The use of the green fluorescent protein as a biomarker for sapstain fungi

To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip^®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non‐transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP‐expressing fungi were easily differentiated from the wild‐type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood.     

Characterization of host–fungus interactions among wood decay fungi associated with Khaya senegalensis (Desr.) A. Juss (Meliaceae) in Singapore

Tree pruning creates wounds that are amenable for wood decay fungi colonization. To characterize the dynamic host–fungus interactions at this location in Senegal mahogany (Khaya senegalensis), in vitro and in vivo pathogenicity tests were conducted with wood decay fungi associated with this tropical tree species. Fomitiporella caryophylii, Hymenochaete murina and Phellinus noxius isolates were included in this experiment following their frequent isolation from Senegal mahogany pruning wounds. The evaluated isolates demonstrated unique host interactions in laboratory tests that suggest equally divergent prognoses for living Senegal mahoganies affected by these fungi. Although all evaluated fungal isolates successfully breached naturally induced reaction zones, P. noxius alone caused significant mass loss to incubated wood blocks. In addition, P. noxius caused extensive wood decay after inoculation in living hosts, successfully illustrating Koch's postulates for this host–fungus relationship. The wood decay ability, invasiveness and facultative parasitism demonstrated by P. noxius suggest its dominant role in wood decay columns below pruning wounds on living Senegal mahoganies. These results highlight the importance of characterizing specific host–fungus interactions and their implications for wood decay severity below pruning wounds in living trees.     

Structure-activity relationships of some congeners of 4-phenyl-3-butenoic acid against aspen decay and stain fungi

Phellinus tremulae is the most important decay fungus on Populus tremuloides. In forest industries using aspen wood chips, two blue-stain fungi Ophiostoma crassivaginatum and Ophiostoma piliferum cause considerable reduction in the value of the end product. Thirty-eight homologues and analogues of 4-phenyl-3-butenoic acid were studied against P. tremulae, O. crassivaginatum, and O. piliferum. Varied inhibition of the in vitro growth of these fungi was observed at 1–1000 μg/ml. The wood-chip colonization by P. tremulae was not affected by the six most active compounds at 1 μg/ml, however, at 10 and 100 μg/ml the growth of P. tremulae on wood chips was totally arrested. 4-Phenyl-3-butenoic acid and 4-phenyl-3-butynoic acid prevented blue-stain development on wood chips at 1 μg/ml.     

Headspace solid‐phase microextraction gas chromatography–mass spectrometry for the detection of volatile organic compounds released from Ganoderma boninense and oil palm wood

Basal stem rot disease caused by the white‐rot fungus Ganoderma boninense is a major threat to the oil palm industry, and hence, the ability to detect infections at an early stage of development is desired. In this study, a headspace solid‐phase microextraction (HS‐SPME) method coupled with gas chromatography–mass spectrometry (GC‐MS) was employed to analyse the volatile organic compounds (VOCs) released from G. boninense cultures and infected oil palm wood. We examined VOCs released from three types of samples: G. boninense mycelium, oil palm wood and oil palm wood colonized by G. boninense. This preliminary study led to the tentative identification of 57 VOCs, including alcohols, alkanes, volatile acids, ketones, aldehydes, esters, sesquiterpenes and polycyclic aromatic hydrocarbon groups. Aliphatic compounds with eight‐carbon atoms, such as 1‐octen‐3‐ol, 3‐octanone, 1‐octanol and (E)‐2‐octenal, were the most abundant constituents of the Ganoderma samples, whereas furfural and hexanal were the major compounds detected in the oil palm wood samples. Chemometric analyses using cluster heat maps and principal component analyses were used to discriminate between the VOC profiles. The results indicated that the novel method described here could be used to detect Ganoderma disease and, more generally, for chemoecological studies of plant–pathogen interactions.     

