Characterization of a New Burrowing Nematode Population,Radopholus citrophilus,from Hawaii

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.     

Genome Similarity Implies that Citrus-Parasitic Burrowing Nematodes do not Represent a Unique Species

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.     

Lectin Binding to Radopholus citrophilus and R. similis Proteins

Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r² = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes.     

A Bioassay to Estimate Root Penetration by Nematodes

An in vitro bioassay with a 96-well microtiter plate was used to study the effect of lectins on burrowing nematode penetration of citrus roots. In each well, one 4-mm root segment, excised from the zone of elongation of rough lemon roots, was buried in 0.88 g dry sand. Addition of a Radopholus citrophilus suspension containing ca. 300 nematodes in 50 μ1 test solution completely moistened the sand in each well. The technique assured uniform treatment concentration throughout the medium. Within 16-24 hours, burrowing nematodes penetrated citrus root pieces, primarily through the cut ends. The lectins (100 μg/ml) Concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), and Lotus tetragonolobus agglutinin (LOT) stimulated an increase in penetration of citrus root segments by Radopholus citrophilus. Concentrations as low as 12.5 μg/ml Con A, LOT, and WGA stimulated burrowing nematode penetration of citrus roots. Heat denaturation of the lectins reversed their effect on penetration; however, incubation of nematodes in lectin (25 μg/ml) with 25 mM competitive sugars did not. The reason for enhanced penetration associated with lectins is unclear.     

Influence of Mycorrhizal Fungus, Phosphorus,and Burrowing Nematode Interactions on Growth of Rough Lemon Citrus Seedlings

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.     

Morphological Differences Between Radopholus citrophilus and R. similis

SEM observations of the external morphology of populations of Radopholus citrophilus and R. similis revealed several diagnostic differences. The cloaco-spicular orifice on males of R. citrophilus had three to seven genital papillae (anterior hypoptygmata), whereas males of R. similis were either smooth or had one or two shorter genital papillae (anterior hypoptygmata). Females of R. citrophilus had four annules in the region of the vulval opening, but R. similis had five annules in the same region. The labial disc and lateral lips appeared to be of diagnostic significance, but these areas were more susceptible to artifacts due to fixation. An unknown population of Radopholus from Puerto Rico with a chromosome number of n = 4 was morphologically similar to R. similis. These morphological differences provide additional support that R. citrophilus and R. similis are distinct species.     

Improved Nematode Extraction from Carrot Disk Culture

Radopholus spp. were reared in carrot tissue culture via established procedures, with slight modification. Several plant tissue maceration enzymes and flotation media (salts and sucrose) were evaluated with regard to nematode toxicity and extraction efficiency. Best extraction of viable nematodes and eggs was attained when carrot tissue infested with Radopholus citrophilus or R. similis was macerated with a mixture of 0.50% driselase and 0.50% cellulysin, w/v each, with 2.5 ml of enzyme solution based for each gram of carrot tissue. Maceration slurries containing carrot tissue and nematodes were maintained in open flasks on a rotary shaker (175 rpm) at 26 C for 24 hours. Nematodes and eggs were extracted from resultant culture slurries by flotation with MgSO₄-7H₂0 (sp gr 1.1). A protocol is presented to extract large quantities of viable burrowing nematodes and their eggs from carrot disk cultures.     

Effect of Endophytic Fusarium oxysporum on Host Preference of Radopholus similis to Tissue Culture Banana Plants

The burrowing nematode Radopholus similis is one of the major constraints to banana (Musa spp.) production worldwide. Resource-poor farmers can potentially manage R. similis by using naturally occurring banana endophytes, such as nonpathogenic Fusarium oxysporum, that are inoculated into tissue culture banana plantlets. At present, it is unclear at what stage in the R. similis infection process the endophytes are most effective. In this study, the effect of three endophytic F. oxysporum isolates (V5w2, Eny1.31i and Eny7.11o) on R. similis host preference of either endophyte-treated or untreated banana plants was investigated. No differences were observed between the proportion of nematodes attracted to either root segments excised from endophyte-treated or untreated plants, or in experiments using endophyte-treated and untreated tissue culture banana plantlets. These results imply that the early processes of banana plant host recognition by R. similis are not affected by endophyte infection.     

