Characterization of the <Emphasis Type="Italic">RMja</Emphasis> gene for resistance to root-knot nematodes in almond: spectrum,location, and interest for <Emphasis Type="Italic">Prunus</Emphasis> breeding

In Prunus spp., resistance genes to root-knot nematodes (RKN), Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne floridensis, confer either a complete spectrum, e.g., the Ma and Rjap genes in Myrobalan and Japanese plums (subgenus Prunophora), respectively, or a more restricted spectrum, e.g., the RMia gene (M. arenaria + M. incognita) in peach (subgenus Amygdalus). We report here characterization data of the RMja gene from the almond Alnem1, another Amygdalus source. The study of its spectrum is hampered by the inability of almond to be propagated by cuttings; we overcame this problem by using F1 and BC1 crosses with previously genotyped Myrobalan plums that conferred their rooting ability to hybrids for simultaneous evaluation to different RKN. As expected from a homozygous dominant resistance, BC1 progenies of Alnem1 segregated for resistance to M. javanica but were uniformly susceptible to M. incognita and M. floridensis, demonstrating that RMja controlled M. javanica but not M. incognita nor M. floridensis. SSR markers covering the Prunus reference map placed RMja on LG7 in the same region as Ma and Rjap and thus showed its independence from the RMia gene (LG2) of the botanically closer peach. The spectrum of this gene allows the theoretical construction of interspecific rootstocks, Myrobalan plum × (almond × peach), which cumulate RMja with Ma and RMia and are protected from each of the predominant RKN affecting Prunus, i.e., M. arenaria, M. incognita, and M. javanica, by at least two genes. This pyramiding strategy should offer to rootstock material an unprecedented guarantee of durable RKN resistance.     

Comparative Studies on Root Invasion,Root Galling,and Fecundity of Three Meloidogyne spp. on a Susceptible Tobacco Cultivar

Root invasion, root galling, and fecundity of Meloidogyne javanica, M. arenaria, and M. incognita on tobacco was compared in greenhouse and controlled environment experiments. Significantly more M. javanica than M. arenaria or M. incognita larvae were found in tobacco roots at 2, 4, and 6 d after inoculation. Eight days after inoculation there were significantly more M. arenaria and M. javanica than M. incognita larvae. Ten days after inoculation no significant differences were found among the three Meloidogyne species inside the roots. Galls induced by a single larva or several larvae of M. javanica were significantly larger than galls induced by M. incognita: M. arenaria galls were intermediate in size. Only slight differences in numbers of egg masses or numbers of eggs produced by the three Meloidogyne species were observed up to 35 d after inoculation.     

Effect of Carbamate,Organophosphate, and Avermectin Nematicides on Oxygen Consumption by Three Meloidogyne spp.

Second-stage juveniles (I2) of Meloidogyne arenaria consumed more oxygen (P ≤ 0.05) than M. incognita J2, which in turn consumed more than M. javanica J2 (4,820, 4,530, and 3,970 μl per hour per g nematode dryweight, respectively). Decrease in oxygen consumption depended on the nematicide used. Except for aldicarb, there was no differential sensitivity among the three nematode species. Meloidogyne javanica had a greater percentage decrease (P ≤ 0.05) in oxygen uptake when treated with aldicarb, relative to the untreated control, than either M. arenaria or M. incognita. Meloidogyne javanica J2 had a greater degree of recovery from fenamiphos or aldicarb intoxication, after subsequent transfer to water, than did M. incognita. This finding may relate to differential sensitivity among Meloidogyne spp. in the field. Degree of respiratory inhibition and loss of nematode motility for M. javanica after exposure to the nematicides were positively correlated (P ≤ 0.05).     

Occurrence of Meloidogyne spp. in Cerrado Vegetations and Reaction of Native Plants to Meloidogyne javanica

