Comparative Metabolomic Analysis of Four Fabaceae and Relationship to In Vitro Nematicidal Activity against Xiphinema index.

The grapevine fanleaf virus (GFLV), responsible for fanleaf degeneration, is spread in vineyards by the soil nematode Xiphinema index. Nematicide molecules were used to limit the spread of the disease until they were banned due to negative environmental impacts. Therefore, there is a growing interest in alternative methods, including plant-derived products with antagonistic effects to X. index. In this work, we evaluated the nematicidal potential of the aerial parts and roots of four Fabaceae: sainfoin (Onobrychis viciifolia), birdsfoot trefoil (Lotus corniculatus), sweet clover (Melilotus albus), and red clover (Trifolium pratense), as well as that of sainfoin-based commercial pellets. For all tested plants, either aerial or root parts, or both of them, exhibited a nematicidal effect on X. index in vitro, pellets being as effective as freshly harvested plants. Comparative metabolomic analyses did not reveal molecules or molecule families specifically associated with antagonistic properties toward X. index, suggesting that the nematicidal effect is the result of a combination of different molecules rather than associated with a single compound. Finally, scanning electron microscope observations did not reveal the visible impact of O. viciifolia extract on X. index cuticle, suggesting that alteration of the cuticle may not be the primary cause of their nematicidal effect.

Selected Genotypes with the Genetic Background of Vitis aestivalis and Vitis labrusca Are Resistant to Xiphinema index.

The ectoparasitic nematode Xiphinema index transmits grapevine fanleaf virus (GFLV) during feeding on grapevine roots, causing fanleaf degeneration in the plant. Hence, resistance breeding is a key to develop novel rootstocks to overcome such threats. In past years, various grapevine species were screened, and a few candidates with partial resistance were identified. However, they were hardly sufficient for viticulture because of their many agronomical defects. To develop reliably resistant rootstocks applicable in viticulture, multiple Vitis spp. genotypes were analyzed using root inoculation with nematodes in glass vials as an early and easy evaluation test. Resistance levels were evaluated 35 days after inoculation based on nematode reproduction factors, focusing on juveniles and eggs. Infection of grapevines with GFLV was analyzed after inoculation with viruliferous X. index. With this fast screening system, putative candidates with resistances against X. index have been identified for future breeding programs. Particularly, genotypes with the genetic background of Vitis aestivalis and Vitis labrusca were found to be nematode-resistant.

Characterization of the Biogenic Volatile Organic Compounds (BVOCs) and Analysis of the PR1 Molecular Marker in Vitis vinifera L. Inoculated with the Nematode Xiphinema index.

Upon pathogen attack, plants very quickly undergo rather complex physico-chemical changes, such as the production of new chemicals or alterations in membrane and cell wall properties, to reduce disease damages. An underestimated threat is represented by root parasitic nematodes. In Vitis vinifera L., the nematode Xiphinema index is the unique vector of Grapevine fanleaf virus, responsible for fanleaf degeneration, one of the most widespread and economically damaging diseases worldwide. The aim of this study was to investigate changes in the emission of biogenic volatile organic compounds (BVOCs) in grapevines attacked by X. index. BVOCs play a role in plant defensive mechanisms and are synthetized in response to biotic damages. In our study, the BVOC profile was altered by the nematode feeding process. We found a decrease in ß-ocimene and limonene monoterpene emissions, as well as an increase in α-farnesene and α-bergamotene sesquiterpene emissions in nematode-treated plants. Moreover, we evaluated the PR1 gene expression. The transcript level of PR1 gene was higher in the nematode-wounded roots, while in the leaf tissues it showed a lower expression compared to control grapevines.

In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index.

Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family.

Nematicidal effect of rhizobacteria on plant-parasitic nematodes associated with vineyards.

The action of metabolites and exoenzymes from rhizobacteria on different plant-parasitic nematodes has an influence on the nematicidal efficacy of the microbe. Seven rhizobacteria, divided into two bacterial groups, were evaluated in vitro for nematicidal activity on Meloidogyne ethiopica and Xiphinema index. The direct effect of their filtrates on egg hatching and juveniles of M. ethiopica as well as mobile stages of X. index was evaluated during a 72-h period. The production of four exoenzymes and two metabolites associated with nematode mortality was investigated. Molecular characterization of three isolates was performed, and the physiological profiles and lipase activity of all isolates were obtained using the BIOLOG EcoPlate system. While chitinase and collagenase were measured using the BIOLOG MT2 plate system, protease, hydrogen cyanide and hydrogen sulphide were directly determined in Petri dishes. Nematode mobile stages exposure to the bacterial filtrate revealed a nematicidal effect up to 93.7% on X. Index and up to 83.3% on M. ethiopica. The control of egg hatching varied between 35 and 85%. A positive correlation was found between the mortality of both nematode mobile stages and the concerted activities of the bacterial enzymes as well as the level of the volatile metabolites. The nematicidal effect of rhizobacteria strains varies by nematode genera and among the developmental stages evaluated.

Nematicidal Activity of a Garlic Extract Formulation against the Grapevine Nematode Xiphinema index.

