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.     

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.     

Morphological and Molecular Characterization of Meloidogyne mayaguensis Isolates from Florida

The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.     

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.     

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.     

A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

Damage Functions for Three Meloidogyne Species on Arachis hypogaea in Texas

The yield response of Florunner peanut to different initial population (Pi) densities of Meloidogyne arenaria, M. javanica, and an undescribed Meloidogyne species (isolate 93-13a) was determined in microplots in 1995 and 1996. Seven Pi''s (0, 0.5, 1, 5, 10, 50, and 100 eggs and J2/500 cm³ soil) were used for each Meloidogyne species in both years. The three species reproduced abundantly on Florunner in both years. In 1995, mean reproduction differed among the three species; mean Rf values were 10,253 for isolate 93-13, 4,256 for M. arenaria, and 513 for M. javanica. In 1996, the reproduction of M. arenaria (mean Rf = 7,820) and isolate 93-13a (mean Rf = 7,506) were similar, and both had greater reproduction on peanut than did M. javanica (mean Rf = 2,325). All three nematode species caused root and pod galling, and a positive relationship was observed between Pi and the percentage of pods galled. Meloidogyne arenaria caused a higher percentage of pod galling than did M. javanica or isolate 93-13a. A negative linear relationship between log₁₀ (Pi + 1) and pod yield was observed for all three nematode species each year. The yield response slopes were similar except for that of M. javanica, which was less negative than that of isolate 93-13a in 1995, and less negative than that of M. arenaria and isolate 93-13a in 1996.     

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.     

Morphological Comparison of Head Shape and Stylet Morphology of Second-stage Juveniles of Meloidogyne Species

Head shape and stylet morphology of second-stage juveniles of one population each of M. incognita, M. javanica, M. arenaria, and M. hapla were compared by light microscopy. Excised stylets of each species were also compared by scanning electron microscopy (SEM). Differences in head morphology were observed only between M. hapla and the other three species. In SEM, differences in stylet size, shape, and relative distance of the dorsal esophageal gland orifice to the base of the stylet were evident. Differences in stylet morphology between M. incognita and M. javanica could not he detected by light microscopy, but M. arenaria and M. hapla could be distinguished from each other and from the other two species. Head shape and styler morphology of second-stage juveniles are considered useful taxonomic characters.     

Diversity of Meloidogyne spp. on Musa in Martinique,Guadeloupe, and French Guiana

Ninety-six isolates of Meloidogyne species collected from banana fields from Martinique, Guadeloupe, and French Guiana, were examined using esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Adult females identified as M. arenaria, M. incognita, M. javanica, M. cruciani, M. hispanica, and Meloidogyne sp. showed species-specific phenotypes only for the esterase enzymes. Intraspecific variability among isolates of M. arenaria, M. incognita, and M. javanica was detected using Est and Mdh. Perineal patterns were used as a complementary tool together with enzyme characterization and were essential for checking the morphological consistency of the identification. The major species of M. arenaria and M. incognita were detected at 61.9% and 34.3% of the total number of isolates, respectively, and the other minor species at 3.8%. The mixed Meloidogyne species were detected in 45.1% of the samples. Genetic analysis was conducted using RAPD markers, which alone or in combination provided reliable polymorphisms both between and within species. RAPD analysis of the data resulted in clustering of species and isolates congruent with esterase phenotype characterization. The intraspecific variability in M. incognita and in M. arenaria represented 14.9% and 61.6% of the amplified polymorphic fragments, respectively. This high level of variation in M. arenaria isolates may indicate multiple origins for populations classified as M. arenaria or more than one species inside the same group, but more detailed morphological and DNA studies will be necessary to test this hypothesis.     

Pepper Rootstock Graft Compatibility and Response to Meloidogyne javanica and M. incognita

Resistance of pepper species (Capsicum annuum, C. baccatum, C. chinense, C. chacoense, and C. frutescens), cultivars and accessions to the root-knot nematodes Meloidogyne incognita race 2 and M. javanica, and their graft compatibility with commercial pepper varieties as rootstocks were evaluated in growth chamber and greenhouse experiments. Most of the plants tested were highly resistant to M. javanica but susceptible to M. incognita. Capsicum annuum AR-96023 and C. frutescens accessions as rootstocks showed moderate and relatively high resistance to M. incognita, respectively. In M. incognita-infested soil in a greenhouse, AR-96023 supported approximately 6-fold less nematode eggs per gram root and produced about 2-fold greater yield compared to a nongrafted commercial variety. The commercial variety grafted on AR-96023 produced a yield as great as the non-grafted variety in the root-knot nematode-free greenhouse. Some resistant varieties and accessions used as rootstocks produced lower yields (P < 0.01) than that of the non-grafted variety in the noninfested greenhouse. Use of rootstocks with nematode-resistance and graft compatibility may be effective for control of root-knot nematodes on susceptible pepper.     

