Population biology and biological control of nematodes.

We studied the population biology of the nematophagous fungus Hirsutella rhossiliensis to understand its potential as a biological control agent. Because the fungus is an infectious and transmissible parasite, we framed our study within an epidemiological context. Field observations, theory, and experiments demonstrated that (i) parasitism of nematodes by H. rhossiliensis is dependent on nematode density, (ii) local populations of the fungus will go extinct unless supplied with some minimum number of nematodes (the host threshold density), and (iii) natural epidemics of this fungus in populations of nematodes develop slowly and only after long periods of high host density. Additional in-depth research on population biology is needed to explain other biological control systems and to guide future research. The most effective research will combine field observation, theory, and experimentation.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.     

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.     

Effects of Microbial and Other Antagonistic Organism and Competition on Entomopathogenic Nematodes

Antagonistic factors, broadly identified as antibiosis, competition and natural enemies, impact on entomopathogenic nematodes. Antibiosis can occur through the release of plant chemicals from the roots into the soil, which may adversely affect the host-finding behavior of the infective stage nematode, or the presence of these chemicals in the host insect may negatively affect nematode reproduction. In laboratory studies, intra-specific and inter-specific competition reduces nematode fitness, and inter-specific competition can cause local extinction of a nematode species. For example, after concomitant infection of a host, a steinernematid species usually excludes a heterorhabditid species. The mechanism for the steinernematid superiority has been postulated to be a bacteriocin(s) produced by Xenorhabdus, the symbiotic bacterium of the steinernematid, which prevents Photorhabdus, the symbiotic bacterium of the heterorhabditid, from multiplying. Inter-specific competition between two steinernematid species shows that both can co-exist in a host, but one species will eventually prevail in the environment. By having different foraging strategies, however, both steinermatid species may co-exist in the same habitat. An important issue is whether the introduction of an exotic entomopathogenic nematode species will competitively displace an indigenous nematode species. Although the environmental risks are small, the recommended policy is that the introduction of exotic nematodes be regulated. With other pathogens, entomopathogenic nematodes can out-compete entomopathogenic fungi, but not Bacillus thuringiensis, for the same host individual when both the nematode and entomopathogen are applied simultaneously. The best studied natural enemy is the nematophagous fungus, Hirsutella rhossiliensis, which causes higher mortality in Steinernema glaseri compared with Heterorhabditis bacteriorphora. Differential susceptibility to the fungus may be associated with the retention of the second-stage cuticle by H. bacteriophora. Invertebrate predators including mites and collembolans feed on entomopathogenic nematodes. Although a number of studies have been conducted with antagonists, there is a dearth of field data. We suggest that long-term research plots be established where natural populations of entomopathogenic nematodes occur and include antagonists as a component of such studies.     

Interaction of the Microbivorous Nematode Teratorhabditis dentifera and the Nematode-Pathogenic Fungus Hirsutella rhossiliensis

The fungus Hirsutella rhossiliensis is an obligate pathogen with a broad host range among nematodes. Microbivorous nematodes are abundant around plant roots and may serve as hosts for the fungus. Our objective was to determine the influence of the bacterial-feeding nematode Teratorhabditis dentifera on the abundance of H. rhossiliensis. Experiments were conducted in a growth chamber with pots containing pasteurized soil, the fungus, and potato plants. The abundance of infectious conidia was compared in pots with and without T. dentifera after 50 or 70 days. The nematode reached high densities (10-40/cm³ soil) but had no effect on the abundance of conidia. Many individuals were dauer juveniles, a stage that acquired conidia but did not become infected. To test whether this life stage could deplete the pool of conidia in soil, different proportions of dauer juveniles with (resistant) and without (susceptible) a sheath were added to H. rhossiliensis-infested soil. The number of conidia in the soil decreased with an increasing proportion of resistant nematodes. Different stages of T. dentifera appear to have opposing effects on H. rhossiliensis; while adults and regular juveniles acquire conidia, become infected, and produce new infectious conidia, dauer juveniles can deplete the supply of conidia.     

