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.     

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.     

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.     

Role of Nematodes,Nematicides, and Crop Rotation on the Productivity and Quality of Potato,Sweet Potato,Peanut, and Grain Sorghum

The objective of this experiment was to determine the effects of fenamiphos 15G and short-cycle potato (PO)-sweet potato (SP) grown continuously and in rotation with peanut (PE)-grain sorghum (GS) on yield, crop quality, and mixed nematode population densities of Meloidogyne arenaria, M. hapla, M. incognita, and Mesocriconema ornatum. Greater root-gall indices and damage by M. hapla and M. incognita occurred on potato than other crops. Most crop yields were higher and root-gall indices lower from fenamiphos-treated plots than untreated plots. The total yield of potato in the PO-SP and PO-SP-PE-GS sequences increased from 1983 to 1985 in plots infested with M. hapla or M. arenaria and M. incognita in combination and decreased in 1986 to 1987 when root-knot nematode populations shifted to M. incognita. The total yields of sweet potato in the PO-SP-PE-GS sequence were similar in 1983 and 1985, and declined each year in the PO-SP sequence as a consequence of M. incognita population density increase in the soil. Yield of peanut from soil infested with M. hapla increased 82% in fenamiphos-treated plots compared to untreated plots. Fenamiphos treatment increased yield of grain sorghum from 5% to 45% over untreated controls. The declining yields of potato and sweet potato observed with both the PO-SP and PO-SP-PE-GS sequences indicate that these crop systems should not be used longer than 3 years in soil infested with M. incognita, M. arenaria, or M. hapla. Under these conditions, these two cropping systems promote a population shift in favor of M. incognita, which is more damaging to potato and sweet potato than M. arenaria and M. hapla.     

Isolation of a Repeated DNA Probe Showing Polymorphism among Meloidogyne incognita Populations

Several Meloidogyne incognita geographic populations were characterized by analysis of the restriction fragment length polymorphisms (RFLP) obtained after digestion of their total DNA and hybridization with a [³²P]-labeled probe. The probe consisted of a 1.7-kb-repeated DNA sequence, isolated from a M. incognita genomic library, that hybridized to multiple BamH I fragments in the genome of each isolate. The patterns showed sufficient polymorphism to enable the accurate differentiation of all the populations tested.     

Relative Damage Functions and Reproductive Potentials of Meloidogyne arenaria and M. hapla on Peanut

The reproductive potential and damage functions for Meloidogyne hapla and M. arenaria race 1 on Virginia-type peanuts (Arachis hypogaea cv. Florigiant) were determined over 2 years in microplot experiments in North Carolina. Peanut yield suppression and damage to pods as a result of galling were greatest in response to M. arenaria (P = 0.01). Damage functions for the two species were adequately described by the quadratic models: yield (g/plot) = 398 - 17.1 (log₁₀[Pi + 1]) - 17.0(log₁₀[Pi + 1])²; (R² = 0.83, P = 0.0001) for M. arenaria; and yield = 388 - 10.2(log₁₀[Pi + 1]) - 7.5(log₁₀[Pi + 1])², (R² = 0.30, P = 0.0001) for M. hapla. Both species caused galling on pods, but this was more severe in response to M. arenaria. Reproduction of M. arenaria race 1 was greater than M. hapla on peanut, which accounts in part for the more severe pod galling. Peanut was an excellent host for both M. arenaria race 1 and for M. hapla, but reproduction by M. hapla was more variable.     

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.     

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.     

Species-Specific Restriction Site Polymorphism in Root-knot Nematode Mitochondrial DNA

Research was initiated to physically characterize the mitochondrial genomes of several Meloidogyne spp. and host-races, to address questions regarding their systematics and dispersal, and to assess the possibility of developing molecular diagnostics for these nematodes. Techniques were developed for purification and rapid detection of mitochondrial DNA from root-knot nematodes. Mitochondrial DNAs among Meloidogyne spp. were demonstrated to exhibit extensive divergence. The potential for using the rapidly diverging mitochondrial genomes as a diagnostic assay for M. incognita, M. hapla, M. arenaria, and M. javanica is discussed.     

