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.     

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.     

Host Deception: Predaceous Fungus,Esteya vermicola,Entices Pine Wood Nematode by Mimicking the Scent of Pine Tree for Nutrient

Background

A nematophagous fungus, Esteya vermicola, is recorded as the first endoparasitic fungus of pine wood nematode (PWN), Bursaphelenchus xylophilus, in last century. E. vermicola exhibited high infectivity toward PWN in the laboratory conditions and conidia spraying of this fungus on Japanese red pine, Pinus densiflora, seedlings in the field protected the pine trees from pine wilt disease to some extent, indicating that it is a potential bio-control agent against PWN. Previous research had demonstrated that the living fungal mycelia of E. vermicola continuously produced certain volatile organic compounds (VOCs), which were responsible for the PWN attraction. However, identity of these VOCs remains unknown.

Methodology/Principal Findings

In this study, we report the identification of α-pinene, β-pinene, and camphor produced by living mycelia of E. vermicola, the same volatile compounds emitted from PWN host pine tree, as the major VOCs for PWN attraction using gas chromatography-mass spectrometry (GC-MS). In addition, we also confirmed the host deception behavior of E. vermicola to PWN by using synthetic VOCs in a straightforward laboratory bioassay.

Conclusions/Significance

This research result has demonstrated that the endoparasitic nematophagous fungus, E. vermicola, mimics the scent of PWN host pine tree to entice PWN for the nutrient. The identification of the attractive VOCs emitted from the fungus E. vermicola is of significance in better understanding parasitic mechanism of the fungus and the co-evolution in the two organisms and will aid management of the pine wilt disease.     

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.     

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.     

Trap Induction and Trapping in Eight Nematode-trapping Fungi (Orbiliaceae) as Affected by Juvenile Stage of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

This study measured trap induction and trapping on agar disks as affected by juvenile stages (J1, J2, J3, and J4) of the nematode Caenorhabditis elegans and by species of nematode-trapping fungi. Eight species of nematode-trapping fungi belonging to the family Orbiliaceae and producing four kinds of traps were studied: adhesive network-forming Arthrobotrys oligospora, A. vermicola, and A. eudermata, constricting ring-forming Drechslerella brochopaga, and Dr. stenobrocha, adhesive column-forming Dactylellina cionopaga, and adhesive knob-forming Da. ellipsospora, and Da. drechsleri. The number of traps induced generally increased with increasing juvenile stages of C. elegans. The ability to capture the juveniles tended to be similar among isolates that produced the same kind of trap but differed among species that produced different kinds of traps. Trapping by Dr. stenobrocha and Da. cionopaga was correlated with trap number and with juvenile stage. A. oligospora and A. vermicola respectively captured more than 92 and 88% of the J1, J3, and J4 but captured a lower percentage of J2. The knob-producing isolates captured more younger than elder juveniles. Partial correlation analyses demonstrated that the trap induction of the most fungal species positively correlated with the juvenile size and motility, which was juvenile stage dependent. Overall, trap induction and trapping correlated with C. elegans juvenile stage (size and motility) in six species of trapping fungi.     

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.     

Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Arthrobotrys conoides</Emphasis>

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2°C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65°C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.     

Ion Beam Mutagenesis in Arthrobotrys oligospora Enhances Nematode-Trapping Ability

The nematode-trapping fungus Arthrobotrys oligospora is able to produce extracellular protease that degrades the body walls of parasitic nematode larvae found in livestock and immobilizes the nematodes. Our aim was to obtain a strain of A. oligospora with a strong ability to trap nematodes by production of high levels of extracellular protease. A wild type strain of A. oligospora was subjected to mutagenic treatments involving low-energy ion beam implantation to generate mutants. Among these mutants, A. oligospora N showed high efficiency in trapping nematodes and was also able to secrete more extracellular protease, helping it to penetrate and digest the body walls of larvae. This work represents the first application of low-energy ion beams to generate mutations in a nematode-trapping fungus, and provides a new method of obtaining a fungus with high potential application.     

A new species of catenaria parasitic on nematodes of sugarcane

Summary A new species,Catenaria vermicola, is named and described. It attacks several plant-parasitic nematodes of economic importance in sugarcane soils of Louisiana. The fungus forms numerous zoospores which encyst on the cuticle, generally near natural openings of the nematode, thereby causing infection. Within the parasitized nematodes hyphae and sporangia quickly form and destroy the victim completely. The following plant-parasitic nematodes were attacked and destroyed:Belonolaimus longicaudatus, Criconemoides rusticum, Hemicycliophora gigas, Hoplolaimus tylenchiformis, Meloidogyne larvae,Pratylenchus brachyurus, P. zeae, Radopholus similis, Tylenchulus semipenetrans larvae,Tylenchorhynchus martini, andXiphinema chambersi.     

Assessment of germination and carnivorous activities of a nematode-trapping fungus Arthrobotrys dactyloides in fungistatic and fungicidal soil environment

Carnivorism is the ability of nematode-trapping fungi to trap and digest the nematodes by sophisticated devices called traps. Delivery of nematode-trapping fungi in soil for bio-control of pest nematodes often fails or gives inconsistent results. Possible reasons for failure could be the effect of soil fungistasis on germination of nematode-trapping fungi in soil environment, use of avirulent species and sensitivity of these fungi to fungicidal residues in soil. Exploitation of nematode-trapping fungi for nematode control demands that it be compatible with fungicides applied in soil or crops and proliferate in soil. This investigation represents is one of the first to evaluate the effect of fungicides on the nematode-trapping fungus Arthrobotrys dactyloides. A. dactyloides showed in vitro carnivorous potential against Meloidogyne incognita, Meloidogyne javanica, Meloidogyne graminicola, Helicotylenchus dihystera and Heterodera cajani. Conidia of A. dactyloides exposed to agricultural soils showed poor germination but formed conidial traps, which captured and killed the soil nematodes. Conidial traps, which trapped the nematodes, grew well in all soils after killing and nutrient absorption from nematode body. Soil amended with 20 mg ai kg⁻¹ of carbendazim and thiram, 30 mg ai kg⁻¹ of mancozeb, 50 mg ai kg⁻¹ of captan, and 100 mg ai kg⁻¹ of carboxin completely checked the conidial trap formation and nematode capturing. 30, 50 and 100 mg ai kg⁻¹ of metalaxyl adversely affected the conidial trap formation and nematode capturing in soil. Propiconazole inhibited 15.2% conidial trap formation up to 50 mg ai kg⁻¹ but caused 93.3% inhibition of conidial traps formation and complete inhibition of nematode capturing at 100 mg ai kg⁻¹. Sulphur, triademefon, and tricyclazole showed least toxic effect on conidial trap formation and nematode capturing activities of A. dactyloides in soil up to 100 mg ai kg⁻¹.     

Purification and cloning of a novel serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Dactylellina varietas</Emphasis> and its potential roles in infection against nematodes

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5°C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method. Jinkui Yang and Lianming Liang contributed equally to this work.     

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.     

Predacity by nematophagous fungi and its relation to the attraction of nematodes

Predacity, the ability of nematophagous fungi to destroy nematodes, was investigated for eight species of fungi by a method using sterilized soil and the nematodePanagrellus redivivus. In addition, the ability of the fungi to attract nematodes was investigated using an agar plate technique. Predacity and attraction were highly correlated (r=0.98) in these tests. The presence of traps in cultures ofArthrobotrys oligospora increased the ability to attract nematodes by a factor of 2.     

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.     

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.     

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.     

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.