Hermaphrodites were detected in diploid and polyploid isolates of population 86-Va of Meloidogyne hapla. Young hermaphrodites are indistinguishable from normal females. Initially, hermaphrodite ovaries are filled with oocytes at various stages of development. Hermaphroditism is expressed later when young oocytes in the early pachytene region of the growth zone suddenly advance to diakinesis and proceed with maturation divisions, resulting in spermatid production. Spermatogenesis may be initiated shortly after the fourth molt, or later, after a female has produced some eggs. Spermatogenesis may occur in one or both gonads, and it may be initiated in one gonad before the other. Once initiated, spermatogenesis continues for the entire reproductive life of the hermaphrodite. Several thousand spermatozoa accumulate in the ovotestis. Because they do not pass through the oviduct into the spermatotheca, they do not take part in reproduction (nonfunctional hermaphroditism). Among the progeny of hermaphrodites, ca. 50% are hermaphroditic, and the remainder are apparently normal females which, however, produce about 50% hermaphroditic progeny. Two temperature regimes (20-23 C and 27-30 C) did not influence the percentage of hermaphrodites among the progeny. Hermaphroditism could not be transmitted to nonhermaphroditic isolates following attempted crosses between males of hermaphroditic and females of nonhermaphroditic isolates. Although this result suggests cytoplasmic rather than nuclear inheritance, this conclusion is not definitive.
cytogenetics; hermaphroditism; Meloidogyne hapla; nematode; reproduction; spermatogenesis
Two tetraploid isolates of Meloidogyne hapla, 86P and E289P, with haploid chromosome numbers of 34 and 28, respectively, were studied cytogenetically and biologically in relation to the diploid populations, 86-Va (n = 17) and E289-Taiwan (n = 14), from which they had been originally isolated. Both isolates were quite stable, converting to diploidy at the low rate of about 2.5%. The tetraploid isolate 86P maintained itself in competition with its diploid counterpart in mixed cultures, although an initial frequency of 50% polyploidy was reduced to about 9% at the end of the sixth generation. Both tetraploid isolates could maintain themselves in greenhouse cultures without artificial selection for at least 2 years. Crosses between diploid females and tetraploid males resulted in a few triploid females that produced mostly nonviable eggs, suggesting partial reproductive isolation between the two ploidy forms. Ten generations of propagation of only polyploid females of isolate 86P that were associated with males failed to yield an obligatorily amphimictic isolate that would not convert at all to diploidy. If one accepts a previous assumption that the present day amphimictic root-knot nematodes are tetraploids derived from diploid ancestors, results of the present study are not inconsistent with an evolutionary trend toward an even higher level of ploidy in Meloidogyne, presumably octaploidy.
cytogenetics; cytology; evolution; Meloidogyne hapla; parthenogenesis; polyploidy; northern root-knot nematode
Controlled crosses of Heterodera glycines were carried out by placing one o r more virgin females of known esterase phenotype on an agar plate and adding, at various time intervals, one or more males of different esterase phenotypes. Progeny (second-stage juveniles) of such crosses were propagated on soybeans, and 30 days later young females were subjected to electrophoretic analysis to determine their esterase phenotype. Esterase phenotypes that represented the heterozygous state of the maternal and paternal genomes confirmed the hybrid nature of the progeny and identified their male parent. When each of 74 females was given the opportunity to mate successively with two males of different esterase phenotypes, 43 mated with a single male and 31 mated with both males. One female mated with three males, i.e., with a male of its own population (sib mating) and the two males provided for the cross. Inseminated females could mate for a second time soon after, or as late as 24 hours after, their first mating. When single males were given the opportunity to mate with many females, about equal numbers of them inseminated zero, one, two, or three females. In greenhouse tests, 12 females were given the opportunity to mate with many males of three different esterase phenotypes. Two females mated with one and possibly more males of the same phenotype, and 10 females mated with males of two different esterase phenotypes. In conclusion, multiple mating appears to be a common behavior of males and females of H. glycines.
biochemical marker; cross; electrophoresis; enzyme; esterase; Heterodera glycines; multiple mating; soybean cyst nematode
Extensive studies during the last 20 years have demonstrated that enzyme phenotypes, especially those of esterases, are species-specific for Meloidogyne and can be used as reliable taxonomic characters for identification of most major and several minor species of this genus. Recent progress in electrophoretic procedures and advanced computer technology have made available automated electrophoretic apparati that can process very thin polyacrylamide slab gels on which the phenotypes of two or more enzymes can be revealed from the protein extract of a single Meloidogyne female. Presently, such apparati facilitate objective species identification. They also are convenient for performing routine field surveys to determine the relative distribution of major Meloidogyne species, conducting population dynamics studies in the field and in microplots, and testing the purity of greenhouse cultures.
electrophoresis; identification; isozyme; Meloidogyne; taxonomy
cryopreservation; cryoprotectant; Heterodera; Meloidogyne; liquid nitrogen
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.
