RAPD linkage map of the genomic region encompassing the root-knot nematode (Meloidogyne javanica) resistance locus in carrot

Inheritance studies have indicated that resistance to the root-knot nematode (Meloidogyne javanica) in carrot inbred line ’Brasilia-1252’ is controlled by the action of one or two (duplicated) dominant gene(s) located at a single genomic region (designated the Mj-1 locus). A systematic search for randomly amplified polymorphic DNA (RAPD) markers linked to Mj-1 was carried out using bulked segregant analysis (BSA). Altogether 1000 ten-mer primers were screened with 69.1% displaying scorable amplicons. A total of approximately 2400 RAPD bands were examined. Four reproducible markers (OP-C2₁₇₀₀, OP-Q6₅₀₀, OP-U12₇₀₀, and OP-AL15₅₀₀) were identified, in coupling-phase linkage, flanking the Mj-1 region. The genetic distances between RAPD markers and the Mj-1 locus, estimated using an F₂ progeny of 412 individuals from ’Brasilia 1252’×’B6274’, ranged from 0.8 to 5.7 cM . The two closest flanking markers (OP-Q6₅₀₀ and OP-AL15₅₀₀) encompassed a region of 2.7 cM . The frequency of these RAPD loci was evaluated in 121 accessions of a broad-based carrot germplasm collection. Only five entries (all resistant to M. javanica and genetically related to ’Brasilia 1252’) exhibited the simultaneous presence of all four markers. An advanced line derived from the same cross, susceptible to M. javanica but relatively resistant to another root-knot nematode species (M. incognita), did not share three of the closest markers. These results suggest that at least some genes controlling resistance to M. incognita and M. javanica in ’Brasilia 1252’ reside at distinct loci. The low number of markers suggests a reduced amount of genetic divergence between the parental lines at the region surrounding the target locus. Nevertheless, the low rate of recombination indicated these markers could be useful landmarks for positional cloning of the resistance gene(s). These RAPD markers could also be used to increase the Mj-1 frequency during recurrent selection cycles and in backcrossing programs to minimize ’linkage drag’ in elite lines employed for the development of resistant F₁ hybrids. Received: 22 June 1999 / Accepted: 6 July 1999     

Meiotic parthenogenesis and heterochromatization in a soft scale,pulvinaria hydrangeae (Coccoidea: Homoptera)

Summary Meiotic parthenogenesis of a type not previously described was found in Pulvinaria hydrangeae Steinweden. During diakinesis 8 bivalents were formed. At prometaphase the spindle was tripolar but anaphase I was bipolar and normal. After completion of division of the primary oocyte, the following sequence occurred: 1. polar body I divided, usually into 3 products; 2. the secondary oocyte divided to yield the egg pronucleus and polar body II; 3. the egg pronucleus divided into its two haploid products; and 4. the second polar body divided. The products of the egg pronucleus fused while dividing to restore the diploid chromosome number; this division may be equated to the first cleavage division. The products of the polar bodies did not take part in the formation of the embryo proper or the mycetocytes.Among the embryos produced by females of two out of the three populations studied some of the embryos showed a heterochromatic chromosome set, characteristic of males in this and related families. The reproductive system of the females as well as the eggs did not contain any sperm; thus the male embryos were apparently produced parthenogenetically.The euchromatic and heterochromatic chromosome sets were genetically identical, since they both originated from the egg pronucleus by mitosis. The heterochromatization of one set but not the other might be due in part to a previous difference in their position in the cytoplasm.The females were completely homozygous yet they produced male and female embryos. Thus it appears that sex determination in the group does not depend on the segregation of genetic factors in either males or females.In addition to male and female embryos, three types of degenerating embryos were observed. It is believed that these embryos were formed by polyploid somatic cells which invaded abnormal eggs and embryos and took over development.     

