The nucleic acid sequences of rDNA ITS1 and the rDNA D2/D3 expansion segment were compared for 57 burrowing nematode isolates collected from Australia, Cameroon, Central America, Cuba, Dominican Republic, Florida, Guadeloupe, Hawaii, Nigeria, Honduras, Indonesia, Ivory Coast, Puerto Rico, South Africa, and Uganda. Of the 57 isolates, 55 were morphologically similar to Radopholus similis and seven were citrus-parasitic. The nucleic acid sequences for PCR-amplified ITS1 and for the D2/D3 expansion segment of the 28S rDNA gene were each identical for all putative R. similis. Sequence divergence for both the ITS1 and the D2/D3 was concordant with morphological differences that distinguish R. similis from other burrowing nematode species. This result substantiates previous observations that the R. similis genome is highly conserved across geographic regions. Autapomorphies that would delimit phylogenetic lineages of non-citrus-parasitic R. similis from those that parasitize citrus were not observed. The data presented herein support the concept that R. similis is comprised of two pathotypes-one that parasitizes citrus and one that does not.
banana; citrus; evolution; genetics; ITS1; 28S; nematode; pathotype; phylogeny; quarantine; race; Radopholus similis; rDNA; species concepts; taxonomy
Karyotype, gametogenesis, and gonad morphology were characterized for 56 Radopholus spp. isolates collected from Africa, Australia, Central America, Cuba, Dominican Republic, Guadeloupe, Puerto Rico, North America (Florida), and Hawaii. Seven of the isolates, all collected from Florida, were citrus-parasitic. The haploid karyotype for all isolates was n = 5, and gonad organization was similar for each. Furthermore, reproduction did not involve parthenogenesis. Initially, spermatids were produced in young adult females and accumulated in the spermatheca prior to differentiation to sperm. At the cessation of spermatogenesis, oogenesis began and continued for the remainder of the nematode's life. Oocytes first entered a mitotic phase, then a transition zone, and remained in pachytene until they reached the proximal end of the ovary. Thus, Radopholus can reproduce as a hermaphrodite when amphigony does not occur. The gonad is actually an ovatestis.
banana; Caenorhabditis elegans; citrus; egg; evolution; genetics; hermaphrodite; karyotype; nematode; oogenesis; ovary; ovatestis; parthenogenesis; polar body; quarantine; Radopholus; sperm
Monoxenic cultures of burrowing nematode populations extracted from banana roots from Belize, Guatemala, Honduras, and Costa Rica were established on carrot discs. Cultures of Radopholus spp. were also obtained from Florida, Puerto Rico, Dominican Republic, and Ivory Coast. The aggressiveness (defined as reproductive fitness and root necrosis) of these populations was evaluated by inoculating banana plants (Musa AAA, cv. Grande Naine) with 200 nematodes/plant. Banana plants produced by tissue culture were grown in 0.4-liter styrofoam cups, containing a 1:1 mix of a coarse and a fine sand, at ca. 27 °C and 80% RH. Banana plants were acclimated and allowed to grow for 4 weeks prior to inoculation. Plant height, fresh shoot and root weights, root necrosis, and nematode population densities were determined 8 weeks after inoculation. Burrowing-nematode populations varied in aggressiveness, and their reproductive fitness was generally related to damage reported in the field. Plant height and fresh shoot and root weight did not reflect damage caused by nematodes under our experimental conditions. Necrosis of primary roots was closely related to the reproductive fitness of the nematode populations. Variation in aggressiveness among nematode populations followed a similar trend in the two susceptible hosts tested, Grande Naine and Pisang mas. All nematode populations had a low reproductive factor (Rf ≤2.5) in the resistant host except for the Ivory Coast population which had a moderate reproductive factor (Rf ≤ 5) on Pisang Jari Buaya. This is the first report of a burrowing nematode population parasitizing this important source of resistance to R. similis.