The Cerrado biome represents a hotspot of biodiversity. Despite this, the nematofauna in this biome has not been well characterized, especially that related to root‐knot nematodes. This work aimed to identify Meloidogyne species present in different cerrado vegetations and to investigate potential hosts of Meloidogyne javanica in this biome. Soil samples (250) were collected in native areas of cerrado vegetation located at the National Park of Brasília (PNB) (125 samples) and Água Limpa Farm (FAL) (125 samples), and transferred to sterile pots. Single tomato plants cv. Santa Clara (susceptible) were transplanted into individual pots and maintained for 90 days under glasshouse. Females of Meloidogyne spp. were extracted from tomato roots and identified based upon esterase phenotypes and confirmed with PCR using specific sequence characterized amplified regions (SCAR) primers. Native plants were inoculated with 10 000 individuals (eggs + J2) of a pure culture of M. javanica and maintained under glasshouse for 6 months. From the 250 samples collected, 57 (22.8%) presented Meloidogyne spp. A total of 66 Meloidogyne populations were identified as follows: M. javanica (75.76%), M. incognita (10.60%), M. hapla (9.1%), M. morocciensis (3.03%) and M. arenaria (1.51%). The following esterase phenotypes were detected: M. javanica (J3 and J2), M. incognita (I1 and I2), M. hapla (H1), M. morocciensis (A3) and M. arenaria (A2). The SCAR primers incK14F/incK14R, Fjav/Rjav and Fh/Rh amplified specific fragments in M. incognita (399 bp), M. javanica (670 bp) and M. hapla (610 bp) and can be used for identification of indigenous Meloidogyne spp. from cerrado. The primer set Far/Rar is not specific for M. arenaria due to the amplification of DNA in M. morocciensis. Mimosa caesalpiniifolia was the only native plant in which M. javanica developed a high reproductive rate, and it is probably a host for this nematode in cerrado.     

Root Penetration by Meloidogyne incognita Juveniles Infected with Bacillus Penetrans

Bacillus penetrans inhibited penetration by Meloidogyne incognita second-stage juveniles (J2) into tomato roots in the laboratory and greenhouse. Spores from this Florida population of B. penetrans attached to J2 of M. javanica, M. incognita, and M. arenaria. A greater proportion of J2 of M. javanica were infected than were J2 of either M. incognita or M. arenaria, and a greater number of spores attached to M. incognita than to M. arenaria.     

Nucleotide Substitution Patterning within the Meloidogyne rDNA D3 Region and Its Evolutionary Implications

Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among acollection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified from genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in an isolate.     

Host Suitability and Response of Asparagus Cultivars to Meloidogyne Species and Races

The host-parasite relationships of asparagus and Meloidogyne spp. were examined under greenhouse and microplot conditions. Meloidogyne species and races differed greatly in their ability to reproduce on asparagus seedlings. Meloidogyne hapla generally failed to reproduce, and M. javanica, M. arenaria race 1, and M. incognita race 3 reproduced poorly, with a reproduction factor (Rf = final population/initial population) usually < 1.0. Only M. arenaria race 2 and M. incognita races 1 and 4 reproduced consistently on all asparagus cultivars tested (Rf typically 1-11). No effect of M. incognita race 4 on host growth was detected. Meloidogyne arenaria race 2 and M. incognita race 1 had slight negative effects (5-10%) on plant and root growth.     

Differences in parasitism of Meloidogyne incognita and two genotypes of M. arenaria on Solanum torvum in Japan

Two genotypes of root‐knot nematode, Meloidogyne arenaria (A2‐O and A2‐J), are found in Japan. They were distinguished from each other based on mitochondrial DNA sequences. The primer set (C2F3/1108) amplified a 1.7‐kb fragment from A2‐J, whereas a 1.1‐kb fragment was amplified from A2‐O. M. arenaria (A2‐O) was detected in local regions of southern Japan, whereas M. arenaria (A2‐J) was widespread from the Kyushu region to the Tohoku region. The distribution of M. arenaria (A2‐J) overlaps with the cultivation area of eggplant. Solanum torvum is used worldwide as a rootstock for eggplant cultivation, and it is resistant to Meloidogyne spp. In particular, it is reported that S. torvum is resistant to M. arenaria outside Japan. In this study, we inoculated S. torvum rootstock cultivars with M. arenaria (A2‐J), M. arenaria (A2‐O) and Meloidogyne incognita populations. Although M. incognita and M. arenaria (A2‐O) produced only a few egg masses on S. torvum, thereby confirming its resistance, the four geographical populations of M. arenaria (A2‐J) produced large numbers of egg masses on S. torvum. This study confirmed that S. torvum is resistant to M. incognita and M. arenaria (A2‐O) populations, but susceptible to populations of M. arenaria (A2‐J) in the eggplant production area of Japan.     

