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.     

Occurrence of Biotypes in 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.     

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.     

The Response of Citrus limon Seedlings to a Symbiont,Glomus etunicatus,and a Pathogen,Radopholus similis

The influences of a vesicular-arbuscular mycorrhiza (Glomus etunicatus) and burrowing nematode (Radophohts similis), alone and in combination, on the growth of rough lemon (Citrus limon) seedlings were studied in the greenhouse. Growth of mycorrhizal seedlings was significantly greater than that of nonmycorrhizal seedlings or seedlings inoculated with R. sindlis. Mycorrhizal stimulation of seedling growth was inhibited by nematode infection. When seedlings were inoculated with G. etunicatus arid R. similis, suppression of seedling growth by R. similis was less on VAM seedlings than on nonmycorrhizal seedlings, Nonmycorrhizal seedlings infected with R. similis were significantly smaller than nonmycorrhizal seedlings free of R. similis. Vesicle formation and mycelia growth were less in nematode-infected roots.     

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.     

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.     

Response of Citrus Seedlings to Radopholus similis in Two Soils

Radopholus similis was less pathogenic to greenhouse-grown citrus seedlings in Leon loamy sand than in Lakeland fine sand. This was not affected by different watering regimes. Seedling growth reduction by the burrowing nematode in either soil, compared with noninfected controls, was significant at the 1% level.     

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.     

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.     

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.     

Nematode–citrus plant interactions: host preference,damage rate and molecular characterization of Citrus root nematode Tylenchulus semipenetrans

• Citrus plants are host to several plant parasitic nematodes (PPNs), which are microscopic organisms. Among PPNs, the citrus root nematode, T. semipenetrans (Cobb 1913) (Tylenchida: Tylenchulidae), causes significant damage to citrus plantations worldwide. Understanding citrus nematode populations, precise identification, host preference among citrus species, and damage threshold are crucial to control T. semipenetrans. The minutiae of citrus plant–nematode interactions, nematode density and molecular nematode identification are not well understood. In this study, nematode species and density in citrus orchards, host specialization, molecular and morphological characteristics of nematodes were assessed.
• Molecular and morphological methods, host–nematode interactions, host (citrus species) preference, damage economic threshold (ET), and economic injury level (EIL) were determined using laboratory methods and field sampling. Citrus plantations in different provinces in the Mediterranean region of Turkey were investigated.
• Nematode species were identified molecularly and morphologically. ITS sequences revealed that samples were infected by citrus root nematode T. semipenetrans. The lowest nematode density was in C. reticulata in Mersin (53 2nd stage juveniles (J2s) 100 g⁻¹ soil), while the highest density was from Hatay in C. sinensis (12173 J2s 100 g⁻¹ soil). Highest citrus nematode population density was on roots of C. reticulata, followed by C. sinensis, C. limon, and C. paradisi.
• The citrus nematode is more common than was thought and population fluctuations change according to specific citrus species. Environmental conditions, host and ecological factors, such as temperature, soil pH, and soil nutrients, might influence nematode populations in citrus orchards. Investigating nematode density in diverse soil ecologies and the responses of different resistant/tolerant citrus species and cultivars to nematode populations is essential in future studies.

     

Effects of Pratylenchus coffeae—Tylenchulus semipenetrans Interactions on Nematode Population Dynamics in Citrus

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.     

Biology and Influence of Pterotylenchus cecidogenus on Desmodium ovalifolium

Pterotylenchus cecidogenus did not inhibit germination of Desmodium ovalifolium but did reduce survival of seedlings and root and stem growth. Stem gall formation was associated with nematode population densities which were positively correlated with plant age at time of inoculation. Nematode population densities increased 100 times in 52 days following inoculation.     

Distribution of Tylenchulus semipenetrans in a Texas Grapefruit Orchard

Distribution of the citrus nematode (Tylenchulus semipenetrans) was studied over 18 months in a 6-year-old orchard of grapefruit (Citrus paradisi cv. Ruby Red) on sour orange (C. aurantium) rootstock. The 1.8-ha orchard was under chemical weed control, no tillage, and flood irrigation. Highest numbers of nematodes were found in the top 15 cm of the soil profile. The nematode population peaked in April and declined to lowest levels in August and September. Numbers of nematodes were negatively correlated (r = -0.95) with soil temperatures above 29 C. Soil populations of nematodes were not correlated with soil moisture. The distribution of the nematode in the field was highly skewed and was described by a negative binomial. In this 1.8-ha block, five soil samples of 12 cores each would provide an estimate within 20% of the true nematode population mean with 95% confidence.     

