Selective staining and visualization of hyphal sheath of a white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 with phloxine B

The hyphal sheath is a morphological feature of many kinds of fungi. Although the fine structures of the sheath have been studied in detail by a number of electron microscopy techniques, the function and physiology of the hyphal sheath are not yet clarified. One reason for this is that the hyphal sheath is a colorless, mucilaginous, and delicate material so that it is not easily identified. We developed a simple method to visualize and identify the hyphal sheath of the white-rot fungus Phanerochaete crassa WD1694. The small mycelial pellets in shaken liquid cultures of P. crassa WD1694 were stained directly with phloxine B. Both the hyphae and the hyphal sheath that filled the gaps between each of the hyphae were visualized and observed by light microscopy. The stained hyphae were further studied by transmission electron microscopy, atomic force microscopy, and fl uorescence microscopy. Based on these observations, we confirmed that the staining of the hyphae was also due to the presence of the hyphal sheath that closely covered the fungal cell wall. These results clearly showed that the hyphal sheath was selectively stained with phloxine B and could be observed and identified by conventional light microscopy. Part of this report was presented at the 50th Lignin Symposium, Nagoya, October 2005     

A major facilitator superfamily transporter in Colletotrichum fructicola (CfMfs1) is required for sugar transport,appressorial turgor pressure,conidiation and pathogenicity

The major facilitator superfamily (MFS) is one of the largest membrane‐protein families. To investigate the role of MFS proteins in the fungal plant anthracnose pathogen Colletotrichum fructicola, the CfMFS1 gene was deleted. This resulted in reduced mycelial growth, conidial yield and decreased virulence on tea oil camellia leaves. In addition, ?Cfmfs1 showed increased sensitivity to osmotic stress and to a cell‐wall stressor. Further analysis revealed that CfMfs1 is required for conidial penetration and appressorial turgor pressure, both important for fungal pathogen invasion. Confocal fluorescence microscopy showed that CfMfs1 is localized to membranes of both hyphae and conidia, suggesting that it may be a membrane transporter. Our study provides evidence that CfMfs1 has a role in conidiation, sugar transport, stress response, conidial penetration, appressorial turgor pressure and virulence against tea oil camellia.     

Infection scene investigation of shot holes: Incomplete but neutral abscission of brown spots on oriental cherry leaves

Shot holes on oriental cherry (Prunus serrulata var. spontanea) leaves were investigated to evaluate their role in the elimination of pathogens using analytical electron microscopy. Small brown spots caused by Mycosphaerella cerasella appeared on the leaf surface and developed into shot holes that were approximately 1–5 mm in diameter. Field emission scanning electron microscopy revealed clusters of fungal conidiophores and hyphal growth on the leaf surface around the shot holes. The fungal structures were mostly located within a radius of 500 μm from the shot holes and confined to the epidermal sector separated by veins. X‐ray microanalysis revealed distinct calcium ring patterns encircling the shot holes. The abscised surfaces consisted of suberized plant cells colonized with filamentous fungi and yeasts. Embedded in the mesophyll tissue, druse crystals were co‐localized with calcium and oxygen. Despite the fungal presence around the shot holes, no further expansion of the shot holes was observed on the leaf surface. These results suggest that shot holes may represent an incomplete but neutral abscission of infection loci in the presence of necrotrophic fungal pathogens. This microscopy study provides insight into the formation and roles of calcium oxalate in plants under pathological conditions.     

Extent of colonization by Raffaelea quercivora of artificially inoculated living and gamma‐ray‐sterilized seedlings of two Japanese and three American oak species

Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.     

