Using the nematophagous fungus Esteya vermicola to control the disastrous pine wilt disease

The present study evaluated the protective effects of the nematophagous fungus Esteya vermicola on the large pine trees of Mt. Wora, Jinju, South Korea for six years. When pine trees were treated with E. vermicola 110 days before artificial normal pinewood nematode (PWN) infection, 30–50% of the trees survived for six years. When pine trees were treated with E. vermicola one week after artificial normal PWN infection, 40% of the trees were saved. In contrast, all of the control trees were killed by pine wilt disease in the first year. Although it has been more than six years since the beginning of this experiment, the existence of E. vermicola inside the treated pine trees was successfully detected using a PCR method with two pairs of specific primers for E. vermicola. These results suggest that E. vermicola possesses great potential as a biocontrol agent to combat the disastrous pine wilt disease. This is the first report of using nematophagous fungi to control pine wilt disease in the field for a duration of over five years.     

Attraction of Pinewood Nematode to Endoparasitic Nematophagous Fungus Esteya vermicola

The investigations on attraction of nematodes to nematophagous fungi have mostly dealt with the nematode-trapping species. Esteya vermicola is the endoparasitic fungus of pinewood nematode (PWN) with high infection activity. In the present study, the attraction of PWNs to E. vermicola was investigated. It was confirmed that the living mycelia and exudative substances of E. vermicola were attractive to PWN. Compared with the nematode-trapping fungus A. brochopaga as well as nematode-feeding fungus B. cinerea, E. vermicola showed the significantly strongest attraction ability to nematode. It therefore appeared that the attraction ability reflects the dependence of the fungi on nematodes for nutrients. Furthermore, a new method was developed and used in the study to confirm the effect of volatile substances for the attraction of nematode to fungi. The results suggested that the attractive substances were consisted of avolatile exudative and volatile diffusing compounds.     

Biological control of the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> by application of the endoparasitic fungus <Emphasis Type="Italic">Esteya vermicola</Emphasis>

Esteya vermicola (Ophiostomataceae) is the first reported endoparasitic fungus of the pinewood nematode (PWN), Bursaphelenchus xylophilus (Nematoda: Aphelenchoidoidea). It has high in vitro infectivity. In this study, the nematocidal effect of E. vermicola in logs was investigated and evaluated. Two months after inoculation of pine wilt-killed Pinus densiflora logs with E. vermicola conidia suspensions of 3 × 10⁸ and 3 × 10⁶ ml⁻¹, the density of nematodes decreased by approximately 79% and 47%, respectively. When the fungus was sprayed on to four-year-old pine seedlings one month before PWN inoculation, the survival index of seedlings reached 0.67 compared with only 0.067 for control seedlings without fungal spraying. These results suggest that conidia spraying of E. vermicola can, to some extent, protect pine trees from wilt disease. Moreover, infected nematodes and hyphae of E. vermicola were observed in the treated wood sections.     

Viability and pathogenicity of Esteya vermicola in pine trees

Esteya vermicola, as the first reported endoparasitic fungus of pinewood nematode (PWN), exhibited high infectivity in vitro and has been patented based on its potential as a bio-control agent against PWN. The isolation substrates and taxonomic status suggested E. vermicola is associated with beetles, saprotrophic and kills nematodes in trees. However, the direct experimental evidence for this was still lacking. In the present studies, beta-tubulin gene was applied to confirm the taxonomic identification of E. vermicola. Furthermore, our results showed that E. vermicola survived resin and other chemicals secreted by pine trees, and reproduced with new lunate conidia to parasitize other migratory PWNs. In order to confirm the pathogenicity of E. vermicola, pine seedlings and large pine trees were inoculated with 300 µL and 40 mL conidia suspensions (10⁹ mL^?1). The results showed that all treated pine trees were healthy with no differences compared to the controls. Furthermore, necrosis or discoloration caused by this fungus was not observed on wood slices. Basal knowledge was provided for the application of E. vermicola to control PWN in vivo.     

