Ophiostoma Floccosum Research Articles

Identification of sapstain fungi from Scots pine pallets and assessment of their staining ability

Wood-staining fungi, moulds and decay fungi colonize freshly cut wood. Of these, only ophiostomatoid fungi are considered major agents of sapstain on logs and freshly sawn lumber because they can cause permanent staining and discoloration. Sapstain in pine pallets significantly reduces their market value and use in the food industry. The aim of this study was to identify the sapstaining fungi that colonize Scots pine wood used in pallet production. In addition, we evaluated the growth and stain intensity of six isolates of ophiostomatoid fungi on freshly cut Scots pine billets. Fungi were isolated from samples of Scots pine and identified based on morphology and DNA sequence comparisons for three gene regions (ITS, β-tubulin, TEF-1α). A total of 1259 isolates representing 31 fungal species were obtained from the pine samples in Poland during July and September. The isolates represented different ecological and taxonomical groups and belonged to the categories of staining fungi, decay fungi and surface moulds. The most frequently isolated fungi were ophiostomatoid fungi (14 species including an unknown Leptographium sp. and Ophiostoma sp.) and moulds (mainly Trichoderma and Mucor spp.). Of the ophiostomatoid species, Ophiostoma floccosum, O. piceae, O. piliferum and Endoconidiophora pinicola were the predominant species. The results of pine billet inoculation showed that among the six fungal species tested, E. pinicola exhibited the fastest growth in all three directions on the billets. Ophiostoma minus and O. piliferum displayed moderate growth rates in pine billets, while O. floccosum, O. piceae, and Leptographium lundbergii grew very slowly, especially in the tangential and radial directions. The information provided in this paper will help develop more effective control strategies for sapstain prevention in Scots pine.

Effect of Albino ophiostoma strains on Eucalyptus nitens extractives

Wood extractives promote pitch formation during pulp and paper manufacturing. To date, this problem has been controlled by extended storage of the chips and/or chemical additives. Biotreatment of the wood prior to pulping provides an alternative that not only decreases the negative impact of the extractives but may also improve the kraft pulping efficiency. This initiative seeks to verify the quantity and chemical composition of Eucalyptus nitens wood extractives following biotreatment with three albino fungi species (Ophiostoma floccosum, Ophiostoma piceae and Ophiostoma piliferum). Eucalyptus nitens wood chips were sprayed with spore suspensions of Ophiostoma piliferum, Ophiostoma piceae and Ophiostoma floccosum albino strains (1 × 108 spore concentration). After 7 and 21 days of fungal treatment, the extractive content was determined via Soxhlet extraction with an 80:20% n-hexane:ethyl acetate solvent mixture. The Ophiostoma floccosum F1A94, Ophiostoma piliferum F2D8 and Ophiostoma piceae F2A68 strains proved to be most capable of bioreduction with reductions of 35,1%, 33,2% and 29,3%, respectively. The chemical composition of the extract was analyzed via gas chromatography coupled with mass spectrometry, which demonstrated that most of the tested strains could reduce the s- sitosterol content.

Effect of growth conditions on Ophiostoma piceae (Münch) H. & P. Syd. and Ophiostoma floccosum Math-Käärik albino strains culture morphology

Blue stain in Pinus radiata D. Don wood is predominantly caused by Ophiostoma genus fungi. Stained wood causes significant economic losses to forestry and timber industries. Currently, different chemical fungicides are used to prevent the occurrence of blue stain fungi. However, recent reports have questioned the environmental friendliness of these fungicides. For this reason, the use of biological control, in particular with albino strains of the Ophiostoma genus, appears to be an environmentally safe and a technically feasible alternative to work with. The potential field application of this technology would use a formulation containing the albino fungus-the bioactive ingredient- in its yeast like form. However, some Ophiostoma strains cultures present a marked filamentous morphology in liquid culture medium, affecting the efficiency in the production of the bioactive ingredient. The hypothesis of this work presumed the possibility to control the morphology of Ophiostoma cultures, through the modification of growth conditions in liquid medium. The effect of inoculum size, growth temperature and agitation rate on the morphology of PcF2A29 Ophiostoma piceae and FlF1A55 Ophiostoma floccosum albino strains in liquid culture medium, was studied. A 23 factorial design was employed. The results showed that the initial inoculum size had a statistically significant effect on yeast-like growth morphology in both strains, whereas the temperature only produced a significant effect in PcF2A29 O. piceae strain.

Quorum sensing activity and control of yeast-mycelium dimorphism in Ophiostoma floccosum.