New insights into the survival strategy of the invasive soilborne pathogen Phytophthora cinnamomi in different natural ecosystems in Western Australia

Despite its importance as one of the most notorious, globally distributed, multihost plant pathogens, knowledge on the survival strategy of Phytophthora cinnamomi in seasonally dry climates is limited. Soil and fine roots were collected from the rhizosphere of severely declining or recently dead specimens of 13 woody species at 11 dieback sites and two dieback spots and from healthy specimens of five woody species at four dieback‐free sites in native forests, woodlands and heathlands of the south‐west of Western Australia (WA). Phytophthora cinnamomi was recovered from 80.4, 78.1 and 100% of tested soil, fine root and soil–debris slurry samples at the 11 dieback sites, in some cases even after 18‐month storage under air‐dry conditions, but not from the small dieback spots and the healthy sites. Direct isolations from soil–debris slurry showed that P. cinnamomi colonies exclusively originated from fine roots and root fragments not from free propagules in the soil. Microscopic investigation of P. cinnamomi‐infected fine and small woody roots and root fragments demonstrated in 68.8, 81.3 and 93.8% of samples from nine woody species the presence of thick‐walled oospores, stromata‐like hyphal aggregations and intracellular hyphae encased by lignitubers, respectively, while thin‐walled putative chlamydospores were found in only 21.2% of samples from five woody species. These findings were confirmed by microscopic examination of fine roots from artificially inoculated young trees of 10 woody species. It is suggested that (i) the main function of chlamydospores is the survival in moderately dry conditions between consecutive rain events and (ii) selfed oospores, hyphal aggregations, and encased hyphae and vesicles in infected root tissue of both host and non‐host species are the major long‐term survival propagules of P. cinnamomi during the extremely dry summer conditions in WA.     

Spatial distribution of Raffaelea quercivora hyphae in transverse sections of seedlings of two Japanese oak species

To compare the distributions of Raffaelea quercivora hyphae within seedlings of a susceptible species, Quercus crispula, and a resistant species, Q. glauca, we examined water conductance at transverse sections. Raffaelea quercivora was inoculated into the stems of seedlings in July (summer) and October (autumn) and observed by light and fluorescence microscopy at 1 and 2 weeks after inoculation. The hyphal distribution patterns expressed as the I_δ index differed between the species at each occasion. The hyphal growth was wider in Q. crispula than in Q. glauca in July inoculation, but did not differ in October inoculation. Non‐conductive sapwood in Q. crispula in transverse section was wider than that in Q. glauca at each occasion. The differences of hyphal growth and hyphal distribution patterns between species as little as 1 week after inoculation reflect differences in host susceptibility to the fungus.     

In vitro interactions in artificial and wood‐based media between fungi colonizing stumps of Sitka spruce

Interspecific interactions between fungi that colonize stumps of Picea sitchensis in Scotland were tested in dual cultures on Norkrans agar, spruce sawdust and in autoclaved blocks cut from roots. Isolates were ranked according to competitive ability on the different media, based on their ability to overgrow competitors or to form deadlock interactions. On the defined medium, Phaeolus schweinitzii was the species most able to overgrow competitors, followed by Stereum sanguinolentum and Heterobasidion annosum; Resinicium bicolor was the least able to overgrow competitors on this medium. By contrast, R. bicolor was the most competitive on spruce sawdust medium. Deadlocking interactions were formed most often in dual cultures on Norkrans agar. Observation of hyphal interactions on Norkrans agar under the microscope identified several different response types including growth of thin hyphae compared to control cultures, hyphal coiling, vacuolation of hyphae, hyphal lysis of one competitor and deposition of crystals in the agar. Hymenomycetes caused varying amounts of decay in autoclaved root blocks. Resinicium bicolor was able to replace other species in most co‐inoculations. Stereum sanguinolentum appeared to be the least competitive species in root block inoculations, being replaced by Melanotus proteus and R. bicolor, although interactions with H. annosum varied widely. These results indicate that substrate has a marked effect on interspecific fungal interactions, with wood‐based, particularly intact woody tissues closely matching competitive behaviour displayed in the field.     

