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.     

Identification of fungi associated with rotylenchulus reniformis

The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.     

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.     

Effects of Nematophagous Fungi on Numbers and Death Rates of Bacterivorous Nematodes in Arable Soil

In a series of microcosm experiments with an arable, sandy loam soil amended with sugarbeet leaf, the short-term (8 weeks) dynamics of numbers of nematodes were measured in untreated soil and in γ-irradiated soil inoculated with either a field population of soil microorganisms and nematodes or a mixed population of laboratory-propagated bacterivorous nematode species. Sugarbeet leaf stimulated an increase in bacterivorous Rhabditidae, Cephalobidae, and a lab-cultivated Panagrolaimus sp. Differences were observed between the growth rates of the nematode population in untreated and γ-irradiated soils, which were caused by two nematophagous fungi, Arthrobotrys oligospora and Dactylaria sp. These fungi lowered the increase in nematode numbers due to the organic enrichment in the untreated soil. We estimated the annually produced bacterivous nematodes to consume 50 kg carbon and 10 kg nitrogen per ha, per year, in the upper, plowed 25 cm of arable soil.     

Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay

Hirsutella rhossiliensis, a nematophagous fungus, has shown potential in biocontrol of plant-parasitic nematodes. Monitoring the population dynamics of a biocontrol agent in soil requires comprehensive techniques and is essential to understand how it works. Bioassay based on the fungal parasitism on the juveniles of soybean cyst nematode, Heterodera glycines, can be used to evaluate the activity of the fungus but fails to quantify fungal biomass in soil. A real-time polymerase chain reaction (PCR) assay was developed to quantify the fungal population density in soil. The assay detected as little as 100 fg of fungal genomic DNA and 40 conidia g⁻¹ soil, respectively. The parasitism bioassay and the real-time PCR assay were carried out to investigate the presence, abundance and activity of H. rhossiliensis in soil after application of different inoculum levels. Both of the percentage of assay nematodes parasitized by H. rhossiliensis based on the parasitism bioassay and the DNA yield of the fungus quantified by real-time PCR increased significantly with the increase of the inoculum levels. The DNA yield of the fungus was positively correlated with the percentage of assay nematodes parasitized by H. rhossiliensis. The combination of the two is useful for monitoring fungal biomass and activity in soil.     

Distribution of the entomopathogenic nematodes from La Rioja (Northern Spain)

Entomopathogenic nematodes (EPNs) distribution in natural areas and crop field edges in La Rioja (Northern Spain) has been studied taking into account environmental and physical-chemical soil factors. Five hundred soil samples from 100 sites of the most representative habitats were assayed for the presence of EPNs. The occurrence of EPNs statistically fitted to a negative binomial distribution, which pointed out that the natural distribution of these nematodes in La Rioja was in aggregates. There were no statistical differences (p < or = 0.05) in the abundance of EPNs to environmental and physical-chemical variables, although, there were statistical differences in the altitude, annual mean air temperature and rainfall, potential vegetation series and moisture percentage recovery frequency. Twenty-seven samples from 14 sites were positive for EPNs. From these samples, twenty isolates were identified to a species level and fifteen strains were selected: 11 Steinernema feltiae, two S. carpocapsae and two S. kraussei strains. S. kraussei was isolated from humid soils of cool and high altitude habitats and S. carpocapsae was found to occur in heavy soils of dry and temperate habitats. S. feltiae was the most common species with a wide range of altitude, temperature, rainfall, pH and soil moisture, although this species preferred sandy soils. The virulence of nematode strains were assessed using G. mellonella as insect host, recording the larval mortality percentage and the time to insect die, as well as the number of infective juveniles produced to evaluate the reproductive potential and the time tooks to leave the insect cadaver to determinate the infection cycle length. The ecological trends and biological results are discussed in relationship with their future use as biological control.     

