Colonization of Japanese beech leaves by phyllosphere fungi

To examine the colonization of living leaves from buds and twigs by phyllosphere fungi of Japanese beech, the mycobiota were investigated on buds and twigs and on leaves covered with well-ventilated bags before budbreak. Ten phyllosphere fungi were isolated from rolled-up leaves within buds, bud scales, and twigs. However, frequencies of phyllosphere fungi on expanded leaves were reduced markedly when the buds were covered with well-ventilated bags before budbreak compared to the leaves that were not covered. This observation suggests that invasion of the fungi to the leaves from buds and twigs may be possible but is not the main route. Horizontal transmission may be common in endophytes and epiphytes of beech leaves. Phyllosphere mycobiota were then compared between sun and shade leaves. Of 13 species recorded as phyllosphere fungi, the frequencies of 2 species were lower and those of 3 species were higher in sun leaves than in shade leaves. Frequencies of the other 8 phyllosphere species were not different between sun and shade leaves. This result indicates that the colonization of leaves by some phyllosphere fungi was affected by the microenvironmental conditions on leaf surfaces.     

Colonization and lignin decomposition of Camellia japonica leaf litter by endophytic fungi

Endophytic fungi occur on various types of leaf litter, but few studies have been done on their roles as saprophytes in decomposition. This study examined the succession of fungi in live, newly shed, and decomposing leaves at 2 months of decomposition of Camellia japonica and chemical changes in decomposing leaves colonized by endophytes. Coccomyces nipponicum, Lophodermium sp., Geniculosporium sp. 1, and Colletotrichum gloeosporioides were isolated from living leaves. Coccomyces nipponicum and Lophodermium sp. were also isolated frequently from newly shed and decomposing leaves. These two fungi caused a decrease of lignin content and bleaching in decomposing leaves under field and laboratory conditions. Total hyphal length in decomposing leaves was higher in bleached portions than in surrounding nonbleached portions, which probably reflected the early onset of hyphal growth of endophytes inside leaf tissue at leaf senescence or death. Incubation of newly shed leaves that were sterilized to exclude previously established endophytes resulted in no occurrence of bleached portions in decomposing leaves on the forest floor. This result indicated that these endophytes were incapable of colonizing leaves directly after litterfall and that the persistence of endophytes from live leaves was crucial for their colonization in decomposing leaves.     

Endophytic and epiphytic phyllosphere fungi of Camellia japonica: seasonal and leaf age-dependent variations

Seasonal and leaf age-dependent variations in the endophytic and epiphytic phyllosphere fungal assemblages of Camellia japonica were examined and compared. Live leaves of C. japonica were collected in four seasons (May, Aug, Nov, Feb), and fungi were isolated from healthy-looking leaves of 0, 1, 2 and 3 y old. The infection rate and total number of endophytic fungi increased May-Feb, and species richness of endophytes increased as leaves aged. In contrast the infection rate of epiphytic fungi was 100% for all leaf ages at every sampling date. The total number of epiphytic fungi isolated was greatest in May and lowest in Aug. The species richness of epiphytes did not differ significantly by season or leaf age. Eight fungal species were recorded as major phyllosphere fungi of C. japonica. Seasonal variations were detected for the frequencies of Colletotrichum gloeosporioides, C. acutatum, and epiphytes Pestalotiopsis sp.1, Aureobasidium pullulans, Phoma sp.1 and Ramichloridium sp., whereas the frequency of the endophyte Geniculosporium sp.1 varied with leaf age. The frequency of the epiphyte Cladosporium cladosporioides varied with both season and leaf age.     

Colonization and succession of fungi during decomposition of Swida controversa leaf litter

Decomposition processes of Swida controversa leaves were investigated in initially sterilized (fungi-excluded) and nonsterilized freshly fallen leaves to examine the relationship between chemical changes and fungal succession during decomposition and the effect of exclusion of previously established phyllosphere fungi from leaves on subsequent decomposition and fungal succession. Fifteen species were isolated frequently from decomposing leaves with surface-disinfection and washing methods. These fungi were divided into early and late colonizers according to their occurrence during decomposition. The 1.5 y decomposition process was divided into three stages characterized by different dominant organic chemical constituents. A clear relationship was demonstrated between chemical changes and fungal succession. Total hyphal length and frequencies of some early colonizers were reduced in initially sterilized leaves at 3 wk, but this had no significant effect on loss of litter mass or chemical changes during the first 3 wk or on the subsequent decomposition and fungal succession.     

