Geographical and seasonal influences on the distribution of fungal endophytes in Laurus nobilis

The species composition of the endophytic mycobiota in bark, leaves and twigs of Laurus nobilis were studied in Western Anatolia. Four sampling sites were selected; one of which was sampled twice, in spring and autumn. About 3892 fungal isolates obtained grouped into 85 morphological types. The 11 dominant species, with isolation frequencies >1.5%, were Alternaria alternata, Botryosphaeria sarmentorum, Colletotrichum dematium, C. gloeosporioides, Hendersonula sp., Paraconiothyrium sp., Phoma sp., three species of Phomopsis and Seimatosporium lichenicola. Numbers of fungal species isolated per tree differed between the sampled sites. Isolation frequencies of the dominant species, and other less frequent species, were dependent on the sampling site. The degree of endophytic infection and the diversity of fungal species were significantly higher in spring. Frequencies of all dominant species depended significantly on season, except for Stemphylium sp. and S. lichenicola. Analysis of the total endophytic mycoflora of the sampled trees suggested that geographical factors affects the endophytic distribution patterns more significantly than seasonal factors.     

Seasonal,tissue and age influences on frequency and biodiversity of endophytic fungi of Citrus sinensis in Iran

A total of 4,009 endophytic fungal isolates were recovered from healthy leaves, bark and xylem of Citrus sinensis in different seasons and age‐classes. Corresponding to those factors, the majorities of fungal isolates were recovered from leaves, 2‐yr‐old trees and winter, respectively. Fungal isolates were initially categorized based on cultural and morphological characters, and representatives of each morphotype were subjected to molecular identifications based on ITS‐rDNA and β‐tubulin sequences. All isolates obtained in this study belonged to the phyla Ascomycota and Basidiomycota assigned to 30 fungal taxa. The most abundant fungal species were Alternaria spp. Species diversity indices of Margalef richness and Shannon–Wiener revealed a high diversity of fungal taxa recovered from leaf and during winter. Frequency (%) of fungal endophytes was highest in winter followed by summer, autumn, and spring significantly, whereas the corresponding sequence was winter, autumn, summer and spring for the diversity of endophytic fungi. Overall, the results of this study based on the community and diversity of endophytic fungal species in C. sinensis showed that the type of plant tissue, season of sampling and age of tree play a driving role on their abundance while their diversity was mainly dependent on the type of plant tissue, season of sampling than on the age of tree.     

Fungi in leaves,twigs and stem bark of Populus tremula from northern Spain

Fungal species were recorded from Populus tremula leaves, twigs and bark (trunk) at eight sites in the northern region of Spain, with the aim of evaluating the effect of sampling site, sampling tissue and isolation method on the frequency and species distribution of the fungi recovered from necrotic and healthy tissues. Two different isolation methods were used: the first consisted of finding fruitbodies on plant tissues after incubating them in wet and warm conditions, and the second included growing mycelia from plant fragments plated on potato dextrose agar (PDA). A total of 48 fungal species were isolated from 960 plant fragments. Cytospora chrysosperma, previously reported to be a pathogen of poplar, was recovered very frequently and was recorded as much in healthy as in dead or dying tissues. Cladosporium maculicola, Elsinoe veneta, Alternaria alternata, Aureobasidium pullulans and Pollaccia radiosa were also frequently isolated. The isolation frequencies from necrotic and healthy plant tissues were significantly different between sites and isolation methods. The incubation in moist chambers revealed significantly more fungal isolates and a higher species richness (38 species) than isolations on PDA. However, 16% of the fungal species were exclusively recorded from cultures. Therefore, a combination of several isolation methods is recommended for surveys of fungal communities associated with trees.     

Temporal variations in endophytic fungal assemblages of Ginkgo biloba L.