绿僵菌侵染光肩星天牛幼虫过程的透射电镜观察

应用透射电镜观察并研究光肩星天牛幼虫感染绿僵菌后体壁和中肠发生的病理变化。结果表明:接菌后16h,寄主表皮层中观察到少量菌丝段,至24h菌丝已普遍侵入,芽管在穿透过程中表现了机械压力和酶活性。36～48h菌丝即可穿透体壁进入血腔。菌丝和菌丝段经血淋巴循环侵入各器官组织。48～72h,随着菌丝和菌丝段在肠壁细胞中迅速大量增殖,肠壁组织逐渐被破坏,中肠微绒毛脱落,肠壁细胞形成空泡,围食膜被分解消失,最终中肠组织崩溃、解体。     

The effect of bagging branches on levels of endophytic fungal infection in Japanese beech leaves

The species composition of the endophytic mycobiota in leaves of Japanese beech trees (Fagus crenata) and the sources for leaf infections were studied in a forest reserve situated in central eastern Honshu, Japan. To clarify the mechanism of infection of leaves, half of the branches were covered with polyethylene bags and species composition and levels of endophytic fungal infection were then compared with those of unbagged controls. Isolations were carried out from the leaves, petioles, and current‐year twigs of both, bagged and unbagged branches. Additionally, species composition was detected in overwintered terminal buds of beech trees and in the leaves of potted seedlings that had been placed in the field in different seasons. The species assemblage of the unbagged leaves, petioles, and current‐year twigs was dominated by Mycosphaerella buna, Ascochyta fagi, Periconiella sp., and Tritirachium sp. Other frequently recovered species were Xylaria sp., Phomopsis sp., and Tubakia dryina. Mycosphaerella buna and A. fagi were never isolated from leaves on bagged branches. A. fagi was, however, detected on both bagged and unbagged petioles and current‐year twigs at comparatively low isolation frequencies. The detection of Periconiella sp. on all occasions in both bagged and unbagged leaves was a characteristic feature that differs from those of the other three dominant endophytic fungi. The fungus was also detected without significant differences in bagged and unbagged petioles and current‐year twigs on most sampling dates. Furthermore, Periconiella sp. was isolated from immature twigs inside the bud scales. Tritirachium sp. was frequently detected in unbagged leaves and petioles and in both bagged and unbagged current‐year twigs, and rarely in bagged leaves and petioles, but was never recovered from terminal buds. The results of the potted seedling experiments revealed that all four dominant species had airborne inocula. The infection of leaves by M. buna occurs exclusively by airborne propagules, i.e. ascospores in spring and conidia in autumn. In Periconiella sp. hyphal growth of the fungus from immature twigs inside the buds into the leaf tissues was suggested in addition to infection by airborne inocula. Tritirachium sp. hyphae were suggested to grow from previous‐ to current‐year twigs. Ascochyta fagi was present in the outermost scales of overwintered terminal buds, but no systemic growth of the fungus into the petioles and current‐year twigs was observed. Our technique of covering the branches before new leaves unfolded was effective in preventing infection by airborne inocula of endophytic fungi.     

Extracellular peroxidase reaction at hyphal tips of white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 and in fungal slime

The distribution of an extracellular peroxidase reaction by white-rot fungus Phanerochaete crassa WD1694 was visualized by peroxidase activity staining. The extracellular peroxidase reaction occurred at the hyphal tips and in the fungal slime filling the gaps between the hyphae. We investigated whether the peroxidase reaction occurred from the hyphal tips or in the slime. The hyphal tips were observed by phase-contrast microscopy, which showed that slime did not exist around the hyphal tips. Time-course observation of hyphal tips showed that peroxidase staining became thick and intense at the tips that did not have fungal slime. Daily observation of the peroxidase staining revealed that the staining was first observed at the hyphal tips. Furthermore, strongly stained hyphae were observed in the stained slime. These results suggested that an active species that oxidizes a peroxidase substrate is first produced at the tips of the hyphae, and then occurs in the slime via diffusion when slime exists around the hyphae. Our results show that the extracellular peroxidase reaction that is important to lignin biodegradation by white-rot fungi occurs directly at the tips of the hyphae and in the slime. Part of this report was presented at the 50th Lignin Symposium, October 19–20, 2005, Nagoya, Japan     

Studies on leaf spot and shoot blight of Eucalyptus caused by Cylindrocladium quinqueseptatum

