Comparison of soil fungal community structure in different peanut rotation sequences using ribosomal intergenic spacer analysis in relation to aflatoxin-producing fungi.

The present study focuses on determining soil fungal community structure in different peanut-cropping sequences by using a high-resolution DNA fingerprinting technique: ribosomal intergenic spacer analysis (RISA). This study was initiated to determine fungal community profiles in four peanut-cropping sequences (continuous peanut, 4 years of continuous bahiagrass followed by peanut, peanut-corn-cotton, and peanut-cotton rotations), with a special focus to evaluate whether the profiles under investigation may have also indicated microbial differences that could affect Aspergillus flavus populations. Results indicated 75% similarities among fungal communities from the same cropping sequences as well as with similar times of sampling. Polymerase chain reaction (PCR)-based detection of A. flavus directly from these soils was carried out using A. flavus-specific primers (FLA1 and FLA2) and also through quantitative estimation on A. flavus and A. parasiticus agar medium. Population levels of A. flavus in soil samples ranged from zero to 1.2 × 10(3) CFU g(-1) of soil (based on culturable methods); however, the fungus was not detected with A. flavus-specific primers. The minimum threshold limit at which these aflatoxin-producing fungi could be detected from the total soil genomic DNA was determined through artificial inoculation of samples with 10-fold increases in concentrations. The results indicated that a minimum population density of 2.6 × 10(6) CFU g(-1) of soil is required for PCR detection in our conditions. These results are useful in further determining the relative population levels of these fungi in peanut soils with other soil fungi. This is a new approach to understanding soil fungal communities and how they might change over time and under different rotation systems.

Field Efficacy of Verticillium lecanii, Sex Pheromone, and Pheromone Analogs as Potential Management Agents for Soybean Cyst Nematode.

A soybean cyst nematode sex pheromone (vanillic acid), chemical analogs of the pheromone, and the fungus Verticillium lecanii were applied in alginate prills (340 kg/ha) to microplots and small-scale field plots as potential management agents for Heterodera glycines on soybean. In 1991 microplot tests, treatment with V. lecanii, vanillic acid, syringic acid plus V. lecanii, or vanillic acid plus V. lecanii lowered midseason cyst numbers compared with the untreated susceptible cultivar control, autoclaved V. lecanii treatment, or aldicarb treatment, At-harvest cyst numbers were lowest with V. lecanii and with vanillic acid treatments. Aldicarb treatment reduced midseason cyst numbers in 1992. There were no differences among seed yields either year. In the field trials, numbers of cysts were reduced one or both years with aldicarb, ferulic acid, syringic acid, vanillic acid, or 4-hydroxy-3-methoxybenzonitfile treatments, or with a resistant cultivar, compared to an untreated susceptible cultivar. Highest yields were recorded after treatment with 4-hydroxy-3-methoxybenzonitrile (1991), methyl vanillate (1992), and aldicarb (1992). These studies indicate that some chemical analogs of vanillic acid have potential for use in soybean cyst nematode management schemes.

Application of a Sex Pheromone, Pheromone Analogs, and Verticillium lecanii for Management of Heterodera glycines.

A mutant strain of the fungus Verticillium lecanii and selected bioregulators of Heterodera glycines were evaluated for their potential to reduce population densities of the nematode on soybean under greenhouse conditions. The bioregulators tested were the H. glycines sex pheromone vanillic acid and the pheromone analogs syringic acid, isovanillic acid, ferulic acid, 4-hydroxy-3-methoxybenzonitrile, and methyl vanillate. A V. lecanii-vanillic acid combination and a V. lecanii-syringic acid combination were also applied as treatments. Syringic acid, 4-hydroxy-3-methoxybenzonitrile, V. lecanii, V. lecanii-vanillic acid, and V. lecanii-syringic acid significantly reduced nematode population densities in the greenhouse tests. Results with vanillic acid, isovanillic acid, and ferulic acid treatments were variable. Methyl vanillate did not significantly reduce cyst nematode population densities in the greenhouse tests.

Optimal Levels of Meloidogyne incognita Inoculum for Infection of Tomato and Peach in Vitro.

Penetration of second-stage juveniles (J2) of Meloidogyne incognita into tomato root explants and in vitro propagated peach plantlet roots were compared. Five inoculum levels were used: 25, 50, 75, 100, and 200 J2 for tomato; and 50, 100, 200, 500, and 1,000J2 for peach. The greatest root penetration into tomato was 30% at the 75 J2 level, but the maximum penetration into peach roots was only 8% at the 200 J2 level. The difference (P = 0.05) in penetration of M. incognita at all inoculum levels into these two hosts indicates that penetration versus inoculum density for in vitro studies need to be determined for different plant species.

