Larry W. Duncan

Nematode communities as indicators of status and processes of a soil ecosystem influenced by agricultural management practices

Abstract Nematode communities were monitored for three years in a citrus soil ecosystem in Central Florida under various agricultural regimes comparing standard vs. reduced-input practices. Differences in agricultural regimes consisted of two fertilization levels, two irrigation levels, and two types of ground cover under the tree (herbicide vs. mulch). While some nematodes were affected sporadically by fertilization and irrigation treatments, mulch had a consistent and frequently significant effect on many bacterivores, fungivores, herbivores, and omnivores. Rhabitidae, Cephalobus, Aphelenchus, and Aphelenchoides had an immediate but temporary response to mulch additions. Acrobeles, Acrobeloides, Eucephalobus, Teratocephalus, Criconemoides, Aporcelaimellus, and Eudorylaimus were always less abundant in mulch-treated plots, whereas Plectus and Belonolaimus were always more abundant. Of various indices of community composition, only maturity indices, unlike diversity indices, indicated the status and intensity of soil processes (decomposition, mineralization). However, different responses of single genera within a trophic group implied unique contributions of nematode genera in soil ecosystem processes on a temporal scale, suggesting that generic or possibly species level of resolution provide the most adequate information about the soil ecosystem.

Nematode parasites of citrus.

Citrus is grown in more than 125 countries in a belt within 35° latitude north or south of the equator. The major limiting factor to citrus production is a requirement that the occurrence of freezing temperatures be of very short duration. Within the family Rutaceae, the genera Citrus (oranges, mandarins, pomelos, grapefruit, lemons, limes and citrons), Fortunella (kumquats) and Poncirus (trifoliate oranges) contain the principal commercial species (Swingle and Reese, 1967). Citrus production worldwide exceeded 96 Mt in 2002. Approximately 68% of the world’s citrus production is consumed as fresh fruits, and about 11% of total production is used in international trade (Anonymous, 2002). Citrus spp. are naturally deep-rooted plants (Ford, 1954a,b), and optimum growth requires deep, well-drained soils because roots will not grow into or remain in saturated zones. Nevertheless, trees can be well managed in areas with high water tables if grown on beds. Citrus grows well under any rainfall regime provided that adequate soil moisture can be maintained. Irrigation of citrus is commonly practised by a variety of methods that range from orchard flooding to low-volume drip or microsprinkler systems. In areas with sporadic rainfall, the ability to manage soil moisture is critical for good production, particularly during the period when fruit are set after the first seasonal flower bloom (Sites et al., 1951). There is a tendency at present in the USA and elsewhere to increase early returns by planting higher density orchards with shorter life expectancies due to such diseases as citrus blight, tristeza and greening (Hearn, 1986).

Subterranean, Herbivore-Induced Plant Volatile Increases Biological Control Activity of Multiple Beneficial Nematode Species in Distinct Habitats

While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9–12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests.

Inheritance of citrus nematode resistance and its linkage with molecular markers.

Abstract Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds.

Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves.

Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.

Wide interguild relationships among entomopathogenic and free-living nematodes in soil as measured by real time qPCR

Entomopathogenic nematodes (EPNs) are promising biological control agents of soil-dwelling insect pests of many crops. These nematodes are ubiquitous in both natural and agricultural areas. Their efficacy against arthropods is affected directly and indirectly by food webs and edaphic conditions. It has long been suggested that a greater understanding of EPN ecology is needed to achieve consistent biological control by these nematodes and the development of molecular tools is helping to overcome obstacles to the study of cryptic organisms and complex interactions. Here we extend the repertoire of molecular tools to characterize soil food webs by describing primers/probe set to quantify certain free-living, bactivorous nematodes (FLBNs) that interact with EPNs in soil. Three FLBN isolates were recovered from soil baited with insect larvae. Morphological and molecular characterization confirmed their identities as Acrobeloides maximum (RT-1-R15C and RT-2-R25A) and Rhabditis rainai (PT-R14B). Laboratory experiments demonstrated the ability of these FLBNs to interfere with the development of Steinernema diaprepesi, Steinernema riobrave and Heterorhabditis indica parasitizing the weevil Diaprepes abbreviatus (P<0.001), perhaps due to resource competition. A molecular probe was developed for the strongest competitor, A. maximum. We selected the highly conserved SSU rDNA sequence to design the primers/probe, because these sequences are more abundantly available for free-living nematodes than ITS sequences that can likely provide better taxonomic resolution. Our molecular probe can identify organisms that share ⩾98% similarity at this locus. The use of this molecular probe to characterize soil communities from samples of nematode DNA collected within a citrus orchard revealed positive correlations (P<0.01) between Acrobeloides-group nematodes and total numbers of EPNs (S. diaprepesi, H. indica and Heterorhabditis zealandica) as well as a complex of nematophagous fungi comprising Catenaria sp. and Monachrosporium gephyropagum that are natural enemies of EPNs. These relationships can be broadly interpreted as supporting Linfords hypothesis, i.e., decomposition of organic matter (here, insect cadavers) greatly increases bactivorous nematodes and their natural enemies.