We isolated endophytic fungi from living healthy leaves, petioles, and current-year twigs of Ginkgo biloba L. from April to November 2004 with the objective of identifying the dominant endophytic fungal taxa, and monitoring their occurrence and frequency. A total of 9 fungal taxa were identified to the genus level. Diversity measures inferred from the Shannon–Wiener, Morisita–Horn, and S?rensen indices indicated that leaves and petioles harbored more diverse endophytic fungal assemblages than twigs, and that fungal taxa involved in twigs shared less with those in leaves and petioles. Among the organs, the occurrence pattern of overall endophytic fungi differed significantly, and two taxa, Phomopsis sp. and Phyllosticta sp., were the most frequently isolated and thus regarded as the dominant endophytic fungi. Phomopsis sp. was isolated frequently from twigs (84.8%) but rather few from leaves (16.1%) and petioles (24.3%). Phyllosticta sp. was isolated frequently from leaves (72.9%) and petioles (65.7%) but was never isolated from twigs. Temporal changes in relative frequency of total endophytic fungi tended to differ among sampling dates for all three organs. The occurrence of Phyllosticta sp. in both leaves and petioles was first detected in August and peaked in October. Phomopsis sp. was detected in twigs throughout the growing season. These results suggest that the distribution of the two dominant endophytic fungi was organ-specific and differed within seasons.     

Differences in composition of endophytic mycobiota in twigs and leaves of healthy and declining Quercus species in Italy

The composition of the endophytic fungal assemblages in Quercus cerris, Q. pubescens and Q. robur, three oak species most susceptible to decline, was investigated in Fagarè (Padova), Ulignano (Pisa), and Radda in Chianti (Firenze). The endophytic assemblages were studied as a function of health status of the whole tree and of twigs and leaves. The effect of station altitude was tested on Q. cerris. Samples were gathered from healthy and declining trees. In April (bud opening time) 20 twigs and 10 leaves were collected from each tree. Fungi were isolated from twig segments and leaf portions. A total of 23 fungal genera with 27 different species were isolated, in addition to a few sterile mycelia. Eleven species were found to be common to all three oak species, while other species were found only on one oak species, suggesting host specificity. Among the fungal species isolated, some were known to be pathogenic (Apiognomonia quercina, Colpoma quercinum, Diplodia mutila, Phomopsis quercina). The colonization frequency (CF) of pathogenic species varied between 0.9% for A. quercina in Q. cerris and 60.2% for P. quercina in Q. robur. Analysis of variance showed a statistically significant difference among the oak species tested. The CF was higher in declining trees in comparison with healthy trees, and also in twigs vs. leaves. The CF was found to be higher for Q. cerris trees growing at sea level as compared with those situated at an elevation of 350–400 m. In stands where, on account of particular ecological conditions, pathogenic behaviour is displayed simultaneously by more than one endophytic fungal species, trees accentuate their decline.     

Identification,diversity and host specificity of the wood-decay fungi in major sawmill depots of north-eastern Bangladesh

The study was carried out to identify wood-decay fungi, and quantify the diversity and host preferences of the fungi in major sawmill depots in north-eastern Bangladesh. A total of 23 fungal species belonging to 15 genera in seven families were recorded and identified. The Polyporaceae was the most dominant family, while Schizophyllum commune was the most abundant species among all species recorded. Other commonly observed fungal species were Daldinia concentrica, Trametes versicolor, Trametes coccinea and Flavodon flavus. The Simpson diversity index (0.93) and Shannon–Wiener index (2.90) showed a wide distribution of the wood-decay fungi in the study areas. The species diversity index (0.036), species evenness index (0.92) and species richness index (3.40) indicated a diverse distribution of the fungal species. Two-thirds of the identified fungal species showed significant preferences for their hosts. The host vulnerability was found to be significantly affected by storage facility, duration of storage, depot yard condition, treated or non-treated wood and shade facility. The findings of this work may help sawmill owners to utilize a scientific approach to management of logs and timber stored in depots, to minimize fungal decay before incurring any economic loss.     