Parasitism of Xiphinema rivesi and X. americanum by Zoosporic Fungi

Living Xiphinema americanum (Xa) and X. rivesi (Xr) extracted from soil samples and stored for 1-5 days at 4 or 20 C contained aseptate fungal hyphae. The fungi directly penetrated the nematode''s cuticle from spores encysted near the head. Penetration through the stoma, vulva, or anus was rare. Catenaria anguillulae (Cat), Lagenidium caudatura (Lag), Aphanomyces sp. (Aph), and Leptolegnia sp. (Lep) were isolated into pure culture from infected nematodes. The pathogenicity of these zoosporic fungi was determined by incubating mixed freshly extracted Xa and Xr in 2% soil extract (pH = 6.7, conductivity = 48 μmhos, 20 ± 2 C) containing zoospores obtained from single-spore isolates. After 4 days, Cat, Lag, Aph, and Lep had infected 78, 18, 13, and 22%, respectively, of the nematodes. Both Xa and Xr were infected by every fungus; however, the relative susceptibility of Xa and Xr to these fungi was not determined. All noninoculated control nematodes remained uninfected and alive. In a second experiment, parasitism of Xa and Xr by Aph and Lep was increased when nematodes were incubated in 2% soil extract for 4 days before exposure to zoospores. In a third experiment, parasitism of Xa and Xr by Cat was greater in diluted saturation soil extract (conductivity = 100-400 μmhos) than in undiluted saturation extract (conductivity = 780 μmhos). Cat produced small zoospores (4-μm-d), bulbous infection hyphae, and assimilative hyphae of varying diameters in nematodes, whereas Lag, Aph, and Lep produced large zoospores (8-μm-d) and tubular, uniform infection and assimilative hyphae in nematodes.     

捕食线虫真菌在北京地区果园中的季节性分布

通过对北京海淀区2个苹果园和2个葡萄园根际捕食线虫真菌2年的季节性变化调查,共分离到199个捕食线虫真菌菌株,隶属3个属16个种,Arthrobotrysdactyloides分离频率最高,其次是A.oligospora,A.conoides和Stylopagesp.。捕食线虫真菌的数量在晚春夏初最丰富,秋季次之,冬夏最少。通过统计分析,捕食线虫真菌数量的季节性变化与温度线性相关,而与线虫总数S形相关,与土壤湿度和植物寄生线虫数量无显著相关。     

Infection of Pratylenchus penetrans by Nematode-pathogenic fungi

Eleven fungal isolates were tested in agar dishes for pathogenicity to Pratylenchus penetrans. Of the fungi that produce adhesive conidia, Hirsutella rhossiliensis was a virulent pathogen; Verticillium balanoides, Drechmeria coniospora, and Nematoctonus sp. were weak or nonpathogens. The trapping fungi, Arthrobotrys dactyloides, A. oligospora, Monacrosporium dlipsosporum, and M. cionopagum, killed most of the P. penetrans adults and juveniles added to the fungus cultures. An isolate of Nematoctonus that forms adhesive knobs trapped only a small proportion of the nematodes. In 17-cm³ vials, soil moisture influenced survival of P. penetrans in the presence of H. rhossiliensis; nematode survival decreased with diminishing soil moisture. Hirsutella rhossiliensis and M. ellipsosporum were equally effective in reducing numbers of P. penetrans by 24-25% after 4 days in sand. After 25 days in soil artificially infested with H. rhossiliensis, numbers of P. penetrans were reduced by 28-53%.     

Biocontrol: Fungal Parasites of Female Cyst Nematodes

Three species of fungi, Catenaria auxiliarls (Kühn) Tribe, Nematophthora gynophila Kerry and Crump, and a Lagenidiaceous fungus have been found attacking female cyst nematodes. All are zoosporic fungi which parasitize females on the root surface, cause the breakdown of the nematode cuticle, and prevent cyst formation. Their identification and some aspects of their biology are reviewed. N. gynophila is widespread in Britain and reduces populations of the cereal cyst nematode, Heterodera avenae Woll., to nondamaging levels. The potential of these nematophagous fungi as biocontrol agents is discussed.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.     

