High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Potential of Leguminous Cover Crops in Management of a Mixed Population of Root-knot Nematodes (Meloidogyne spp.)

Root-knot nematode is an important pest in agricultural production worldwide. Crop rotation is the only management strategy in some production systems, especially for resource poor farmers in developing countries. A series of experiments was conducted in the laboratory with several leguminous cover crops to investigate their potential for managing a mixture of root-knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica). The root-knot nematode mixture failed to multiply on Mucuna pruriens and Crotalaria spectabilis but on Dolichos lablab the population increased more than 2- fold when inoculated with 500 and 1,000 nematodes per plant. There was no root-galling on M. pruriens and C. spectabilis but the gall rating was noted on D. lablab. Greater mortality of juvenile root-knot nematodes occurred when exposed to eluants of roots and leaves of leguminous crops than those of tomato; 48.7% of juveniles died after 72 h exposure to root eluant of C. spectabilis. The leaf eluant of D. lablab was toxic to nematodes but the root eluant was not. Thus, different parts of a botanical contain different active ingredients or different concentrations of the same active ingredient. The numbers of root-knot nematode eggs that hatched in root exudates of M. pruriens and C. spectabilis were significantly lower (20% and 26%) than in distilled water, tomato and P. vulgaris root exudates (83%, 72% and 89%) respectively. Tomato lacks nematotoxic compounds found in M. pruriens and C. spectabilis. Three months after inoculating plants with 1,000 root-knot nematode juveniles the populations in pots with M. pruriens, C. spectabilis and C. retusa had been reduced by approximately 79%, 85% and 86% respectively; compared with an increase of 262% nematodes in pots with Phaseolus vulgaris. There was significant reduction of 90% nematodes in fallow pots with no growing plant. The results from this study demonstrate that some leguminous species contain compounds that either kill root-knot nematodes or interfere with hatching and affect their capacity to invade and develop within their roots. M. pruriens, C. spectabilis and C. retusa could be used with effect to decrease a mixed field populations of root-knot nematodes.     

红麻种质资源根结线虫病抗性鉴定

为鉴定我国红麻种质资源对根结线虫病的抗病性,在对120份红麻遗传资源进行田间自然发病鉴定的基础上,选择25份有代表性(22份高抗和3份感病)的种质进行盆栽根结线虫病接种鉴定。田间自然发病鉴定结果:120份红麻遗传资源中5个高感、19个中感、25个中抗、71个高抗;其侵染的根结线虫主要为南方根结线虫(Meloidogyne incognita)、爪哇根结线虫(Meloidogyne javanica)和花生根结线虫(Meloidogyne arenria)的混合种群。进行盆栽接种鉴定的25份遗传资源中,7份为中抗,其余分别为中感和高感品种,未发现高抗或免疫品种。本研究可为红麻种质资源发掘利用提供科学依据。     

海南岛象耳豆根结线虫的种类鉴定及其rDNA-ITS序列分析

【背景】植物根结线虫病是世界性分布的土传病害,常造成农作物的重大经济损失。目前分布于热带亚热带区域的象耳豆根结线虫,由于其致病性和分子特征以及与植物互作关系的独特性,被认为是一种对农作物具有潜在危害性的重要病原根结线虫,因而引起国内外植物寄生线虫学者的广泛关注。【方法】用形态学、同工酶技术和分子生物学技术相结合的方法,对从海南岛农作物上采集到的10个根结线虫纯化种群进行分类鉴定。【结果】象耳豆根结线虫在海南岛大面积栽培的10种农作物和南药植物,包括黄瓜、南瓜、苦瓜、丝瓜、葫芦瓜、辣椒、番石榴、海巴戟、沈香和丁香上均有寄生,其形态学、酯酶表型和mtDNA-PCR扩增产物均有别于常见的根结线虫种类;用引物18S和28S扩增象耳豆根结线虫种群的rDNA-ITS区序列,并对其进行克隆、测序和比对分析,结果表明,象耳豆根结线虫4HBJ种群分别与南方根结线虫、爪哇根结线虫和花生根结线虫的同源性均仅为88%左右。【结论与意义】本文准确鉴定了象耳豆根结线虫,首次阐述其对海南岛多种农作物的致害性;阐释了象耳豆根结线虫的形态和分子特征,并明确了其与3种常见根结线虫的系统发育关系。本研究对今后进一步开展象耳豆根结线虫的基础研究和防治工作具有重要参考价值。     

Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.

RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures.     