Penetration of Susceptible and Resistant Tobacco Cultivars by Meloidogyne Juveniles

Rates of penetration of Meloidogyne incognita, M. arenaria, and M. javanica into tobacco cultivars NC2326 (susceptible to all three species) and K399 (resistant to M. incognita) and a breeding line that had been selected for resistance to M. incognita were compared. Meloidogyne incognita penetrated NC2326 rapidly during the first 24 hours after inoculation. Numbers of M. incognita continued to increase gradually through the 14-day experiment. Higher numbers of M. incognita were observed in the roots of K399 during the first 24 hours than were observed in NC2326. The number of M. incognita in K399 peaked 4 days after inoculation, then declined rapidly as the nematodes that were unable to establish a feeding site left the root or died. Numbers of M. incognita in the breeding line followed the same pattern as with K399, but in lower numbers. Numbers of M. arenaria showed little difference between cultivars until 7 days after inoculation, then numbers increased in NC2326. Numbers of M. javanica fluctuated in all cultivars, resulting in patterns of root population different from those observed for M. incognita or M. arenaria. Resistance to M. incognita appears to be expressed primarily as an inability to establish a feeding site rather than as a barrier to penetration. Some resistance to M. arenaria may also be present in K399 and the breeding line.     

Genetic dissection of resistance to root-knot nematodes Meloidogyne spp. in plum, peach, almond, and apricot from various segregating interspecific Prunus progenies

Sources of resistance in Prunus spp. exhibit different spectra to the root-knot nematodes (RKN) Meloidogyne incognita, Meloidogyne javanica and Meloidogyne floridensis. In this Prunus genus, two dominant genes, Ma with a complete spectrum from the heterozygous Myrobalan plums P.2175 and P.2980 (section Euprunus; subgenus Prunophora) and RMia with a more restricted spectrum from the peaches Nemared and Shalil (subgenus Amygdalus), have been identified. This study characterizes the resistance spectra of interspecific crosses involving (1) previous Myrobalan and peach sources, (2) two Alnem almonds (subgenus Amygdalus) resistant to M. javanica, and (3) the apricot A.3923, representing a species considered RKN-resistant (section Armeniaca; Prunophora). For both latter species, genetic data could be obtained through F1 crosses with genetically characterized Myrobalans that conferred their rooting ability for clonal multiplication of the hybrids and permitted their simultaneous evaluation to the three RKN. Crosses involving either Ma or RMia or both generated the expected resistance spectra. Nemared confirmed the species-specific resistance to M. incognita conferred by RMia. This rootstock, also previously considered resistant to M. javanica, was susceptible to the M. javanica isolate used, what illustrates an isolate-specific resistance to this species. Alnem accessions were shown homozygous resistant to M. javanica. In the progeny P.2980 × A.3923, Ma markers allowed to distinguish resistant individuals carrying that gene from resistant individuals lacking it. Distribution of non-Ma individuals in this cross suggested, in the apricot parent, (1) the absence of a major gene allelic to Ma and (2) the presence of a non RKN specific polygenic resistance.     

Suitability of Zucchini and Cucumber Genotypes to Populations of Meloidogyne arenaria,M. incognita,and M. javanica

The host suitability of five zucchini and three cucumber genotypes to Meloidogyne incognita (MiPM26) and M. javanica (Mj05) was determined in pot experiments in a greenhouse. The number of egg masses (EM) did not differ among the genotypes of zucchini or cucumber, but the eggs/plant and reproduction factor (Rf) did slightly. M. incognita MiPM26 showed lower EM, eggs/plant, and Rf than M. javanica Mj05. Examination of the zucchini galls for nematode postinfection development revealed unsuitable conditions for M. incognita MiPM26 as only 22% of the females produced EM compared to 95% of the M. javanica females. As far as cucumber was concerned, 86% of the M. incognita and 99% of the M. javanica females produced EM, respectively. In a second type of experiments, several populations of M. arenaria, M. incognita, and M. javanica were tested on zucchini cv. Amalthee and cucumber cv. Dasher II to assess the parasitic variation among species and populations of Meloidogyne. A greater parasitic variation was observed in zucchini than cucumber. Zucchini responded as a poor host for M. incognita MiPM26, MiAL09, and MiAL48, but as a good host for MiAL10 and MiAL15. Intraspecific variation was not observed among the M. javanica or M. arenaria populations. Cucumber was a good host for all the tested populations. Overall, both cucurbits were suitable hosts for Meloidogyne but zucchini was a poorer host than the cucumber.     

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     

Identification of Single Meloidogyne Juveniles by Polymerase Chain Reaction Amplification of Mitochondrial DNA

Polymerase chain reaction (PCR) was used to amplify a specific 1.8-kb sequence of mitochondrial DNA from single juveniles and eggs from 17 populations of Meloidogyne incognita, M. hapla, M. javanica, and M. arenaria. Approximately 2 μg amplified product were produced per reaction. Restriction digestion of the amplified product with HinfI permitted discrimination of clonal lineages of the four species. Meloidogyne javanica, however, could not be separated from M. hapla by the enzymes used in these experiments. Various amplification conditions and nematode lysis procedures were examined in order to optimize the speed and quality of identifications.     