Quorum sensing (QS) activity in Ophiostoma fungi has not been described. We have examined the growth conditions on the control of dimorphism in Ophiostoma floccosum, an attractive biocontrol agent against blue-stain fungi, and its relationship with QS activity. In a defined culture medium with L-proline as the N source, a high inoculum size (10(7)c.f.u.ml(-1)) was the principal factor that promoted yeast-like growth. Inoculum size effect can be explained by the secretion of a QS molecule(s) (QSMs) responsible for inducing yeast morphology. QSM candidates were extracted from spent medium and their structure was determined by GC-MS. Three cyclic sesquiterpenes were found. The most abundant molecule, and therefore the principal candidate to be the QSM responsible for yeast growth of O. floccosum, was 1,1,4a-trimethyl-5,6-dimethylene-decalin (C15H24). Other two compounds were also detected.

6-Pentyl-α-pyrone as an anti-sapstain compound produced by Trichoderma gamsii KUC1747 inhibits the germination of ophiostomatoid fungi

Abstract The capability of Trichoderma gamsii to biologically control sapstain fungi has been investigated. Six ophiostomatoids, including Ophiostoma floccosum, O. piceae, O. piliferum, O. ips, Grosmannia koreana, and G. piceaperda, were inhibited by the extracts of T. gamsii. Of the various cultivation periods, 7 days of T. gamsii cultivation produced antifungal compounds most efficiently. The crude extracts of T. gamsii inhibited O. ips, G. koreana, and G. piceaperda by over 99%. The crude extracts of T. gamsii were isolated, and the compounds were purified by thin layer chromatography (TLC), open column chromatography, ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) and NMR spectroscopy. The active fractions were separated based on the degree of T. gamsii antifungal activity. The compound 6-pentyl-α-pyrone (6PAP) was evaluated as a biological control agent for sapstain fungi.

Ophiostomatoid fungi isolated from fallen shoots of Scots pine pruned by Tomicus species in Poland

Ophiostomatoid fungi are known to be associated with <em>Tomicus</em> spp. on <em>Pinus sylvestris</em>. However, very little is actually known about the fungi present in the pine shoots damaged by these insects. The aim of this study was to survey species of <em>Ophiostoma</em> s.lato associated with fallen shoots of Scots pine pruned by <em>Tomicus</em> spp. in Poland. The study was conducted in four pure Scots pine stands in southern and south-western part of the country. Fungi were identified based on morphology and DNA sequence comparison for two gene (the ITS rDNA region and ß–tubulin). In total, 64 isolates obtained, represent seven species of ophiostomatoid fungi, including their asexual states of the genera. Two of them, <em>Ophiostoma</em> sp. 1 and <em>Sporothrix</em> sp. 1 probably represent new taxa. All species were found at very low frequencies. Among them, <em>Ophiostoma minus, Ophiostoma</em> sp. 1 and <em>Sporothrix</em> sp. 1 were the most frequently isolated, with a frequency of 2.0%. Occasionally, isolated species included <em>Leptographium piriforme, Ophiostoma canum, Ophiostoma floccosum</em> and <em>Grosmannia cucullata</em>-like. Association of species of Ophiostoma s. lato with <em>Tomicus</em> spp. and the taxonomic status of two new species are discussed.

Large Shift in Symbiont Assemblage in the Invasive Red Turpentine Beetle

Changes in symbiont assemblages can affect the success and impact of invasive species, and may provide knowledge regarding the invasion histories of their vectors. Bark beetle symbioses are ideal systems to study changes in symbiont assemblages resulting from invasions. The red turpentine beetle (Dendroctonus valens) is a bark beetle species that recently invaded China from its native range in North America. It is associated with ophiostomatalean fungi in both locations, although the fungi have previously been well-surveyed only in China. We surveyed the ophiostomatalean fungi associated with D. valens in eastern and western North America, and identified the fungal species using multi-gene phylogenies. From the 307 collected isolates (147 in eastern North America and 160 in western North America), we identified 20 species: 11 in eastern North America and 13 in western North America. Four species were shared between eastern North America and western North America, one species (Ophiostoma floccosum) was shared between western North America and China, and three species (Grosmannia koreana, Leptographium procerum, and Ophiostoma abietinum) were shared between eastern North America and China. Ophiostoma floccosum and O. abietinum have worldwide distributions, and were rarely isolated from D. valens. However, G. koreana and L. procerum are primarily limited to Asia and North America respectively. Leptographium procerum, which is thought to be native to North America, represented >45% of the symbionts of D. valens in eastern North America and China, suggesting D. valens may have been introduced to China from eastern North America. These results are surprising, as previous population genetics studies on D. valens based on the cytochrome oxidase I gene have suggested that the insect was introduced into China from western North America.

