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.     

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.     

Fungal colonization patterns in necrotic rootstocks and stem bases of dieback‐affected Fraxinus excelsior L.

European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.     

Fungal endophytes associated with Sonneratia (Sonneratiaceae) mangrove plants on the south coast of China

The aim of this study was to describe endophytic fungal communities residing in five mangrove species (Sonneratia caseolaris, Sonneratia hainanensis, Sonneratia ovata, Sonneratia Paracaseolaris, Sonneratia apetala) collected at the south coast of China. The colonization frequency (CF) of endophytes in roots, stems and leaves was measured and the structures of endophytic communities were investigated, using endophytic fungal diversity indices and similarity coefficients. We found high biodiversity and tissue specificity of endophytic fungi. A total of 391 fungal isolates were identified using morphological and molecular methods. We recovered a total of 39 distinct endophytic species, of which Cytospora, Diaporthe, Fusarium, Glomerella, Mycosphaerella, Phoma, Phomopsis and Stemphylium were the dominant fungal taxa. Most recovered species were found on more than one host species but the dominant species differed according to host and tissue type.     

Oak decline: Aerobic bacteria associated with declining Quercus cerris in Central Italy

Declining Turkey oaks (Quercus cerris) in Central Italy were studied. Bacteria were isolated from woody tissues between necrotic areas and healthy tissues beneath the bark, from apparently healthy tissues and from exudates oozing from the trunk in spring. A total of 98 bacterial isolates were collected. The determination of the bacteria was by fatty acid profiling and biochemical tests. Erwinia herbicola was frequently found both in necrotic and healthy tissues. Ice-nucleation active species were present. None of the species isolated was considered responsible for the decline     

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.     

Mortierella species from declining Araucaria araucana trees in Patagonia,Argentina

Since 2015, Araucaria araucana, an ecologically and economically important conifer native to Argentina and Chile, has suffered an unusual partial death of the crown throughout almost all of the distribution range in Argentina. No primary pathogen or pest was evident, associated with the phenomenon. Isolates of Mortierella, a poorly studied fungal genus in Patagonia, were obtained from the margins of necrotic phloem tissue of symptomatic trees. Five species of Mortierella were isolated from affected tissues. In inoculation tests, Mortierella alpina and M. aff. basiparvispora were pathogenic to A. araucana. These species caused necrosis of phloem, leading to chlorosis, foliar desiccation and eventually death, demonstrating that Mortierellales may play a role in the decline of the tree.     

Pathogenic fungi associated with pre‐ and post‐emergence seedling blight of pine and cypress in Fars Province,Iran

Seed and root rot of pine and cypress seedlings cause heavy annual losses to forest nurseries in Fars Province. Root and crown samples of various species of conifers, such as Tehran pine (Pinus eldarica), brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazian cypress (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crown rot symptoms were collected from pine and cypress nurseries across Fars province at various time intervals. Infected tissues were washed and cultured on acidified potato dextrose agar (PDA) and corn meal agar (CMA) amended with Delvocide and ampicillin, with and without surface sterilization, respectively, and incubated at 25°C for 3–5 days. Decaying seeds of all plant species were also collected from nursery seed stocks and cultured on PDA. Hyphal tip isolates were used for further studies. Species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomatic seedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates of Phytophthora, Pythium and Rhizoctonia was confirmed on seedlings of all plant species, whereas Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pine and Arizona cypress. Virulent isolates identified as Phytophthora nicotiana var. parasitica, Pythium ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia‐like fungus caused root and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identified as Fusarium proliferatum also caused pre‐emergence seed decay of pine and cypress. Isolates of Phytophthora and Pythium were the most virulent of these fungal isolates.     

The importance of wild plant species as potential inoculum reservoirs of white root rot disease

Rosellinia necatrix is a soilborne plant pathogenic fungus belonging to the phylum Ascomycota. The fungus is distributed widely in temperate zones and often causes huge economic losses on numerous crop plants. To determine the potential for wild plants to serve as inoculum reservoirs of white root rot disease, eight woody and two herbaceous plant species belonging to nine families were inoculated with two R. necatrix isolates. The species used were Actinidia polygama, Broussonetia kazinoki, Camellia sinensis, Castanea crenata, Hydrangea serrata, Magnolia obovata, Pteridium aquilinum, Petasites japonicus, Quercus serrata and Rosa multiflora, all common flora in the cool temperate forests of Japan. The mortality of plants varied between species, independent of the effect of fungal isolates. Although some of the plants survived fungal infection, all plant species tested were more or less susceptible, having rotting lesions on the inner bark and cortex. For the woody plants, most of the mycelial strands in the diseased tissues became plumose mycelia, growing compressed between the outer and inner barks. However, mycelia found in diseased tissues of the two herbaceous plants were not in the form of plumose mycelia. All plant species harboured mycelial strands on the surface of their root systems and underground main stem, and the amount of the mycelial strands did not correlate with the mortality of the plants. Even if plants exhibited no external symptoms, some of them harboured abundant mycelial strands. In conclusion, wild plants can serve as alternative host plants for R. necatrix and function as persistent inoculum reservoirs, irrespective of their apparent health status. Thus, assessments of such latent host plants must be considered in the course of managing white root rot.     

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.     