Comparison of Sequences from the Ribosomal DNA Intergenic Region of Meloidogyne mayaguensis and Other Major Tropical Root-Knot Nematodes

The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications.     

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).     

Identification of Meloidogyne Species on the Basis of Head Shape and,Stylet Morphology of the Male

Head shape and stylet morphology of males of 90 populations of M. arenaria, M. hapla, M. incognita, and M. javanica from geographic regions of the world were compared by light microscopy (LM). In addition, stylets of one population each of M. arenaria, M. incognita, and M. javanica and three different chromosomal forms of M. hapla race A and two of race B were excised and examined with a scanning electron microscope (SEM). Differences among species occurred in both head and stylet morphology. Head morphology differed in size and shape of the head cap, annulation of the head region, and width of the head region relative to the first body annule. Differences in stylets occurred in size and shape of the cone, shaft, and knobs. All populations of M. hapla, except one, had similar head morphology, but stylet morphology was different between cytological races A and B. Populations of M. javanica varied with respect to the presence of head annulations. Head shape and stylet morphology of males are recommended as additional characters useful in the identification of root-knot nematodes.     

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.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

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.     

Resistance to Meloidogyne spp. in Allohexaploid Wheat Derived from Triticum turgidum and Aegilops squarrosa

Expression of resistance to Meloidogyne incognita and M. javanica from Aegilops squarrosa was studied in a synthetic allohexaploid produced from Triticum turgidum var. durum cv. Produra and Ae. squarrosa G 3489. The reproductive rate of different races of M. incognita and M. javanica, expressed in eggs per gram of fresh root, was low (P < 0.05) on the synthetic allohexaploid and the resistant parent, Ae. squarrosa G 3489, compared with different bread and durum wheat cultivars. Reproduction of race 2 and race 3 of M. incognita and an isolate of M. javanica was studied on the synthetic allohexaploid and seven cultivars of T. aestivum: Anza, Coker 747, Coker 68-15, Delta Queen, Double Crop, McNair 1813, and Southern Bell. The latter six cultivars are grown in the southeastern United States and reportedly were resistant to M. incognita. Significant differences (P < 0.05) were detected in nematode reproduction on the seven bread wheat cultivars. Reproduction of M. incognita race 3 and M. javanica was highest on Anza. Reproductive rates on the six southeastern United States bread wheat cultivars varied both within and among nematode isolates. The lowest reproductive rates of the three root-knot isolates were detected in the synthetic allohexaploid.     

Response of Peach Scion Cultivars and Rootstocks to Meloidogyne incognita in Vitro and in Microplots

The response of the peach scion cultivars, Jerseyqueen, Redhaven, Compact Redhaven, and Rio Oso Gem and rootstocks ''Lovely and ''Nemaguard'' to inoculation with Meloidogyne incognita was compared in vitro and in microplots. One or more parameters monitored in vitro correlated with at least one parameter monitored in microplots, 4 years after tree planting (1989). A range of responses was observed from highlysusceptible in Lovell to resistant in Nemaguard. In vitro and microplot data suggest high and moderate levels of resistance to M. incognita in Compact Redhaven and Redhaven, respectively. Both Jerseyqueen and Rio Oso Gem were susceptible to M. incognita, but not as susceptible as Lovell. The response of self-rooted peach cultivars and rootstocks to M. incognita in vitro appears to be a reliable method for predicting the reaction of each to these nematodes under field conditions.     

Morphological Comparison of Meloidogyne Males by Scanning Electron Microscopy

Males of five populations of Meloidogyne hapla were compared by scanning electron microscopy (SEM). Three populations of race A had haploid chromosome numbers of 15, 16, and 17 and reproduced by facultative parthenogenesis. Race B consisted of two mitotically parthenogenetic populations with somatic chromosome numbers of 45 and 48. Males of one population each of M. arenaria, M. incognita, and M. javanica were also examined to delineate species differences. The populations of M. arenaria, M. incognita, and M. javanica had 54, 41-43, and 44 chromosomes, respectively, and reproduction was by mitotic parthenogenesis. Observations were made on head structures, lateral field, excretory pore, and tail. The expression of labial and cephalic sensilla, shape and proportion of labial disc and lips, and markings on the head region were distinctly different for each species. The head morphology of the two cytological races of M. hapla was dissimilar. Populations of race A were different from each other and showed intrapopulation variation. Populations of race B were morphologically similar and stable in head morphology. The structure of the lateral field, excretory pore, and tail was of little value in distinguishing species or populations because of inter- and intrapopulation variation. The results are discussed in relation to earlier SEM observations of second-stage juveniles of the same populations.