Autoradiography of Developing Syncytia in Cotton Roots Infected with Meloidogyne incognita

Cotton (Gossypium hirsutum) seedlings, uniformly infected with Meloidogyne incognita, were exposed for periods of 1-15 days to a nutrient solution containing tritium-labelled thymidine. Syncytium formation began with the amalgamation of cells near the nematode head, and was followed by synchronized mitoses of the nuclei which had been incorporated into a single cell. Syncytial nuclei synthesized DNA in roots harvested 3, 6, 9, 12, and 15 days after inoculation. Seedlings transferred from unlabelled to labelled nutrient solution 9 days after inoculation, and grown for 6 more days, contained some syncytial nuclei which did not become labelled. Giant-cell nuclei increased in size and, in many cases, all nuclei in one giant cell of a set showed active DNA synthesis at about the time the nematode molted to the adult stage.     

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.     

Characterization of Citrus Rootstock Responses to Tylenchulus semipenetrans (Cobb)

Citrus rootstocks which significantly limited the reproduction of Tylenchulus semipenetrans (Cobb) "Citrus" and "Poncirus" biotypes responded to infection by producing a hypersensitive-type response in the root hypodermis, wound periderm and/or cavities in the root cortex, and/or abnormal vacuoles in nurse cell cytoplasm. Rootstocks which limited nematode reproduction also had significantly fewer nematodes in the rhizoplane within 8 d of inoculation than did rootstocks which did not limit reproduction. Germplasm sources of the cellular responses which limited citrus nematode reproduction were identified.     

Eggs of Tylenchulus semipenetrans Inhibit Growth of Phytophthora nicotianae and Fusarium solani in vitro

In previous greenhouse and laboratory studies, citrus seedlings infested with the citrus nematode Tylenchulus semipenetrans and later inoculated with the fungus Phylophthora nicotianae grew larger and contained less fungal protein in root tissues than plants infected by only the fungus, demonstrating antagonism of the nematode to the fungus. In this study, we determined whether eggs of the citrus nematode T. semipenetrans and root-knot nematode Meloidogyne arenaria affected mycelial growth of P. nicotianae and Fusarium solani in vitro. Approximately 35,000 live or heat-killed (60°C, 10 minutes) eggs of each nematode species were surface-sterilized with cupric sulfate, mercuric chloride, and streptomycin sulfate and placed in 5-pl drops onto the center of nutrient agar plates. Nutrient agar plugs from actively growing colonies of P. nicotianae or F. solani were placed on top of the eggs for 48 hours after which fungal colony growth was determined. Live citrus nematode eggs suppressed mycelial growth of P. nicotianae and F. solani (P ≤ 0.05) compared to heat-killed eggs and water controls. Reaction of the fungi to heat-killed eggs was variable. Root-knot nematode eggs had no effect on either P. nicotianae or F. solani mycelial growth. The experiment demonstrated a species-specific, direct effect of the eggs of the citrus nematode on P, nicotianae and F. solani.     

Effects of Oxamyl on the Citrus Nematode,Tylenchulus semipenetrans,and on Infection of Sweet Orange

Foliar sprays of 4 μg/ml oxamyl on sweet orange trees in a greenhouse slightly depressed the number of Tylenchulus semipenetrans larvae obtained from roots and soil, but similar treatments were not effective in two orchards. Soil drench treatments decreased the number of citrus nematode larvae obtained from roots or soil of citrus plants grown itt a greenhouse and in orchards. Exposure to 5-10 μg/ml of oxamyl in water was lethal to only a few second-stage larvae treated 10 days, and many second-stage larvae in 2.0 μg/ml oxamyl recovered motility when transferred to fresh water. Aqueous solutions of 50 and 100 μg/ml of oxamyl were toxic to citrus nematode larvae. Additional observations indicate that oxamyl interfered with hatch of citrus nematode larvae and was nematistatic and/or protected sweet orange roots from infection. Oxamyl degraded at different rates in two soils. The number of citrus nematode larvae that infected and developed on sweet orange roots was increased by an undetermined product of the degradation of oxamyl in soil, water, and possibly within plants. This product apparently was translocated in roots.     

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.