banana; burrowing nematode; host resistance; Musa spp.; nematode; Radopholus; reproductive fitness
The genetic variability of 12 burrowing nematode (Radopholus sp.) isolates from Central America, the Caribbean, and Florida, and one isolate from Ivory Coast were compared with RAPD analysis. A high degree of genetic similarity (>0.82) was determined for isolates from the Western Hemisphere. Genome similarity was greatest among isolates collected within a country. Among isolates collected in Central America and the Caribbean, burrowing nematodes from Belize and Guatemala were genetically more distant. However, the genome of the isolate from Ivory Coast was most dissimilar (>0.30). These results suggest that African and American burrowing-nematode isolates may have had different origins or that they have been geographically isolated for a sufficient amount of time to have accumulated genetic changes detectable by RAPD analysis. No relationship was found between the genomic similarity and extent of reproduction or damage to banana or citrus roots. Morphometric analysis involving eight of the isolates indicated that they were morphologically identical and values for morphometric parameters were well within the range previously published for banana and citrusparasitic burrowing nematodes.
Banana; biogeography; citrus; genetic diversity; molecular biology; Musa; nematode; Radopholus; RAPD
Burrowing nematodes from Central America, Dominican Republic, Florida, Guadeloupe, Hawaii, and Puerto Rico were characterized for their ability to parasitize citrus, but citrus parasites were found only in Florida. Sequence tag sites originally amplified from a citrus-parasitic burrowing nematode were polymorphic among 37 burrowing nematode isolates and were not correlated with citrus parasitism, nematode isolate collection site, or amplification of a 2.4-kb sequence tag site (DK#1). Results of a RAPD analysis and characterization of the isozymes phosphoglucose isomerase, lactate dehydrogenase, and malate dehydrogenase indicated that the burrowing nematode isolates were highly similar. Citrus parasitism in Florida appears to be associated with limited changes in the burrowing nematode genome. Findings did not substantiate a previous report that R. citrophilus was present in Hawaii. Overall, these data do not support assignment of sibling species status to burrowing nematodes that differ with respect to citrus parasitism.
anthurium; banana; citrus; evolution; genetics; isozymes; molecular biology; nematode; phylogeny; quarantine; Radopholus; RAPD; STS; taxonomy
Males of citrus-parasitic Radopholus citrophilus (FL1) were mated with non-citrus-parasitic R. similis (FL5) females. Progeny inherited a 2.4-kb sequence tag site (DK#1) and the ability to reproduce in citrus from the paternal parent (FLl); both traits were absent in the maternal line (FL5). The hybrid progeny produced offspring in roots of citrus seedlings over an 8-month period and therefore were considered reproductively viable. Genomic DNA hybridization studies indicated that one or more copies of DK#1 were present in R. citrophilus FL1. It is not likely that DK#1 represents a citrus parasitism gene because it was amplified from some burrowing nematode isolates that did not parasitize citrus and because DK#1 contains no open reading frames. Inability to reliably test individual nematodes for their ability to parasitize citrus was a constraint to obtaining F2 data required for definitive genetic characterization of citrus parasitism in burrowing nematodes, and alternate approaches will be required. Although the physical relationship of DK#1 and the citrus parasitism locus remains undefined, results of controlled mating studies using these parameters as genetic markers enabled us to identify hybrid F₁ progeny. Therefore, R. similis and R. citrophilus are not sibling species since gene flow between the two does not appear to be restricted via geographic isolation (sympatric in Florida) or by genetics.
banana; citrus; Florida; genetics; hybrid; nematode; PCR; quarantine; Radopholus citrophilus; Radopholus similis; RAPD; STS; taxonomy
The closely related soybean (Glycine max) cultivars Centennial and Pickett 71 were confirmed to be resistant and susceptible, respectively, to the root-knot nematode Meloidogryne incognita. Increases in superoxide dismutase (SOD) activity were detected in roots of both soybean cultivars 48 hours following inoculation. Superoxide dismutase activity increased in roots of the susceptible cultivar overall, but declined after 96 hours in roots of the resistant cultivar. The isoelectric points of SOD isolated from preparasitic and parasitic developmental stages of the nematode appeared to differ. The SOD activity increased dramatically as nematodes matured and enlarged. Plant and nematode SOD were present as ca. 40-kDa cuprozinc dimers. Initial increases in SOD activity in infected tissue appeared to involve nematode regulation of plant gene expression. However, as the nematode enlarged, SOD activity could be detected within the female body only.
