Role of Phialemonium curvatum as a potential biological control agent against a blue stain fungus on aspen

Ophiostoma crassivaginatum is the most important blue stain fungus on Populus tremuloides and causes considerable reduction in the value of the end product. The effect of a hyphomycetous fungus, Phialemonium curvatum, on the in vitro growth and wood chip colonization by O. crassivaginatum was studied. The in vitro growth of O. crassivaginatum was significantly inhibited when grown in dual culture or when treated with culture filtrate of P. curvatum. Ophiostoma crassivaginatum failed to colonize P. tremuloides wood chips when they were pre-inoculated with mycelial suspension of P. curvatum. The potential use of P. curvatum as a biological control agent against O. crassivaginatum is suggested.     

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.     

The occurrence of Phellinus tremulae (Bond.) Bond. and Borisov as a primary parasite in Populus tremula L.

Phellinus tremulae was inoculated on Populus tremula. It spread in the sapwood and inner wood of a living tree without other organisms being present. Plating experiments with wood meal from different parts of a sound tree showed that there were very few, if any, propagules of micro organisms in the sound wood, neither were there any organisms found in front of the decay of P. tremulae at the top of the decay column. These results support the theory that P. tremulae is a primary parasite in aspen. Decay fungi grown on fresh and autoclaved wood of aspen had very different growth rates. Most of the fungi tested grew best on autoclaved sapvvood. The difference in growth rate on fresh and autoclaved heartwood was lcss significant, suggesting that there is a barrier in the living wood which is destroyed by autoclaving. P. tremulae was thc only fungus that grew best on fresh inner wood.     

Fungal decomposition of sapwood and heartwood of European aspen,Populus tremula L.

In contrast to the woodrotting fungi which can only attack Populus tremula after felling, Phellinus tremulae successfully invades the central part of the living tree. Possible causes for this difference were investigated. It was found that sterile wood samples were decomposed more rapidly by saprophytic fungi than by parasitic fungi. With both types of fungi, heartwood was more resistant to decay than sapwood. It is postulated that living sapwood inhibits infection by most woodrotting fungi, but that P. tremulae can successfully overcome this barrier. This postulate cannot be confirmed by experiments using sterilized wood samples.     

Biocontrol of decay in seasoning utility poles. I. Growth rate and colonizing ability of bluestain and decay fungi in vivo and in vitro

Seasoning (air drying) of utility poles for 6–12 months is essential before preservative treatment can be achieved. However, during seasoning, pine sapwood is often colonized by decay fungi, thereby compromising the performance and service life of the poles. This study investigated the potential of bluestain fungi to act as short‐term biocontrol agents against decay during seasoning. An important attribute for biocontrol is rapid growth, so growth rates of common bluestain (Ceratocystis coerulescens, Ophiostoma minus, Ophiostoma piceae, Ophiostoma piliferum, Sphaeropsis sapinea) and decay fungi (Heterobasidion annosum, Phlebiopsis gigantea, Stereum sanguinolentum) were compared on agar medium and pine in logs at various temperatures. On agar, the growth temperature optimum of most bluestain fungi and all the decay fungi was ～25°C, with little growth at ≤5°C or above 32.5°C. Overall, the fastest growing were S. sapinea and O. minus. In logs, the most effective colonizers were S. sapinea and O. minus with pathogenic abilities that made them well fitted to colonize the sapwood of freshly felled pine. Within these species, certain isolates produced much larger lesions in phloem and the sapwood tangential plane than all the decay fungi. Notably, there was significant variation in colonizing ability between different isolates within a species, emphasizing the need for testing a range of isolates when selecting a potential biocontrol agent.     

云杉八齿小蠹体内外和坑道内真菌多样性初步研究

2009年8月在黑龙江丰林国家级保护区云杉八齿小蠹危害的红皮云杉林内,对云杉八齿小蠹体内外和坑道内真菌进行了分离.研究结果表明,黑龙江丰林国家级自然保护区内云杉八齿小蠹体内外和坑道内栖息真菌多样性比较丰富,获得真菌13属26种,其中Alternaria、Fusarium、Hypocrea、Ophiostoma、Peni...     