English

Ophiostoma floccosum is one of blue stain fungi that cause cosmetic damage in softwood. To develop a molecular maker for the detection of O. floccosum, we designed Oflo1 and Oflo2 primers based on the β-tubulin gene. PCR assay with eleven isolates ofO. floccosum and nine other blue stain and discoloring fungal species (moulds) proved that the designed primers specifically amplified a 333 bp sized DNA band only from genomic DNA of O. floccosum isolates. Together with microwave heating extraction of spore DNA, we could detect O. floccosum within 2 h on artificially inoculated wood chip. We believed that the developed PCR method will be useful where detection of target species such as O. floccosum is required.   Key words: Ophiostoma floccosum, Species-specific primer, β-tubulin gene.

Diversity of ophiostomatoid fungi associated with the large pine weevil, Hylobius abietis, and infested Scots pine seedlings in Poland

Bark beetles are known to be associated with fungi, especially the ophiostomatoid fungi. However, very little is known about role of pine weevils, e.g., Hylobius abietis, as a vector of these fungi in Europe. The aims of our study were to demonstrate the effectiveness of H. abietis as a vector of ophiostomatoid fungi in Poland and to identify these fungi in Scots pine seedlings damaged by weevil maturation feeding. Insects and damaged Scots pine seedlings were collected from 21 reforestation sites in Poland. The fungi were identified based on morphology, DNA sequence comparisons for two gene regions (ITS, β-tubulin) and phylogenetic analyses. Sixteen of the ophiostomatoid species were isolated and identified. In all insect populations, Leptographium procerum was the most commonly isolated fungus (84 %). Ophiostoma quercus was also found at a relatively high frequency (16 %). Other ophiostomatoid fungi were found only rarely. Among these rarer fungi, four species, Leptographium lundbergii, Ophiostoma floccosum, Ophiostoma piliferum and Sporothrix inflata, were isolated above 3 %. L. procerum was isolated most frequently and was found in 88 % of the damaged seedlings. S. inflata was isolated from 26 %, while O. quercus occurred in 10 % of the seedlings. This study confirmed that L. procerum and O. quercus were common associates of H. abietis, while others species were found inconsistently and in low numbers, indicating causal associations. H. abietis also acted as an effective vector transmitting ophiostomatoid species, especially L. procerum and S. inflata, to Scots pine seedlings.

Albino strains of Ophiostoma spp. fungi effect on radiata pine permeability

Albino strains of Ophiostoma piliferum, Ophiostoma piceae and Ophiostoma floccosum used as single strain and in mixtures are tested for their abilities to increase the permeability of radiata pine sapwood after 2 and 4 weeks of incubation. The bio-treatments yielded permeability increments up to 1.9 and 2.4 times controls after 2 and 4 weeks, respectively. The fungus type involved in the treatments as well as the incubation time appeared as significant factors. Results also suggest that combinations of albino Ophiostoma spp. fungi could increase permeability to higher levels than individual ones, hinting at a possible synergistic relationship among them. Furthermore, treatments with a high concentration of O. floccosum seem particularly successful. Simultaneously to the increment of permeability such bio-treatment might also offer protection against blue sap-stain. Further research is recommended to refine and find an optimum time of incubation, as well as the most effective combination and concentration of fungi.

Fungal associates of the lodgepole pine beetle, Dendroctonus murrayanae

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74-100% of all beetles) followed closely by Ophiostoma abietinum (29-75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0-13%), Ophiostoma ips (0-15%), Ophiostoma piliferum (0-11%), a Pesotum sp. (0-11%) and Ophiostoma floccosum (0-1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.

Characterization of a Sapstaining Fungus, Ophiostoma floccosum, Isolated from the Sapwood of Pi mis thunbergii in Korea

An Ophiostoma fungus was isolated from a stump of Pinus thunbergii in a forest on the West coast of Korea. Mieroseopie analysis using a light microscope, a stereo microscope, and a scanning electron microscope revealed that it had morphological features of Pesotum and Sporothix synanarmorphs. Based on the β-tubulin gene sequence analysis, the fungus was identified as the anamorph of Ophiostoma floccosum. Myeologieal properties of the species including its growth properties on different culture media were described.

Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I ( amy1) gene from Ophiostoma floccosum

UV mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein production after repeated rounds of mutagenic treatment. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the increase in total secreted protein was about six times greater than the parental strain. Secreted proteinase and lipase activities of the parental strain and four key mutants were also investigated. N-terminal sequencing of the five dominant protein bands separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an α-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the α-amylase amino acid sequence. The amplified fragment contained the entire gene encoding α-amylase ( amy1) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single α-amylase gene locus.

