrepancies in body size between the female lectotype and other Florida populations. In the present study, a topotype sample from Silver Springs, Florida was examined using an integrative approach combining morphological data and molecular analyses of sequences from the 18S rRNA, D2-D3 expansion segments of 28S rRNA and partial COI genes. With exception of a population from north Florida that was analysed morphologically and molecularly, like the topotype specimens, the several populations collected from other Florida localities were analysed only molecularly. Photomicrographs of selected morphological features were also taken. Morphological comparisons revealed that some adult females from the topotype sample exceeded 3 mm in body length, consistent with the lectotype and larger than those reported for other populations. Stylet lengths in both topotype females and males were greater than those of the lectotypes but remained within the range observed in other Florida populations. This notable intraspecific variability, especially in the populations from south Florida, was reflected in COI gene sequences. Despite these differences, the results of combined morphological and molecular analyses did not support significant differentiation among the studied populations and confirmed their taxonomic status as representative of a single species, D. heterocephalus. Overall, these findings provide updated reference data that improves diagnostic accuracy of this awl nematode.
Bursaphelenchus paragerberae n. sp., isolated in acacia wood chips from Vietnam is described. It is characterised by a slim body (a = 23-31), lateral field with three lines, excretory pore posterior to metacorpus, vulva at 72% of total body length, a very small vulval flap in lateral view, post-uterine sac well developed, extending to half of the vulva-anus distance, tail conical, smoothly tapering and slightly ventrally bent, 3.1-4.2 anal body diam. long, with a sharply pointed terminus, male spicules mitten-shaped, short and straight, with high and rounded condylus, pointed rostrum at the middle of the spicules, cucullus absent, a single precloacal papilla (P1) and three pairs of ventro-sublateral papillae present (P2-P4). It belongs to the hofmanni group of the genus Bursaphelenchus. Phylogenetic analyses based on partial 18S, ITS, and 28S D2-D3 regions confirm its status as a new species.
Insect-associated nematodes are an ecologically important yet understudied component of soil biodiversity, particularly in tropical and subtropical agroecosystems. Here, we report the first molecular characterisation and comparative analysis of Metarhabditis costai from India by combining morphological and molecular approaches. The studied population was isolated from the rhizosphere of Sorghum bicolor L. in Jaipur, Rajasthan, using the Galleria mellonella baiting method. The studied population of the species is characterised by a body length of 0.87-1.25 mm length in females; a finely straited cuticle; lateral fields with five incisures; a lip region continuous with the adjacent body, small, oval amphidial openings; and a rhabditoid stoma with a buccal tube approximately four times longer than wide. The pharynx possesses a slightly swollen metacorpus, with nerve ring and excretory pore located at level of the isthmus. Females are didelphic-amphidelphic, with a slightly protruding vulva, a rectum measuring 0.7-1.4 times the anal body width, a conical-elongate tail with an acute terminus (84-114 & micro;m, c = 9.1-14.2, c ' = 3.8-4.5), and phasmids located at 26-42% of tail length. Males are monorchic, with spicules 25-38 & micro;m long, with a truncated manubrium, and a straight lamina with a ventrally bent tip; gubernaculum 11-18 & micro;m long; bursa leptoderan with eight genital papillae (1 + 1 + 1/3 + 2) plus a fine phasmid posterior to GP8, and a conical tail (20-32 & micro;m, c = 21-34, c ' = 0.8-1.5) with a filiform extension beyond the bursa. Compared with the type population, intraspecies variations were observed in stoma length, distance from anterior end to the nerve ring and pharynx length in females. In males, variations were observed in pharynx length, maximum body diam., tail length, gubernaculum and total body length. Molecular phylogenetic analyses based on 18S and ITS rDNA sequences were conducted for the first time for this species. In Bayesian phylogenetic reconstructions, the newly generated sequences from the Indian population clustered with available sequences of the genus in both datasets, forming well-supported clades.