In vitro screening of biocontrol and biodegradation potential of selected hymenomycetes against Ganoderma boninense and infected oil palm waste

The antagonistic and enzymatic activities of non‐pathogenic hymenomycetes naturally occurring on oil palm (Elaeis guineensis) trunks were assessed to determine their potential as biocontrol agents against G. boninense. Twenty‐five Basidiomycota were isolated on a Ganoderma‐selective medium and identified using internal transcribed spacer (ITS) sequencing. The three fungi with potentially higher antagonistic activity against G. boninense, based on the percentage inhibition of radial mycelial growth (PIRG) in dual culture, were Pycnoporus sanguineus (84%), Trametes lactinea (82%) and Grammothele fuligo (81%). In addition to these three isolates, Lentinus tigrinus and a Rigidoporus sp. were also able to invade sterilized oil palm blocks previously colonized by G. boninense despite having lower PIRG values. Colonization of oil palm blocks by G. fuligo, P. sanguineus, Rigidoporus sp., T. lactinea and L. tigrinus resulted in mass losses of 32.50, 30.78, 27.64, 25.20 and 19.33%, respectively, probably due to an ability to produce one or more lignocellulolytic enzyme. Further work is needed to determine the enzymatic degradation potential of these fungi under natural conditions and their possible use in the prevention of colonization of oil palm by G. boninense.     

Associations between Pityogenes bidentatus and fungi in young managed Scots pine stands in Poland

The association between Pityogenes bidentatus and fungi was studied in young, managed Pinus sylvestris stands in Poland. Fungi were isolated from emerged adults and their galleries collected from four populations. In total, 2089 fungal isolates including 42 species, were obtained. Penicillium sp. 1 and Geosmithia sp. 1 were the most commonly isolated fungi from beetles (49% and 41% of beetles respectively). Geosmithia sp. 1 species was the dominant species in P. bidentatus galleries with a frequency of occurrence of 57.9%. Hormonema dematioides was the second most abundant fungus in gallery systems (17.1% of wood samples). Two of the isolated Geosmithia species were previously undescribed. Pityogenes bidentatus also vectored three ophiostomatoid species: Ophiostoma minus, O. piceae and Graphium sp. ‘W’. These species were occasionally isolated from beetles and their galleries, suggesting a non-specific relationship.     

Field tests on biological control of Heterobasidion annosum by Phaeotheca dimorphospora in comparison with Phlebiopsis gigantea

In field tests conducted on freshly cut red pine (Pinus resinosa) logs, the antagonistic fungus Phaeotheca dimorphospora formulated as pre‐colonized rye grains reduced colonization of wood tissue by Heterobasidion annosum to less than 2% over the 2 months following inoculation. However, compared to controls, a water suspension of P. dimorphospora did not reduce the infection, but rather promoted colonization by H. annosum after a 2‐month exposure. Production of antifungal metabolites by P. dimorphospora in the rye grains and better colonization of the logs by the antagonist were observed with the rye grain formulation. In this field test, the most effective treatment to control H. annosum was the application of an oidial suspension of Phlebiopsis gigantea. This fungus completely inhibited colonization by H. annosum mostly by rapid colonization of wood tissue. All treatments with P. gigantea, P. dimorphospora and rye grains influenced the naturally occurring mycoflora. The most important species composing this mycoflora were Hormonema sp., Mortierella spp., Mucor hiemalis f. hiemalis, Nectria sp., Pesotum sp., P. gigantea, Tympanis spp., Trichoderma spp. and an unknown basidiomycete.     

Efficacy of radio frequency treatment and its potential for control of sapstain and wood decay fungi on red oak,poplar, and southern yellow pine wood species

The effectiveness of radio frequency (RF) treatment in the control of wood decay fungi (Gloeophyllum trabeum, Ganoderma lucidum, and Irpex lacteus) and sapstain fungus (Ceratocystis fimbriata) in red oak (Quercus spp.), poplar (Populus alba), and southern yellow pine (Pinus spp.) was evaluated in the laboratory as an alternative to methyl bromide (MB) treatment. Wood samples (15.5 x 10 x 10 cm) were inoculated with fungi from a 7-day culture by dipping them to a depth of one face deep (2 cm) into inoculum and incubating them at 25°C for 14 days. Identical wood samples were left uninoculated as controls. Subsequent to incubation, the wood blocks were exposed to RF radiation in an industrial 40-kW dielectric oven at temperatures between 60° and 70°C for 2 min. The test fungi were recovered and reisolated from all of the control wood blocks but not from RF-treated wood blocks. RF treatment resulted in complete inhibition of the fungus in 98%-100% of the wood samples. Moisture content loss (≥1%) was noted after wood had been exposed to RF treatment. Moisture content may be an important factor to consider with RF treatments. RF treatment can, therefore, potentially provide an effective and rapid quarantine treatment as an alternative to MB fumigation for certain pathogen-wood combinations. This article reports the results of research only. Mention of a product does not constitute an endorsement or recommendation by USDA for its use.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.     