Comparisons of Isozyme Phenotypes in Five Meloidogyne spp. with Isoelectric Focusing

Meloidogyne incognita race 1, M. javanica, M. arenaria race 1, M. hapla, and an undescribed Meloidogyne sp. were analyzed by comparing isozyme phenotypes of esterase, malate dehydrogenase, phosphoglucomutase, isocitrate dehydrogenase, and α-glycerophosphate dehydrogenase. Isozyme phenotypes were obtained from single mature females by isoelectric focusing electrophoresis. Of these five isozymes, only esterase and phosphoglucomutase could be used to separate all five Meloidogyne spp.; however, the single esterase electromorphs were similar for M. incognita and M. hapla. Yet when both nematodes were run on the same gel, differences in their esterase phenotypes were detectable. Isozyme phenotypes from the other three isozymes revealed a great deal of similarity among M. incognita, M. javanica, M. arenaria, and the undescribed Meloidogyne sp.     

Identification of Sources of Resistance to Four Species of Root-knot Nematodes in Tobacco

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.     

Evaluation of High‐resolution Melting Curve Analysis as a New Tool for Root‐knot Nematode Diagnostics

In the detection of plant pests, speed and accuracy are vital. High‐resolution melting curve (HRMC) analysis was therefore evaluated as a new tool for the identification of root‐knot nematodes (Meloidogyne spp.). On the basis of the second intergenic spacer (IGS2) region of the ribosomal DNA cistron, Meloidogyne chitwoodi, M. fallax and M. hapla were successfully distinguished from each other and the group of the three tropical species, M. incognita, M. arenaria and M. javanica. Conversely, it was shown that the IGS2 region is not suitable for the tropical species M. enterolobii (senior synonym of M. mayaguensis) as the amplification of multiple fragments of different lengths prevented a reliable HRMC analysis. However, the obtained results provide a proof of principle that HRMC analysis can be a suitable single‐tube assay for fast and accurate root‐knot nematode identification.     

植物罹病组织中南方、爪哇、花生根结线虫的LAMP快速检测

【目的】建立一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,从植物罹病组织中直接检测3种常见的根结线虫,为根结线虫的监测和防治提供技术支持。【方法】分别采用3种根结线虫的种类特异性引物对所选择的根结线虫的DNA片段进行PCR扩增,扩增产物纯化、回收并测序。根据3种根结线虫的测序结果,针对种类特异区段,采用PrimerExplorerV4软件,分别设计3种根结线虫的LAMP引物。设计的引物组人工合成后,以提取的纯化种群线虫DNA为模板,分别进行引物组的特异性测试,筛选出分别针对3种根结线虫的最佳引物组。【结果】研究设计的3种根结线虫的LAMP特异性引物能够直接从植物根结中检测出南方、花生、爪哇3种常见根结线虫,LAMP快速检测体系为:dNTPS浓度为1 mmol·L~(-1),Mg~(2+)的浓度为5 mmol·L~(-1),不添加甜菜碱,反应时间为45 min。【结论】本实验建立的南方、花生、爪哇根结线虫LAMP快速分子检测方法,具有特异性强、灵敏度高、简单、快速、经济等特征,能够从罹病植物组织中快速准确地检测出南方、花生和爪哇根结线虫,具有极高的实践应用价值。     

Genotypic Differentiation of Meloidogyne Populations by Detection of Restriction Fragment Length Difference in Total DNA

Detection of EcoRI restriction fragment length differences in repetitive DNA sequences permitted the rapid diagnosis, by genotype, of randomly selected populations of Meloidogyne incognita, Races 1, 2, 3, and 4; M. javanica; M. arenaria, Races 1 and 2; and M. hapla, Races A and B.     

Application of Isoelectric Focusing to the Taxonomic Identification of Meloidogyne spp.

Meloidogyne incognita, M. arenaria, M. hapla, and M. javanica were distinguishable from each other by isoelectric focusing (IEF) of nematode egg proteins. Proteins extracted from larvae and adults of Hoplolaimus columbus and from eggs of Heterodera glycines had distinctive profiles, also. Protein profiles from eggs, preparasitic larvae and egg-laying adults of M. incognita showed differences. It was necessary to compare samples run at the same time to ensure reliability.     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.