New evidence for broad trophic status of leaf endophytic fungi of Quercus gambelii

Endophytic fungi inhabit the living tissues of every terrestrial plant species thus far examined. In at least some cases they significantly improve stress tolerance of their hosts. We asked whether endophytic fungi play other ecological roles, specifically whether the leaf endophytes of Quercus gambelii persist during the course of leaf decomposition, requiring a transition from a biotrophic to a saprotrophic mode of nutrition. Using automated ribosomal intergenic spacer analysis (ARISA), we found that endophyte fungal OTU diversity declined as decomposition commenced, but some endophytes persisted for months during which leaves were decomposing. In contrast, saprotroph fungi OTU diversity increased as decomposition progressed. These results are consistent with the hypothesis that some biotrophic endophytes persist in leaves during decomposition by becoming saprotrophic, and that the niche occupied by them is broader than expected.     

Role of phyllosphere fungi of forest trees in the development of decomposer fungal communities and decomposition processes of leaf litter

The ecology of endophytic and epiphytic phyllosphere fungi of forest trees is reviewed with special emphasis on the development of decomposer fungal communities and decomposition processes of leaf litter. A total of 41 genera of phyllosphere fungi have been reported to occur on leaf litter of tree species in 19 genera. The relative proportion of phyllosphere fungi in decomposer fungal communities ranges from 2% to 100%. Phyllosphere fungi generally disappear in the early stages of decomposition, although a few species persist until the late stages. Phyllosphere fungi have the ability to utilize various organic compounds as carbon sources, and the marked decomposing ability is associated with ligninolytic activity. The role of phyllosphere fungi in the decomposition of soluble components during the early stages is relatively small in spite of their frequent occurrence. Recently, the roles of phyllosphere fungi in the decomposition of structural components have been documented with reference to lignin and cellulose decomposition, nutrient dynamics, and accumulation and decomposition of soil organic matter. It is clear from this review that several of the common phyllosphere fungi of forest trees are primarily saprobic, being specifically adapted to colonize and utilize dead host tissue, and that some phyllosphere fungi with marked abilities to decompose litter components play important roles in decomposition of structural components, nutrient dynamics, and soil organic matter accumulation.     

Seasonal and leaf age-dependent changes in occurrence of phyllosphere fungi of giant dogwood

To examine the relative importance of leaf age and season on the occurrence of phyllosphere fungi, temporal patterns of epiphytic and endophytic phyllosphere fungi of giant dogwood (Swida controversa) were studied with reference to leaf emergence at first occurrence and in the middle of the growing season. A total of 15 and 44 species were isolated from the surface and interior of leaves, respectively. On the leaf surface, detection rate of fungi was consistently 100% and their frequencies increased during the growing season, whereas in the leaf interior, detection rate of fungi and their frequencies were low at leaf emergence and gradually increased during the growing season. Six epiphytic and two endophytic fungi were observed frequently. A white sterile mycelium was frequent only on the surface of newly emerged leaves in the first-order shoot in May. The other 7 species increased during the growing season. The frequencies of Phomopsis sp., Pestalotiopsis sp. 1, and Trichoderma viride were higher on the leaves of first-order shoots than those of higher-order shoots that emerged between July and September, suggesting the possible effects of leaf age on their occurrence. On the other hand, the frequencies of Colletotrichum gloeosporioides, Clonostachys rosea, Cladosporium cladosporioides, and Phoma sp. 1 were not different between the first- and higher-order shoots, suggesting the negligible effect of leaf age. The influence of phenological patterns of leaf emergence of deciduous trees on the diversity and composition of assemblages of phyllosphere fungi is discussed.     