The nematicidal activity of garlic extracts is known on root-knot nematodes but never investigated on the grapevine nematode Xiphinema index. In this study, the nematicidal activity of a commercial garlic extract formulate (GEF) was assessed on X. index, both in vitro and in a pot assay. In the in vitro assays, mixed specimens of X. index were exposed to a 0-4 mL L-1 range of GEF concentrations, checking nematode immotility and mortality after 2, 4 or 8 h. In the experiments on potted grapevines, plants cultivated in soil infested by X. index were irrigated twice at a 15-day interval with 0.05, 0.2 and 0.5 mL L-1 solutions of GEF, including nontreated soil as a control. An almost complete mortality of X. index specimens occurred after a 2 h exposure to a 2 mL L-1 GEF concentration, while an 8 h exposure to even the 0.0312 and 0.0156 mL L-1 solutions resulted in about 50% and 30% mortality, respectively. Soil treatment with a 0.5 mL L-1 GEF solution significantly reduced the population of X. index and increased the grapevine root growth compared to nontreated soil or soil treated with the lower dosages. Results of this study indicated that garlic-based nematicides could be an effective tool for X. index management in organic and integrated vineyards.

Activity of Saponins from Medicago Species against Phytoparasitic Nematodes.

Content of bioactive saponins of Medicago species suggests that they may also exert, as previously demonstrated on M. sativa, nematicidal properties exploitable for the formulation of new products for sustainable phytoparasitic nematode management. This study was addressed to highlight the bioactivity of saponins from five different Medicago species still poorly known for their biological efficacy, i.e., M. heyniana, M. hybrida, M. lupulina, M. murex and M. truncatula, against the plant parasitic nematodes Meloidogyne incognita, Xiphinema index and Globodera rostochiensis. The bioactivity of the extracts from the five Medicago species was assessed by in vitro assays on the juveniles (J2) and eggs of M. incognita and G. rostochiensis and the adult females of X. index. The suppressiveness to M. incognita of soil treatments with the Medicago plant biomasses was also investigated in a tomato experiment. The nematicidal activity of the five Medicago species was reported and discussed in relation to their phytochemical profile.

Detection of Multiple Variants of Grapevine Fanleaf Virus in Single Xiphinema index Nematodes.

Grapevine fanleaf virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The virus is transmitted from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of virus diversity.

A draft genome sequence of Pseudomonas veronii R4: a grapevine (Vitis vinifera L.) root-associated strain with high biocontrol potential.

A new plant commensal Pseudomonas veronii isolate (strain R4) was identified from a Xiphinema index biocontrol screen. Isolated from grapevine roots from vineyards in central Chile, the strain R4 exhibited a slower yet equivalently effective nematicide activity as the well-characterized P. protegens CHA0. Whole genome sequencing of strain R4 and comparative analysis among the available Pseudomonas spp. genomes allowed for the identification of gene clusters that encode putative extracellular proteases and lipase synthesis and secretion systems, which are proposed to mediate-at least in part-the observed nematicidal activity. In addition, R4 strain presented relevant gene clusters related to metal tolerance, which is typical in P. veronii. Bioinformatics analyses also showed gene clusters associated with plant growth promoting activity, such as indole-3-acetic acid synthesis. In addition, the strain R4 genome presented a metabolic gene clusters associated with phosphate and ammonia biotransformation from soil, which could improve their availability for plants.

Nematicidal activity of allyl bromide and dibromo(nitro)methane under laboratory conditions.

BACKGROUND: Restrictions on soil fumigants are prompting the development of new compounds for controlling nematodes, other soilborne pathogens and weeds. We evaluated the nematicidal activity of five bromine compounds against Meloidogyne javanica in vitro, and tested the two most effective ones against Pratylenchus penetrans and Xiphinema index in vitro and in soil. RESULTS: Only allyl bromide and dibromo(nitro)methane showed nematicidal activity against M. javanica juveniles in vitro at <320 mg L(-1) . Allyl bromide killed M. javanica and P. penetrans at 20 mg L(-1), and X. index at 10 mg L(-1), whereas 320 mg dibromo(nitro)methane L(-1) was required to kill P. penetrans. Allyl bromide also showed higher nematicidal activity than dibromo(nitro)methane against M. javanica and P. penetrans in soil. Allyl bromide at 40 and 20 mg L(-1) soil eliminated root galls and nematode eggs on tomato roots grown in M. javanica-inoculated loess and sandy soils respectively, showing higher nematicidal activity than 1,3-dichloropropene. No P. penetrans were recovered from soil treated with 80 mg allyl bromide L(-1) soil or 320 mg dibromo(nitro)methane L(-1) soil. CONCLUSION: Allyl bromide showed high nematicidal activity against all three nematode species, and nematicidal activity of dibromo(nitro)methane was discovered. These compounds could serve as new fumigation nematicides, pending further experiments.

Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index.

The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.

Effects of Seasonal and Site Factors on Xiphinema index Populations in Two California Vineyards.