Polyploidy and reproductive patterns in the root-knot nematode Meloidogyne hapla

A total of 32 populations and egg mass isolates of Meloidogyne hapla obtained from various geographical areas were studied cytologically and with respect to their mode of reproduction. In 29, maturation of oocytes is by regular meiosis. The reduced chromosome number at metaphase I is 17 in 18 populations, 16 in 8, and 15 in 3 populations. Reproduction in all these populations is by cross-fertilization, although nonfertilized eggs can develop by parthenogenesis. In the latter case, the two groups of telophase chromosomes of the second maturation division become enclosed in the same pronucleus, thus reestablishing the somatic chromosome number. Maturation of spermatocytes in three populations studied is by regular meiosis and the reduced chromosome number appears to be equal to that of the oocytes. In the remaining three populations, no synapsis takes place and the somatic number of 45 chromosomes is observed at metaphase of the single maturation division of both oocytes and spermatocytes. Reproduction is by obligatory mitotic parthenogensis. It is postulated that the basic chromosome number for the genus is nine and that the facultatively parthenogenetic populations are tetraploid, whereas, the obligatorily parthenogenetic populations are pentaploid. A preliminary scheme of the phylogeny in the family Heteroderidae is given.     

Post-transcriptional gene silencing of root-knot nematode in transformed soybean roots

RNAi constructs targeted to four different genes were examined to determine their efficacy to reduce galls formed by Meloidogyne incognita in soybean roots. These genes have high similarity with essential soybean cyst nematode (Heterodera glycines) and Caenorhabditis elegans genes. Transformed roots were challenged with M. incognita. Two constructs, targeted to genes encoding tyrosine phosphatase (TP) and mitochondrial stress-70 protein precursor (MSP), respectively, strongly interfered with M. incognita gall formation. The number of galls formed on roots transformed with constructs targeting the M. incognita TP and MSP genes was reduced by 92% and 94.7%, respectively. The diameter of M. incognita inside these transformed roots was 5.4 and 6.5 times less than the diameter of M. incognita found inside control plants transformed with the empty vector. These results indicate that silencing the genes encoding TP and MSP can greatly decrease gall formation and shows a promising solution for broadening resistance of plants against this plant-parasitic nematode.     

Rice root-knot nematode (Meloidogyne graminicola) infestation in rice

Rice (Oryza sativa) is an important staple food crop for majority of human population in the world in general and in Asia in particular. However, among various pests and diseases which constitute important constraints in the successful crop production, plant parasitic nematodes play an important role and account for yield losses to the extent of 90%. The major nematode pests associated with rice are Ditylenchus angustus, Aphelenchoides besseyi, Hirschmanniella spp., Heterodera oryzicola and Meloidogyne graminicola. However, rice root-knot nematode (M. graminicola) happens to be the most important pest and is prevalent in major rice producing countries of the world. In India, the distribution of M. graminicola in rice growing areas of different states has been documented in nematode distribution atlas prepared by All India Coordinated Research Project (Nematodes) and published by Directorate of Information and Publications of Agriculture, Indian Council of Agricultural Research, New Delhi, India during 2010. M. graminicola affected rice plants show stunting and chlorosis due to the characteristic terminal swellings/galls on the roots which ultimately result in severe reduction in growth and yield. Number of eco-friendly management technologies against M. graminicola have been developed and demonstrated, including the use of bioagents for minimising the losses due to rice root-knot nematode. This review is focused on collating information to understand the current scenario of rice root-knot nematodes with greater emphasis on its ecological requirements, damage symptoms, biology, morphology, host range and management strategies.     

Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein

Our laboratory has demonstrated previously that Bacillus thuringiensis (Bt) crystal (Cry) proteins present in the Cry5 and Cry6 subclades intoxicate free-living nematodes. In this study, we tested whether the expression of nematicidal Cry6A in transgenic plants provided protection against plant-parasitic nematodes. As bacterial codon usage is incompatible with expression in plants, two different codon-modified cry6A genes were synthesized for expression in plants. One was designed by maintaining codon diversity whilst removing codons not common in plants, and the other was designed by selecting the optimal codon for each amino acid based on the Arabidopsis genome. Both versions of the cry6A gene, driven by the constitutive cauliflower mosaic virus 35S promoter, were introduced into tomato roots via Agrobacterium rhizogenes . Although both were found to express Cry6A protein, the codon diversity gene generated superior expression. These Cry6A-expressing roots were then challenged with root-knot nematode, Meloidogyne incognita . Three different infection parameters were compared between Cry6A-expressing roots and control roots transformed with empty vector or green fluorescent protein (GFP). These data demonstrated that M. incognita was able to ingest the 54-kDa Cry6A, and that Cry6A intoxicated the parasitic nematode, as indicated by a decrease in progeny production of up to fourfold. These results indicate, for the first time, that a Bt Cry protein can confer plant resistance to an endoparasitic nematode, and that Cry proteins have the potential to control plant-parasitic nematodes in transgenic plants.     

Location of independent root-knot nematode resistance genes in plum and peach

Prunus species express different ranges and levels of resistance to the root-knot nematodes (RKN) Meloidogyne spp. In Myrobalan plum (Prunus cerasifera), the dominant Ma gene confers a high-level and wide-spectrum resistance to the predominant RKN, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica and the isolate Meloidogyne sp. Florida which overcomes the resistance of the Amygdalus sources. In Japanese plum (Prunus salicina), a similar wide-spectrum dominant resistance gene, termed R _jap , has been hypothesized from an intraspecific segregating cross. In peach, two crosses segregating for resistance to both M. incognita and M. arenaria were used to identify single genes that each control both RKN species in the Shalil (R _Mia557 ) and Nemared (R _MiaNem ) sources. Localisation of these genes was made possible using the RFLP and SSR- saturated reference Prunus map T×E, combined with a BSA approach applied to some of the genes. The Ma1 allele carried by the Myrobalan plum accession P.2175 was localised on the linkage group 7 at an approximate distance of 2 cM from the SSR marker pchgms6. In the Japanese plum accession J.222, the gene R _jap was mapped at the same position in co-segregation with the SSR markers pchgms6 and CPPCT022. The peach genes R _Mia557 and R _MiaNem , carried by two a priori unrelated resistance sources, were co-localized in a subtelomeric position on linkage group 2. This location was different from the more centromeric position previously proposed by Lu et al. (1999) for the resistance gene Mij to M. incognita and M. javanica in Nemared, near the SSR pchgms1 and the STS EAA/MCAT10. By contrast, R _Mia557 and R _MiaNem were flanked by STS markers obtained by Yamamoto and Hayashi (2002) for the resistance gene Mia to M. incognita in the Japanese peach source Juseitou. Concordant results for the three independent sources, Shalil, Nemared and Juseitou, suggest that these peach RKN sources share at least one major gene resistance to M. incognita located in this subtelomeric position. We showed that plum and peach genes are independent and, thus, can be pyramided into interspecific hybrid rootstocks based on the plum and peach species.Communicated by H.C. Becker     

Inheritance of resistance to the root-knot nematode Meloidogyne javanica in lettuce

Resistance to the root-knot nematodes Meloidogyne spp. would be a valuable attribute of lettuce Lactuca sativa L. cultivars grown in tropical regions. The looseleaf lettuce 'Grand Rapids' is resistant to both M. incognita and M. javanica. Resistance to M. incognita has a high heritability, under the control of a single gene locus, in which the 'Grand Rapids' allele, responsible for resistance (Me), has predominantly additive gene action, and has incomplete penetrance and variable expressivity. We studied the inheritance of the resistance of 'Grand Rapids' (P(2)) to M. javanica in a cross with a standard nematode-susceptible cultivar Regina-71 (P(1)). F(1)(Regina-71 x Grand Rapids) and F(2) seed were obtained, and the F(2) inoculated, along with the parental cultivars, with a known isolate of M. javanica to evaluate nematode resistance. A high broad sense heritability estimate (0.798) was obtained for gall indices. Class distributions of gall indices for generations P(1), P(2), and F(2) were in agreement with theoretical distributions based on a monogenic inheritance model for the range of assumed degrees of dominance between approximately -0.20 and 0.20. M. javanica resistance appears to be under control of a single gene locus, with predominantly additive gene action. Whether or not the Grand Rapids allele imparting resistance to M. javanica is the same Me allele imparting resistance to M. incognita remains to be determined.     