Fungal Epiphytes and Endophytes of Coffee Leaves (Coffea arabica)

Plants harbor diverse communities of fungi and other microorganisms. Fungi are known to occur both on plant surfaces (epiphytes) and inside plant tissues (endophytes), but the two communities have rarely been compared. We compared epiphytic and endophytic fungal communities associated with leaves of coffee (Coffea arabica) in Puerto Rico. We asked whether the dominant fungi are the same in both communities, whether endophyte and epiphyte communities are equally diverse, and whether epiphytes and endophytes exhibit similar patterns of spatial heterogeneity among sites. Leaves of naturalized coffee plants were collected from six sites in Puerto Rico. Epiphytic and endophytic fungi were isolated by placing leaf pieces on potato dextrose agar without and with surface sterilization, respectively. A total of 821 colonies were isolated and grouped into 131 morphospecies. The taxonomic affinities of the four most common nonsporulating fungi were determined by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region: two grouped with Xylaria and one each with Botryosphaeria and Guignardia. Of the most common genera, Pestalotia and Botryosphaeria were significantly more common as epiphytes; Colletotrichum, Xylaria, and Guignardia were significantly more common as endophytes. Suprisingly, more morphospecies occurred as endophytes than as epiphytes. Differences among sites in number of fungi per plant were significant. Thus epiphytic and endophytic communities differed greatly on a single leaf, despite living only millimeters apart, and both communities differed from site to site. Significant correlations between occurrence of fungal morphospecies suggested that fungi may have positive or negative effects on their neighbors. This is the first quantitative comparison of epiphytic and endophytic fungal floras in any plant, and the first to examine endophytic fungi or epiphytic fungi in leaves of coffee, one of the world’s most valuable crops.     

Diversity and ubiquity of xylariaceous endophytes in live and dead leaves of temperate forest trees

To test the hypothesis that xylariaceous endophytes were ubiquitous on live and dead leaves of various tree species in the field, xylariaceous fungi were isolated from live leaves and bleached and nonbleached portions of dead leaves of a total of 94 tree species in a cool temperate forest in Japan. The biodiversity of xylariaceous endophytes was evaluated as the richness of operational taxonomic units (OTUs) determined by phylogenetic analysis of the nucleotide sequence of the D1/D2 region of the LSU rDNA of fungal isolates. A total of 326 isolates of xylariaceous fungi were isolated from live and dead leaves and classified into 15 OTUs. The three major OTUs, Xylaria sp.1, Nemania sp., and Biscogniauxia sp., accounted for 94% (308 isolates) of the total number of isolates, and were isolated from various live and dead leaves. Xylaria sp.1 was frequently encountered on bleached portions (which were produced due to the selective decomposition of lignin) of dead leaves of broad-leaved deciduous tree species. The results suggest that xylariaceous endophytes did not show host specificity and had a saprobic phase on dead leaves in their life cycles and that Xylaria sp.1 was capable of decomposing lignin in the field conditions.     

Endophytic and epiphytic phyllosphere fungi of red-osier dogwood (<Emphasis Type="Italic">Cornus stolonifera</Emphasis>) in British Columbia

Endophytic and epiphytic phyllosphere fungi associated with red-osier dogwood (Cornus stolonifera), a deciduous shrub, were examined in coastal British Columbia, Canada. Current-year shoots were divided into four types based on the absence or presence of inflorescence and secondary elongated shoots at the apex of primary shoots. Leaves on these shoots were then classified into six categories so as to examine the effect of flowering, secondary shoot elongation, and shoot order within current-year shoots on the occurrence of phyllosphere fungi. Species composition of fungi was markedly different between the interior and surface of leaves, whereas it was relatively similar among the six leaf categories in the interior or on the surface. Frequencies of the eight major species were not different between leaves on flowering and nonflowering shoots. The frequency of Colletotrichum gloeosporioides in the leaf interior was greater on leaves on the primary shoots that elongated the secondary shoots than on those that did not, and was greater on leaves on the primary shoots than on those on the secondary shoots. On the other hand, secondary shoot elongation and shoot order had no effect on the frequencies of C. gloeosporioides and the other seven epiphytes on leaf surfaces.     