Host Status of Endophyte-Infected and Noninfected Tall Fescue Grass to Meloidogyne spp

Tall fescue grass cultivars with or without endophytes were evaluated for their susceptibility to Meloidogyne incognita in the greenhouse. Tall fescue cultivars evaluated included, i) wild-type Jesup (E+, ergot-producing endophyte present), ii) endophyte-free Jesup (E-, no endophyte present), iii) Jesup (Max-Q, non-ergot producing endophyte) and iv) Georgia 5 (E+). Peach was included as the control. Peach supported greater (P ≤ 0.05) reproduction of M. incognita than all tall fescue cultivars. Differences in reproduction were not detected among the tall fescue cultivars and all cultivars were rated as either poor or nonhosts for M. incognita. Suppression of M. incognita reproduction was not influenced by endophyte status. In two other greenhouse experiments, host susceptibility of tall fescue grasses to two M. incognita isolates (BY-peach isolate and GA-peach isolate) did not appear to be related to fungal endophyte strain [i.e., Jesup (Max-Q; nontoxic endophyte strain) vs. Bulldog 51 (toxic endophyte strain)]. Host status of tall fescue varied with species of root-knot nematode. Jesup (Max-Q) was rated as a nonhost for M. incognita (BY-peach isolate and GA-peach isolate) and M. hapla, a poor host for M. javanica and a good host for M. arenaria. Bulldog 51 tall fescue was also a good host for M. arenaria and M. javanica, but not M. incognita. Jesup (Max-Q) tall fescue may have potential as a preplant control strategy for M. incognita and M. hapla in southeastern and northeastern United States, respectively.     

杨梅根结线虫病及其病原鉴定

本文报道对杨梅根结线虫病的研究及病原鉴定结果。该病症状为:病树根部形成大小不一的根结,内有乳白色囊状雌虫及棕色卵囊;后期根结腐烂,病树叶片黄化脱落,梢枯乃至死亡。病原鉴定确认,引起该病的根结线虫有3个种:爪哇根结线虫(Meloidogyne javanica)、南方根结线虫(M.incognita)和北方根结线虫(M. hapla)。爪哇根结线虫为优势种。     

Rapid identification of cyst (Heterodera spp., Globodera spp.) and root-knot (Meloidogyne spp.) nematodes on the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) in cultures and field samples

Cyst and root-knot nematodes show high levels of gross morphological similarity. This presents difficulties for the study of their ecology in natural ecosystems. In this study, cyst and root-knot nematode species, as well as some ectoparasitic nematode species, were identified using the second internal transcribed spacer (ITS2) sequence variation detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). The ITS2 region was sufficiently variable within the taxa investigated to allow species to be separated on the basis of minor sequence variation. The PCR primers used in this study were effective for 12 species with three genera within the Heteroderinae (Globodera pallida, G. rostochiensis, Heterodera arenaria/avenae, H. ciceri, H. daverti, H. hordecalis, H. mani, H. schachtii, H. trifolii, Meloidogyne ardenensis, M. duytsi and M. maritima). However, pathotypes of Globodera pallida and G. rostochiensis could not be distinguished. The method was tested at two coastal dune locations in The Netherlands (one in the lime-poor dunes of the north and one in calcareous dunes of the south) to determine the population structure of cyst nematodes. At each site, cyst nematodes were associated with three plant species: two plant species on the foredune (Elymus farctus and Ammophila arenaria) and one plant species occurring further inland (Calamagrostis epigejos). Two species of cyst nematodes, H. arenaria and H. hordecalis, were found. H. arenaria associated with vigorous A. arenaria and H. hordecalis in association with degenerating A. arenaria and C. epigejos. The field survey demonstrated that in coastal dunes abiotic factors may be the important affecting the distribution of cyst nematodes.     

Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne

Among root knot nematodes of the genus Meloidogyne, the polyploid obligate mitotic parthenogens M. arenaria, M. javanica, and M. incognita are widespread and common agricultural pests. Although these named forms are distinguishable by closely related mitochondrial DNA (mtDNA) haplotypes, detailed sequence analyses of internal transcribed spacers (ITSs) of nuclear ribosomal genes reveal extremely high diversity, even within individual nematodes. This ITS diversity is broadly structured into two very different groups that are 12%-18% divergent: one with low diversity (< 1.0%) and one with high diversity (6%-7%). In both of these groups, identical sequences can be found within individual nematodes of different mtDNA haplotypes (i.e., among species). Analysis of genetic variance indicates that more than 90% of ITS diversity can be found within an individual nematode, with small but statistically significant (5%-10%; P < 0.05) variance distributed among mtDNA lineages. The evolutionarily distinct parthenogen M. hapla shows a similar pattern of ITS diversity, with two divergent groups of ITSs within each individual. In contrast, two diploid amphimictic species have only one lineage of ITSs with low diversity (< 0.2%). The presence of divergent lineages of rDNA in the apomictic taxa is unlikely to be due to differences among pseudogenes. Instead, we suggest that the diversity of ITSs in M. arenaria, M. javanica, and M. incognita is due to hybrid origins from closely related females (as inferred from mtDNA) and combinations of more diverse paternal lineages.     

