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.     

Sequence Tag Site and Host Range Assays Demonstrate that Radapholus similis and R. citraphilus are not Reproductively Isolated

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.     

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.     

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.     

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.     

Root Tissue Response of Two Related Soybean Cultivars to Infection by Lectin-treated Meloidogyne spp.

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.     

Control of Root-knot Nematodes on Tomato by Lectins

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.     

Influence of Lectins on Constricting Ring Formation by Arthrobotrys dactyloides

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.     

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.     

Competition between the Plant-parasitic Nematodes Pratylenchus neglectus and Meloidogyne chitwoodi

In experiments on competition between Pratylenchus neglectus and Meloidogyne chitwoodi in barley, the species that parasitized the roots first inhibited penetration by the latter species. Prior presence of P. neglectus impeded the development of M. chitwoodi. Pratylenchus neglectus reduced egg production, final population levels, and reproductive index of M. chitwoodi. The reduction was linearly related to initial population densities of P. neglectus. Initial population densities of M. chitwoodi had no effect on final population levels of P. neglectus. Carbon assimilation by barley plants was reduced when either nematode species was present alone, but not when both were present together. Both nematode species assimilated lower amounts of carbon when present together than when present alone. A split-root experiment demonstrated that translocatable chemicals were not involved in the competition between the two species.     

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.     

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.     

Population Development of Meloidogyne incognita on Soybean Defoliated by Pseudoplusia includens

Greenhouse studies examined population densities of Meloidogyne incognita race 4 on soybean (Glycine max ''Davis'') defoliated by larvae of soybean looper (Pseudoplusia indudens (Walker)). Plants were defoliated over a 2-week period beginning 5 weeks after seedlings were transplanted. Four groups of plants were infested with nematodes (5,000 eggs/pot) at 2-week intervals to allow harvesting of plants at 0, 2, 4, and 6 weeks postdefoliation (WPD). Plants in each group were harvested 4 weeks after nematode infestation. Root and nodule weights of defoliated plants were suppressed at 0 WPD, but differences were not detectable at 2, 4, and 6 WPD. Population densities of M. incognita were similar on defoliated and control plants at 0 WPD but were greater on defoliated plants at 4 and 6 WPD. Percentage hatching of eggs produced on the latter plants also was higher. Effects of insect-induced defoliation on development of M. incognita remained detectable even after soybean plant growth apparently returned to normal.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

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.     

Interactions between Nematodes and Earthworms: Enhanced Dispersal of Steinernema carpocapsae

Dispersal of the nematode Steinernema carpocapsae (All strain), applied on the top or the bottom of soil columns, was tested in the presence or absence of two earthworm species, Lumbricus terrestris or Aporrectodea trapezoides. Nematode dispersal was estimated after a 2-week period with a bioassay against the greater wax moth, Galleria mellonella. Vertical dispersal of nematodes was increased in the presence of earthworms. When nematodes were placed on the surface of soil columns, significantly more nematodes dispersed to the lower half of the columns when either earthworm species was present than when earthworms were not present. When nematodes were placed on the bottom of soil columns, significantly more nematodes dispersed to the upper half of the columns when L. terrestris was present than when A. trapezoides was present or in the absence of earthworms. Because nematodes were found on the exterior and in the interior of earthworms, nematode dispersal may be enhanced by direct contact with the earthworms.     

Nodulation of Soybeans as Affected by Half-root Infection with Heterodera glycines

A split-root technique was applied to soybean, Glycine max (L.) Merr. cv. Lee 68, to characterize the nature of the nodulation suppression by race 1 of the soybean cyst nematode (SCN), Heterodera glycines. Root-halves of each split-root plant were inoculated with Rhizobium japonicum, and one root-half only was inoculated with various numbers of SCN eggs. Nodulation (indicated by nodule number, nodule weights, and ratio of nodule weight to root weight) and nitrogen-fixing capacity (indicated by rate of acetylene reduction) were systemically and variously suppressed on both root-halves of the split-root plant 5 weeks after half-root inoculation with 12,500 SCN eggs. Inoculation with 500 eggs caused this suppression only on the SCN-infected (+NE) root-half; nodulation on the companion uninfected (-NE) root-half was stimulated slightly. The +NE root-halves inoculated with 5,000 eggs were excised at 2-week intervals; nodulation on the remaining -NE root-halves was not different from that of the noninoculated control when measured 6 weeks after the SCN inoculation. Thus, the systemic suppression of nodulation was reversible upon the removal of the SCN. Similarly, application of various levels of KNO₃ to the -NE root-halves of the split-root plant did not alleviate the suppressed nodulation on the companion +NE root-halves, even though plant growth was much improved at certain levels of nitrogen (125 μg N/g soil). This indicated that the localized suppression of nodulation by SCN was caused by factors in addition to poor plant growth.     

Tests for Transmission of Prunus Necrotic Ringspot and Two Nepoviruses by Criconemella xenoplax

In two of three trials, detectable color reactions in ELISA for Prunus necrotic ringspot virus (PNRSV) were observed for Criconemella xenoplax handpicked from the root zone of infected peach trees. Criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with PNRSV failed to transmit the virus to cucumber or peach seedlings. The nematode also failed to transmit tomato ringspot (TomRSV) or tobacco ringspot viruses between cucumbers, although Xiphinema americanum transmitted TomRSV under the same conditions. Plants of peach, cucumber, Chenopodium quinoa, and Catharanthus roseus were not infected by PNRSV when grown in soil containing C. xenoplax collected from root zones of PNRSV-infected trees. Shirofugen cherry scions budded on Mazzard cherry seedling rootstocks remained symptomless when transplanted into root zones of PNRSV-infected trees. Virus transmission was not detected by ELISA when C. xenoplax individuals were observed to feed on cucumber root explants that were infected with PNRSV and subsequently fed on roots of Prunus besseyi in agar cultures. Even if virus transmission by C. xenoplax occurs via contamination rather than by a specific mechanism, it must be rare.