In glasshouse experiments, Cylindrocladium quinqueseptatum, which was associated with outbreaks of leaf spot and shoot blight in a young plantation of Eucalpytus microcorys in north Queensland during the 1982 and 1983 autumns, was pathogenic on seedlings of E. microcorys and caused varying degrees of leaf spot and shoot blight in ten species of Eucalyptus from major taxonomic groupings of the genus, When the outbreaks occurred, the weather was unusually showery with minimum temperatures above 16°C. In vitro studies showed that free water was essential for conidial germination, which occurred between 14–32°C. The optimum for mycelial growth was 28.5°C. In vivo studies showed that leaf penetration. by germinated conidia was via the stomata. Mancozeb was more effective at controlling the disease than benomyl, captan or copper oxychloride. The potential importance of C. quinqueseptatum and options for control in young Eucalyptus plantations are discussed.     

Electron-dense particles in wood decayed by Ganoderma applanatum

Summary Hemlock sawdust samples degraded by Ganoderma applanatum showed no electron-dense particles either in hyphae or in wood cell walls after aldehyde/OsO₄ fixation. After KMnO₄ fixation at early stage of attack, particles were in hyphae, hyphal sheath and wood cell walls. In samples prepared by a cytochemical technique which localizes cellulase activity at the ultrastructural level, particles were in hyphae, hyphal sheath and wood cell walls. The smallest diameter range of the particles lay between 3 and 7 nm which corresponds to the size of cellulases. Larger diameter particles were peresent which are probably aggregates of the smaller units. We believe that particles present in hyphal cytoplasm and hyphal sheath are cellulolytic enzymes. Whether particles present in attacked wood cell walls are enzymes or degradation products cannot be determined by this study. Nevertheles, the particles reveal the decay pattern in wood by the white-rot fungus G. applanatum.     

Induction of morphogenesis,branching and conidiogenesis in Fomes annosus

In Fomes annosus the addition of L-threonine induces specifically branching and septum formation in the leading hyphae of the mycelium. The formation of conidia is stimulated by the combination of light and dark phases and starvation.     

Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash in northern Sweden

Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash was studied in northern Sweden during 2003 and 2004. Once a month between September 2003 and April 2004, two to three trees were cut down and debranched. Shoots with pycnidia were sampled at the felling date and then at every consecutive month. The percentage of germinated conidia from each shoot was calculated after 24, 48 and 72 h incubation. The vitality of G. abietina pycnidia in the slash remained high the whole period. Intact pycnidia were found on slash several months after the time of conidial sporulation, which indicates that new pycnidia may be produced on dead pine branches. Sampling of shoots from slash on 13‐ to 18‐month‐old clear‐cuts showed conidial germination capacity as high as in pycnidia collected in fresh slash. Due to survival of G. abietina in slash it is recommended to postpone planting of P. sylvestris seedlings in northern boreal areas to the third vegetation period after sanitary clear‐cuts.     

RETRACTED ARTICLE: Abundance of Collembola collected from ectomycorrhizal hyphal mats and fruiting bodies of Tricholoma matsutake

Collembolans collected from hyphal mat soil and fruit bodies of Tricholoma matsutake were examined to investigate whether mycophagous microarthropods are a potential insect pest of this fungus in forest soils. The number of collembolans collected in hyphal mat soil did not differ significantly from that in adjacent nonmat soil. Fungal materials contained in the gut of collembolans consisted mostly of hyphal fragments of dematiaceous fungi and unknown basidiomycetes. There were few collembolans on the fruit bodies of T. matsutake, which has the largest fruit body of the fungi at the study site. Our findings suggested that collembolans are not significant feeders on the hyphal mat and fruit body of T. matsutake. An erratum to this article is available at .     

Isolation and growth of single genotype axenic cultures of Cronartium quercuum f. sp. fusiforme using hyphal fragments from multi-genotype stock colonies

A technique was developed to isolate and grow single genotype axenic cultures of Cronartium quercuum f. sp. fusiforme from individual hyphal fragments.Multi-genotype colonies established from multiple basidiospores served as stock material for single genotype establishments via hyphal fragments. multi-genotype colonies, subcultured fifteen days earlier, were minced via scalpel chopping and streaked on fresh medium. Single fragments of hyphae liberated from the minced inoculum were dispersed along the streak line, and those fragments widely separated from others were chosen as potential precursors for single genotype cultures. Three days after streaking, the widely separated fragments of hyphae were transferred along with a small amount of supporting agar to fresh medium. On fresh medium, single genotype cultures developed from individual hyphal fragments in 1.5 to 2 months. The addition of BSA (bovine serum albumin) to the growth medium greatly enhanced establishment of cultures. Of several media tested, HGYP with 0.4% or 1% BSA yielded superior establishment frequencies of 79% and 77%, respectively.This work represents a portion of a M.S. thesis submitted to North Carolina State University by the senior author.     