Variation in Radopholus citrophilus Population Densities in the Citrus Rootstock Carrizo Citrange

Seedlings of the hybrid citrus rootstock, Carrizo citrange (Citrus sinensis × Poncirus trifoliata) do not uniformly limit development of the citrus burrowing nematode, Radopholus citrophilus. Variation in nematode population densities in roots of seedlings germinating from the same seed suggests that factors responsible for nematode incompatibility are not functional or are not inherited uniformly among progeny. Seeds which produced a single seedling were more likely to produce plants which suppressed citrus burrowing nematode population increase than were seeds which produced two or three seedlings.     

Antagonists of Plant-parasitic Nematodes in Florida Citrus

In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were recovered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyces lilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and Dactylella ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous arthropods also were detected. Nematode-trapping fungi and nematophagous arthropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare.     

Potential of Foliar,Dip, and Injection Applications of Avermectins for Control of Plant-Parasitic Nematodes

Studies were conducted to determine the potential of two avermectin compounds, abamectin and emamectin benzoate, for controlling plant-parasitic nematodes when applied by three methods: foliar spray, root dip, and pseudostem injection. Experiments were conducted against Meloidogyne incognita on tomato, M. javanica on banana, and Radopholus similis on banana. Foliar applications of both avermectins to banana and tomato were not effective for controlling any of the nematodes evaluated. Root dips of banana and tomato were moderately effective for controlling M. incognita on tomato and R. similis on banana. Injections (1 ml) of avermectins into banana pseudostems were effective for controlling M. javanica and R similis, and were comparable to control achieved with a conventional chemical nematicide, fenamiphos. Injections of 125 to 2,000 μg/plant effectively controlled one or both nematodes on banana; abamectin was more effective than emamectin benzoate for controlling nematodes.     

Population Densities of Five Migratory Endoparasitic Nematodes in Carrot Disk Cultures

Numbers of nematodes recovered per culture varied greatly among five species cultured on carrot disks. Radopholus similis and Pratylenchus vulnus showed the highest population densities, with 23,400-fold and 16,600-fold increases, respectively, in 90 days. Final populations of P. thornei and Zygotytenchus guevarai were similar but lower than those of R. similis and P. vulnus. The population of P. neglectus increased 74 times. Species with the greatest reproduction in this study reproduce sexually.     

Influence of Metalaxyl on Three Nematodes of Citrus

Metalaxyl significantly reduced population of Pratylenchus coffeae, Radopholus similis, and Tylenchulus semipenetrans in roots of Citrus limon (rough lemon) under greenhouse conditions. Postinoculation treatment of rough lemon seedlings was not as effective i n reducing nematode populations as was treatment before inoculation. Fewer nematodes infected metalaxyl-treated roots than nontreated roots. However, incubation of nematodes in metalaxyl did not inhibit nematode motility or their ability to locate and infect roots. Cellular responses to nematode injection differed between treated and nontreated tissues. Metalaxyl appeared to confer nematode contraol by modifying citrus roots such that a normally susceptible rootstock became tolerant.     

Effect of Conditioning Treatments on the Survival of Radopholus similis at High Temperatures

Heat treatments are an environmentally safe method for eliminating quarantine pests from tropical foliage. Conditioning heat treatments can induce thermotolerance against subsequent and otherwise phytotoxic temperatures in tropical foliage, allowing heat treatments to be even more effective. However, if thermotolerance is also induced in nematodes of quarantine significance like Radopholus similis, heat treatments would be rendered ineffective. A lethal thermal death point (LT_99.9) was established for R. similis by recording mortality at 25 (control temperature), 43°C, 45°C, 47°C, or 49°C after a 0, 1-, 2-, 4-, 6-, 8-, 10-, 12-, or 15-minute exposure. In a second experiment, nematodes were conditioned at 35, 40, or 45°C for 0, 15, 30, 60, 120, and 180 minutes, allowed to rest for 3 hours, and then challenged at 47°C for 5 minutes. No nematodes survived the challenge heat treatment; rather, nematode mortality was hastened by the conditioning treatment itself. In a third experiment, R. similis inside anthurium roots were conditioned at 25°C or 40°C for 15 minutes and then treated at 45°C for up to 8 minutes. Mortality of conditioned and unconditioned nematodes was similar (P > 0.1). Conditioning treatments increase plant thermotolerance but do not induce thermotolerance in R. similis. Heat treatments have promise as disinfection protocols for quarantines.     