Endohyphal bacteria from fungal endophytes of the Mediterranean cypress (Cupressus sempervirens) exhibit in vitro bioactivity

Endohyphal organisms of endophytic fungi can induce, or promote, beneficial effects of their respective host fungi to the host plant that harbours them. The Cupressaceae plant family (Coniferales) hosts highly bioactive endophytic fungi. Here, we show that a fraction of such endophytic fungi harbours bioactive endohyphal bacteria with a non‐obligatory symbiotic lifestyle. Indeed, 5 of 16 (31.25%) endophytic fungi of Cupressus sempervirens harboured endofungal bacterial strains of Bacillaceae (G⁺, Bacilli) and Sphingomonadaceae (G^?, α‐Proteobacteria), that is Bacillus pumilus (from the fungi Leptosphaeria CSE₂₁₁ and Pyrenochaeta CSE₁₃₄), Bacillus subtilis (from the fungi Leptosphaeria CSE₂₁₂ and Ascorhizoctonia CSE₁₄₈) and Sphingomonas paucimobilis (from the fungus Ascorhizoctonia CSE₁₉₅). Notably, each endophytic fungal species contained only one endofungal bacterial species that was stably maintained in symbiosis over several rounds of subculturing. Moreover, we investigated whether cypress endofungal bacteria (CEB) could benefit their host fungus competing with other fungi and bacteria or the host plant against the invading microorganisms. In vitro assays indicated that CEB possessed antagonistic activity against cypress endophytic and pathogenic microbiome. Also, CEB metabolites and volatile compounds (VOCs) exhibited antifungal and antibacterial activity against the target microbiome. Bioactivity of CEB was less than that of the endophytic microbiome of Cupressaceae, on which we reported earlier. In conclusion, our work is the first to document endohyphal bacteria of fungal endophytes of C. sempervirens and the bioactivity of such endohyphal symbionts. These findings implicate a complicated interrelationship among host plant, endophytic microbiome and endofungal bacteria, which might be of high importance for evolutionary, as well as environmental and agricultural studies. Eventually, endohyphal bacteria may be introduced as a novel source for lead molecule discovery.     

Diversity and bioactivity of bacterial endophyte community of Cupressaceae

The plant family Cupressaceae (Coniferales) harbours diverse endophytic fungi with antifungal, antibacterial and antiproliferative activities. Here, endophytic association of a broad bacterial community with the healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens, Juniperus communis and Thuja orientalis (Cupressaceae) is shown. We isolated over 69 endophytic bacterial strains of Proteobacteria, Bacilli and Actinobacteria from Cupressaceae. The initial screening for antifungal activity against Pyricularia oryzae identified eleven superior bacterial strains which were identified as Brevundimonas diminuta CAE₂₄ (G^?, Caulobacteraceae, Alphaproteobacteria); Stenotrophomonas maltophilia CAE₂₃, CSE₄, CSE₁₂, CSE₄₂, CSE₄₉ and CSE₆₂ (G^?, Xanthomonadaceae, Gammaproteobacteria); Bacillus pumilus CSE₆₆ and Bacillus subtilis POE₂₆ (G⁺, Bacillaceae, Bacilli); and Microbacterium resistens CSE₁₉ (G⁺, Microbacteriaceae, Actinobacteria). The dominant isolated bacterial species was S. maltophilia. This species represented 63.6% of the superior strains which was ubiquitous and also non‐host specific. In addition, the superior bacterial strains produced bioactive secondary metabolites and volatile compounds (VOCs) with antifungal activity against the fungal pathogens of Cupressaceae, that is Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. The antagonistic activity of the endophytic bacteria on the target fungi was also confirmed in vitro. To our knowledge, this is the first to document such an endophytic bacterial community in Cupressaceae and its bioactivity. These findings may find application in organic agroforestry for plant disease biocontrol and in biopharmacy for lead molecule discovery.     

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.     