Inheritance of resistance to the root-knot nematode Meloidogyne arenaria in Myrobalan plum

The inheritance of resistance of the self-incompatible Myrobalan plum Prunus cerasifera to the root-knot nematode Meloidogyne arenaria was studied using first a diallel cross between five parents of variable host suitability (including two highly resistant clones P.1079 and P.2175, a moderate host P.2032, a good host P.2646 and an excellent host P.16.5), followed by the G2 crosses P.16.5 × (P.2646 × P.1079) and P.2646 × (P.16.5 × P.1079). A total of 355 G1 and 72 G2 clones obtained from hard-wood cuttings sampled from trees in the field experimental design, then rooted in the nursery and inoculated individually in containers (5–10 replicates per clone) under greenhouse conditions, were evaluated for their host suitability based on a 0–5 gall-index rating under a high and durable inoculum pressure of the nematode. In the crosses involving the resistant P.1079 and P.2175 and the hosts P.2646 and P.16.5: (1) all of the G1 crosses of P.1079 were resistant while the G2 crosses segregated 1 resistant to 1 host, (2) the G1 crosses between P.2175 and either P.2646 or P.16.5 segregated 1 resistant to 1 host, and (3) all of the G1 progeny between P.2646 and P.16.5 were host. These results indicate that resistance is conferred by a single major dominant resistance gene (homozygous) in P.1079, and the same, or an allelic or a different, major dominant gene (heterozygous) in P.2175, and that P.2646 and P.16.5 are recessive for this (these) major resistance gene(s). As expected according to the hypothesis of a recessive genotype for P.2032, all of its hybrids with P.1079 were resistant, all of its hybrids with P.2646 and P.16.5 were host, and its hybrids with P.2175 segregated for resistance. Nevertheless, the 32 segregation ratio of these latter hybrids suggests that clones bearing the P.2175 gene would have a selective advantage. Both resistance genes are completely dominant and confer a non-host behaviour that totally prevents the multiplication of the nematode. This is the first reported evidence of major nematode resistance genes towards M. arenaria in a species of the subgenus Prunophora in the genus Prunus. The symbols Ma1 for the P.2175 gene and Ma2 for the P.1079 gene are proposed.     

Interactions between the root-knot nematode Meloidogyne incognita and Glomus mosseae in banana

The effects of the interaction between the arbuscular mycorrhizal fungus Glomus mosseae and the root-knot nematode Meloidogyne incognita on growth and nutrition of micropropagated ;Grand Naine banana (Musa AAA) cultivar was studied under greenhouse conditions. Inoculation with two G. mosseae isolates significantly increased growth of plants in relation to non-mycorrhizal plants. Response to mycorrhizae was as effective as with an optimum P fertilization in promoting plant development for most growth parameters. Meloidogyne incognita had no apparent effect on the percentage of root colonization in mycorrhizal plants. In contrast, G. mosseae suppressed root galling and nematode buildup in the roots. The percentage of mycorrhizal colonization was high (over 80%) in low P fertilized plants, but optimum P rates for bananas (four times higher than low P) significantly reduced mycorrhizal colonization. Most elements were within sufficiency levels for banana with exception of N which was low for all treatments. Mycorrhizal plants fertilized with a low P rate showed higher N, P, K, Ca, and Mg contents as compared to non-mycorrhizal plants low in P with or without the nematode. Inoculation with G. mosseae favours growth of banana plants by enhancing plant nutrition and by suppressing nematode reproduction and galling during the early stages of plant development.     