Sphaeropsis canker,a new disease of cypress in Greece

The fungus Sphaeropsis sp. is reported for first time in Greece to cause cankers on Cupressus sempervirens. The cultural characteristics on PDA, spore shape and size and canker morphology were identical to those of the fungus described as Diplodia pinea f. sp. cupressi in Israel. The cankers of Sphaeropsis sp. are characterized by resin exudation, with fissuring of the bark over a dry sector of the wood. The pathogenicity of the Sphaeropsis sp. was proved by artificial inoculations on selected cypress clones resistant and susceptible to Seiridium cardinale. The mean canker length of the susceptible clone was significantly more than that of the resistant clones. It appears that the defence reaction in cypress against both fungi is similar. In artificial inoculations on Pinus halepensis and Pinus pinea, Sphaeropsis sp. was nonpathogenic. During a 3‐year period, in an experimental plot in western Peloponnese, the fungus spread from one, initially, to 13 cypress clones. Drought stress during the summer appears to be the main factor predisposing the cypress plants to become susceptible to the fungus.     

Relationship between production of hydroxyl radicals and degradation of wood, crystalline cellulose, and a lignin-related compound or accumulation of oxalic acid in cultures of brown-rot fungi

The degradation of wood, filter paper cellulose, and a lignin-substructure model, was measured in cultures of seven fungi usually regarded as brown-rot fungi. Hydroxyl radical production and the accumulation of oxalic acid in the cultures were also measured. Four of the fungi, Gloeophyllum trabeum, Tyromyces palustris, Laetiporus sulphureus, and Postia placenta, were typical brown-rot fungi, in that they preferentially degraded and eliminated the polysaccharides in wood and produced large amounts of hydroxyl radical. The rates of hydroxyl radical generation in cultures of the four fungi were directly proportional to the degradation rates of wood, cellulose, and the lignin-related compound, and inversely proportional to the amount of oxalic acid in the cultures. Two of the fungi, Daedalea dickinsii and Lentinus lepideus, did not degrade any of the substrates significantly and produced very little hydroxyl radical. Coniophora puteana had the highest rate of cellulose degradation, but did not degrade wood or the lignin model significantly and produced only negligible amounts of hydroxyl radical. These results indicate that brown-rot fungi produce large amounts of hydroxyl radical for the degradation of wood and crystalline cellulose.     

Anatomical characterization of decayed wood in standing light red meranti and identification of the fungi isolated from the decayed area

To further our understanding of wood decay in living light red meranti (Shorea smithiana) trees, microscopic characteristics of the cell and cell wall degradations of S. smithiana wood in the presence of the decay fungi, the identity of the causal fungi, and the decay potential and pattern by an isolated fungus were investigated. Cell wall degradations, including cell wall thinning, bore holes formation, rounded pit erosion, and eroded channel opening were clearly observed under light and scanning electron microscopy. In transverse view, many large voids resulting from a coalition of degraded wood tissue appeared in the decayed canker zone. All these observations suggest the well-known simultaneous decay pattern caused by white-rot fungi. By phylogenetic analysis based on the sequences of internal transcribed spacer region of ribosomal DNA, a basidiomycete fungus isolated from the decayed wood was identified as Schizophyllum commune. The degradation caused by this fungus on sound S. smithiana wood in an in situ laboratory decay test was classified as the early stage of simultaneous decay, and showed a similar pattern to that observed in the wood samples naturally decayed.     

Interaction between Epicoccum purpurascens and xylophagous basidiomycetes on wood blocks

Epicoccum purpurascens, a component of the bark micromycota, was evaluated as a potential biocontrol agent for three decay‐causing hymenomycetes frequently found in Buenos Aires city trees, Ganoderma platense, Inonotus rickii and Rigidoporus ulmarius. Treatments consisted of dual cultures of E. purpurascens and xylophagous fungi on box elder (Acer negundo) wood blocks. Two inoculation sequences were tested: (i) the biocontrol agent as the first microorganism inoculated followed by the pathogen; and (ii) the xylophagous fungus as the first microorganism inoculated followed by the biocontrol agent. After 3 months of incubation, percentage dry weight loss was calculated. When E. purpurascens was allowed to colonize wood blocks before the introduction of the xylophagous fungus, dry weight loss was significantly reduced in all instances. However, as a secondary colonizer, E. purpurascens mildly reduced the weight loss caused by G. platense, had no effect on degradation by I. rickii and increased wood weight loss by R. ulmarius.     