Susceptibility of the boll weevil to Steinernema riobrave and other entomopathogenic nematodes

The susceptibility of the boll weevil (BW), Anthonomus grandis Boheman, to Steinernema riobrave and other nematode species in petri dishes, soil (Hidalgo sandy clay loam), and cotton bolls and squares was investigated. Third instar weevils were susceptible to entomopathogenic nematode (EN) species and strains in petri dish bioassays at 30 degrees C. Lower LC(50)'s occurred with S. riobrave TX- 355 (2 nematodes per weevil), S. glaseri NC (3), Heterorhabditis indicus HOM-1 (5), and H. bacteriophora HbL (7) than H. bacteriophora IN (13), S. riobrave TX (14), and H. bacteriophora HP88 (21). When infective juveniles (IJs) of S. riobrave were applied to weevils on filter paper at 25 degrees C, the LC(50) of S. riobrave TX for first, second, and third instars, pupae, and 1-day-old and 10-days-old adult weevils were 4, 5, 4, 12, 13, and 11IJs per weevil, respectively. The mean time to death, from lowest to highest concentration, for the first instar (2.07 and 1.27days) and second instar (2.55 and 1.39days) weevils were faster than older weevil stages. But, at concentrations of 50 and 100IJs/weevil, the mean time to death for the third instar, pupa and adult weevils were similar (1.84 and 2.67days). One hundred percent weevil mortality (all weevil stages) occurred 3days after exposure to 100IJs per weevil. Invasion efficiency rankings for nematode concentration were inconsistent and changed with weevil stage from 15 to 100% when weevils were exposed to 100 and 1IJs/weevil, respectively. However, there was a consistent relationship between male:female nematode sex ratio (1:1.6) and nematode concentration in all infected weevil stages. Nematode production per weevil cadaver increased with increased nematode concentrations. The overall mean yield of nematodes per weevil was 7680IJs. In potted soil experiments (30 degrees C), nematode concentration and soil moisture greatly influenced the nematode efficacy. At the most effective concentrations of 200,000 and 400,000IJs/m(2) in buried bolls or squares, higher insect mortalities resulted in pots with 20% soil moisture either in bolls (94 and 97% parasitism) or squares (92 and 100% parasitism) than those of 10% soil moisture in bolls (44 and 58% parasitism) or squares (0 and 13% parasitism). Similar results were obtained when nematodes were sprayed on the bolls and squares on the soil surface. This paper presents the first data on the efficacy of S. riobrave against the boll weevil, establishes the potential of EN to control the BW inside abscised squares and bolls that lay on the ground or buried in the soil.     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.     

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.     

Substrate modulation, group effects and the behavioral responses of entomopathogenic nematodes to nematophagous fungi

Laboratory experiments were conducted on the behavioral responses of five species of entomopathogenic nematodes (EPNs; Steinernema diaprepesi, Steinernema sp. glaseri-group, Steinernema riobrave, Heterorhabditis zealandica, Heterorhabditis indica) to three species of nematophagous fungi (NF; trapping fungus Arthrobotrys gephyropaga; endoparasites Myzocytium sp., Catenaria sp.). We hypothesized that EPN responses to NF and their putative semiochemicals might reflect the relative susceptibility of EPNs to particular NF species. EPN responses to “activated” NF (i.e., induced to form traps or sporangia by previous interactions with nematodes) versus controls of non-activated NF or heat-killed EPNs were compared in choice experiments on water agar in Petri dishes (dia = 9 cm) and in horizontal sand columns (8 cm L × 2.7 cm dia). On agar, all EPN species were attracted to all activated NF species except for S. riobrave, which was neutral. In sand, all EPN species were repelled by activated Arthrobotrys but attracted to activated Myzocytium and Catenaria, except H. indica (neutral to Myzocytium) and Steinernema sp. (neutral to Catenaria). EPN behavioral responses appeared unrelated to relative susceptibility to NF except that H. indica exhibited low susceptibility and a neutral response to Myzocytium in sand whereas the remaining EPNs were highly susceptible and attracted. These results indicate potential complexity (i.e., mixed responses, aggregation or group movement) and species specificity in the responses of EPNs to NF, demonstrate that results on agar can differ markedly from those in sand, and underline the potential importance of utilizing natural substrates to properly assess the role of semiochemicals in nematode-fungus interactions.     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.     

捕食线虫真菌捕食器相关基因标记Ⅰ.——REMI转化捕食线虫真菌及转化子特征分析

采用REMI技术转化了少孢节丛孢、指状节丛孢和贵州节丛孢3种捕食线虫真菌,并对转化条件、转化子的形态特征、胞外蛋白酶的分泌差异、抗性稳定性进行了测定,分析了转化子对线虫的致病性和对土壤抑菌作用的忍耐性。