enzyme; Glycine max; host-parasite interaction; Meloidogyne incognita; nematode; resistance; root; soybean; superoxide dismutase
Unproductive > 7-year-old greenhouse cultures of citrus nematode (Tylenchulus semipenetrans) had a well-developed soil invertebrate fauna that included nematophagous mite species characteristic of Florida citrus groves. Nematophagous mite densities in box cultures were 285 ± 42 mites/liter, 2.5 to 25 times higher than densities in citrus nematode-infested groves. Vigorous root-knot nematode (Meloidogyne incognita) cultures grown in steam-pasteurized soil had few nematophagous mites until more than 3 months after inoculation. Mite species diversity had a significant (P < 0.0001) positive linear relationship with culture age that explained about one-half the variance in species number. Nematophagous mite densities rose and then fell with culture age. In root-knot cultures > 3-months-old, mite densities often exceeded 1,000 mites/liter. Twelve species of nematophagous fungi also were isolated from greenhouse nematode cultures.
biological control; culture; fungus; mite; nematode; nematophagous fungus; Meloidogyne incognita; Tylenchulus semipenetrans
Incubation of Arthrobotrys dactyloides conidia in the presence of Radopholus citrophilus in lectin solutions with their corresponding sugars did not alter the stimulation of trap formation in solutions containing lectins alone. The lack of inhibition of lectin-stimulated trap formation by sugars or by lectin denaturation and the lack of lectin specificity indicate that the carbohydrate-binding regions of the particular lectins studied are not the stimulatory moieties of these macromolecules.
Arthrobotrys; biological control; carbohydrate; fungus; lectin; Radopholus citrophilus; recognition
Treatment of second-stage juveniles (J2) of Meloidogyne incognita race 1 and M. javanica with soybean agglutinin, Concanavalin A, wheat germ agglutinin, Lotus tetragonolobus agglutinin, or Limax flavus agglutinin or the corresponding competitive sugars for each of these lectins did not alter normal root tissue response of soybean cultivars Centennial and Pickett 71 to infection by M. incognita race 1 or M. javanica. Giant cells were frequently induced in Centennial and Pickett 71 roots 5 and 20 days after inoculation of roots with untreated J2 of a population of M. incognita race 3. Treatment of J2 of M. incognita race 3 with the lectins or carbohydrates listed above caused Centennial, but not Pickett 71, root tissue to respond in a hypersensitive manner to infection by M. incognita race 3. Penetration of soybean roots by J2 of Meloidogyne spp. was strongly inhibited in the presence of 0.1 M sialic acid. Treatment of J2 with sialic acid was not lethal to nematodes, and the inhibitory activity of sialic acid was apparently not caused by low pH. These results suggest that carbohydrates may influence plant-nematode interactions.
carbohydrate; giant cell; Glycine max; hypersensitive reaction; lectin; Meloidogyne incognita; Meloidogyne javanica; resistance; sialic acid; soybean; specificity
Rough lemon seedlings were grown in mycorrhizal-infested or phosphorus-amended soil (25 and 300 mg P/kg) in greenhouse experiments. Plants Were inoculated with the citrus burrowing nematode, Radopholus citrophilus (0, 50, 100, or 200 nematodes per pot). Six months later, mycorrhizal plants and nonmycorrhizal, high-P plants had larger shoot and root weights than did non-mycorrhizal, low-P plants. Burrowing nematode population densities were lower in roots of mycorrhizal or nonmycorrhizal, high-P plants than in roots of nonmycorrhizal, low-P plants; however, differences in plant growth between mycorrhizal and nonmycorrhizal plants were not significant with respect to initial nematode inoculum densities. Phosphorus content in leaf tissue was significantly greater in mycorrhizal and nonmycorrhizal, high-P plants compared with nonmycorrhizal, low-P plants. Nutrient concentrations of K, Mg, and Zn were unaffected by nematode parasitism, whereas P, Ca, Fe, and Mn were less in nematode-infected plants. Enhanced growth associated with root colonization by the mycorrhizal fungus appeared to result from improved P nutrition and not antagonism between the fungus and the nematode.