Low amounts of herbivory by root-knot nematodes affect microbial community dynamics and carbon allocation in the rhizosphere

Increased carbon translocation to the rhizosphere via 'leakage' induced by low amounts of plant parasitic nematodes can foster microorganisms. The effects of the root-knot nematode Meloidogyne incognita on microbial biomass (C(mic)) and community structure (phospholipid fatty acids) in the rhizosphere of barley were studied. Inoculation densities of 2000, 4000, and 8000 nematodes were well below the threshold level for plant damage. A (13)CO(2) pulse-labelling was performed to assess the distribution of assimilated (13)C in the rhizosphere. Infection with M. incognita increased the carbon concentration in shoots, and enhanced root biomass slightly. The presence of nematodes did not affect microbial biomass, but significantly changed the allocation of the recent photosynthate. Less plant carbon was sequestered by microorganisms with increasing nematode abundance. Microbial community structure was distinctly altered in the early stages of the plant-nematode interactions. Both, bacteria and fungi, showed a positive response with 2000, and a negative one with 4000 and 8000 M. incognita added. The results suggest that low-level root herbivory still imposes a considerable carbon demand, and that proliferation of microorganisms due to increased rhizodeposition may be short-termed. The carbon flow to rhizosphere microbial communities is likely dependent on the specific nematode-plant association and the developmental stage of the nematode in the host.     

Resolving tylenchid evolutionary relationships through multiple gene analysis derived from EST data

Sequence-based phylogenetic analyses typically are based on a small number of character sets and report gene trees which may not reflect the true species tree. We employed an EST mining strategy to suppress such incongruencies, and recovered the most robust phylogeny for five species of plant-parasitic nematode (Meloidogyne arenaria, M. chitwoodi, M. hapla, M. incognita, and M. javanica), three closely related tylenchid taxa (Heterodera glycines, Globodera pallida, and G. rostochiensis) and a distant taxon, Caenorhabditis elegans. Our multiple-gene approach is based on sampling more than 80,000 publicly available tylenchid EST sequences to identify phylum-wide orthologues. Bayesian inference, minimum evolution, maximum likelihood and protein distance methods were employed for phylogenetic reconstruction and hypothesis tests were constructed to elucidate differential selective pressures across the phylogeny for each gene. Our results place M. incognita and M. javanica as sister taxa, with M. arenaria as the next closely related nematode. Significant differences in selective pressure were revealed for some genes under some hypotheses, though all but one gene are exclusively under purifying selection, indicating conservation across the orthologous groups. This EST-based multi-gene analysis is a first step towards accomplishing genome-wide coverage for tylenchid evolutionary analyses.     

The heat-stable root-knot nematode resistance gene <Emphasis Type="Italic">Mi-9</Emphasis> from Lycopersicon peruvianum is localized on the short arm of chromosome 6

The tomato gene Mi-1 confers resistance to three species of root-knot nematodes, Meloidogyne spp. However, the resistance mediated by Mi-1 is inactive at soil temperatures above 28 degrees C. Previously, we identified and mapped a novel heat-stable nematode resistance gene from the wild species Lycopersicon peruvianum accession LA2157 on to chromosome 6. Here we report further characterization of this heat-stable resistance against three Mi-1-avirulent biotypes of Meloidogyne javanica, Meloidogyne arenaria and Meloidogyne incognita. Screening segregating F(2) and F(3) progenies, derived from an intraspecific cross between susceptible LA392 and resistant LA2157, for nematode resistance at 25 degrees C and 32 degrees C, revealed a simple dominant monogenic inheritance with all the biotypes tested. We designate this gene as Mi-9. As a first step towards cloning of Mi-9, we constructed a linkage map around this gene. A total of 216 F(2) progeny from the cross between LA392 and LA2157 were screened with M. javanica at 32 degrees C and with CT119 and Aps-1, markers that flank the genetic interval that contains the Mi-1 gene. DNA marker analysis indicated that these markers also flank Mi-9. Further mapping of recombinants with both RFLP and PCR-based markers localized Mi-9 to the short arm of chromosome 6 and within the same genetic interval that spans the Mi-1 region.     

