Root-knot nematodes (RKN; Meloidogyne spp.) can parasitize over 2,000 plant species and are generally considered to be the most agriculturally damaging group of plant-parasitic nematodes worldwide. Infective juveniles (J2) are non-feeding and must locate and invade a host before their reserves are depleted. However, what attracts J2 to appropriate root entry sites is not known. An aim of this research is to identify semiochemicals that attract RKN to roots. J2 of the three RKN species tested are highly attracted to root tips of both tomato and Medicago truncatula. For both hosts, mutants defective in ethylene signaling were found to be more attractive than those of wild type. We determined that cell-free exudates collected from tomato and M. truncatula seedling root tips were highly attractive to M. javanica J2. Using a pluronic gel-based microassay to monitor chemical fractionation, we determined that for both plant species the active component fractionated similarly and had a mass of ~400 based on size-exclusion chromatography. This characterization is a first step toward identification of a potent and specific attractant from host roots that attracts RKN. Such a compound is potentially a valuable tool for developing novel and safe control strategies.

Recognition of specific molecule signatures of microbes, including pathogens, induces innate immune responses in plants, as well as in animals. Analogously, a nematode pheromone, the ascaroside ascr#18, induces hallmark plant defences including activation of (a) mitogen‐activated protein kinases, (b) salicylic acid‐ and jasmonic acid‐mediated defence signalling pathways and (c) defence gene expression and provides protection to a broad spectrum of pathogens. Ascr#18 is a member of an evolutionarily conserved family of nematode signalling molecules and is the major ascaroside secreted by plant–parasitic nematodes. Here, we report the effects of ascr#18 on resistance in four of the major economically important crops: maize, rice, wheat and soybean to some of their associated pathogens. Treatment with low nanomolar to low micromolar concentrations of ascr#18 provided from partial to strong protection in seven of eight plant–pathogen systems tested with viruses, bacteria, fungi, oomycetes and nematodes. This research may have potential to improve agricultural sustainability by reducing use of potentially harmful agrochemicals and enhance food security worldwide.

Meloidogyne haplanaria is a species originally found infesting peanut in Texas and, more recently, in Arkansas. In this study, we confirmed the presence of M. haplanaria in Florida based on morphological and molecular characterization. This species was identified from a sample submitted for diagnosis collected from Mi-resistant tomato rootstock grown in Naples, FL. The major diagnostic criteria to distinguish M. haplanaria from other closely related root-knot nematode (RKN) species are based on morphological differences and host range tests, which are time consuming and labor intensive and require living or well-preserved specimens. In our study, we provide an easy diagnostic strategy to distinguish M. haplanaria from other RKN species based on amplification of two mitochondrial DNA regions. These regions span the intergenic spacer and part of the adjacent large subunit ribosomal RNA gene (lrDNA) and sequence polymorphisms in lrDNA revealed by the restriction pattern following digestion with the restriction enzymes HinfI and MnlI. A unique haplotype pattern, which has not been observed in any of the RKN species described thus far, was observed in M. haplanaria. The outcome of molecular analysis of M. haplanaria aligned with morphological measurement and characteristics as well as perineal pattern originally described for M. haplanaria.

The in vitro nematicidal effects of an aqueous garlic extract, salicylaldehyde, a nonylphenol ethoxylate surfactant and a formulation containing these constituents were evaluated against the potato cyst nematode, Globodera pallida. Newly hatched, infective second-stage juveniles (J2) were placed for 24, 48 and 72 h in solutions containing concentrations of the formulation from 30.0-80.0 μl l −1 with 20% (v/v) potato root leachate and sterile distilled water controls. The garlic extract, salicylaldehyde and surfactant treatments were assessed at concentrations proportional to their occurrence in the formulation. Hatching assays involved a series of experiments in which G. pallida cysts were incubated for 8 weeks in potato root leachate solution containing different concentrations of the test substances. A second set of experiments involved incubating cysts in different concentrations of the test substances for 2, 4 and 8 weeks prior to hatching in potato root leachate solution to determine how prior exposure to these substances influences hatching and inegg viability. The formulation caused 100% mortality at 75.0 μl l −1 with an LC 50 of 43.6 μl l −1 after 24 h exposure. Salicylaldehyde was the most toxic constituent of the formulation with an LC 50 of 6.5 μl l −1 after 24 h, while the garlic extract achieved 50% J2 mortality at 983.0 μl l −1 , demonstrating that the formulation and salicylaldehyde are more toxic to G. pallida in vitro than oxamyl but less toxic when compared with aldicarb. The surfactant showed no dose-dependent toxic effects on J2 when compared with the controls. Emergence of J2 from the cysts was significantly reduced by concentrations of the formulation above 688.0 μl l −1 and its equivalent concentration of salicylaldehyde, while concentrations of the formulation above 2752.0 μl l −1 and the corresponding salicylaldehyde concentrations resulted in complete irreversible hatch inhibition. Concentrations of the garlic extract below 137.6 μl l −1 caused 26% more J2 hatch in comparison to the potato root leachate solution. This study has shown that salicylaldehyde is more toxic to nematodes than the garlic extract, and is the first report of a hatch stimulatory effect of a garlic extract on G. pallida under in vitro conditions.