An In Vitro Test for Temperature Sensitivity and Resistance to Meloidogyne incognita in Tomato.

An in vitro root explant tissue culture technique is described for determining susceptibility of tomato (Lycopersicon esculentum Mill.) breeding lines and cultivars to the root-knot nematode Meloidogyne incognita. Root explants were taken from 2-day-old seedlings cultured for 30 days at 28 C on Gamborg's B-5 medium with or without nematode inoculum. The remaining portion of the root and stem from the excised root explants was transferred to soil in pots and grown to maturity in the greenhouse. In vitro root explants were evaluated for growth and occurrence of juveniles, adults, and egg masses. The regenerated plants were used to produce more seed, The proposed technique is simple, reliable, and adapted to routine screening of large numbers of F and F samples, and it utilizes less space than tests performed on intact plants in the greenhouse or growth chamber. Evidence is presented also on the breakdown of resistance to M. incognita under high temperature stress using this in vitro root explant technique.

Response of Peach Scion Cultivars and Rootstocks to Meloidogyne incognitain Vitro and in Microplots.

The response of the peach scion cultivars, Jerseyqueen, Redhaven, Compact Redhaven, and Rio Oso Gem and rootstocks 'Lovely and 'Nemaguard' to inoculation with Meloidogyne incognita was compared in vitro and in microplots. One or more parameters monitored in vitro correlated with at least one parameter monitored in microplots, 4 years after tree planting (1989). A range of responses was observed from highlysusceptible in Lovell to resistant in Nemaguard. In vitro and microplot data suggest high and moderate levels of resistance to M. incognita in Compact Redhaven and Redhaven, respectively. Both Jerseyqueen and Rio Oso Gem were susceptible to M. incognita, but not as susceptible as Lovell. The response of self-rooted peach cultivars and rootstocks to M. incognita in vitro appears to be a reliable method for predicting the reaction of each to these nematodes under field conditions.

Description and SEM Observations of Meloidogyne sasseri n. sp. (Nematoda: Meloidogynidae), Parasitizing Beachgrasses.

Meloidogyne sasseri n. sp. is described and illustrated from American beachgrass (Ammophila breviliffulata) originally collected from Henlopen State Park and Fenwick Island near the Maryland state line in Delaware, United States (6). Its relationship to M. graminis, M. spartinae, and M. californiensis is discussed. Primary distinctive characters of the female perineal pattern were a high to rounded arch with shoulders, widely spaced lateral lines interrupting transverse striations, a sunken vulva and anus, and coarse broken striae around the anal area. Second-stage juvenile body length was 554 mum (470-550), stylet length 14 mum (13-14.5), tail length 93 mum (83-115), tapering to a finely rounded terminus. Male stylet length 20 mum (19-21.5), spicule length 33 mum (30-36). Scanning electron microscope observations provided additional details of perineal patterns and face views of the female, male, and J2 head. Wheat, rice, oat, Ammophila sp., Panicum sp., bermudagrass, zoysiagrass and St. Augustinegrass were tested as hosts. Distribution of the species was the coasts of Delaware and Maryland. The common name "beachgrass root-knot" is proposed for M. sasseri n. sp.

Resistance in Zea mays to Heterodera zeae.

Twenty-three precommercial field corn lines (Zea mays) were screened in the greenhouse and in vitro for the ability to support reproduction of Heterodera zeae. Although H. zeae reproduced on all corn lines, reproduction was only 0.4 to 4.5% on the five least suitable corn lines in greenhouse tests compared with the susceptible check line Pioneer brand 3184. The least suitable experimental line supported an average of 30 cysts plus females after 8 weeks growth, whereas the susceptible check, Pioneer brand 3184, averaged 8,183 cysts plus females per pot. Reproduction of H. zeae in in vitro root cultures of the 23 lines and susceptible check cultivar, Iochief, was too low to be of any value in detecting resistance to this nematode under the conditions of these tests.

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes.

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 mug benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 mug/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 mug/ml, but some hyphae grew on medium amended with 1,000 mug/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 mug/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.

Isolation of Fungi from Heterodera glycines and in vitro Bioassays for Their Antagonism to Eggs.