Cot analysis; DNA; Meloidogyne; repetitive DNA; root-knot nematode; unique DNA
The genetic basis of esterase polymorphism in Heterodera glycines was investigated through controlled matings and analysis of F₁ and F₂ progeny. Three nematode lines, each fixed for a different esterase phenotype, were isolated and purified through repeated directional selection and inbreeding. Each phenotype was characterized by its distinct pair of closely spaced bands of esterase activity. Single-female single-male crosses were conducted according to a modified agar-plate mating technique. F₁ progeny were homogeneous, exhibiting both parental esterase phenotypes (codominant heterozygotes) but no hybrid bands. Approximately 1,500 F₂ progeny segregated in a 1:2:1 ratio for expression of the esterase phenotypes of the female parental line, the heterozygote, and the male parental line. Apparently the three esterase phenotypes correspond to three codominant alleles of a single esterase locus. Reciprocal crosses gave similar results, suggesting no maternal inheritance.
esterase; genetics; Glycine max; Heterodera glycines; hybridization; inheritance; isozyme; soybean cyst nematode
Phenol extraction and cesium trifluoroacetate ultracentrifugation were compared for efficiency in the extraction of DNA from eggs and second-stage juveniles of four species of Meloidogyne. The second method proved to be more satisfactory in that it yielded larger amounts of DNA, shortened the extraction period, and reduced sample handling by eliminating phenol and ether extraction and RNAse treatment. It also made possible the extraction of DNA: from more than one sample at a time. The mean base compositions (% GC) of the total DNA of M. incognita, M. javanica, M. arenaria, and M. hapla, as determined by thermal denaturation tests, were quite similar, as they ranged only between 31 and 33%. Similarly, the thermal stability of the DNA of all four species covered a narrow range from 82.97 to 83.63 C.
Meloidogyne; DNA; genome; thermal denaturation; GC content; thermal stability
Thirty populations of Meloidogyne of diverse geographic origin representing 10 nominal species and various reproductive, cytological, and physiological forms known to exist in the genus were examined to determine their enzymatic relationships. The 184 bands resolved in the study of 27 enzymes were considered as independent characters. Pair-wise comparisons of populations were performed in all possible combinations to estimate the enzymatic distances (ED) and coefficients of similarity (S). A phylogenetic tree was constructed. The apomictic species M. arenaria, M. microcephala, M. javanica, and M. incognita shared a common lineage. M. arenaria was highly polytypic, whereas conspecific populations of M. javanica and M. incognita were largely monomorphic. The mitotic and meiotic forms of M. hapla were very similar (S = 0.93), suggesting that the apomictic race B evolved only recently from the meiotic race A. The five remaining meiotic species (M. chitwoodi, M. graminicola, M. graminis, M. microtyla, and M. naasi - each represented by a single population) were not closely related to each other or to the mitotic species.
biochemistry; biosystematics; electrophoresis; enzymes; evolution; Meloidogyne spp.; root-knot nematode; phylogeny; taxonomy
Four populations of Meloidogyne spartinae from the coast of North and South Carolina were identical cytogenetically. Fourteen rod-shaped chromosomes were present in oogonia and spermatogonia, whereas seven bivalents were observed in oocytes and spermatocytes. There were no distinguishable sex chromosomes. Chromosome behavior was similar to that of other Meloidogyne species. A slight deviation in morphology of prometaphase bivalents was attributed to an increase in frequency of chiasmata that may be associated with the obligatorily amphimictic reproduction of this nematode. The anatomy of the oviduct-spermatotheca region and most cytogenetic features studied suggested that M. spartinae can be regarded as a root-knot nematode. Its position in the genus Meloidogyne or Hypsoperine can be decided by taxonomists. Its small chromosome number (n = 7) compared to the larger number (n = 13-19) of other Meloidogyne species suggests that, cytologically, M. spartinae stands closer to the ancestral form from which the prescent day root-knot nematodes have evolved.