Ophiostomatoid fungi isolated from three different pine species in Argentinian Patagonia

Pine plantations in Argentinian Patagonia cover ca. 95,000 ha in Chubut, Río Negro and Neuquén provinces. Exotic bark beetles (Orthotomicus laricis, Hylastes ater and Hylurgus ligniperda) commonly occur in freshly cut logs, stumps and slash. These beetles are vectors of “ophiostomatoid” fungi which include primary tree pathogens as well as important agents of blue stain. The aim of this study was to identify these beetle‐associated fungi. Sawing mills and pine plantations were surveyed three consecutive years. Fungal isolates from stained logs, processed wood and insect galleries were identified based on morphological and DNA sequence comparisons of ITS and β‐tubulin gene regions. Two Grosmannia, one Graphilbum and three Ophiostoma species were identified. Ophiostoma piliferum and O. peregrinum sp. nov. were the most frequently isolated taxa. O. peregrinum occurred in all provinces, colonizing different conifer species and, interestingly, also the native broadleaved species Nothofagus dombeyi. Pine plantation forestry in southern South America includes Argentina, Brazil, Chile, Paraguay and Uruguay. Emerging data from Argentina, Chile and Uruguay revealed some coincidences between these countries, but also several differences, probably, as a result of multiple introduction events.     

The decay pattern of Phellinus tremulae (Bond.) Bond,et Borisov in Populus tremula L.

The distribution of Phellinus tremulae in stems of Populus tremula was studied visually, macroscopically as well as microscopically, and by means of isolation of the fungus in pure culture. Evidence suggests that P. tremulae occurs alone in most of the tree. Microorganisms associated with P. tremulae in the wood are suggested to be independent invaders or secondary invaders. A dark reaction zone was formed in certain parts of the wood and consisted microscopically of melanized hyphes and brown material in the ray cells. An inner wood, formed as a result of the death of nearby brandies, is apparently the main part infected by P. tremulae, and its outer border may basically limit the spread of the fungus in the trunk. The formation of “heartwood” in aspen is discussed, and a suggested outline shown for the infection and decay processes caused by the fungus.     

Comparative virulence of blue-stain fungi isolated from Japanese red pine

 We made inoculations to determine the comparative virulence of blue-stain fungi isolated from Japanese red pine (Pinus densiflora Sieb. & Zucc.). Based on the symptoms caused by each fungus, the most virulent fungus was Leptographium wingfieldii. Also, Ophiostoma minus was relatively virulent while lesions induced by some species did not differ from those in the control treatment. Based on the results, we concluded that the pathogenicity of the blue-stain fungi varied widely, and thus their impact on the host most likely varies too. Received: June 7, 2002 / Accepted: October 4, 2002 Present address: JST Domestic Research Fellow, Forest Pathology Laboratory, Tohoku Research Center, Forestry and Forest Products Research Institute, 92-25 Nabeyashiki, Shimo-Kuriyagawa, Morioka 020-0123, Japan Tel +81-19-648-3961; Fax +81-19-641-6747 e-mail: H_masu@hotmail.com Acknowledgments We thank Drs. Ohtaka and Usuki, Plant Pathology and Mycology Laboratory, Institute of Agriculture and Forestry, University of Tsukuba, for technical assistance. Contribution No. 174, Plant Pathology and Mycology Laboratory, Institute of Agriculture and Forestry, University of Tsukuba. Correspondence to:H. Masuya     

Blue stain fungi associated withTomicus piniperda (Coleoptera: Scolytidae) on Japanese red pine

