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.     

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.     

Early embryogenesis of the pinewood nematode Bursaphelenchus xylophilus

The early embryogenesis and cell lineage of the pinewood nematode Bursaphelenchus xylophilus was followed from a single-cell zygote to a 46-cell embryo under Nomarski optics, and elongation of the microtubules was studied by immunostaining. As a B. xylophilus oocyte matures, it passes through a passage connecting the oviduct with the quadricolumella, the distal part of the uterus, and reaches the quadricolumella where it stays for a few minutes and is fertilized. After fertilization, the germinal vesicle disappears, an eggshell is formed, and the male and female pronuclei appear. The pronuclei move toward each other and fuse at the center of the egg. Around this time, the microtubule-organizing center appears. The presumptive region of sperm entry into the oocyte becomes the future anterior portion of the embryo. This anterior-posterior axis determination is opposite to that of Caenorhabditis elegans, where the sperm entry site becomes the posterior portion of the embryo. The optimal growth temperatures of these two nematodes also differ in that temperatures of about 30 degrees C afford the fastest growth rate and highest hatching frequency in B. xylophilus. Otherwise, the lineage resembles that of C. elegans with respect to timing, positioning and the axis orientation of each cell division.     

Nematicidal effects of piperine on the pinewood nematode Bursaphelenchus xylophilus

The plant-parasitic nematode Bursaphelenchus xylophilus is a notorious forest pest and the cause of pine-wilt disease which is becoming a serious issue in Asia and Europe. Few available pesticides or nematicides effectively kill B. xylophilus and the topic is the subject of on-going research. This study describes the killing effects of three constituents (piperine, piperlongumine, and piperonal) of the long pepper on the pinewood nematode juveniles. Piperine at 50 µg/mL achieved killing of B. xylophilus juveniles in 6 h, and uniquely, the pattern of killing involved rapid curling (1 h), aggregation (2 h), and death (6 h). Also, piperine adversely affected the fecundity and locomotor traits in B. xylophilus while piperine had marginal effect on the growth of plant seed germination. Computational studies showed piperine interacts with the Gln²¹⁹ residue of glutamate gated chloride ion channel (GluCl) receptor, a residue associated with receptor activation. Furthermore, the mode of action of piperine resembled that of ivermectin in in silico studies. These results suggest that piperine (the major component of peppers) be regarded a potential nematicide for pinewood nematodes and warrant further field investigations.     

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.     

Antinematodal activity of some tropical rainforest plants against the pinewood nematode, Bursaphelenchus xylophilus

Sixty five methanolic extracts of Sumatran rainforest plants representing 63 species of 21 families were assayed in vivo for antinematodal activity against Bursaphelenchus xylophilus using our cotton ball-fungal mat method. Extracts of 27 plants species from 14 families exhibited antinematodal activity, while 37 species were inactive. Among them, three extracts of Bischofia javanica, Knema hookeriana and Areca catechu exhibited very strong activity at minimum effective dose (MED) of 0.7 mg/cotton ball (mg/bl.). Eight extracts from Allamanda cathartica, Ervatamia corymbosa, Hoya diversifolia, Bischofia javanica, Derris malacensis, Melastoma melabathricum, Ophiorriza konsteleary and Brucea sumatrana also showed strong activity (MED, 5 mg/bl.).     

Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2–70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.     

Pathogenicity of the pinewood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings

Pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings was tested with inoculation experiments under nursery conditions. Water suspensions of nematodes (mixed stages cultured on Botrytis cinerea or dispersal fourth-stage juveniles (DJ4) extracted from the adult Japanese pine sawyer, Monochamus alternatus) were injected into the stems of 2- and 3-year-old Japanese larch and Japanese black pine, Pinus thunbergii, seedlings growing in a nursery. In another treatment, Japanese pine sawyer adults holding DJ4 were released under a net that covered the upper half of the seedlings. Regardless of nematode inoculation method, Japanese larch seedlings were as susceptible as Japanese black pine seedlings to B. xylophilus under nursery conditions. The rate of disease development was similar on larch and pine seedlings. Nematode population densities were lower in the stems of dead larch seedlings than in the stems of dead pine seedlings. Histopathological observations revealed that the distribution of nematodes in the stems of dead larch seedlings was mostly limited to the cortex, phloem and cambial zone. Traumatic resin canal formation was one of the most characteristic symptoms in larch seedlings which was dissimilar to that in pine seedlings.     

Identification of cdc25 gene in pinewood nematode, Bursaphelenchus xylophilus, and its function in reproduction

The cdc25 gene, which is highly conserved in many eukaryotes, encodes a phosphatase that plays essential roles in cell cycle regulation. We identified a cdc25 ortholog in the pinewood nematode, Bursaphelenchus xylophilus. The B. xylophilus ortholog (Bx-cdc25) was found to be highly similar to Caenorhabditis elegans cdc-25.2 in sequence as well as in gene structure, both having long intron 1. The Bx-cdc25 gene was determined to be composed of seven exons and six introns in a 2,580 bp region, and was shown to encode 360 amino acids of a protein containing a highly-conserved phosphatase domain. Bx-cdc25 mRNA was hardly detectable throughout the juvenile stages but was highly expressed in eggs and in both female and male adults. Functional conservation during germline development between C. elegans cdc25 and Bx-cdc25 was revealed by Bx-cdc25 RNA interference in C. elegans.     