Diversity and decomposition potential of endophytes in leaves of a Cinnamomum camphora plantation in China

This study was carried out to improve our understanding of the diversity and decomposition potential of endophytes in the leaves of Cinnamomum camphora trees grown in a subtropical region of China. We isolated and identified endophytic fungi from senescent leaves of C. camphora and tested their role in decomposition through pure-culture and pre-colonization. A total of 2,861 endophytic fungi isolated from 69 leaves of C. camphora were grouped into 39 taxa comprising 36 Ascomycetes and 3 Basidiomycetes based on sporulation and ITS sequence analysis. Of these, Colletotrichum gloeosporioides was the most common species (69% relative abundance and 96% colonization frequency), followed by Cladosporium sp.1, Colletotrichum sp. and Chaetomium sp. All 39 endophytes had the ability to decompose C. camphora leaf litter in pure culture, and a few exhibited >20% litter mass loss in 2 months. In most cases, single endopyhytic species showed lower mass loss than mixed microbial groups from active soil after 60 or 120 days. In pre-inoculation, endophytic fungi like Chaetomium sp., Cladosporium sp.1, C. gloeosporioides, Colletotrichum sp. and Guignardia sp. exhibited higher abundance and caused greater mass loss, indicating the potential of these groups to enter and significantly accelerate the process of decomposition. This study concludes that, after entering the decomposition process, selected endophytic fungi with high abundance could influence significantly the decomposition process and thus probably affect carbon and nutrient cycling in C. camphora plantations.     

Fungal community composition shifts along a leaf degradation gradient in a European beech forest

Aims

The fungal communities in living and decomposed leaves of European Beech (Fagus sylvatica) were compared to identify the phyllosphere fungi involved in litter decomposition at a site in Bavaria, Germany.

Methods

New primers were designed to cover a broad range of fungal ribosomal DNA sequence diversity. Following ‘environmental PCR’, clone libraries from each of five samples of living leaves (surface-sterilized and untreated), freshly fallen, initially and highly decomposed leaves, were screened using RFLP fingerprinting.

Results

Statistical analysis (ANOSIM) revealed that the fungal communities colonizing living (a) and initially decomposed leaves (c) significantly differed between each other and from freshly fallen (b) and highly decomposed leaves (d). Fungal assemblages of a and d were statistically indistinguishable from each other and from the endophyllous fungal community in living leaves.

Conclusions

The results showed that endophyllous fungi play a role throughout the whole decomposition process of beech leaf litter. Therefore, clarification of the life cycle of certain endophytic and/or soil fungi may only be achieved by considering both phyllosphere and soil habitats.     

Distribution of phyllosphere fungi within the canopy of giant dogwood

Distribution of phyllosphere fungi within the canopy of giant dogwood (Swida controversa) was examined. Canopies of two dogwood trees (about 8m height) were divided into five parts in relation to the order of shoots within the current-year shoot, the height of leaf layers, and the distance from the main trunk, and leaves were collected from the five positions. A total of 13 and 33 species were isolated from the interior and surface of leaves by surface sterilization and washing methods, respectively. Species composition of fungi was different markedly between interior and surface of an individual leaf, whereas it was similar among five canopy positions in the interior or on the surface. Of 13 frequent species regarded as phyllosphere fungi, 6 species showed no difference in frequencies among five positions within the canopy. The other 7 species showed significant preference within the current-year shoot, between the leaf layer, and/or at the distance from the trunk. The probable effect of leaf properties was detected on 2 of the 7 species, while the frequencies of the other 5 species were not related to the leaf properties but were affected by the order of shoots (leaf age), the height of leaf layer, and/or the distance from the trunk (sunlight intensity).     

A comparative study of endophytic and epiphytic fungal association with leaf of <Emphasis Type="Italic">Eucalyptus citriodora</Emphasis> Hook., and their antimicrobial activity

Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don’t produce sexual spores. The Sorensen’s index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

Endophytic fungi in leaves of Lycopersicon esculentum Mill

Endophytic fungi were isolated from healthy tomato leaves from field-grown plants. Several surface-sterilization techniques were assayed for removing and killing epiphytes on tomato leaves. Surface-sterilization with an undiluted commercial solution of sodium hypochlorite was employed. To determine the spectrum of endophytes, samples were collected in February 1998 and 1999 from tomato plants (cultivar Tommy) cultivated in the experimental fields of Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Buenos Aires, Argentina. In general, different spectra of species were isolated in 1998 and 1999.Alternaria alternata was the fungus most frequently isolated from tomato leaves in 1999, but it was the second most common species in 1998. In contrast, Colletotrichum gloeosporioides was the fungus most frequently isolated in 1998, but it was not found in 1999. Species of other genera, such as Cladosporium and Penicillium, were isolated in both years.     