Rme1 is necessary for Mi-1-mediated resistance and acts early in the resistance pathway

The tomato gene Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphid, and whitefly. Using genetic screens, we have isolated a mutant, rme1 (resistance to Meloidogyne spp.), compromised in resistance to M. javanica and potato aphid. Here, we show that the rme1 mutant is also compromised in resistance to M. incognita, M. arenaria, and whitefly. In addition, using an Agrobacterium-mediated transient assay in leaves to express constitutive gain-of-function mutant Pto(L205D), we demonstrated that the rme1 mutation is not compromised in Pto-mediated hypersensitive response. Moreover, the mutation in rme1 does not result in increased virulence of pathogenic Pseudomonas syringae or Mi-1-virulent M. incognita. Using a chimeric Mi-1 construct, Mi-DS4, which confers constitutive cell death phenotype and A. rhizogenes root transformation, we showed that the Mi-1-mediated cell death pathway is intact in this mutant. Our results indicate that Rme1 is required for Mi-1-mediated resistance and acts either at the same step in the signal transduction pathway as Mi-1 or upstream of Mi-1.     

Evaluation of an Antibiotic-Producing Strain of Pseudomonas fluorescens for Suppression of Plant-Parasitic Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.     

Possible utilization of weeds for the management of plant parasitic nematodes infesting some vegetable crops

Leaf extracts of noxious weeds such as Solanum xanthocarpum and Argemone maxicana were used as bare-root dip treatment for the management of three important plant-parasitic nematodes, Meloidogyne incognita, Rotylenchulus reniformis and Tylenchorhynchus brassicae infesting tomato (Lycopersicon esculantum ) and chilli (Capsicum annuum) plants. Significant reduction was observed in the root-knot development caused by M. incognita, multiplication of nematode populations of R. reniformis and T. brassicae on both the test plants. Larval penetration of second stage juveniles of M. incognita was also inhibited at various concentrations of leaf extracts and dip durations. Leaf extract of S. xanthocarpum caused relatively more inhibition in root-knot development in case of root-knot nematode, nematode multiplication of reniform and stunt nematodes than that of A. maxicana. Because of dip treatment in leaf extracts of Argemone maxicana and Solanum xanthocarpum, the plants show better growth and at the same time the populations of nematodes such as M. incognita, R. reniformis and T. brassicae significantly decreased, which naturally improved plant growth. The efficacy of root-dip treatment with respect to improvement in plant weight and reduction in root-knot development and nematode populations, increased with increasing the concentration of leaf extracts and dip durations.     

Identification of proteins expressing differences among isolates of Meloidogyne spp. (Nematoda: Meloidogynidae) by nano-liquid chromatography coupled to ion-trap mass spectrometry

Total protein variation (up to ninety-five different positions) was revealed by two-dimensional electrophoresis (2-DE) in 18 isolates from populations of M. arenaria (6 isolates), M. incognita (10), M. javanica (1) plus an unclassified isolate in a previously reported study. Isolates of M. arenaria, M. javanica, Meloidogyne sp., and M. incognita formed two separate groups defined on the basis of two sets of protein positions that could be considered as diagnostic characters, but we could not identify these proteins by MALDI-TOF. To identify these marker positions, nano-liquid chromatography as peptides separation method was coupled to an ion-trap mass spectrometer for induced real-time fragmentation of eluted peptides. Group diagnostic proteins for M. incognita and M. arenaria were in-gel digested and on line analyzed by tandem mass spectrometry (LC-MS/MS). Six proteins out of seven selected spots were unambiguously identified by the analysis of the corresponding MS/MS (MS2) spectrum from parent ions fragmentation: Actin, Enolase, CG3752-PA protein similar to Aldehyde Dehydrogenase, HSP-60 and Translation initiation factor elF-4A. In M. incognita sample, de novo sequencing experiment of doubly charged ion at m/z=936.9 Da in spot 29 identified as enolase, reveals three residue substitutions (K to T, N to T, and D to E) when tentative sequence was compared with that of Anisakis simplex and Onchocerca volvulus enolase, thus three SNPs (single nucleotide polymorphisms) were also possibly identified.     

Utilization of noxious weeds for the management of plant-parasitic nematodes infesting some vegetable crops: Einsatz von schädlichen unkräutern zur bekämpfung von pflanzenparasitären nematoden auf verschiedenen gëmusekulturen

Significant reduction was observed in the population of plant-parasitic nematodes, Meloidogyne incognita, Rotylenchulus reniformis and Tylenchorhynchus brassicae infesting eggplant and cauliflower when given root-dip treatment in the leaf extracts of Argemone maxicana and Solanum xanthocarpum at different concentrations and dip durations. The root-knot development and larval penetration of second stage juveniles of M. incognita were also inhibited, may be due to bare-root dip treatment in leaf extracts of both the weed plants. Leaf extracts of S. xanthocarpum caused more inhibition in root-knot development, nematode multiplication of reniform and stunt nematodes than that of A. maxicana. Plant growth improvement was noted which seems to be due to dip treatment and reduction in the population of parasitic nematodes. The efficacy of root-dip treatment with respect to improvement in plant growth of eggplant and cauliflower and reduction in root-knot development and nematode population, increased with increasing the concentration of leaf extracts and dip durations.