oogenesis; spermatogenesis; taxonomy; cytotaxonomy; chromosomes; reproduction; Meloidogyne spartinae
Enzyme phenotypes were obtained for 291 populations from 16 species of Meloidogyne originating from 65 countries. Soluble proteins from macerates of individual egg-laying females were separated by electrophoresis in 0.7-mm-thick polyacrylamide gels. Enzymes investigated were nonspecific esterases, malate dehydrogenase, superoxide dismutase, and glutamate-oxaloacetate transaminase. Esterases were polymorphic and most useful in identification of major species. About 94% of the populations of M. hapla, 98% of M. incognita, and 100% of M. javanica could be identified to species on the basis of esterase phenotypes alone. About 84% of the populations of M. arenaria exhibited three distinct phenotypes. Two of them were highly species specific (accuracy of identification 98-100%). The third, and least prevalent, phenotype occurred also in two other species. Another 12 less common Meloidogyne species, of which only one or a few populations of each were studied, exhibited a variety of esterase phenotypes, some of which may prove to be species specific. Superoxide dismutase phenotypes similarly were helpful in the characterization of certain species; however, the same phenotype was often observed in more than one species. The remaining two enzymes, with few exceptions, proved to be less useful for identification of Meloidogyne species. Multienzyme phenotypes represented by two or more enzymes often offered biochemical profiles more valuable for definitive characterization of Meloidogyne species than single enzymes.
electrophoresis; isozymes; root-knot nematodes; taxonomy; biosystematics
Studies of oogenesis and spermatogenesis revealed that Meloidogyne nataliei is a diploid, amphimictic species with four (n), relatively large chromosomes, and possibly with an XX ♀-XY ♂ mechanism of sex determination. It differs considerably from all other amphimictic, or meiotically parthenogenetic, species of Meloidogyne which have 13-18 smaller chromosomes and from Meloidogyne (Hypsoperine) spartinae which has seven. Consequently, the taxonomic position of M. nataliei needs to be re-evaluated. The chromosomes of M. nataliei and their behavior during gametogenesis resemble more closely chromosomes of the genus Heterodera than those of the genus Meloidogyne. This resemblance, however, may not imply a closer phyletic relationship of M. nataliei to heteroderid nematodes.
oogenesis; spermatogenesis; phylogeny; taxonomy; cytotaxonomy
The genetic nature of resistance and its epidemiologic effects on two Meloidogyne incognita populations were assessed in the F₁ hybrid tomato cv. Small Fry. The progeny of a Small Fry × Small Fry cross segregated in a 3:1 resistant:susceptible ratio, indicating the presence of a single, completely dominant resistance gene (LMiR₂) in Small Fry. In a subsequent experiment, infection frequency and the rate of development of primary infection on resistant Small Fry × Small Fry segregates were compared to those on susceptible segregates and the susceptible cultivar Rutgers. Suppression in both infection frequency and rate of development of primary infection was entirely attributable to gene LMiR₂. A single egg-mass population of M. incognita propagated for 12 generations on Small Fry showed an increased ability over the wild type population to parasitize plants containing the LMiR₂ gene but failed to completely overcome resistance. The relationship of this phenomenon to the genetics of the Lycopersicon esculentum-M. incognita interaction is discussed.
infection frequency; primary infection; selection; rate of development
220 populations of Meloidogyne incognita and related forms from 46 countries reproduced by mitotic parthenogenesis (apomixis). Determination of somatic chromosome numbers from oogonia and oocytes revealed the existence of a predominant, possibly triploid race A with 3n = 40 to 46 and a rare, diploid race B with 2n = 32 to 36 chromosomes. There is no correlation between cytological races and the four recognized host races of this species. The characteristic behavior of prophase I chromosomes of maturing oocytes, which results in a prolonged prophase stage, is a unifying feature of all forms of M. incognita and supports monophyletic evolution, distinct from that of other Meloidogyne species. Extensive chromosomal polymorphism detected among populations can be helpful in elucidating the cytological pathway of evolution of the species.