Effect of Nutrition and Environmental Factors on the Endoparasitic Fungus Esteya vermicola, a Biocontrol Agent Against Pine Wilt Disease

The nematophagous fungus Esteya vermicola has tremendous potential for biological control. This species exhibits strong infectious activity against pinewood nematodes, whereas the study on the effect of nutrition and environmental factors is still of paucity. Carbon (C), nitrogen (N), pH value, temperature, and water activity have great impact on the fungal growth, sporulation, and germination. In nutrition study, the greatest number of conidia (2.36 × 10⁹ per colony) was obtained at the C:N ratio of 100:1 with a carbon concentration 32 g l^?1. In addition, the germination rate and radial growth of E. vermicola were used to evaluate the effects of environmental conditions and they were optimized as following: pH 5.5, 26 °C and water activity of 0.98. Our results also confirmed that variation of environmental factors has a detrimental influence on the efficacy of active conidia and growth of fungus. Moreover, under above optimal condition, the biocontrol efficacy was significantly improved in regard to the increase of adhesive and mortality rate, which highlight the study on the application of E. vermicola as pine wilt disease biocontrol agent.     

Comparison between conidia and blastospores of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.     

A Method for the Enhancement of Environmental Stress Resistance of Endoparasitic Fungus Esteya vermicola

Esteya vermicola is the first recorded endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus, which is the causal agent for the pine wilt disease. Culture on modified agar media with herbal extraction (0.5%) was found to be able to induce resistance to UV radiation, heat and drought conditions in Esteya vermicola. Herba Houttuyniae, Tatraxacum officinale and Scutellaria baicalensis Georgi exhibited the highest improvement on environmental competence of Esteya vermicola at all the tested time points under the stress conditions. In addition, improved quality and effective viability of Esteya vermicola were observed amended with the three herbal extractions in culture media. Enhanced stress resistance was associated with herbal metabolites. These findings provided a green, feasible, economical method for developing an open‐field spay application of fungal biocontrol agents against pine wilt disease.     

Protective formula and preservation conditions for the endoparasitic fungus, <Emphasis Type="Italic">Esteya vermicola</Emphasis>

The endoparasitic nematophagous fungus, Esteya vermicola, is a bio-control agent with demonstrated ability to attack pinewood nematode (Bursaphelenchus xylophilus). An optimized solution for the protection and preservation of E. vermicola conidia is needed in order to ensure their survival during transportation, preservation, and application. Five protectants, kaolin, arabinose, sorbitol, PEG8000, and Span 80, were selected from 34 agents. These were incorporated into calcium alginate gel capsules at the following concentrations: 10% kaolin, 0.1% Span 80, 1% arabinose, 5% sorbitol, and 5% PEG8000. The improved diffluent formula contained 69.9% soluble starch, 14% wheat flour, 5% PEG8000, 0.1% span 80, 1% arabinose and 10% skim milk. The viability of E. vermicola conidia preserved in the protectant (5% sorbitol and 20% PEG8000) at six temperatures,–70,–20, 4, 26, 37°C, and room temperature (uncontrolled), was also assessed. The highest viability after storage for one month was achieved at–70°C.     

The ratio and concentration of two monoterpenes mediate fecundity of the pinewood nematode and growth of its associated fungi