(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes.     

In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode

Esophageal secretions from endoparasitic sedentary nematodes are thought to play key roles throughout plant parasitism, in particular during the invasion of the root tissue and the initiation and maintenance of the nematode feeding site (NFS) essential for nematode development. The secretion in planta of esophageal cell-wall-degrading enzymes by migratory juveniles has been shown, suggesting a role for these enzymes in the invasion phase. Nevertheless, the secretion of an esophageal gland protein into the NFS by nematode sedentary stages has never been demonstrated. The calreticulin Mi-CRT is a protein synthesized in the esophageal glands of the root-knot nematode Meloidogyne incognita. After three-dimensional modeling of the Mi-CRT protein, a surface peptide was selected to raise specific antibodies. In planta immunolocalization showed that Mi-CRT is secreted by migratory and sedentary stage nematodes, suggesting a role for Mi-CRT throughout parasitism. During the maintenance of the NFS, the secreted Mi-CRT was localized outside the nematode at the tip of the stylet. In addition, Mi-CRT accumulation was observed along the cell wall of the giant cells that compose the feeding site, providing evidence for a nematode esophageal protein secretion into the NFS.     

Physiological and genetic mapping study of tolerance to root-knot nematode in rice

The root-knot nematode Meloidogyne graminicola is an obligate biotrophic parasite and a major pest of rice (Oryza sativa) for which resistant varieties are not currently available. Quantitative trait loci (QTLs) for partial resistance to M. graminicola were identified using a mapping population based on two rice varieties, Bala x Azucena. Experiments were carried out to investigate the interactions between M. graminicola and these two varieties in terms of nematode establishment, reproduction and effect on rice yield. Nematode establishment was also assessed in the mapping population. Meloidogyne graminicola consistently caused more galling and had higher reproductive success in Azucena than in Bala. M. graminicola did not significantly reduce yield in Bala, but caused a yield reduction of almost half in Azucena, suggesting that the partial resistance to nematode establishment was related to nematode tolerance. A total of six significant or putative QTLs for nematode tolerance were detected. For two of the QTLs detected, Azucena was the donor of the tolerance alleles, suggesting it may be possible to breed plants with greater tolerance than Bala.     

基于GIS的南方根结线虫在陕西省越冬区划分析

土壤温度是影响南方根结线虫(Meloidogyne incognita)越冬的重要因子。通过自动温度记录仪,从2009—2012年的每年冬季(11月至次年3月),对陕西省延安、商洛、杨凌和大荔4个生态区的气温和不同种植模式下的土壤温度进行数据采集和模拟统计,建立土温与气温关系的数学模型,根据模型将陕西省96个气象站点的气温数据转换为土温数据。利用GIS的克里金(Kriging)空间插值功能,结合实验室测得的南方根结线虫存活的最低温度,生成南方根结线虫在拱棚、地膜覆盖和露地3种种植条件下的越冬区划图并进行分析。研究表明,(1)0℃以下低温对南方根结线虫有明显抑制作用,南方根结线虫在低于-1℃低温持续32 d以上时无法越冬。(2)土壤温度和气温呈线性相关关系,4种种植条件下土壤温度(Y)与气温(X)的关系方程分别为露地:Y=0.8125X+1.9325,R=0.934;地膜覆盖:Y=0.7943X+1.8563,R=0.918;拱棚:Y=0.7046X+6.2685,R=0.907;温室:Y=0.302X+14.519,R=0.597。(3)最冷月土壤均温低于-1℃的概率在70%—80%的区域可以认为是南方根结线虫的越冬界线,越冬界线在露地、地膜覆盖和拱棚条件下依次北移,在温室条件下可在全省范围内越冬。     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.