Twenty fungi were assayed in vitro for antagonism to eggs of Heterodera glycines. Eight of the fungi were isolated from cysts or eggs of H. glycines during the current study, one was isolated from Panagrellus redivivus, and eleven were obtained from other researchers or collections. The bioassays were conducted on eggs from nematodes that had been grown monoxenically on excised root tips. Phoma chrysanthemicola, one strain of Verticillium chlamydosporium, and one strain of V. lecanii caused a decrease (P < 0.01, P < 0.05, P < 0.05, respectively) in the number of viable eggs, although no hyphae were observed colonizing live eggs. Trichoderma polysporum infected live eggs but enhanced (P < 0.05) egg survival. Acremonium bacillisporum, Chaetomium sp., Drechmeria coniospora (two strains), Epicoccum sp., Exophiala jeanselmei, Fusarium sp., Neocosmospora vasinfecta, Scytalidium fulvum, Trichoderma harzianum (two strains), V. chlamydosporium (one strain), V. lecanii (three strains), and an unidentified fungus did not measurably affect egg viability, even though hyphae of five of these fungi were seen in live eggs. The bioassay provides a useful step in the selection of a biological control agent for this major nematode pest.

Isolation and identification of a compound from soybean cyst nematode,Heterodera glycines, with sex pheromone activity.

A single compound with sex pheromone activity was isolated from the female soybean cyst nematode,Heterodera glycines, by a sequence of four high-performance liquid chromatographic steps and identified as vanillic acid by a combination of ultraviolet spectroscopy and chromatography. The structure was confirmed by gas chromatography-mass spectrometry. Both attractancy and coiling behavior in male soybean cyst nematode were elicited by authentic vanillic acid.

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.

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode, Heterodera glycines.

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.

Chemical communicators in nematodes.

Chemical signals released by one organism and perceived by another organism are classified as semiochemicals. Semiochemicals are divided into pheromones, which elicit intraspecific responses, and allelochemics, which elicit interspecific responses. Nematodes utilize and (or) recognize signals from both categories of semiochemicals. The existence of pheromones, specifically sex and aggregation pheromones, has been demonstrated in numerous plant and animal parasitic and free-living nematodes. Sex pheromones have been isolated and purified from Nippostrongylus brasiliensis and Heterodera glycines, and epidietic pheromones have been shown to be responsible for initiation of dauer juvenile formation in Caenorhabditis elegans. Allelochemics cause interspecific responses in insects and other invertebrates but are only postulated to occur in nematodes. Food-finding behavior of nematodes is almost certainly caused by host-released allelochemic messengers. Understanding of the behavioral responses and the chemical messengers that affect bioregulation of various processes in nematodes will influence future management strategies.

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.

Population Dynamics, Root Penetration, and Feeding Behavior of Pratylenchus agilis in Monoxenic Root Cultures of Corn, Tomato, and Soybean.

Population dynamics, rate of root penetration, and external root feeding behavior of Pratylenchus agilis (Pa) in monoxenic cultures of intact corn seedlings and root explants of corn, tomato, and soybean were studied. In descending order of suitability as hosts were I. O. Chief corn, Rutgers tomato, and Williams soybean. Soybean entries Kent, Pickett 71, PI 90763, and Essex were poor hosts. Numbers of eggs and vermiform Pa in the agar medium indicated total fecundity and host suitability. Agar, sand, or soil as support media did not appear to affect Pa root penetration, but the rate of corn root growth did. Whereas most vermiform Pa and eggs were in roots, substantial numbers appeared able to feed and complete their life cycle as ectoparasites on root epidermal cells and root hairs.

Biochemical Identification of the Two Races of Radopholus similis by Starch Gel Electrophoresis.

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, alpha + beta esterase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, and phosphoglucose isomerase are diagnostic markers of the races.

Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis.

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.

Parthenogenesis in the Two Races of Radopholus similis from Florida.

Single larval inoculations of both the citrus and banana races of Radopholus similis from Florida indicate parthenogenetic reproduction is possible. These races normally reproduce amphimictieally. Both races produced all-female populations from single larva inoculations after 80 d and male-female populations after 180 d. The all-female populations produced female progeny with viable eggs, but no spermatozoa were present in the spermatheca, indicating tychoparthenogenesis occurred. Uninseminated females in the latter study produced both male and female progeny. Approximately 95% of the female progeny of the male-female populations were inseminated, indicating that cross-fertilization had occurred. The proportion of males to females was as expected in a normal bisexual population. No intersex males were observed. The mechanism of sex determination is not fully understood but assumed to be enviromnentally induced.