Assessing RNAi frequency and efficiency in Ophiostoma floccosum and O. piceae

Ophiostoma species are an economically important group of saprophytic and pathogenic fungi that grow in trees or wood. Ophiostoma like O. piceae and O. floccosum produce melanin, a pigment that stains lumber and logs. We used such species as model organisms for characterizing the molecular mechanisms in fungal melanin production. Because homologous recombination is rare in the Ophiostoma, identifying gene function in this group is challenging. We addressed this by assessing RNA interference (RNAi) as an alternative to gene replacement. For this, we built different inverted repeat transgene (IRT) constructs to down-regulate the polyketide synthase ( PKS1) gene of the melanin pathway in O. piceae and O. floccosum. Transformation with IRT-PKS reduced mRNA levels for the PKS1 gene, and consequently decreased pigmentation in transformants. We showed that the PKS1 RNAi efficiency was proportional to the length of the dsRNA expressed from IRT constructs. These results indicated that RNAi is an appropriate tool for functional analysis of genes in Ophiostoma.

Microscopic, Biochemical and Physiological Assessment of the Effect of Methylene Bisthiocyanate on the Sapstain Fungus Ophiostoma floccosum

In vitro effects of methylene bisthiocyanate (MBT) on hyphal morphology and ultra-structure of Ophiostoma floccosum were examined using differential interference contrast, epifluorescence and transmission electron microscopy (TEM). To understand the mode of action of MBT, experiments were undertaken to measure potassium ion (K+) leakage from cells, oxygen consumption, glucose and ATP levels. Differential interference contrast microscopy indicated that MBT caused rapid changes in O. floccosum hyphae resulting in extensive vaculoation and accumulation of granular materials within the cytoplasm. Epifluorescence microscopy provided evidence that MBT treatment causes a loss in the permeability properties of the plasma membrane. TEM showed retraction of the plasma membrane from the cell wall, aggregation of cytoplasmic contents, vesiculation of membranous components, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. There was a rapid efflux of intracellular K+ ions from cells, a substantial loss in K+ ions occurring within the first 5 min of MBT treatment. The rate of K+ leakage was MBT concentration treatment-time dependent. The study also showed that the effect of lower concentrations of MBT (0.01 and 0.1 mM) on respiratory activity was negligible. However, at the same concentrations, glucose consumption and ATP production were affected. Taken together, these observations suggest that the target site of MBT in O. floccosum alters membrane properties and uncouples oxidative phosphorylation from the respiratory chain.

A survey of Ophiostoma species vectored by Hylastes ater to pine seedlings in New Zealand

SummaryEcological studies in New Zealand require consideration of the impact of exotic (introduced) organisms from disparate environments. A survey of Ophiostoma species isolated and identified from Pinus radiata stumps, the bark beetle Hylastes ater collected from these stumps and from P. radiata seedlings following sublethal damage by H. ater, was undertaken in 10 reforestation sites in New Zealand. Nine Ophiostoma species were isolated and identified in this survey. Ophiostoma galeiformis and Ophiostoma huntii were isolated most frequently from all the substrates sampled. Ophiostoma floccosum, Ophiostoma setosum, Leptographium procerum and Leptographium truncatum were also isolated from stumps, damaged seedlings and beetles. Ophiostoma quercus was only isolated from stumps and beetles and Ophiostoma ips only from beetles. A strong relationship between the severity of H. ater feeding damage, and the presence and number of species of Ophiostoma was found. Eight Ophiostoma species were transmitted by H. ater to P. radiata seedlings in laboratory transmission experiments. From the results of this field survey and the laboratory experiments we confirm that H. ater is a vector of Ophiostoma species to P. radiata seedlings in New Zealand.

Ophiostomatoid fungi isolated fromPinus radiatalogs imported from New Zealand to Korea