Response of maple sapwood to injury and infection

In sapwood challenge experiments in Acer rubrum, columns of discolouration initiated by wounding and inoculation with pioneer fungi (Cephalosporium sp., Phialophora sp.) were similar in size to untreated wounds. Inoculation with decay fungi (Pleurotus ostreatus, Trametes versicolor) produced larger columns of wound-initiated discolouration. The removal of bark around a bore wound caused a significantly larger column to form compared to the sum of the columns inititiated by separate wounds. Stage-I discoloured wood, not associated with obviously rotted wood, had concentrations of mobile cations and soluble phenols similar to sapwood. Stage-II discoloured wood, spatially associated with rotted wood, was frequently bounded by a chemically distinct boundary layer and the discoloured wood contained significantly greater concentrations of mobile cations and soluble phenols than stage-I discoloured wood.     

Blue stain fungi associated withIps cembrae (Coleoptera: Scolytidae) in Japanese larch in connection with their brood development

The association between blue stain fungi andIps cembrae Heer (Coleoptera: Scolytidae) was investigated in the Japanese larch,Larix leptolepis Gordon, in the Nagoya University Forest, Aichi Prefecture, central Japan.I. cembrae had one or two generation(s) in a year in this study area. Two blue stain fungi,Ophiostoma piceae andLeptographium sp., were isolated from the body surface of both male adults in mating chambers and female adults in parent galleries, suggesting that this beetle species was a vector of these fungi. Although no blue stain fungi were isolated from non-stained wood, both fungi were isolated from the mating chambers, the center and the uppermost end of the galleries throughout the season. The fact thatO. piceae was consistently isolated with high frequency from adults and from their galleries strongly suggested that this species would be the principal blue stain fungus infecting the beetle-attacked larch trees.     

Fungal colonisation of outside weathered modified wood

Specimens of Scots pine sapwood (Pinus sylvestris L.) and beech wood (Fagus sylvatica L.) were treated with an amino-alkyl-functional oligomeric siloxane, a sodium water glass solution and 1,3-dimethylol-4,5-dihydroxyethylene urea (DMDHEU). Treated and untreated wood specimens were exposed outdoors without ground contact. After 9?months of outside exposure, all specimens showed discolouration caused by infestations of mould and staining fungi on the exposed wood surface. Fungi grown on the sample surface were isolated and identified by microscopic technique and sequencing of PCR-amplified DNA from the ITS region. Primarily, an infestation by ascomycetes and related deuteromycetes was found. The most dominant fungi were Trichoderma sp. and Epicoccum sp.. An infestation of Aureobasidium pullulans was only detected on untreated and DMDHEU-treated samples. There were only marginal differences of fungal infestation between the two wood species.     

Persistence of some pine pathogens in coarse woody debris and cones in a Pinus pinea forest

The persistence of Sphaeropsis sapinea, Leptographium serpens and Heterobasidion annosum s.s. in artificially inoculated pine branch pieces (S. sapinea and L. serpens) and wood blocks (L. serpens and H. annosum s.s.) was investigated in order to discuss the alternative of leaving coarse woody debris in stands of Italian stone pine (Pinus pinea). Also, natural colonization by S. sapinea of pine cones of different ages was assessed. Methods used for inoculating branch pieces and wood blocks were highly effective for all fungi. Type of a forest stand in which branch pieces and wood blocks have been incubated did not affect the persistence of the pathogens in the inoculated samples. For branch pieces, the success of re-isolation of L. serpens dropped as the sample incubation time increased, while S. sapinea was always successfully (100%) re-isolated (even 12 months after the inoculation). L. serpens and H. annosum s.s. were re-isolated from most of the buried wood blocks (from more than 95% samples) up to 3 months following the inoculation. Of the observed P. pinea cones (in most cases, more than 2 years old), 74% were naturally infected byS. sapinea. All three investigated pathogens were able to survive in dead plant tissues for long periods of time (at least for several (3–12) months). The persistence of these pine-pathogenic species in dead plant material questions the feasibility of leaving coarse woody debris in managed Italian stone pine forests meant for landscape conservation and leisure activities.     