A number of various species of blue-stain fungi were isolated fromTomicus piniperda adults at various stages of development, as well as from the galleries, pupal chambers and sapwood underneath galleries on Japanese red pine. This study was an attempt to identify the species, composition of blue-stain fungi associated withT. piniperda, the frequency of occurrence of the fungi, and their role in the sapwood-staining of Japanese red pine in Tsukuba City, central Japan. Among the seven species of blue-stain fungi isolated, an undescribed species ofOphiostoma together withO. minus were the dominant species and closely associated withT. piniperda. These two species occurred on newly emerging adults more frequently than the overwintered adults.Hormonema dematioides was also associated with the beetle, however, its frequency of occurrence from the emerged new adults was very low. Although the two other species,O. ips andGraphium sp. were also isolated from emerged beetles, the frequency of these fungi from gallery systems suggested that they were accidentally carried byT. piniperda. Leptographium wingfieldii, known to be associated with the beetle in Europe, was also isolated at a very low frequency and the fungus seemed not to be closely associated with the beetle.Ophiostoma sp. andO. minus appear to be the most important causes of blue-stain of Japanese red pine sapwood after infestation byT. piniperda.     

Mechanical characterization of Pinus massoniana cell walls infected by blue-stain fungi using in situ nanoindentation

Characterizing the mechanical properties of wood cell walls will lead to better understanding and optimization of modifications made to wood infected by the blue-stain fungi.In this study,in situ nanoindentation was used to characterize the mechanical properties of the cell walls of Pinus massoniana infected by blue-stain fungi at the cellular level.The results show that in situ nanoindentation is an effective method for this purpose and that blue-stain fungi penetrate wood structures and degrade wood cell walls,significantly reducing the mechanical properties of the cell walls.The method can also be used to evaluate and improve the properties of other wood species infected by blue-stain fungi.     

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

Context

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.

Aims

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.

Methods

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.

Results

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.

Conclusion

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.     

Mountain pine beetle-associated blue-stain fungi are differentially adapted to boreal temperatures

Mountain pine beetles (MPB) are the most serious pest of lodgepole pine in Canada and are likely to invade boreal jack pine forests. MPB vector three blue-stain fungi, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum, which contribute to beetle success. Fungal survival at extreme boreal temperatures will contribute to their success in jack pine. Growth, sporulation and survival of the three fungi at −20 to 37°C were tested in vitro. Overwintering survival of G. clavigera and O. montium was assessed in vivo. All species grew at 5–30°C, with optimal growth at 20–25°C. Grosmannia clavigera and L. longiclavatum survived at −20°C, but O. montium died. Growth of G. clavigera and L. longiclavatum was inhibited at 30°C, but O. montium grew well. Grosmannia clavigera and O. montium overwintered in living pines. These results suggest that G. clavigera and L. longiclavatum were adapted to cold boreal winters but not hot summers, with the converse true for O. montium. Temperature tolerance varied among G. clavigera isolates. British Columbian and Californian isolates grew faster at 25°C than Albertan isolates. Isolates from Alberta and Idaho/Montana grew optimally at 20°C, while British Columbian and Californian isolates grew optimally at 25°C.     

Automated image processing for quantification of blue-stain discolouration of Norway spruce wood

Bioincising is a promising method for enhancing liquid uptake (e.g. preservatives or wood-modification agents) in refractory wood. Incubation with the white-rot fungus, Physisporinus vitreus, which selectively degrades pit membranes, results in deeper and more homogeneous penetration of liquids. Conventional methods of assessing the degree of fungal discolouration of wood after treatment with preservatives (e.g. European standard EN 152) are partly based on a subjective rating scale, which gives a rough value of the surface colonisation by blue-stain fungi. Hence, an automated image processing (AIP) procedure was developed for standardised quantification of the segmentation thresholds of discolouration and tested against manual segmentation analysis. Using the red filter in the AIP method revealed high correlation (R ² 0.95) and allowed for more user friendly and objective determination of blue staining of wood.     

Sapstain fungi on <Emphasis Type="Italic">Pinus radiata</Emphasis> 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.     

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.     