Non-mycorrhizal fungal endophytes in two orchids of Kaiga forest (Western Ghats), India

We used standard isolation protocols to explore the endophytic fungal communities in three tissue types of two dominant orchids (Bulbophyllum neilgherrense and Vanda testacea) of the Kaiga forest ofthe Western Ghats. We surface sterilized and assessed 90 segments ofeach orchid for the occurrence and diversity of endophytic fungal taxa.The 118 fungal isolates were obtained from root, bulb and leaves of B.neilgherrense, consisting of 17 anamorphic taxa (range, 10 15 taxa) with 1.3 fungal taxa per segment (range, 1.2 1.4 taxa). Four taxa (Aspergillus flavus, A. niger, Penicillium sp. and morpho sp. 1) belonged to the coregroup (11.1% 32.2%). The relative abundance of A. flavus and morpho sp.1 was more than 10%. A total of 130 fungal isolates from roots, stems and leaves of V. testacea yielded 20 anamorphic taxa (range, 11 15 taxa)with 1.4 fungal taxa per segment (range, 1.4 1.5 taxa). Aspergillus flavus,A. niger, A. ochraceus, Gliocladium viride, Penicillium sp. and morphosp. 1 belonged to the core group. Relative abundance exceeded 10% for A. flavus, A. niger, and morpho sp. 1. The Simpson and Shannon diversity indices were higher in leaf than root or bulb/stem of both orchids.Jaccard’s similarity coefficient was higher between root and leaf in both orchids (56.3% 60%) than between other pairs. Our study revealed thatthe endophytic fungal assemblage and diversity of B. neilgherrense and V.testacea of Kaiga forest of the Western Ghats were relatively similar between orchids and their tissues.     

Biodiversity of endophytic fungi from seven herbaceous medicinal plants of Malnad region,Western Ghats,southern India

A total of 3611 fungal isolates were recovered from 4200 leaf segments incubated from 7 medicinal herbs during monsoon, winter and summer seasons. These fungal isolates belonged to teleomorphic Asco-mycota （23.5%）, anamorphic Ascomycota producing conidiomata （17.4%）, anamorphic Ascomycota without conidiomata （46.9%）, Zygo-mycota （1.42%） and sterile forms （10.6%）. Chaetomium globosum, As-pergillus niger, Aureobasidium pullulans, Curvularia lunata, Fusarium spp., Penicillium spp., Pestalotiopsis spp., Trichoderma viridae, Cladosporium cladosporioides, were frequently isolated from more than one host plant. The number of endophytic isolates was higher in winter than in monsoon and summer seasons.     

Identification of endophytic fungi of grapevine with emphasis on the causal agents of grapevine trunk disease in the vineyards of Zanjan Province in Iran

Trunk disease is a major problem on grapevine in Zanjan province, causing serious decline, despite which its aetiology remains unknown. The purpose of this study was to investigate factors involved in grapevine decline in vineyards of Zanjan province. Samples were collected from twigs and branches of grapevines in the region between October and November 2018. Fungal isolates were identified based on morphological characteristics and ITS-rDNA sequence data for selected isolates. The frequency and diversity of the fungal community recovered from grapevines in Darreh Sejin in Zanjan province were higher than in other regions. A total of 112 fungal isolates comprising 22 species were recovered. Phaeoacremonium minimum, Microsphaeropsis olivacea and Kalmusia variispora were identified as the dominant species in the region examined and could be considered the main trunk pathogens of grapevines in Zanjan region. In inoculation tests, M. olivacea was proved to be pathogenic on grapevine for the first time in this study.     

Fungal endophytes in woody roots of Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa)

The fungal community inhabiting large woody roots of healthy conifers has not been well documented. To provide more information about such communities, a survey was conducted using increment cores from the woody roots of symptomless Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) growing in dry forests on the eastern slope of the Cascade Mountains in Washington state, USA. Fungal isolates were cultured on standard media, and then were identified using a combination of molecular and morphological methods. Fungal genera and species identified in this study will provide baseline data for future surveys of fungal endophytes. Examination of internal transcribed spacer (ITS1 and ITS2) and 5.8S rDNA sequences and morphology of cultured fungi identified 27 fungal genera. Two groups predominated: Byssochlamys nivea Westling (20.4% of isolations) and Umbelopsis species (10.4% of isolations). This is the first report of B. nivea within large woody roots of conifers. Both taxa have been previously identified as potential biological control agents. Although some trends were noted, this study found no significant evidence of host species or plant association effects on total recovery of fungal endophytes or recovery of specific fungal taxa.     