Gaseous treatment with allyl isothiocyanate to control established microbial infestation on wood

Applicability of gaseous treatment with allyl isothiocyanate (AIT) was evaluated for controlling the established microbial infestation of wood in the laboratory. Small sapwood specimens from five wood species measuring 20 mm (T)×20 mm (R)×10 mm (L) were first preincubated on 2% malt-agar medium with placing them on a fully grown monoculture of seven test fungi. After 7 weeks of preincubation they were transferred to fresh medium and exposed to AIT fumigant at concentrations of 2360 and 23600ppm AIT in the air of a petri dish to determine threshold values of exposure periods for each test fungus. The 2360 ppm concentration was not effective forPenicillium funiculosum, Gliocladium virens, orRhizopus stolonifer for any of the wood species-exposure period combinations. Those test fungi could grow even at 23600ppm after 48h exposure whenCryptomeria japonica andFagus crenata were used as wood substrate. Growth of other test fungi was inhibited at 2360ppm with a few exceptions in the case ofAspergillus niger. The required periods of exposure to suppress microbial regrowth were different with wood species-test fungi combinations. As two wood-decaying basidiomycetes,Trametes versicolor andFomitopsis palustris, were easily controlled at 2360ppm after 24h exposure regardless of wood species, AIT treatment proved applicable to the control of internal decay of utility poles and other relevant products in service.     

Fungi isolated from Picea abies infested by the bark beetle Ips typographus in the Białowieża forest in north‐eastern Poland

The assemblage of fungi occurring in the sapwood of Norway spruce (Picea abies) and in bark beetle galleries following attack by the Eurasian spruce bark beetle Ips typographus was investigated in the Bia?owie?a forest in north‐eastern Poland. Fungi were isolated from blue‐stained sapwood of beetle‐infested spruce trees in June 2002, and a few isolates were also obtained from ascospores and conidia taken from perithecia and asexual structures occurring in the gallery systems of the insects. The mycobiota of I. typographus in the Bia?owie?a forest was dominated by ophiostomatoid fungi, which were represented by seven species. Four species, including Ceratocystis polonica, Grosmannia penicillata, Ophiostoma ainoae and Ophiostoma bicolor were isolated at high frequencies, whereas three other taxa, Ceratocystiopsis minuta, Ceratocystiopsis alba and a Pesotum sp. were rare. The anamorphic fungus Graphium fimbriisporum and yeasts also occurred occasionally. In addition, the basidiomycete Gloeocystidium ipidophilum was relatively common. The pathogenic blue‐stain fungus C. polonica was the dominant fungal associate of I. typographus in the Bia?owie?a forest, which is consistent with a previous study at this area in the 1930s. Ceratocystis polonica was the most frequently isolated species at the leading edge of fungal colonization in the sapwood and had on an average penetrated deeper into the wood than other fungal associates. This suggests that it acts as a primary invader into the sapwood after attack by I. typographus in the Bia?owie?a forest, followed by O. bicolor, O. ainoae, G. ipidophilum and G. penicillata. Thus far, the Bia?owie?a forest is one of the few areas in Europe, where C. polonica has been reported as a dominate fungal associate of I. typographus.     

Comparative study on the durability of heat-treated White Birch (Betula papyrifera) subjected to the attack of brown and white rot fungi

Abstract

The effect of heat treatment on decay resistance of white birch was evaluated for different incubation periods ranging from 2 to 12 weeks using three species of brown rot and one species of white rot fungus. The results of weight loss tests showed that the white rot fungus, Trametes versicolor, effectively degraded the untreated wood (73.5%). While the degradation of untreated wood by brown rot fungi species, Gloephyllum trabeum (11.6%) and Conifora puteana (6.2%), was considerably less compared to T. versicolor, the third brown rot fungi studied, Poria placenta, caused an appreciable degradation of the same species (52.4%). The results clearly showed that the heat treatment reduced the effect of fungi attack on white birch. Increasing the heat treatment temperature from 195 to 215°C resulted in reduction of weight loss, consequently, reduction in fungal attack. As an example, the weight loss reductions due to T. versicolor, P. placenta, G. trabeum and C. puteana attack was 62.2%, 71.3%, 89.6% and 100%, respectively, compared to the weight loss of untreated wood when it is heat treated at 215°C. Thus, these results confirmed that the heat treatment increased the biological resistance of white birch.