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.

Sending Mixed Messages: A Trophic Cascade Produced by a Belowground Herbivore-Induced Cue

Plants defend themselves against herbivores both directly (chemical toxins and physical barriers) and indirectly (attracting natural enemies of their herbivores). Previous work has shown that plant roots of citrus defend against root herbivores by releasing an herbivore-induced plant volatile (HIPV), pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene), that attracts naturally occurring entomopathogenic nematodes (EPNs) to Diaprepes abbreviatus larvae when applied in the field. However, the soil community is complex and contains a diversity of interspecific relationships that modulate food web assemblages. Herein, we tested the hypothesis that other nematode types beyond EPNs, as well as, nematophagous fungi are affected by the same HIPV that attracts EPNs to herbivore-damaged roots. We employed molecular probes designed to detect and quantify nematodes from the Acrobeloides–group (free-living bacterivorous nematodes, FLBNs), some of which compete with EPNs by ‘hyperparasitizing’ insect cadavers, and five species of nematophagous fungi (NF), which attack and kill EPNs. In two different agricultural systems (citrus and blueberry), we detected diverse species of nematodes and fungi; however, only the behavior of FLBNs was affected in a manner similar to that reported previously for EPNs. Although detected, NF abundance was not statistically affected by the presence of the belowground HIPV. We provide the first evidence showing subterranean HIPVs behave much the same as those aboveground, attracting not only parasitoids, but also hyperparasites and other food web members.

Application and Evaluation of Entomopathogens for Citrus Pest Control

Originally from Southeast Asia, citrus has been introduced into many arid or humid, subtropical to tropical regions around the world where it is cultivated in a diverse array of quasi-permanent tree-crop plantings that range from small interplanted hillside patches to vast monocultural enterprises (Webber, 1948). Numerous arthropods infest citrus and some are considered serious pests locally or more globally either because of the direct damage they cause or because they transmit or otherwise facilitate important plant pathogens (e.g., the brown citrus aphid, Toxoptera citricida, vectors the citrus tristeza virus (CTV), Poprawski et al., 1999).

Molecular and morphological consilience in the characterisation and delimitation of five nematode species from Florida belonging to the Xiphinema americanum -group

Taxonomic keys and original descriptions were used to identify 26 Xiphinema americanum-group populations from Florida comprising X. georgianum (eight populations), X. citricolum (six), X. floridae (six), X. laevistriatum (five) and X. tarjanense (one). Principal component analysis of a subset of 19 morphometric characters accorded with the species designations; discriminant analysis of six characters assigned 93% (111 of 119) of the specimens to the correct putative species. A phylogeny of these populations estimated from analyses of rDNA sequences (ITS and D2D3) was also congruent with species designations from taxonomic keys and PCA. The D2D3 sequences revealed very little intraspecific variation whereas each population sampled produced a unique ITS sequence. Intraspecific variation in the suites of character code values from polytomous keys resulted mainly from minor discrepancies between population character means and reported character ranges for the species. We show that, for these taxa, species delimitation based on the requirement that sister taxa evolve autapomorphies distinguishes intraspecific variation from phylogeny and, as applied to molecular characters, delimits the same taxa as those predicted by morphological keys and PCA.