Culture‐based methods using low‐temperature incubation revealed cold‐adapted fungal endophytes from semiarid forests

Fungal endophytes are an important part of the plant microbiome and participate in maintaining the stability of ecosystems. Culture‐based methods are frequently considered in studying fungal communities because of their advantages in providing cultures for further experiments and their low cost. In comparison with next‐generation sequencing methods, their ability to assess fungal diversity is questionable. Here, we show how using two different incubation temperatures can improve the yield of fungal endophytes by culture‐based methods. Diversity, organ preference and spatiotemporal variability of endophytic fungal communities living within Quercus brantii L. were investigated using two different incubation temperatures. To test the effects of using low‐temperature incubation on diversity estimations, twigs and leaves of Quercus brantii were incubated under 25°C and 4°C conditions. Isolation rate and Hill's series of diversity were calculated for each sample. Model‐based analysis was used to evaluate the effects of (a) incubation temperature, (b) organ type and (c) spatiotemporal variations on fungal isolation rate, fungal diversity and community composition. Our results showed a strong organ specificity and temporal dynamics among endophytic fungal communities, but the location of the forest stand had only a limited effect on fungal community composition. Low‐temperature incubation enabled us to improve culture‐based methods by discovering the diversity of cold‐adapted fungal endophytes and a significantly different part of the endophytic communities that is usually missed by customary culturing temperature. The isolation of cold‐adapted endophytes from a semiarid environment opens a new window to further studies on this kind of fungi in such ecosystems.     

Root-associated fungi of healthy-looking Pinus sylvestris and Picea abies seedlings in Swedish forest nurseries

The aim of this study was to assess fungal communities in roots of healthy-looking Pinus sylvestris L. and Picea abies (L.) Karst. seedlings in nine forest nurseries in Sweden using a combination of traditional culturing and direct sequencing of internal transcribed spacer of fungal ribosomal RNA (ITS rRNA) from the roots. Culturing from 1800 surface-sterilised root segments resulted in 2387 fungal cultures representing 42 different taxa. Direct sequencing from 180 root segments resulted in 119 ITS rRNA sequences representing 25 different taxa. In total, 55 different fungal taxa were detected using both methods. Although direct sequencing was more efficient than culturing in detecting different fungal taxa, both methods provided complementary information about fungal communities in roots since each detected rather different groups of fungi. The most dominant taxa detected by culturing were Trichoderma viride Pers. (19.5%), Phoma mucivora Davey & Currah (19.1%), Phialocephala fortinii Wang & Wilcox (17.4%) and Meliniomyces variabilis Hambl. & Sigler (10.2%), while Thelephora terrestris Ehrh. (26.1%), Unidentified sp. NS126 (25.2%) and Heliotales sp. C20 (10.1%) were most commonly detected by direct sequencing. In conclusion, results showed that forest nurseries in Sweden harbour diverse communities of fungi associated with the roots of healthy-looking P. sylvestris and P. abies seedlings. Although fungal communities were often dominated by saprotrophs and endophytes, several facultative pathogens were also detected indicating that under suitable conditions they may be a potential threat to the plants.     

Biodiversity of Arbuscular Mycorrhizal (AM) fungi in mangroves of Goa in West India

Seventeen mangrove species of eight families at seven riverine and fringe habitats in Goa West India were surveyed for Arbuscular Mycorrhizal (AM) fungal diversity. Sixteen species were found to be mycorrhizal and one species showed no AM fungal colonization. AM root colonization was recorded at all seven sites and ranged from 6%-77%. Maximum root colonization was recorded in Excoecaria agallocha (77%) and minimum colonization in Avicennia marina (6%). Paris-type colonization was predominant at all sites. Auxiliary cells were recorded in roots of Acanthus ilicifolius, Ceriops tagal and Sonneretia alba. AM fungal root colonization and spore density varied by plant species and site. Site average spore density ranged from 1.84 spores·g-1 to 0.54 spores·g-1 of soil. In total, 28 AM fungal species of five genera, viz. Glomus, Acaulospora, Scutellospora, Gigaspora and Entrophospora, were recovered. Glomus was the dominant genus, three species of which were sporocarpic forms. Maximum site species richness (SR) ranged from 16 to 5. Species richness was maximum in A. ilicifolius where seven species of three genera were recovered. Based on relative abundance (RA) and isolation frequency (IF), two common species, viz. G. intraradices and A. laevis, were recovered from all seven sites.     