Aims To generate single spore lines of a population of bacterial parasite of root‐knot nematode (RKN), Pasteuria penetrans, isolated from Florida and examine genotypic variation and virulence characteristics exist within the population. Methods and Results Six single spore lines (SSP), 16SSP, 17SSP, 18SSP, 25SSP, 26SSP and 30SSP were generated. Genetic variability was evaluated by comparing single‐nucleotide polymorphisms (SNPs) in six protein‐coding genes and the 16S rRNA gene. An average of one SNP was observed for every 69 bp in the 16S rRNA, whereas no SNPs were observed in the protein‐coding sequences. Hierarchical cluster analysis of 16S rRNA sequences placed the clones into three distinct clades. Bio‐efficacy analysis revealed significant heterogeneity in the level virulence and host specificity between the individual clones. Conclusions The SNP markers developed to the 5′ hypervariable region of the 16S rRNA gene may be useful in biotype differentiation within a population of P. penetrans. Significance and Impact of the Study This study demonstrates an efficient method for generating single spore lines of P. penetrans and gives a deep insight into genetic heterogeneity and varying level of virulence exists within a population parasitizing a specific Meloidogyne sp. host. The results also suggest that the application of generalist spore lines in nematode management may achieve broad RKN control.

In vitro and glasshouse studies were initiated to determine the mechanisms of action of an aqueous garlic extract, salicylaldehyde, a nonylphenol ethoxylate surfactant and a formulation (G8014S) containing these substances on the potato cyst nematode Globodera pallida. The effects of these substances on host finding and migration of second-stage juveniles (J2) were assessed by investigating their ability to migrate through a sand column and towards potato roots in Pluronic F-127 gel. The garlic extract was shown to be the least disruptive to J2 host finding, reducing attraction by between 13 and 24% at concentrations of 7840 ß\ 1~' and 983 (Ul r ', respectively. By contrast, a reduction of approximately 38% in J2 attraction to roots was achieved by treating J2 with 344 /xl r' of G8014S. Of all the substances tested, salicylaldehyde was the least attractive to G. pallida J2 (4.1%) while the garlic extract and oxamyl treatments attracted 43.5 and 28.9% J2, respectively. In glasshouse experiments, different rates of the test substances were compared with the nematicide oxamyl for their effects on the in-soil hatch, egg viability and multiplication of G. pallida. The effect of treatment with 220.16 1 ha~' of G8014S on G. pallida multiplication in the glasshouse was comparable to that of oxamyl causing a five-fold reduction in PCN multiphcation relative to the water control. These results provide evidence to support the potential of the formulation for the control of G. pallida in potatoes.

Meloidogyne spp. (root‐knot nematodes) are the most damaging plant‐parasitic nematodes to commercial agriculture. Currently used nematicides are of limited effectiveness as well as toxic to other organisms. The present study examines whether disruption of cyclic nucleotide signaling pathways using phosphodiesterase (PDE) inhibitors could serve as the basis for novel nematicides with greater efficacy and reduced environmental impact. To explore this, we carried out an evolutionary analysis of nematode phosphodiesterases (PDEs) and found that nematodes contain only 6 of the 11 members of the vertebrate PDE superfamily. Evolutionary trace analysis reveals nematode‐specific amino acid residues in the drug binding pocket of several nematode PDEs, suggesting the feasibility of developing compounds that selectively inhibit individual nematode PDEs. We also examined the physiological effects of PDE inhibitors on both C. elegans and M. hapla. We found that IBMX (non‐selective), cilostazol and milrinone (PDE3‐selective), rolipram (PDE4‐selective), and papaverine (PDE10 inhibitor) can reduce C. elegans dauer locomotion velocity by 30‐50%. IBMX, cilostazol, and rolipram also reduce by 50‐70% the chemotactic response of C. elegans to salt. Treatment of juvenile M. hapla with cilostazol or rolipram reduced by 蠅50% their ability to invade plant roots. We conclude that inhibitors that target individual PDE families present in nematodes can disrupt locomotion, chemosensation, and/or processes required to invade plant roots, and thus may have nematicidal applications.This work was supported by the New Hampshire Agricultural Experiment Station (USDA).

The cover image, by Daniel F. Klessig et al., is based on the Original Article Nematode ascaroside enhances resistance in a broad spectrum of plant–pathogen systems. DOI: . Photo credit: Cover image © Aardra Kachroo University of Kentucky Images