Comparison of bluestain fungi grown in vitro and in freshly cut pine billets

Both moulds and bluestain fungi cause serious economic losses and much research is aimed at finding environmentally and economically acceptable methods of control. Despite this, there has been little study of the growth of these fungi in freshly cut wood unaltered by drying or sterilization and which therefore resembles the substratum normally invaded under natural field conditions. Thus, the growth of six sapstain fungi was compared at 20°C in freshly cut pine billets and on three types of artificial media (MEA, TWA and Pine Sapwood agar). The fungi comprised Ceratocystis coerulescens, Leptographium wingfieldii, Ophiostoma minus, Ophiostoma piceae, Phacidium coniferarum and Sphaeropsis sapinea. The six species varied markedly in their linear growth rate on agar media. In pine billets, they extended at different rates in longitudinal, radial and tangential directions, showing different pathogenic ability and patterns of colonization. Some species appeared to be ‘xylem preferring’ while others colonized the phloem tissue more readily. The growth of C. coerulescens in pine billets was very rapid (19.4 mm/day) and on average was more than twice as fast as on MEA, suggesting it was strongly stimulated by living pine tissue. The fungi also tended to grow more slowly in logs cut between autumn and spring compared with those harvested in mid- to late summer.     

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.     

Long‐term survival and non‐vector spread of the pinewood nematode,Bursaphelenchus xylophilus,via wood chips

The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is transmitted to new host trees by beetles of the genus Monochamus. The increasing interest in imported wood chips from North America for paper production and energy purposes and the corresponding phytosanitary risk of non‐vector transmission of B. xylophilus has been discussed since 1984, the year of the first interception of B. xylophilus in wood chips in the European Union. The long‐term survival of B. xylophilus in wood chips and its non‐vector spread from infested wood chips to non‐infested trees were studied. Pinus sylvestris logs were inoculated with a suspension of B. xylophilus to produce infested wood chips. During the long‐term storage test, B. xylophilus in P. sylvestris wood chips were examined. Four variants, including sealed and openly stored wood chips at both 15°C and 25°C, were studied. For the test of non‐vector spread, B. xylophilus ‐infested wood chips were placed on three‐ to four‐year‐old P. sylvestris saplings under different conditions. Bursaphelenchus xylophilus survived for more than 1 year at both temperatures in the sealed wood chips, which was significantly longer than for the openly stored variant at 25°C. Temperature, tree condition and wood chip location all influenced non‐vector spread through wood chips. Of the 480 trees that were in contact with infested wood chips and showed clear symptoms of pine wilt disease, B. xylophilus were extracted from 42 pines at 25°C and one pine at 15°C. The highest B. xylophilus infestation rates resulting in clear pine wilt disease symptoms (75%) were found in infested wood chips directly attached to stem‐wounded trees at 25°C. However, more variants exhibited B. xylophilus infestation at this temperature; trees with stem or root injuries plus direct contact with infested wood chips to the wounded part were primarily affected. Moreover, non‐vector spread was also detected in stem‐ and root‐injured pines without any direct contact with infested wood chips. Our results confirmed that B. xylophilus can survive for long periods in wood chips and can be transmitted from infested wood chips to damaged trees, but the likelihood of such PWN establishment should be low compared to spread through vectors. These findings must be considered in the pest risk analysis of B. xylophilus, and studies using outdoor trials should be carried out to complete this pest risk analysis.     

Influence of biotic factors on the mechanical properties of wood,taking into account the time of harvesting

The aim of the present paper is to investigate the influence of biotic factors (fungi and insects) on the mechanical properties of wood through the effect of blue-stain, taking into account the time of harvesting and the time of stay of wood in the forest. Specifically, the resistance to axial compression and to bending (modulus of rupture (MOR)) was studied using infected specimens of Scots Pine (Pinus sylvestris) and Norway Spruce (Picea abies) (the usual types of wood used in woodwork). The specimens were obtained from logs of Scots Pine and Norway Spruce that were harvested in three different seasons of year, namely in July 2012, November 2012 and June 2013, respectively, in the forest of Elatia-Greece, and the attack pace by biotic factors with respect to the time of logging was studied. The placement of the experimental surfaces of each type of tree was made on skid road and in the stand. Totally, 120 laboratory measurements in axial compression and 120 measurements in bending (MOR) took place. The results proved that blue-stain hardly affect the mechanical properties of both wooden species and particularly the specimens that were derived during the winter logging.