Ophiostomatoid fungi discolor lumber, logs, and tree sapwood. Stained wood has a lower market value and can be refused by importing customers because such products can potentially carry pathogenic fungi. Little information is available on the ophiostomatoid fungi that colonize Pinus radiata D. Don (radiata pine) logs imported from New Zealand into Korea. In this work, we attempted to identify the native and non-native fungi colonizing wood imported into Korea. At least 12 species of ophiostomatoid fungi, including an unknown Ophiostoma sp. A, were identified among the fungi-staining radiata pine. They were Leptographium procerum (Kendr.) Wingf., Leptographium bistatum Kim & Kim, Ophiostoma floccosum Mathiesen, Ophiostoma huntii (Rob.) Hoog & Scheff., Ophiostoma ips (Rumbold) Nannf., Ophiostoma nigrocarpum (David.) Hoog, Ophiostoma piceae (Münch) H. & P. Sydow, Ophiostoma piliferum (Fries) H. & P. Sydow, Ophiostoma quercus (Georgév.) Nannf., Ophiostoma radiaticola Kim et al., and Ophiostoma setosum Uzunovic et al. Of these species, O. floccosum was the dominant species in both logs and boards. We confirmed that many of the sapstain species that we isolated have been previously reported in Korea. However, four species, O. radiatcola, O. setosum, O. huntii, and O. nigrocarpum have not been reported previously in Korea. We also found the new species, L. bistatum, along with an unknown Ophiostoma sp. A.Key words: radiata pine, ophiostomatoid fungi, New Zealand, non-native organisms, β-tubulin gene.

Sapstain fungi on Pinus radiata logs – from New Zealand Forest to export in Japan

Supply of clear, unstained logs from an export country is important economically as well as in reducing the biosecurity risk to an importing country such as Japan. Although conditions found within the holds of ships containing logs are thought to be ideal for rapid colonization of sapstain fungi, no research has been conducted. Research-focussed log export trials were designed to determine the extent of sapstain colonisation at specific points in the processing of logs from harvesting to arrival at the export destination. Two trials were established, in the austral summer and winter, in which mature Pinus radiata logs were harvested in New Zealand and shipped to export ports in Japan. Data loggers placed above and below deck of the ships recorded microclimatic conditions. Nine species of sapstain fungi were isolated from logs during the summer trial; the most common species isolated were Ophiostoma floccosum, Ophiostoma querci, and Ophiostoma setosum. In contrast, ten sapstain species were detected during the winter trial, with Sphaeropsis sapinea, O. querci, O. floccosum, and O. setosum most commonly isolated. This research was the first successful attempt at measuring visual sapstain development and isolating sapstain fungi from the time of harvesting to arrival at an export destination.

Effects of New Zealand sapstaining fungi on structural integrity of unseasoned radiata pine

Four sets of green, side-matched specimens of radiata pine (Pinus radiata D. Don) sapwood were incubated with individual isolates of Ophiostoma floccosum, O. pluriannulatum, O. ips, O. piceae, Leptographium procerum and Sphaeropsis sapinea for 8 or 16 weeks, along with non-inoculated control samples. Three decay fungi, Gloeophyllum trabeum, Phlebiopsis gigantea and Schizophyllum commune, were included in the tests to serve as references for weight and toughness loss. Overall toughness and dry weight were not significantly (p<0.05) different between wood specimens inoculated with sapstaining fungi and the controls, except for O. ips #308 causing 18% toughness loss, but not weight loss, in one out of three experiments. In contrast, G. trabeum reduced toughness by 61% and caused a weight loss of 8% whereas S. commune and P. gigantea produced toughness losses of 32% and 16%, respectively, without any significant weight losses. None of the sapstaining fungi tested degraded lignin or structural carbohydrates in radiata pine sapwood after 16 weeks' incubation. Of the isolates tested for their ability to degrade extractives O. floccosum was most effective (54% reduction) and O. pluriannulatum least (0% reduction) effective.

A second reductase gene involved in melanin biosynthesis in the sap-staining fungus Ophiostoma floccosum.

Two different DNA sequences amplified by PCR from Ophiostoma floccosum show high homology to reductase genes of the fungal melanin biosynthetic pathway and suggest that two reductases may participate in this pathway in O. floccosum. Previously, one reductase gene, THN1 had been isolated from O. floccosum and proved to be involved in melanin biosynthesis. The second reductase gene, THN2, encodes a protein of 284 amino acids and is the longest fungal melanin reductase so far reported. The protein sequences deduced from the two THN genes show 44% identity. The products of THN1 and THN2 belong to two different groups of melanin reductases based on a comparative analysis of sequences from several fungal species. We confirmed the function of the THN2 gene by complementing dihydoxynaphthalene (DHN)-melanin-deficient, non-pathogenic mutants of Colletotrichum lagenarium and Magnaporthe grisea that lack the 1,3,8-trihydroxynaphthalene reductase. THN2 transcripts were detected in these transformants. These melanin-producing transformants showed mycelial and appressorium melanization. C. lagenarium transformants that formed melanin exhibited pathogenicity. This suggests that the cloned THN2 gene product functions as the 1,3,8-trihydroxynaphthalene reductase in the DHN melanin biosynthetic pathway in both C. lagenarium and M. grisea.