Screening Isolates of Hirsutella Species for Biocontrol of Heterodera glycines

The fungi, Hirsutella rhossiliensis and Hirsutella minnesotensis, are two endoparasites of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines. The objective of this study was to screen for effective fungal isolates to control the nematode in laboratory and greenhouse assays. A total of 93 isolates of H. rhossiliensis and 25 isolates of H. minnesotensis were evaluated for parasitism of SCN J2 on cornmeal agar. Percentage of SCN J2 parasitized by the fungi varied among the fungal isolates. Most H. rhossiliensis isolates parasitized a high percentage of J2. The isolates of H. rhossiliensis obtained from bacteria-feeding nematodes, however, generally did not parasitize J2 on agar. H. minnesotensis parasitized fewer J2 on agar than did H. rhossiliensis . Forty isolates of H. rhossiliensis and four isolates of H. minnesotensis that parasitized a high percentage of J2 on agar were evaluated for their biocontrol potential in soil treated with microwave heating. Most isolates selected from the agar assay also parasitized a high percentage of J2 in the soil but there was variation among isolates. Pathogenicity of 14 isolates of H. rhossiliensis and four isolates of H. minnesotensis to the SCN was also investigated in the greenhouse using untreated field soil. Most isolates of H. rhossiliensis reduced SCN population density and increased plant growth when compared with 1% corn-grits control (culture media). One isolate (OWVT-1) of H. rhossiliensis reduced the SCN egg density by 95% and J2 density by 98% when compared with the control. Isolates of H. minnesotensis, however, neither reduced SCN density nor increased plant growth in the greenhouse.     

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.     

The interaction of the novel 30C02 cyst nematode effector protein with a plant β-1,3-endoglucanase may suppress host defence to promote parasitism

Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H. schachtii. The 30C02 protein interacted with a specific (AT4G16260) host plant β-1,3-endoglucanase in both yeast and plant cells, possibly to interfere with its role as a plant pathogenesis-related protein. Interestingly, the peak expression of 30C02 in the nematode and peak expression of At4g16260 in plant roots coincided at around 3-5 d after root infection by the nematode, after which the relative expression of At4g16260 declined significantly. An Arabidopsis At4g16260 T-DNA mutant showed increased susceptibility to cyst nematode infection, and plants that overexpressed At4g16260 were reduced in nematode susceptibility, suggesting a potential role of host β-1,3-endoglucanase in the defence response against H. schachtii infection. Arabidopsis plants that expressed dsRNA and its processed small interfering RNA complementary to the Hg30C02 sequence were not phenotypically different from non-transformed plants, but they exhibited a strong RNA interference-mediated resistance to infection by H. schachtii. The collective results suggest that, as with other pathogens, active suppression of host defence is a critical component for successful parasitism by nematodes and a vulnerable target to disrupt the parasitic cycle.     

Control of Root-Knot Nematodes on Tomato by the Endoparasitic Fungus Meria coniospora

The endoparasitic nematophagous fungus Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trials. The fungus was introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematodes infected with M. coniospora to the soil; both methods reduced root galling by root-knot nematodes. These studies represent a part of a recently initiated effort to evaluate the potential of endoparasitic nematophagous fungi for biocontrol of nematodes.     

Differential gene expression in Arabidopsis following infection by plant-parasitic nematodes Meloidogyne incognita and Heterodera schachtii

Whole genome microarrays were used to study plant gene expression in mature Meloidogyne incognita -induced galls in Arabidopsis. We found 959 genes to be significantly differentially expressed, and two-thirds of these were down-regulated. Microarray results were confirmed by qRT-PCR. The temporal and spatial responses of four differentially expressed genes were analysed using GUS reporter plants following infection with M. incognita and the cyst nematode Heterodera schachtii . The ammonium transporter gene AtAMT1;2 was consistently and locally repressed in response to both nematodes at all developmental stages. The lateral organ boundary domain gene LBD41 showed up-regulation in the feeding sites of both nematode species, although there was variation in expression in saccate H. schachtii female feeding sites. Expression of an actin depolymerizing factor ADF3 and a lipid transfer protein was induced in feeding sites of both nematodes at the fusiform stage and this persisted in feeding sites of saccate M. incognita . These results contribute to the knowledge of how plant gene expression responds to parasitism by these nematodes as well as highlighting further differences in the mechanisms of development and maintenance of these feeding site structures.     

Impact of the nematophagous fungus Pochonia chlamydosporia on nematode and microbial populations

The microbial and nematode populations associated with two plants (tomato and cabbage) inoculated with the nematophagous fungus, Pochonia chlamydosporia var. chlamydosporia or root knot nematode (Meloidogyne incognita), or both, were compared with those in unplanted controls. The dominant factor affecting culturable microbial populations was found to be the presence or absence of tomato plants. Generally microbial colony counts were lowest in unplanted soil, small increases were associated with cabbage and significantly greater numbers with tomato plants. Differences in microbial diversity (estimated from community profiles of carbon substrate utlisation, using Biolog) were observed between planted and unplanted soils, however, there were few differences between soils with either of the two plants. The presence of P. chlamydosporia was associated with a reduction in the numbers of plant parasitic nematodes (51%-78%) including the migratory ectoparasites, whereas free-living nematodes, culturable bacteria and bacterial populations assessed by Biolog were unaffected by the application of fungus.     