Host specificity of co‐infecting Botryosphaeriaceae on ornamental and forest trees in the Western Balkans

The Botryosphaeriaceae is a diverse family of endophytes and fungal pathogens of mainly woody plants. We considered the host range and distribution of these fungi by sampling diseased ornamental and forest trees and shrubs in Serbia, Montenegro, Bosnia and Herzegovina, spanning a Mediterranean and a Continental climatic region. In total, ten Botryosphaeriaceae species were identified in the Western Balkans and with the exception of Sphaeropsis visci and Phaeobotryon cupressi, which occurred on one host, all the species had a broader host range. Phaeobotryon cupressi was found only in the Mediterranean region and S. visci, Dothiorella sp., Dothiorella sarmentorum and Diplodia seriata were present only in the Continental region. Pathogenicity tests were conducted on a variety of hosts from which the Botryosphaeriaceae species were isolated. These included leaves and/or stems of seedlings of 21 hosts, and cut leaves and/or branches of six hosts. Moreover, stems of seedlings of Chamaecyparis lawsoniana, Cedrus deodara, Picea omorika, Pinus patula and Eucalyptus grandis were inoculated as hosts from which some or all of the Botryosphaeriaceae species used for inoculation were not isolated. Inoculations showed that the majority of these fungi could also co‐infect hosts other than those from which they were isolated. The results suggest that most of the species have broad host ranges and can potentially cause disease on a broad range of tree species under certain conditions.     

The hosts and geographic range of Dothistroma needle blight in Slovakia

The occurrence and distribution of Dothistroma needle blight (DNB) were studied in 2014–2017 around Slovakia. A total of 84 localities, both native and planted, were investigated, and the presence of DNB was confirmed in 73 of them. In all positive locations, symptoms typical of DNB were observed and the Dothistroma species was confirmed using species‐specific primers either from fungal cultures or directly from needles. Both Dothistroma species—D. septosporum and D. pini—were identified. Both species occurred together in 29 locations, only D. septosporum in 42 and only D. pini in two locations. The host range of D. septosporum included 10 pine species and two spruce species. The host range of D. pini comprised the same number of pine hosts but only one spruce species. Five pine hosts, P. aristata, P. coulteri, P. densiflora, P. jeffreyi, P. × schwerinii, and one spruce host P. abies are new hosts species of D. pini. P. densiflora and Picea pungens have earlier been reported to be susceptible for DNB. In this study, D. septosporum was found from both tree species.     

Variation of endophytic cork oak‐associated fungal communities in relation to plant health and water stress

The main objective of this study was to obtain more comprehensive knowledge about the effect of water stress on endophytic fungal communities in asymptomatic and declining cork oak trees. Six asymptomatic and six declining cork oak trees were randomly selected in a natural cork oak forest located in Sardinia, Italy. In February 2003, the soil around three asymptomatic and three declining trees was covered with a circular plastic film to reduce rain water supply with the intention to induce water stress. The remaining six trees served as controls. Predawn xylematic water potential (PWP) was used as water status indicator and measured seasonally. Between July 2003 and June 2004, fungal endophytes were isolated every 2–4 months from twigs, branches and woody tissues. Significant differences in PWP between covered and control trees were detected mainly in autumn. Gas exchange was greatest in asymptomatic control plants. All tissues were colonized by endophytic fungi. Nineteen fungal species were isolated from 1620 plant fragments. Biscogniauxia mediterranea was the most frequently isolated fungus. Its isolation frequency was significantly higher in declining covered trees than in control trees (p < 0.05). Presence of this fungus in asymptomatic control trees was significantly higher in winter than in summer. Water stress seems to reduce species diversity of the endophytic mycobiota in cork oak and to promote proliferation of some potentially pathogenic endophytes.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.