symbiont; tolerance; Glomus intraradices; Radopholus citrophilus; Citrus limon; burrowing nematode; rough lemon
Fluorescent conjugates of the lectins soybean agglutinin (SBA), Concanavalin A (Con A), wheat germ agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and Limulus polyphemus agglutinin (LPA) bound primarily to amphidial openings and amphidial secretions of viable, preinfective second-stage juveniles (J2) of Meloidogyne incognita races 1 and 3 (Mil, Mi3) and M. javanica (Mj). No substantial difference in fluorescent lectin binding was observed among the populations examined. Binding of only LOT and LPA were inhibited in the presence of 0.1 M competitive sugar. Structural differences in amphidial carbohydrate complexes among populations of Mi 1, Mi3, and Mj were revealed by glycohydrolase treatment of preinfective J2 and subsequent labeling with fluorescent lectins. A quantitative microfiltration enzyme-linked lectin assay revealed previously undetected differences in lectin binding to nonglycohydrolase-treated J2. Freinfective J2 of Mj bound the greatest amount of SBA, LOT, and WGA, whereas J2 of Mil bound the most LPA.
enzyme-linked lectin; fluorescent lectin; glycohydrolase; lectin; Meloidogyne incognita; Meloidogyne javanica; microfilter plate; recognition; root-knot nematode; specificity
Seven species of plant parasitic nematodes were found to be associated with leatherleaf fern (Rumohra adiantiformis) in central Florida. Of these, Pratylenchus penetrans, Tylenchorhynchus claytoni, and Criconemoides curvatum were commonly encountered. Nematode communities generally included two or three species of plant parasitic nematodes, with greatest diversity in nematode species occurring in ferneries shaded by oak trees. Species diversity was not correlated with fernery age. Leatherleaf fern was tolerant of P. penetrans and T. claytoni in microplot tests.
species diversity; tolerance; survey; pathogenicity
Karyotype, host preference, isozyzme patterns, morphometrics, and mating behavior of two burrowing nematode populations from Hawaii, one infecting Anthurium sp. and the second infecting Musa sp., were compared with Radopholus similis and R. citrophilus populations from Florida. The population from Anthurium sp. had five chromosomes (n = 5), and that from Musa sp. had four (n = 4). Neither of the Hawaiian nematode populations persisted in roots of Citrus limon or C. aurantium. Anthurium clarinerivum and A. hookeri were hosts of the burrowing nematode population from anthurium in Hawaii and of R. citrophilus from Florida, whereas the two anthurium species were poor hosts of the population from Musa sp. in Hawaii and R. similis from Florida. The isozyme pattern of the population isolated from anthurium was identical to that of R. citrophigus, whereas the pattern of the population from banana in Hawaii was identical to that of R. similis. Mating behavior between the burrowing nematode population isolated from Anthurium sp. and a Florida population of R. citrophilus supports their close taxonomic relationship. Mating was observed between the population from Anthurium sp. and the Florida population of R. citrophilus but not between the Hawaiian burrowing nematode population isolated from Musa sp. and a Florida population of R. citrophilus. These findings indicate that a previously unidentified population of R. citrophilus which does not parasitize citrus occurs in Hawaii.
anthurium; banana; biotype; citrus; ornamental; race; Radopholus similis; Radopholus citrophilus
Two morphologically and karyotypically identical populations of the citrus burrowing nematode, Radopholus citrophilus, differed in their ability to damage and reproduce in roots of citrus rootstocks previously identified as either resistant or tolerant. These populations are considered to be biotypes, and their occurrence may explain the appearance of spreading decline symptoms in plantings of rootstocks previously considered resistant.
burrowing nematode; citrus; control; resistance; taxonomy
Some aspects of the host-parasite relationships of two heteroderid nematodes are described. Meloidodera floridensis induced formation of single uninucleate giant cells in the stelar parenchyma tissue of sand pine (Pinus clausa) roots. Wrinkling and yellowing of the cuticle were associated with maturation of the adult female (cystoid stage). The mode of parasitism of different life stages of Verutus volvingentis on buttonweed (Diodia virginiana) is described. The nematode caused extensive necrosis during penetration and the formation of a large feeding site consisting of nonhypertrophied parenchyma cells with enlarged nuclei and thickened cell walls in the cortex. Walls between cells within the feeding site degenerated, resulting in the formation of a syncytium. Two citrus rootstocks, rough lemon (Citrus lirnon) and trifoliate orange (Poncirus trifoliata), were not hosts of V. volvingentis.