Molecular and Biochemical Diversity Among Isolates of Radopholus spp. from Different Areas of the World

Eleven isolates of Radopholus similis from various banana-growing areas around the world and one isolate of R. bridgei from turmeric in Indonesia were compared using DNA and isoenzyme analysis. The polymerase chain reaction (PCR) was used to amplify a fragment of ribosomal DNA (rDNA), comprising the two internal transcribed spacers (ITS) and the 5.8S gene. Restriction fragment length polymorphisms (RFLPs) in this rDNA fragment were used to compare the 10 isolates. The analysis of this rDNA region revealed little variation among the isolates tested. However, data also were obtained by random amplified polymorphic DNA (RAPD) analysis of total DNA, and a hierarchical cluster analysis of these data arranged the R. similis isolates into two clusters. The first cluster consisted of isolates from Nigeria, Cameroon, Queensland, and Costa Rica; the second was comprised of isolates from Guinea, Guadeloupe, the Ivory Coast, Uganda, and Sri Lanka. The isolate of R. bridgei from turmeric in Indonesia appeared to be more divergent. This grouping was consistent with that obtained when phosphate glucose isomerase (PGI) isoenzyme patterns were used to compare the R. similis isolates. The results from both RAPD analysis and PGI isoenzyme studies indicate that two gene pools might exist within the R. similis isolates studied. No correlation could be detected between the genomic diversity as determined by RAPD analysis and either geographic distribution of the isolates or differences in their pathogenicity. The results support the hypothesis that R. similis isolates have been spread with banana-planting material.     

An endosymbiotic bacterium in a plant-parasitic nematode: Member of a new Wolbachia supergroup

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electron microscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults.16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.     

Morphological and karyotypic differences within and among populations of Radopholus similis

Twenty populations of Radopholus similis from three countries and different hosts (19 populations from ornamental plants and one population from ginger) were compared using morphological characters, morphometrics and karyotype between progeny from both single females and 30 females of each population. Morphological diversity existed in and among the populations, even within the progeny nematodes from single nematodes compared to that of 30 females. The labial disc shape, the number of head annuli, the terminated position of lateral lips, the number of genital papillae before cloacal apertures and female and male tail terminal shape showed variation. In addition, genital papillae arranged in a double row before cloacal apertures was first found in two ornamental populations. The karyotype of all the 20 populations was n = 5. Combining our results and previous studies, we support that Radopholus citrophilus is a synonym of Radopholus similis, and that it is not possible to distinguish physiological races or pathotypes of Radopholus similis according to morphological characters or karyotype.     

Parasitism of Nematodes by the Fungus Hirsutella rhossiliensis as Affected by Certain Organic Amendments

Experiments were conducted to determine whether the addition of organic matter to soil increased numbers of bacterivorous nematodes and parasitic activity of the nematophagous fungus Hirsutella rhossiliensis. In a peach orchard on loamy sand, parasitism of the plant-parasitic nematode Criconemella xenoplax by H. rhossiliensis was slightly suppressed and numbers of C. xenoplax were not affected by addition of 73 metric tons of composted chicken manure/ha. In the laboratory, numbers of bacterivorous nematodes (especially Acrobeloides spp.) and fungivorous nematodes increased but parasitism of nematodes by H. rhossiliensis usually decreased with addition of wheat straw or composted cow manure to a loamy sand naturally infested with H. rhossiliensis. These results do not support the hypothesis that organic amendments will enhance parasitism of nematodes by H. rhossiliensis.     

Aggressiveness and Damage Potential of Central American and Caribbean Populations of Radopholus spp. in Banana

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.     

Rearing Migratory Endoparasitic Nematodes in Citrus Callus and Roots Produced from Citrus Leaves

Radopholus similis and Pratylenchus coffeae were reared on callus and roots developed from citrus leaves. Callus formed best when leaf petioles were immersed in Astatula fine sand and the leaves were sprayed daily with 4 ppm 2,4-D solution and maintained at 25 or 30 C. The nematodes completed one generation in 20 days at 25 C. Highest populations of R. similis (1,127) occurred after 50 days, and the highest for P. coffeae (619) after 70 days. Leaf-callus cultures from R. similis-resistant citrus rootstocks showed the same degree of infection as susceptible rough lemon callus after 30 days.