The pinewood nematode (PWN) Bursaphelenchus xylophilus, vectored primarily by the sawyer beetle, Monochamus alternatus, is an important invasive pest and causal agent of pine wilt disease of Chinese Masson pine, Pinus massoniana. Previous work demonstrated that the ratios and concentrations of α-pinene∶β-pinene differed between healthy trees and those trees containing blue-stain fungus (and M. alternatus pupae). However, the potential influence of the altered monoterpene ratios and concentrations on PWN and associated fungi remained unknown. Our current results show that low concentrations of the monoterpenes within petri dishes reduced PWN propagation, whereas the highest concentration of the monoterpenes increased PWN propagation. The propagation rate of PWN treated with the monoterpene ratio representative of blue-stain infected pine (α-pinene∶β-pinene = 1∶0.8, 137.6 mg/ml) was significantly higher than that (α-pinene∶β-pinene = 1∶0.1, 137.6 mg/ml) representative of healthy pines or those damaged by M. alternatus feeding, but without blue stain. Furthermore, inhibition of mycelial growth of associated fungi increased with the concentration of the monoterpenes α-pinene and β-pinene. Additionally, higher levels of β-pinene (α-pinene∶β-pinene = 1∶0.8) resulted in greater inhibition of the growth of the associated fungi Sporothrix sp.2 and Ophiostoma ips strains, but had no significant effects on the growth of Sporothrix sp.1, which is the best food resource for PWN. These results suggest that host monoterpenes generally reduce the reproduction of PWN. However, PWN utilizes high monoterpene concentrations and native blue-stain fungus Sporothrix sp.1 to improve its own propagation and overcome host resistance, which may provide clues to understanding the ecological mechanisms of PWN''s successful invasion.     

Laboratory studies on the development of a conidial formulation of Esteya vermicola

The endoparasitic nematophagous fungus, Esteya vermicola, has potential as a biocontrol agent against pinewood nematode, Bursaphelenchus xylophilus. An E. vermicola conidial formulation was developed to improve conidial resistance to ultraviolet (UV), drought and heat stress. The effective concentration of each protective additive [UV protectant [fulvic acid (FA) and skim milk (SM)]; drought protectant (sorbitol) and heat protectant (calcium chloride)] was determined based on the germination rate of E. vermicola conidia after exposure to the different stressors. A combination of 0.2% FA and 4% SM, 5% sorbitol and 0.05% calcium chloride provided the most effective protection. In addition, the concentrations of spreader–sticker and antibiotic were also decided. The final formulation could be used to improve the resistance of E. vermicola conidia to multiple stressors and to increase nematode mortality compared with unformulated conidia.     

Morphological,molecular and biological characterization of Esteya vermicola,a nematophagous fungus isolated from intercepted wood packing materials exported from Brazil

The nematophagous fungus Esteya vermicola, strain NKF 13222, was purified from an isolate of Bursaphelenchus rainulfi which was intercepted from wood packaging materials originating in Brazil and arriving at Tianjin port in China. The fungus produced two types of conidiogenous cells and conidia, each with different germination modes. More lunate adhesive conidia than bacilloid conidia were produced on nutrient-poor water agar medium. Morphological comparisons revealed the NKF 13222 strain closely resembled the Taiwan strain E. vermicola (ATCC 74485) previously isolated from the pinewood nematode B. xylophilus. Phylogenetic analysis of the β-tubulin and elongation factor 1-α genes indicated that the NKF 13222 grouped with other strains of E. vermicola including the Taiwan strain. This was the first record of E. vermicola from B. rainulfi in South America. Infection tests demonstrated that NKF 13222 was more infective to aphelenchid than tylenchid nematodes and that only lunate adhesive conidia were infectious. The results suggest that the fungus might be a pathogen of plant parasitic nematodes with a broad distribution and provide new information for the potential biocontrol of plant diseases caused by B. xylophilus, Aphelenchoides spp. and Ditylenchus destructor.     