cytology; reproduction; polyploidy; polymorphism; races; evolution
The external morphology of female heads of three populations of each of two cytological races of Meloidogyne hapla (race A-meiotic, race B-mitotic) and single populations of M. arenaria, M. incognita, and M. javanica was compared by light (LM) and scanning electron microscopy (SEM). Perineal patterns of all nine populations were observed with a LM and then examined with a SEM. In addition, female stylets of each population were excised, viewed with a SEM, and compared with observations made with a LM. Head morphology of the females, including shape of medial and lateral lips, expression of sensilla, and head annulation, was distinct for each species, each race of M. hapla, and each population of M. hapla race A. The morphology of a given perineal pattern appeared similar with the SEM and the LM. The SEM emphasized surface details, whereas the LM revealed subcuticular structure as well. Stylet morphology was unique for each species but similar in all populations of M. hapla. There were differences between species in the shape of the cone, shaft, and knobs and in the distance of the dorsal esophageal gland orifice from the stylet knob base. Several of the morphological characters first detected in the SEM were seen subsequently with the LM and are helpful in species identification.
cytological races; root-knot nematodes; Meloidogyne hapla; M. arenaria; M. incognita; M. javanica; scanning electron microscopy
A tetraploid single-cyst isolate of Heterodera glycines from a field population from Indiana has been propagated in the greenhouse on Lee soybeans since its discovery, in 1973. The tetraploid isolate has n = 18 chromosomes, compared with n = 9 of the diploid H. glycines; it has larger cysts and larvae, but shows the same level of parasitism and host range as the diploid population from which it apparently evolved. Association of chromosomes is irregular at metaphase I, with quadrivalents, trivalents, and univalents often observed in addition to the bivalents. The second maturation division is usually normal. About 80% of the mature oocytes (just before fertilization) have n = 18, and the other 20% have n = 17 or 19. Reproduction of the tetraploid isolate is exclusively by cross-fertilization. The discovery of such a tetraploid provides an experimental tool for the study of polyploidy in nematodes. Many amphimictic plant-parasitic nematodes are suspected of representing polyploids.
soybean cyst nematode; crossfertilization
Nuclear changes occurring in male and female gonads of Aphelenchus avenae during postembryogenesis were studied in relation to time and feeding periods on Rhizoetonia solani. Development of the female gonad was similar to that in other nematode species, but development of the male gonad followed the growth pattern of female rather than male gonads. This deviation was explained by the assumption that males in antphimictic populations have appeared as the resnlt of recent evolntion of such populations to sexuality from originally parthenogenctic ancestors. A certain, period of feeding of larvae (16 h for L-2 and L-3, but 12 h for L-4) was required before molting. Cell divisions were confined to the periods of lethargus during the second and third molts, but started during the larval stage in fourth-stage larvae. Crosses in various combinations demonstrated that temperature-induced males do inseminate fentales of the amphimictic and some parthenogenetic populations, but their spermatozoa are nonfunctional. Similarly, males of the amphimictic population inseminated females of a parthenogenetic population, but the sperm did not penetrate the oocytes.
postembryogenesis; sexuality; hybridization
Four field populations of Heterodera glycines tested for ability to reproduce on three host differentials were each classified into one of the recognized races. A fifth population represented a new race. Genetic analysis indicated that the designated races are actually field populations that differ from each other primarily in the frequencies of three groups of genes (genes for parasitism) that act quantitatively and control the ability of the nematode to reproduce on resistant P.I. 88788, Pickett, and P.I. 90763 soybeans. Populations of race-3 have none of these genes for parasitism, or they have some in low frequency that results in an index of parasitism of less than 10 on any one of the resistant soybeans. Race-1 has a high frequency of one group of genes that enable it to reproduce on P.I. 88788. Race-2 has two groups of genes for parasitism in high frequency; one for P.I. 88788, and one for Pickett. Based on these findings, it was assumed that race-4 has three groups of genes for parasitism; one for P.I. 88788, one for Pickett, and one for P.I. 90763. Additional races may be recognized when new genes are identified, or when new gene combinations are discovered. The ability to reproduce on P. I. 88788 is inherited independently from the ability to reproduce on Pickett. Although the genetic structure of field populations does not provide a solid foundation for race designation, recognizing races under the present system may be useful when it clearly characterizes the behavior of field populations. Race designations, however, should be regarded as provisional since gene frequencies change with time in response to selection forces and, therefore, the race status of a population may change accordingly.
index of parasitism; genes for parasitism; cyst nematodes
Twelve bisexual species of Heterodera reproduced by amphimixis and had the same number of n=9 (2n=18) chromosomes in maturing oocytes. H. schachtii had slightly larger chromosomes than all other species. Only sperm nuclei with n=9 chromosomes were observed inside maturing oocytes and no specialized sex chromosomes were detected in any case. A "supernumerary" chromosome was observed occasionally in oocytes of H. schachtii and H. weissi and was transmitted regularly to one-half of the progeny of the nematodes that possessed it. Cytological characteristics were not very instructive in differentiating amphimictic tleterodera species. Such karyotypic uniformity indicates cytogenetic stability of the genus and close interrelationship among its members.