Microsatellite variation in the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle in South Korea

The pinewood nematode Bursaphelenchus xylophilus is the causative agent of pine wilt disease, which has caused heavy economic losses to the South Korean forest industry. In this study, we investigated the genetic variation among South Korean pinewood nematodes using newly developed microsatellite loci. In order to ensure sufficient templates for the amplification of multiple loci required for individual identification, we employed an amplifying step of restricted fragments during the microsatellite development procedure. We found atypical genetic patterns in this non-native pest species: high allelic diversity and population structure. The large number of alleles may be the result of continuous and/or large-scaled introduction, which apparently went unnoticed before the first official report of pine wilt disease in Korea in 1998, or may come from gene pools of closely related species through genetic introgression after hybridization. Ecological properties of this species, such as a vector-mediated life cycle, may have contributed to its population structure, which may be enhanced by governmental efforts to prevent dispersal of this disease. As a geographic population structure was not observed, geographic patterns of genetic variation appear to be more affected by anthropogenic mediation than by natural dispersion through vector insects. And genotypes of Korean populations were compared to genotypes found in neighboring countries such as China and Japan.     

Nematicidal activity of essential oils and volatiles derived from Portuguese aromatic flora against the pinewood nematode, Bursaphelenchus xylophilus

Twenty seven essential oils, isolated from plants representing 11 families of Portuguese flora, were screened for their nematicidal activity against the pinewood nematode (PWN), Bursaphelenchus xylophilus. The essential oils were isolated by hydrodistillation and the volatiles by distillation-extraction, and both were analysed by GC and GC-MS. High nematicidal activity was achieved with essential oils from Chamaespartium tridentatum, Origanum vulgare, Satureja montana, Thymbra capitata, and Thymus caespititius. All of these essential oils had an estimated minimum inhibitory concentration ranging between 0.097 and 0.374 mg/ml and a lethal concentration necessary to kill 100% of the population (LC(100)) between 0.858 and 1.984 mg/ml. Good nematicidal activity was also obtained with the essential oil from Cymbopogon citratus. The dominant components of the effective oils were 1-octen-3-ol (9%), n-nonanal, and linalool (both 7%) in C. tridentatum, geranial (43%), neral (29%), and β-myrcene (25%) in C. citratus, carvacrol (36% and 39%), γ-terpinene (24% and 40%), and p-cymene (14% and 7%) in O. vulgare and S. montana, respectively, and carvacrol (75% and 65%, respectively) in T. capitata and T. caespititius. The other essential oils obtained from Portuguese flora yielded weak or no activity. Five essential oils with nematicidal activity against PWN are reported for the first time.     

Inhibition of developmental processes by flavone in Caenorhabditis elegans and its application to the pinewood nematode, Bursaphelenchus xylophilus

Flavone (2-phenyl chromone) is a well-known plant flavonoid, but its bioactivity has been little explored. Treatment of Caenorhabditis elegans or C. brissage with flavones induced embryonic and larval lethality that was pronounced in early larval stages. This anti-nematodal effect was also observed in the pinewood nematode, B. xylophilus. LD(50) values were approximately 100 muM for both B. xylophilus and C. elegans. Our results indicate that flavone is an active nematicidal compound that should be further investigated with the aim of developing a potent drug against B. xylophilus.     

Detection and characterisation of a Bacillus thuringiensis crystal protein with nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus

Nematicidal Bacillus thuringiensis (Bt) strains were isolated from forests in Zhejiang, China for further characterisation. PCR analysis was performed with nine pairs of primers specific for cry1, cry2, cry3, cry4, cry5, cry6, cry9, cry11 and cry13 to characterise and classify cry gene groups from Bt isolates. The isolates from individual cry groups were tested for nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus, which is implicated in pine wilt disease. PCR identified 14 different categories of cry gene combinations, indicating a large diversity of cry genes. The cry1 gene was by far the most abundant in Bt isolates and was found in 68% of samples. The Bt isolates zjfc85 and zjfc392 were from two distinct classes, but shared the same cry5 amplification profile and the same ~130 kDa protein; they had the highest nematicidal activity against pinewood nematode during the 48 h exposure tests, resulting in 90 and 59% mortality (9% of mortality under control conditions), respectively. The ~130 kDa Cry protein from isolate zjfc85 was purified and named as Cry5Ba3. Bioassay results indicated pinewood nematode was highly susceptible to Cry5Ba3 and exhibited profound growth abnormalities after exposure to Cry5Ba3. Our results are a novel finding and provide a potential strategy to manage pine wilt disease caused by B. xylophilus based on a nematicidal Bt.     

Mitochondrial genome of the nematode endoparasitic fungus Hirsutella vermicola reveals a high level of synteny in the family Ophiocordycipitaceae

Ophiocordycipitaceae is a diverse fungal family comprising multiple ecologically, economically, medicinally, and culturally important fungal species; however, only four species of the family have available mitochondrial genomes (mitogenomes). In this study, the complete mitogenome of the nematode endoparasitic fungus Hirsutella vermicola in Ophiocordycipitaceae was sequenced, and a comparative mitogenomic analysis of Ophiocordycipitaceae was performed. We found that the 53,793-bp circular mitogenome of H. vermicola, except for standard fungal mitochondrial genes, harbors seven introns acquired possibly through lateral transfer from other fungi and three free-standing open reading frames (ORFs) coding for hypothetical proteins. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirmed its placement in Ophiocordycipitaceae. Comparison on five mitogenomes of Ophiocordycipitaceae revealed great variation on their sizes, from 35.2 kb in Tolypocladium ophioglossoides to 157.5 kb in Ophiocordyceps sinensis, mainly due to variable numbers of introns (from 7 to 54) as well as variable lengths of intergenic regions. The five mitogenomes, however, are highly syntenic to each other in terms of gene order, the presence of an intronic ORF encoding ribosomal protein S3 within rnl, and the nad2/nad3 joining pattern. Our study is the first report of the mitogenome of H. vermicola and has facilitated the understanding of mitogenome evolution of Ophiocordycipitaceae.

     

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.