Population dynamics of pinewood nematode and the endoparasitic fungus Esteya vermicola: interactions under experimental conditions

Esteya vermicola (Ophiostomataceae), an endoparasitic fungus, exhibits great potential as a biological control agent against pinewood nematodes (PWNs). The present study reports the interaction between PWNs and E. vermicola at different spore concentrations, number of PWNs and the time of culture. The addition of PWNs enhanced the sporulation of E. vermicola after 10 days of culture. The 5-day-old cultures of E. vermicola prior to addition of PWNs increased the highest amount of sporulation than that of 10- or 15-day-old cultures. The PWNs were completely killed by E. vermicola in the pine tree powder culture medium at the concentrations of 10⁷ and 10⁸ colony-forming-units (CFU) per ml. The interaction of the PWNs and E. vermicola was that PWNs provide nutrition to E. vermicola, however, the PWNs can also feed on mycelium of E. vermicola. The effect of E. vermicola on control of PWNs was determined by the population size, time of pest infection and the duration of co-infection.     

Rapid molecular detection of Esteya vermicola based on specific primers and the FTA-DNA extraction method

Esteya vermicola is an endoparasitic fungus of the pinewood nematode and thus has great biocontrol value. At present, the detection of this fungus is still based on microscopic observations and morphological identification, and the sampling is notably inconvenient and inefficient. In the present study, a pair of specific primers (upstream primer, 5′-GTGCCTCTACCAAGACTCGC-3′; downstream primer, 5′-CGCCAAATGTCAAGATCCGC-3′) was designed to detect E. vermicola. The analysis of the PCR amplification and the agarose gel electrophoresis results led to the establishment of a new method for the detection of E. vermicola through the presence of a 176-bp specific fragment. In addition, the use of a FTA-DNA direct extraction method for the detection of E. vermicola was explored. The results suggest that the proposed method can be effectively used for the rapid detection of E. vermicola and may provide important technical support for follow-up studies of the fungus in field experiments.     

Two Cyclic Dipeptides from Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode and Their Toxicities to Japanese Black Pine Suspension Cells and Seedlings in vitro

Pseudomonas fluorescens GcM5-1A, isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, was cultured in Luria Broth medium (LB). The clarified culture was extracted with ethyl acetate, and two dipeptides were purified from the extract. The chemical structures of 1 and 2 were identified as cyclo(-Pro-Val-)and cyclo(-Pro-Tyr-), respectively, by MS, ¹H NMR, ¹³C NMR,¹H-¹H COSY, 1H -¹³C COSY spectra. Bioassay results showed that the two compounds were toxic to both suspension cells and seedlings of Pinus thunbergii, which may offer some clues to research the mechanism of pine wilt disease caused by PWN.     

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.     

Effects of mineral salts on the growth,sporulation and virulence of Esteya vermicola,an endoparasitic fungus of the pinewood nematode,Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study, various mineral supplements, such as chloride salts (KCl, CaCl₂, MgCl₂, FeCl₂, and FeCl₃) and calcium salts (CaCl₂, CaCO₃, and CaSO₄) were evaluated for their ability to enhance the growth, sporulation and virulence of E. vermicola. Of the cations tested, CaCl₂ provided the greatest enhancement of growth speed and sporulation. Of the anions tested, CaCO₃ produced the highest proportion of lunate conidia, and CaCl₂ produced the highest adhesive rate and mortality against the nematode, Bursaphelenchus xylophilus. The optimum concentration of CaCl₂ for optimization of sporulation and virulence was 0.4–0.6%. In conclusion, CaCl₂ is highly effective in enhancing growth, sporulation and virulence of Esteya vermicola.     

Pathogenic Cellulase Assay of Pine Wilt Disease and Immunological Localization

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.     

Impact of Pinewood Nematode in North America: Present and Future

Bursaphelenchus xylophilus, pinewood nematode (PWN), is the most serious pest of pine forests in Japan, but in North America its role in pine wilt disease is still being studied. The PWN is known to infest many species of Pinus, with P. nigra, P. sylvestris, and P. thunbergii the most susceptible in the eastern United States. Because of its potential, several European countries (Finland, Norway, and Sweden) and Korea have established embargoes against the importation of coniferous wood from regions of the world known to be infested with the PWN. Although the PWN is not considered an economic pest in North American forests, the recent embargoes have established an impact on current forest management practices and an economic impact on North American export trade.