reproduction; supernumerary chromosomes; chromosome number; cyst nematodes
Second-stage larvae of Meloidodera floridensis kept in tap water developed without feeding into small, slender males. They completed three molts, and the molted cuticles remained superimposed. All organ systems were well developed in third- and fourth-stage male larvae. Structures in the head region such as cephalic framework, styler and esophagus were smaller and differed morphologically from those of second-stage larvae. Development of the male reproductive system was similar to that of other tylenchids, and sex was recognizable at the end of the second molt. Second-stage larvae, developing in pine roots, increased in size and molted three times to become ovoid to spherical females. Each larval stage had a stylet and fed actively. Sex could be determined at the end of the second molt. Detailed observations were made on the development of the reproductive system, cuticle, esophagus and tail region.
morphology; pine cystoid nematode
Larvae of Meloidodera floridensis develop as females after feeding on pine roots, but become males under conditions of starvation. Seventy to 80% of the larvae kept in tap water at 23 C for 4 months underwent one or two molts, developing as males, and more than 50% became adult males. Ninety-six percent of the larvae that entered pine roots became females and only 4% developed as males. There is evidence that the latter did not feed on the roots. In comparison with tap water, solutions of cholesterol, testosterone propionate and β-estradiol did not significantly affect the percentage of larvae that developed into males. Larvae kept in soil without a host plant did not develop into males. Most of them exhausted their energy supply and died without undergoing any development. We conclude that sex expression in M. floridensis is to a large extent controlled by environmental factors. Under natural conditions of feeding on a host plant, larvae develop as females according to their genetic constitution (thelytokous organism). Under conditions of starvation, however, sexual differentiation proceeds toward the male direction, probably as a result of alteration of the hormonal balance of the larvae and the subsequent activation of different sites of genetic function.
postembryogenesis; development; hormones
Oogenesis and spermatogenesis of seven populations of Meloidogyne graminis and one population of M. ottersoni (formerly Hypsoperine spp.) were of the meiotic type. When males were abundant, reproduction was by amphirnixis. In most greenhouse cultures, however, males were rare and reproduction was by meiotic parthenogenesis. M. graminis and M. ottersoni are closely related to each other and to M. graminicola and M. naasi, but differ in some respect from other Meloidogyne species. It is suggested that these four species be treated together as a group of species, either in the genus Meloidogyne or in the genus Hypsoperine.
Parthenogenesis; polyploidy; chromosomes; phylogeny; Hypsoperine
The relative DNA content of hypodermal nuclei of preparasitic, 2nd-stage larvae was determined cytophotometrically in 19 populations belonging to 13 species of Meloidogyne, Heterodera and Meloidodera. In Meloidogyne hapla, M. arenaria, M. incognita and M. javanica, total DNA content per nucleus is proportional to their chromosome number, indicating that chromosomal forms with high chromosome numbers are truly polyploid. M. graminicola, M. grarninis and M. ottersoni have a DNA content per chromosome significantly lower than that of the other Meloidogyne species. Within Heterodera, species with high chromosome numbers have proportionally higher DNA content, indicating again polyploidy. DNA content per chromosome in Meloidogyne is one third that of Heterodera and one haft that of Meloidodera floridensis. The karyotypic relationships of the three genera are still not clearly understood.
aneuploidy; parthenogenesis; nematodes
Four populations of Heterodera glycines from four different states differed considerably in numbers of adult females developed on five resistant soybean cultivars, mungbean and lespedeza. Differences were observed also in body, tail and tail terminus lengths of second-stage larvae. No attempt was made to assign these populations to recognized races, and it is suggested that race designations should be applied only to representative samples of field populations and not to selected greenhouse populations or isolates.
Three Meloidodera floridensis populations of different geographic or host origin all reproduced by mitotic parthenogenesis. One of them from pine had a somatic chromosome number of 26, whereas, another population from pine and one from azalea had 2n = 27 chromosomes. All are considered to be triploid forms derived from an amphimictic ancestor with n = 9 chromosomes, the basic number in the closely related genus Heterodera. Evidence is presented which suggests that during division the chromosomes of the germ-line cells of the developing embryo behave differently than the chromosomes of all other blastomeres.
Reproduction; Cytology; Germ-line cells