杉木球果炭疽病病原菌及其生物学特性的研究

１９９５～１９９６年对尤溪经营林场杉木种子园的球果病害进行研究,结果表明：病原菌Ｃｏｌｅｔｏｔｒｉｃｈｕｍｓｐ．〔有性阶段是Ｇｌｏｍｅｒｅｌａｃｉｎｇｕｌａｔａ（Ｓｔｏｎｅｍ．）Ｓｐａｕｌｄ．ｅｔ．Ｓｃｈｒｅｎｋ．〕可造成杉木球果炭疽病。病原菌生长的适宜温度范围是２２～３０℃,以２６℃为菌丝最适生长温度,２４℃为分生孢子萌发最适温度;可生长的酸碱度范围为ｐＨ５～８,以ｐＨ７为最适酸碱度;分生孢子以在水滴和１００％相对湿度中萌发率最高;菌丝在常见碳源蔗糖、常见氮源蛋白胨上生长良好。     

Influence of environmental,host and cultural factors on conidium morphology of Marssonina species pathogenic to poplars

Laboratory studies investigating the influence of environmental, host and cultural factors on conidium morphology of isolates of Marssonina brunnea, M. castagnei and M. populi established that conidial features (length and breadth dimensions, shape, conidium ratios, L:B, LS:B and septum location) were remarkably stable. Significant differences in length and breadth dimensions and ratios were induced by culture media and host but not by media pH, incubation temperature, photoperiod, leaf age, leaf surface and infection severity. Significant differences in conidial dimensions and ratios of M. brunnea were observed following examination of monthly collections from a single field-grown tree. The variation in conidial features induced by environmental, host and cultural factors in no instance obscured taxonomic differences between species.     

Molecular identification of the powdery mildew fungus infecting rubber trees in China

Powdery mildew is a foliar disease of rubber trees. It is one of the most important leaf diseases in rubber plantations worldwide. The causal agent of this powdery mildew, previously known as Oidium heveae, has been identified under various names. To precisely identify the causal agents of this disease, 57 conidial samples were collected from 2013 to 2017 in Hainan, Yunnan and Guangdong provinces, the main rubber tree planting regions in China. Based on morphological observations using light and scanning electron microscopy, pathogenicity testing, and nrDNA ITS and 28S sequences, Erysiphe quercicola is suggested as the only causal agent of rubber tree powdery mildew in China.     

Identification and pathogenicity of Alternaria alternata causing leaf spot and blight disease of Ailanthus excelsa in India

Alternaria leaf spot of Ailanthus excelsa is generally considered as minor disease in India. Recently, severe disease outbreaks were recorded in the nursery of the Forest Research Institute, Dehradun, progeny trial at Jhumpa, Haryana, and in a nearby farm field. Leaf symptoms included small circular, brown, necrotic spots with a chlorotic halo. With severe infections, leaf spots coalesced and resulted in leaf blight. A small‐spored Alternaria with concatenated conidia was isolated consistently from the leaf samples with spot symptoms. Sequence analyses of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and the translation elongation factor 1‐alpha (tef‐1α) gene region of two fungal isolates confirmed the species as A. alternata. In detached leaf assays, both isolates produced symptoms similar to those observed on the nursery/field‐grown plants. To validate the detached leaf assay result, pathogenicity was also demonstrated on whole plants in a glasshouse. Koch's postulates were fulfilled by re‐isolating A. alternata from the inoculated leaves. This work is the first to confirm that A. alternata is associated with leaf spot and blight disease of A. excelsa in India.