Fungal species diversity in juvenile and adult leaves of Eucalyptus globulus from plantations affected by Mycosphaerella leaf disease

In recent years, Mycosphaerella leaf disease (MLD) has become very common in Eucalyptus globulus plantations in Galicia, northwest Spain. The aetiology of MLD is complex and is associated with several species of Mycosphaerella and Teratosphaeria. A survey of the fungal mycobiota associated with juvenile and adult leaves and with leaf litter of the same trees in MLD‐affected plantations was made. The goal was to identify pathogens and endophytes, to determine whether the mycobiota of each leaf type differed and whether leaf litter might be a reservoir of MLD inoculum. Fungi belonging to 113 different species were isolated from the leaves of juvenile and adult trees sampled at 10 locations; 81 species occurred in juvenile and 65 in adult leaves. The average number of species obtained from juvenile leaves was significantly greater (P > 0.01) compared to adult leaves. This difference suggested that juvenile leaves are not only more susceptible to a group of pathogens, but to a wide range of fungi. Therefore, a general resistance mechanism might be lacking or be less effective in juvenile than in adult leaves. Several pathogenic species were identified in both leaf types. Leaf litter and living leaf mycobiotas were very different. However, some of the species they shared were MLD pathogens, suggesting that leaf litter could contribute to the inoculum of MLD.     

Diversity and distribution of fungal foliar endophytes in New Zealand Podocarpaceae

The diversity and distribution of fungal endophytes in the leaves of four podocarps (Dacrydium cupressinum, Prumnopitys ferruginea, Dacrycarpus dacrydioides, and Podocarpus totara, all Podocarpaceae) and an angiosperm (Kunzea ericoides, Myrtaceae) occurring in close stands were studied. The effects of host species, locality, and season on endophyte assemblages were investigated. Host species was the major factor shaping endophyte assemblages. The spatial separation of sites and seasonal differences played significant but lesser roles. The mycobiota of each host species included both generalist and largely host-specialised fungi. The host-specialists were often observed at low frequencies on some of the other hosts. There was no clear evidence for family-level specialisation across the Podocarpaceae. Of the 17 species found at similar frequencies on several of the podocarp species, 15 were found also on Kunzea. Many of the endophytes isolated appear to represent species of fungi not previously recognised from New Zealand.     

Fungal Diversity of Norway Spruce Litter: Effects of Site Conditions and Premature Leaf Fall Caused By Bark Beetle Outbreak

Fungi play an important role in leaf litter decomposition due to their ability to break down the lignocellulose matrix, which other organisms are unable to digest. However, little is known regarding the factors affecting components of fungal diversity. Here, we quantified richness of internal fungi in relation to litter nutrient and phenolic concentrations, sampling season (spring or fall), and premature leaf shedding due to low precipitation and infestation of bark beetles (mainly Ips typographus and Ips duplicatus). The study was conducted in 37-year-old Norway spruce [Picea abies (L.) Karst.] stands, with three plots each in mixed forest (MF) and coniferous forest (CF) site conditions in south-central Poland. Fifty-four species of sporulating fungi were identified in 2,330 freshly fallen needles sampled during 2003-2005, including 45 species in MF and 31 in CF. The significantly higher number of species in MF was likely related to moister conditions at that site. Among isolated fungi, 22% (12 species) were identified as endophytes of Norway spruce in prior studies. During spring of 2005, we found less than half the number of isolates and fungal species at each forest site as compared to fall for the two prior years. This pattern was observed in typical soil fungi (e.g., Penicillium daleae, Penicillium purpurogenum) and endophytes/epiphytes (e.g., Aureobasidium pullulans, Alternaria alternata, Cladosporium spp., and Lophodermium piceae). Premature shedding of needles was the most likely cause of this decline because it shortened the time period for fungi to infect green needles while on the tree. For all sites and sampling periods, richness of internal fungi was strongly and positively related to the age of freshly fallen litter (assessed using needle Ca concentration as a needle age tracer) and was also negatively related to litter phenolic concentration. Richness of internal fungi in freshly fallen litter may be adversely affected by low soil moisture status, natural inhibitors slowing fungal colonization (e.g., phenolics) and biotic (e.g., insect infestation) and abiotic (e.g., drought) factors that shorten leaf life span.