Sampling of Xiphinema index for 2 years (1993-95) in two California vineyards indicated that a greater number of nematodes occurred during the winter months. The number of juveniles increased four-fold from December 1993 to January 1994, indicating a high reproductive rate during this time. Extremely high or low soil temperatures corresponded to low nematode numbers. Samples were taken from 0 to 31 cm and 31 to 62 cm deep both within and between the vine rows. Numbers of nematodes were greatest at the 0- to 31-cm depth in one vineyard with a loamy sand soil, and at a depth of 31 to 62 cm in the second vineyard, which had a silt loam soil. In both vineyards, X. index population densities were greater within the vine row.

Influence of four nematodes on root and shoot growth parameters in grape.

Two grape cultivars, susceptible French Colombard and tolerant Rubired, and four nematodes, Meloidogyne incognita, Pratylenchus vulnus, Tylenchulus semipenetrans, and Xiphinema index, were used to quantify the equilibrium between root (R) and shoot (S) growth. Root and shoot growth of French Colombard was retarded by M. incognita, P. vulnus, and X. index but not by T. semipenetrans. Although the root growth of Rubired was limited by all the nematodes, the shoot growth was limited only by X. index. The R:S ratios of Rubired were higher than those of French Colombard. The reduced R:S ratios of Rubired were primarily an expression of reduction in root systems without an equal reduction in shoot growth, whereas in French Colombard the reduced R:S ratios were due to a reduction in both shoot growth and root growth and to a greater reduction in root growth than shoot growth. All nematodes reproduced equally well on both cultivars. Both foliage and root growth of French Colombard were significantly reduced by M. incognita and P. vulnus. Nematodes reduced the shoot length by reducing the internode length. Accumulative R:S ratios in inoculated plants were significantly smaller than those in controls in all nematode treatments but not at individual harvest dates. Bud break was delayed by X. index and was initiated earlier by P. vulnus and M. incognita. All buds in nematode treatments were less vigorous than in controls.

A Collagenolytic Fungus, Cunninghamella elegans, for Biological Control of Plant-parasitic Nematodes.

The root-galling index of tomatoes inoculated with Meloidogyne javanica was decreased 70% when collagen was used as a soil amendment (0.1% w/w) and 90% when the amendment was supplemented with the collagenolytic fungus Cunninghamella elegans. The root-galling index was reduced 80% when the fungus was homogenized in collagen culture medium and added to soil without collagen supplement. Culture filtrates of the fungus C. elegans, grown on collagen as a single source of carbon and nitrogen, immobilized M. javanica second-stage juveniles and inhibited egg hatch. Root galling was reduced when tomato plants were inoculated with filtrate-treated juveniles. Culture filtrates reduced the motility of Rotylenchulus reniformis and Xiphinema index, but they had less effect on Anguina tritici and almost no effect on Ditylenchus dipsaci. Cunninghamella elegans had collagenolytic, elastolytic, keratinolytic, and nonspecific proteolytic activities when grown on collagen media, but only chitinolytic activity when grown on chitin media.

Antioxidant enzymes in phytoparasitic nematodes.

Presence of different antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate, p-phenilendiamine-pyrocathecol (PPD-PC), o-dianisidine, and guaiacol isoperoxidases, was shown in the phytoparasific nematode species Meloidogyne incognita, M. hapla, Globodera rostochiensis, G. pallida, Heterodera schachtii, H. carotae, and Xiphinema index. The activity of the enzymes tested differed among the life stages examined. SOD was present in cysts but was not detected in Meloidogyne egg masses. Catalase activity of Meloidogyne females was higher than that of preparasitic stages and cyst-nematode females. For the first time, ascorbate peroxidase was found to occur commonly in phytoparasitic nematodes, with the highest activity in the invading life-stages. In all the life stages examined, the antioxidant enzyme activities of M. hapla were markedly higher than those of M. incognita. Glutathione peroxidase was not found in the species examined.

Characterization of sialyl and galactosyl residues on the body wall of different plant parasitic nematodes.

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-beta-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.

Survey of crop losses in response to phytoparasitic nematodes in the United States for 1994.

Previous reports of crop losses to plant-parasitic nematodes have relied on published results of survey data based on certain commodities, including tobacco, peanuts, cotton, and soybean. Reports on crop-loss assessment by land-grant universities and many commodity groups generally are no longer available, with the exception of the University of Georgia, the Beltwide Cotton Conference, and selected groups concerned with soybean. The Society of Nematologists Extension Committee contacted extension personnel in 49 U.S. states for information on estimated crop losses caused by plant-parasitic nematodes in major crops for the year 1994. Included in this paper are survey results from 35 states on various crops including corn, cotton, soybean, peanut, wheat, rice, sugarcane, sorghum, tobacco, numerous vegetable crops, fruit and nut crops, and golf greens. The data are reported systematically by state and include the estimated loss, hectarage of production, source of information, nematode species or taxon when available, and crop value. The major genera of phytoparasitic nematodes reported to cause crop losses were Heterodera, Hoplolaimus, Meloidogyne, Pratylenchus, Rotylenchulus, and Xiphinema.