histopathology; pine cystoid nematode; buttonweed; syncytium; uninucleate giant cell
Parasitism by Trophotylenchulus floridensis Raski, 1957 was studied on roots of sand pine (Pinus clausa [Chapm.] Vasey). Different life stages of the nematode were observed to be covered by dark, spherical, brittle, capsule-like structures which protruded from the root surface. The mature capsule enveloped a single sedentary female with a gelatinous matrix containing an average of 76 (44-117) eggs. The capsule was composed of a single layer of cells which appeared to be of plant origin. The anterior end of the nematode was embedded superficially in the root tissue where it created a feeding site comprised of a small number of discrete stelar parenchyma cells with dense cytoplasm and enlarged nuclei and nucleoli. The nematode also infected slash pine, Pinus elliottii, loblolly pine, P. taeda, red oak, Quercus falcata, post oak, Q. stellada, and sweet gum, Liquidambar styraciflua, in four different locations in central Florida. The taxonomic relationship between Tr. floridensis and Tylenchulus spp. is discussed. Based on differences in the tail and lip regions, position of the excretory pore, type of obesity and especially in the mode of plant parasitism, the genus Trophotylenchulus Raski, 1957 is upheld, and the transfer of Tylenchulus clavicaudatus Colbran, 1966, Ty. rnangenoti Luc, 1957, and Ty. obscurus Colbran, 1961 to Trophotylenchulus is proposed.
pine; citrus nematode; histopathology; taxonomy
Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.
proteins; nematode races; burrowing nematode; biosystematics
Analysis of genetic variation between the banana and the citrus races of Radopholus similis by starch gel eleclrophoresis demonstrated that 7 of 16 enzyme-encoding loci could be used for their diagnostic separation. The two races are closely related arid share approximately 75% of the enzymes evaluated. The level of dissimilarities o1 inherited bands indicates that no gene flow occurs between the races. Aldolase, α + β esterase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, and phosphoglucose isomerase are diagnostic markers of the races.
isozyme; nematode races; burrowing nematode; genetics; biosystematics
The distributions of Pratylenchus coffeae and Tylenchulus semipenetrans in a central Florida citrus grove were mutually exclusive. In a challenge experiment conducted in the grove, indigenous populations of either species did not prevlude infection by the other species. Inoculation with either T. semipenetrans or P. coffeae tended to reduce the population size of the other nematode species. In greenhouse tests, individual feeder roots were parasitized predominantly by one or the other of the two species. Host response to parasitism in dual infections did not differ from response to single infection by either species.
nematode distribution; infection; parasitism; histology
Anhydrobiotic nematodes were fixed and extracted from dry Mojave desert soils with hot and cold fixatives (5% formalin and 4% gluteraldehyde). Morphologically, extracted nematodes were tightly coiled and shrunken in size. Various concentrations of KCl, ethylene glycol, and sucrose solutions were compared for their effectiveness in extracting viable nematodes still in the anhydrobiotic state. Approximately 80-95% of the anhydrobiotic nematodes extracted with 1.25 M and 1.5 M sucrose were tightly coiled and shrunken in a manner similar to those fixed and extracted in formalin. Anhydrobiotic nematodes can be maintained in this form for up to 24 days in 1.25 M sucrose solution and still be revived. All molarities of ethylene glycol and KC1 tested were ineffective in recovering and maintaining nematodes in the natural anhydrobiotic state. Straight, air-dried, and active nematodes served as controls and did not coil when placed in hot or cold fixatives or in any concentration of KCI, ethylene glycol, or sucrose. Anhydrobiosis, as represented by the coiled form of nematodes from desert soils, was not confined to any particular life stage or trophic group.
cryptobiosis; nematode survival