Interrelationships of Meloidogyne Species with Flue-cured Tobacco

Microplot and field experiments were conducted to determine relationships of population densities of Meloidogyne spp. to performance of flue-cured tobacco. A 3-yr microplot study of these interactions involved varying initial nematode numbers (P_i).and use of ethoprop to re-establish ranges of nematode densities. Field experiments included various nematicides at different locations. Regression analyses of microplot data from a loamy sand showed that cured-leaf yield losses on ''Coker 319'' for each 10-fold increase in P_i were as follows: M. javanica and M. arenaria—-13-19%; M. incognita—5-10%; M. hapla—3.4-5%; and 3% for M. incognita on resistant ''Speight G-28'' tobacco. A P_i of 750 eggs and larvae/500 cm³ of soil of all species except M. hapla caused a significant yield loss; only large numbers of M. hapla effected a loss. M. arenaria was the most tolerant species to ethoprop. Root-gall indices for microplot and most field-nematicide tests also were correlated negatively with yield. Relationships of P_i(s) and necrosis indices to yield were best characterized by linear regression models, whereas midseason numbers of eggs plus larvae (P_m) and sometimes gall indices vs. yield were better characterized by quadratic models. The relation of field P_m and yield was also adequately described by the Seinhorst model. Degrees of root galling, root necrosis, yield losses, and basic rates of reproduction on tobacco generally increased from M. hapla to M. incognita to M. arenaria to M. javanica.     

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.     

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.     

Morphological and Molecular Evaluation of a Meloidogyne hapla Population Damaging Coffee (Coffea arabica) in Maui,Hawaii

An unusual population of Meloidogyne hapla, earlier thought to be an undescribed species, was found causing large galls, without adventitious roots, and substantial damage to coffee in Maui, Hawaii. Only in Brazil had similar damage to coffee been reported by this species. Unlike M. exigua from South and Central America, this population reproduced well on coffee cv. Mokka and M. incognita-susceptible tomato but poorly on tomato with the Mi resistance gene. Characterization included SEM images, esterase isozymes, and five DNA sequences: i) the D3 segment of the large subunit (LSU-D3 or 28S) rDNA, ii) internal transcribed spacer (ITS-1) rDNA, iii) intergenic spacer (IGS) rDNA, iv) the mitochondrial interval from cytochrome oxidase (CO II) to 16S mtDNA, and v) the nuclear gene Hsp90. Sequences for ITS-1, IGS, and COII were similar to other M. hapla populations, but within species ITS-1 variability was not less than among species. One LSU-D3 haplotype was similar to a previously analyzed population with two minor haplotypes. Hsp90 exhibited some variation between Maryland and Hawaiian populations distinct from other species. Females were narrow with wide vulval slits, large interphasmidial distances, and more posterior excretory pores; 20% of perineal patterns had atypical perivulval lines. Males had a low b ratio (<12 µm). Juveniles had a short distance between stylet and dorsal gland orifice. Juvenile body length was short (<355 µm) and was different between summer and winter populations.     

DNA Complexity of the Root-knot Nematode (Meloidogyne spp.) Genome

Cot curves derived from renaturation kinetics of sheared denatured DNA indicated that the genome of six populations representing the four most common root-knot nematode species (Meloidogyne incognita, M. arenaria, M. javanica, and M. hapla) is composed of 20% repetitive and 80% nonrepetitive sequences of DNA. Cot curves were almost identical, indicating that all populations had a haploid genome of approximately the same size. Calculations from an average Cot curve gave an estimate of 0.51 x 108 nucleotide base pairs for the haploid genome of the four Meloidogyne species. This genome is about 12-13 times larger than the genome of the E. coli strain used as a control.     

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.     

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.     

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.     

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.     

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.     

Morphological Comparison of Second-Stage Juveniles of Six Populations of Meloidogyne hapla by SEM

External morphology of second-stage juveniles of six populations of Meloidogyne hapla, hclonging to two cytological races (A and B), and one population each of M. arenaria, M. incognita, and M. javanica was compared by scanning electron microscopy (SEM). Race A of M. hapla included three facultatively parthenogenetic populations with haploid chromosome numbers of 15. 16, and 17; race B consisted of three mitotically parthenogenetic populations with somalic chromosome numhers of 45, 45, and 48. The mitotically parthenogenetic populations of M. arenaria, M. incognita, and M. javanica had 54, 41-43, and 44 chromosomes, respectively. Observations were made on head structures, lateral field, excretory pore, anal opening, and tail. Head morphology, including shape and proportion of labial disc and lips, expression of labial and cephalic sensilla, and markings on head region, was distinctly different for each species. M. hapla populations of race A were distinct from each other but showed much intrapopulatiou variation in head morphology. Populations of race B were different from those of race A and were very stable and quite similar in head morphology. Considerable inter- and intrapopulatiou variation made the structure of the lateral field, excretory pore, anal opening, and tail of little value in distinguishing species or populations. The results are discussed in relation to earlier SEM observations on the genus Helerodera.