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.     

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.     

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.     

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.     

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.     

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×10⁵ chlamydospores mL^?1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×10⁵ chlamydospores mL^?1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.     

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.     

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.     

THE EFFECT OF SELENITE ON THE PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE BLUE-GREEN ALGA PHORMIDIUM LURIDUM VAR. OLIVACEA1

Phormidium luridum cultures were treated with sodium selenite in concentrations ranging from 10^?6 M to 10^?2 M. In contrast to the increasing culture turbidity of control and 10^?6 M selenite cultures, the turbidity of the other selenite cultures declined in proportion to time and selenite concentration. Chlorophyll extraction revealed similar results. Photosynthetic activity was inhibited within 6 hr in all cultures except control and 10^?6 M selenite. Phormidium at concentrations greater than 10^?6 M selenite showed a gradual loss of the bright green color and turned semitransparent. Cell-associated granules of reduced selenium were observed at higher selenite concentrations. Other structural changes observed were the presence of intracellular and intercellular spaces, spheroplast formation, and gradual cell lysis. Protein analyses of total cell samples and supernatant fractions confirmed cellular breakdown of selenite-treated algal cells.     

Influence of culture age on the phytochemical content and pharmacological activities of five Scenedesmus strains

Five axenic Scenedesmus strains (MACC-411, MACC-422, MACC-493, MACC-720, and MACC-727) were cultured and harvested after 5 and 10 days in culture. Using colorimetric methods, the concentrations of total phenolic, condensed tannin, and iridoids in 50 % methanol extracts from both 5- and 10-day-old cultures were quantified. Different solvent extracts from the strains were also tested for antioxidant, acetylcholinesterase inhibitory (AChEI), and antimicrobial activities using various in vitro test systems. Phenolic content was highest (3.6?±?0.42 mg GAE g^?1 DW) in 10-day-old MACC-727. This was approximately fourfold and significantly higher than in the 5-day-old cultures of MACC-727. Among the tested Scenedesmus strains, 5-day-old MACC-411 had the highest iridoid content (3.4?±?0.3 mg HE g^?1 DW), and this was significantly higher than the level detected in the 10-day-old MACC-411. Scenedesmus strains showed better antioxidant potential in the β-carotene–linoleic acid model compared to the DPPH free radical scavenging assay. The AChEI activity (IC₅₀?μg mL^?1) in all strains (besides MACC-422) was higher in 10-day-old cultures compared to the 5-day-old cultures. Although a broad-spectrum of antibacterial activity was observed, the tested microalgae strains demonstrated varying degrees of antimicrobial potential depending on the harvest time, strain-type, and extracting solvent. Thus, the Scenedesmus strain and time of harvest played a significant role in determining their phytochemical content and resultant pharmacological activity. The promising bioactivity in the tested Scenedesmus strains indicates their potential as possible sources of novel/alternative antioxidants and AChE inhibitors.     

Effects of Indole-3-acetic Acid and Gibberellin on Synchronous Cultures of Chlorella fusca

The applicability of synchronous cultures of Chlorella fusca as a reproducible experimental system for the study of growth regulators has been investigated using indole-3-acetic acid (IAA) and gibberellin.

• 1 None of these compounds stimulated growth or sporulation.
• 2 IAA inhibited growth and sporulation at concentrations higher than 6 × 10^?5M, the effect increasing with increasing acidity. Gibberellin had no effect.

     

Sporulation,storage and infectivity of obligate aphid pathogen Pandora nouryi grown on novel granules of broomcorn millet and polymer gel

Aims: Producing granular cultures of obligate aphid pathogen Pandora nouryi for improved sporulation and storage. Methods and Results: Small millet–gel granules were made of the mixtures of 80–95% millet powder with 5–20% polymer gel (polyacrylamide, polyacrylate or acrylate‐acrylamide copolymer) and inoculated with mycelia at 30 mg biomass g^?1 dry granules plus 87·5% water, followed by static incubation at 20°C for 4–12 days. The fungus grew well on 12 preparations but best on that including 10% copolymer. An 8‐day culture of this preparation discharged maximally 58·5 × 10⁴ conidia mg^?1 granule at 100% RH and was capable of ejecting conidia at the nonsaturated regimes of 86–97% RH. During storage at 6°C, granular cultures with >85% water content had twofold longevity (120 days) and half‐decline period (34–36 days) of those stored at room temperature. The steadily high water content preserved the cultures better than that decreasing at 6°C. However, conidia from 70‐day‐stored granules were less infective to Myzus persicae nymphs than those from fresh ones based on their LC₅₀s. Conclusions: The millet–gel granules had higher sporulation capacity than reported Pandora cultures and a capability of spore discharge at nonsaturated humidity. Significance and Impact of the Study: The granular cultures are more useful for aphid control.     

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.     

Catabolite-Induced Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores. Conversely, excess catabolites inhibit sporulation. It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB. Culture medium acidification was also shown to inhibit Ï^H-dependent sporulation gene expression. The studies reported here investigate the effects of AbrB-mediated pH sensing on B. subtilis developmental competence. We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation. Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation. These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation. The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways.     

Effects of Leaf Age, Inoculum Dose and Freezing on Development of Chocolate Spot (Botrytis fabae) Lesions on Field Bean (Vicia faba) Leaves

Botrytis fabae spore suspensions containing c. 1, 10, 10², 10³, 10⁴, 10⁵, or 10⁶ spores/ml were used to inoculate 5, 17 or 30-day-old field bean leaves. The percentages of the leaf areas covered by, chocolate spot lesions and the percentages of the leaf areas bearing conidiophores were assessed 1, 6, 12, 14, and 19 days after inoculation. The percentage of the area covered by lesions and the percentage of the area bearing conidiophores (logit-transformed) increased linearly with increasing spore concentration (log₁₀-transformed). The proportions of leaf areas covered by lesions and bearing conidiophores were both greater on 17 and 30-day-old leaves than on 5-day-old leaves. The rate of lesion growth increased with both increasing inoculum dose and increasing leaf age. Generally there was no interaction between the effects of leaf age and the effects of inoculum dose on either lesion growth or sporulation. Two days after inoculation with suspensions of either 10⁴ or 10⁶ spores/ml, 7-day-old leaves grown at 15°C were transferred to –16°C or 2.5°C or kept at 15°C for 4 days. Two days later more spores had been produced on leaves which had been frozen (–16°C) than on, leaves kept at 2.5°C.     

Optimization of storage condition for maintaining long-term viability of nematophagous fungus Esteya vermicola as biocontrol agent against pinewood nematode

The fungus, Esteya vermicola has been proposed as biocontrol agent against pine wilting disease caused by Bursaphelenchus xylophilus. In this study, we reported the effects of temperature and different additives on the viability and biocontrol efficacy of E. vermicola formulated by alginate-clay. The viability of the E. vermicola formulation was determined for six consecutive months at temperature ranged from ?70 to 25 °C. The fresh conidia without any treatment were used as control. Under the optimal storage conditions with E. vermicola alginate-clay formulation, the results suggested that E. vermicola alginate-clay formulation with a long shelf life could be a non-vacuum-packed formulation that contains 2 % sodium alginate and 5 % clay at 4 °C. Three conidial formulations prepared with additives of 15 % glycerol, 0.5 % yeast extract and 0.5 % herbal extraction, respectively significantly improved the shelf life. In addition, these tested formulations retained the same biocontrol efficacy as the fresh conidial against pinewood nematode. This study provided a tractable and low-cost method to preserve the shelf life of E. vermicola.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

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.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.