Suppression of the Root-knot Nematode Meloidogyne javanica by Alginate Pellets Containing the Nematophagous Fungi Hirsutella rhossiliensis , Monacrosporium cionopagum and M. ellipsosporum

The endoparasitic fungus Hirsutella rhossiliensis and the nematode-trapping fungi Monacrosporium cionopagum and M. ellipsosporum were formulated as hyphae in alginate pellets. In a soil microcosm experiment, dried pellets of all three fungi decreased the invasion of cabbage seedlings by the root-knot nematode Meloidogyne javanica when juvenile nematodes were placed 2 cm from roots; M. cionopagum was more effective than the other two fungi, reducing nematode invasion by 40-95% with 0.24-0.94 pellets cm - 3 of soil. In a field microplot experiment, in which neither H. rhossiliensis nor M. ellipsosporum suppressed nematodes, 0.5 pellets of M. cionopagum cm - 3 of soil suppressed M. javanica invasion of tomato seedlings by 73%. In a second microplot experiment with only M. cionopagum , again at 0.5 pellets cm - 3 of soil, the fungus suppressed the invasion of tomato seedlings whether the pellets were added 0, 5 or 14 days before planting; the population density of M. cionopagum increased to nearly 3000 propagules g - 1 of soil by day 8 and then declined to less than 300 by day 22. Enchytraeid worms were observed in and around damaged and apparently destroyed pellets in both microplot experiments. Whether enchytraeids consumed the fungi or otherwise affected biological control requires additional research.     

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.     

Conidia of the nematophagous fungus Drechmeria coniospora adhere to but barely infect Acrobeloides buetschilii

Abstract Conidia of the endoparasitic fungus Drechmeria coniospora were able to adhere to the nematode Acrobeloides buetschilii ; approximately 70% of the nematodes were carrying spores 16 h after their addition to the fungal culture. Young nematodes were preferentially affected; adhesion mainly took place in the head region of the animal. Full infection, characterized by colonization of the nematode body and production of conidiophores and conidia, was observed, albeit at low frequencies. In case of failure, a second adhesive knob was often formed on the germinated spore. The low frequencies of infection are not related to a loss of virulence, due to prolonged in vitro culturing of the fungus. Fresh spores, obtained from cultures which were passed several times through the host, did not show higher infection frequencies.     

Functional response of the fungus Hirsutella rhossiliensis to the nematode,Heterodera glycines

Functional response is a key index in determining the population fluctuation in predation. However, the lack of operable research system limits the studies on functional response of fungal predators. Hirsutella rhossiliensis is a dominant parasite of the soybean cyst nematode, Heterodera glycines. In a soil microcosm bioassay, we determined fungal biomass at different days within 21 days after inoculation, and parasitism rate of H. glycines by the fungus was determined. The functional response of H. rhossiliensis to H. glycines was established and found to be Holling's type III, which was influenced by mycelial densities. Meanwhile, we conducted anti-fungal analysis of metabolic fractions extracted from H. rhossiliensis to explain the potential mechanism of the intraspecific competition illustrated by functional response. The result of anti-fungal experiments indicated that the fungal predators had more complicated interaction at population level than expected, which might be regulated by self-inhibition metabolite(s). This study was the first functional response study of fungal predators in microcosm. With the increasing recognition of emerging fungal threats to animal, plant, and ecosystem health, the methodologies and hypotheses proposed in this study might inspire further research in fungal ecology.     

江苏省不同种植年限的西/甜瓜农田土壤线虫群落特征

在江苏省主要西/甜瓜产区,采集不同种植年限的西/甜瓜农田土壤,采用线虫形态学的鉴定方法,分析了不同种植年限西/甜瓜农田土壤线虫的数量、群落结构及相关生态指数的变化。结果表明:所有调查样点中,共发现土壤线虫54属,平均682条·100 g^-1干土,其中原杆属线虫、拟丽突属线虫和矮化属线虫在不同种植年限的西/甜瓜农田土壤中均为优势属;随着种植年限的增加,土壤线虫的总数量虽无显著变化,但其中食细菌类线虫和杂食-捕食类线虫数量显著减少,而植物寄生类线虫数量显著增加;植物寄生类线虫中的矮化属、根结属线虫数量不断增加;随着种植年限的增加,土壤线虫的多样性指数(H)、瓦斯乐斯卡指数(WI)、自由生活线虫成熟度指数(MI)显著降低,线虫通道指数(NCR)、植物寄生线虫成熟度指数(PPI)、PPI/MI显著升高,其他指数无显著变化;相关性分析结果显示,土壤线虫数量与土壤pH、速效磷、有效锌和有效硼均呈显著正相关;土壤p H、有机质、全氮、速效钾、有效硼和有效锌对土壤优势属线虫的影响较大;连续种植西/甜瓜会增加土壤植物寄生类线虫数量,进而可能造成西/甜瓜连作障碍的发生;连续种植6~10年,土壤线虫的多样性最低、稳定性最差,土壤环境已趋于恶化。