Histology of Diplodia pinea in diseased and latently infected Pinus nigra shoots

Diplodia tip blight is a serious disease of >30 conifer species worldwide. Symptoms are particularly severe on non‐native, two‐needled Pinus species, and typically include stunted, necrotic needles and shoots and a general decline of the tree. Latent Diplodia pinea infections occur in current‐year shoots of some symptomless pines, and in some apparently healthy current‐year shoots of diseased pines. Latent infections also occur in symptomless terminal buds. A histological approach was used to investigate the nature of latent infections in shoot stems and terminal buds of landscape Pinus nigra. Fungal colonization was compared in healthy, diseased and latently infected tissues. A nested‐PCR technique that is specific for D. pinea was used to differentiate latently infected tissues from uninfected ones. Latent D. pinea infections were localized in the outer stem cortex, usually in the vicinity of needle scales at leaf axes. In contrast, pathogenic D. pinea infections were characterized by fungal colonization throughout the shoot stem tissues, even very early in symptom development. The presence of necrophylatic periderms in two of the latently infected samples suggests that host defences play a role in the production and maintenance of latent stem infections. Latent infections of terminal buds appeared to originate from the distal bud scales of axillary buds in the terminal bud cluster, and not from the subtending shoot. Fungal tissues were never observed inside asymptomatic, PCR‐negative shoots.     

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.     

Pathogenic ice‐nucleation active bacteria in willows for short rotation forestry

Extensive sampling of endophytically growing bacteria from shoots of diseased Salix viminalis clones were performed in trials at two experimental sites in central Sweden. Field, greenhouse and laboratory studies were conducted to identify and characterize the plant‐associated bacteria and determine their possible role in the severe dieback appearing in short rotation forestry (SRF) plantations in Sweden during the last few years. The most frequently isolated types were found to be non‐fluorescent Pseudomonas fluorescens (biotype A, C, G) and Sphingomonas spp. (23%). Erwinia spp., P. fluorescens, P. syringae and Xanthomonas spp. each constituted 11–14% of the bacterial isolates respectively. After biochemical characterization and screening for ice‐nucleation activity (INA) 78 of the originally 279 isolated bacterial strains were selected for pathogenicity tests and further analyses. Of the 78 selected isolates, 95% were found to be pathogenic, giving different degrees of necrotic reactions after inoculation to Salix plants. Half of these pathogens (46%) also showed strong INA (at temperatures between ?2.5 and ?5°C). With respect to both isolation frequency, clonal distribution in the field, ice‐nucleation ability and necrotic reactions of willow in laboratory tests, we consider the following types to be the most important pathogens in the field: Erwinia spp., P. syringae, Sphingomonas/P. fluorescens spp. (biotype A, C, G) and Xanthomonas spp. Reported damage and dieback in many SRF‐plantations are, according to our results, very likely to be a consequence of bacterial infections, particularly of pathogenic INA‐strains, which both initiate freezing stress and aggravate the frost damage of plants by subsequent invasion of plant tissues leading to complete death of plants under field conditions.     

Fungi associated with Ips typographus on Picea abies in southern Poland and their succession into the phloem and sapwood of beetle‐infested trees and logs

This study dealt with the species distribution and frequency of fungi associated with the bark beetle Ips typographus (Scolytidae) on spruce trees of various states of health in southern Poland. The spruce trees were assessed by their degree of defoliation and damage of their crowns and trunks after attack by I. typographus. The state of health of trees from which samples were obtained was related to varying stages of brood development of I. typographus. Fungi were isolated from phloem taken from and around insect galleries and the sapwood underneath brood systems. Samples were taken from ‘healthy‐looking’, weakened, wind‐fallen and wind‐broken trees as well as from trap trees. The mycobiota associated with I. typographus was quite diverse in respect of the number of detected species, 65 fungal taxa were obtained from the phloem of trees infested by I. typographus, and 36 taxa occurred in the sapwood underneath insect galleries. The spectrum of fungi mainly consisted of ascomycetes and anamorphic fungi. The ophiostomatoid fungi were represented by 14 species and were the most numerously represented group in all niches examined. The most frequent ophiostomatoid species were Ceratocystis polonica, Ophiostoma ainoae, O. bicolor, O. penicillatum, O. piceae and O. piceaperdum. The frequency of occurrence of ophiostomatoid fungi differed significantly between the phloem and sapwood as well as in relation to the varying states of health of the spruce trees. These quantitative differences in the mycobiota of I. typographus between spruce trees belonging to different health categories can be explained by successional patterns of fungal colonization of host tissues following attack by I. typographus. The pathogenic species C. polonica was the primary invader, occurring most frequently in the sapwood of ‘healthy‐looking’ trees. Ophiostoma bicolor, O. penicillatum and O. piceaperdum also occurred during the early stages of brood development of I. typographus on ‘healthy‐looking’ trees, but they mainly colonized the phloem. In contrast, O. ainoae, O. minuta and O. piceae likely follow the aforementioned species as secondary and tertiary invaders into the phloem and the sapwood of spruce trees.     

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.     

两种杨树树皮内生真菌多样性及优势种群动态变化

为了解健杨94(转基因抗虫杨94)、三倍体毛白杨2个杨树品种干部树皮内生真菌区系及其优势种群的季节变化情况,本研究利用组织分离法从2个杨树品种996块组织中分离内生真菌1 175株,健杨94和三倍体毛白杨分别分离612、563株.利用形态特征和分子生物学方法鉴定为15个属、35个分类单元,包括担子菌1个分类单元,子囊菌34个分类单元.2个杨树品种内生真菌优势种群包括链格孢、葡萄座腔菌、镰孢属真菌、间座壳属真菌等,其中,仅有链格孢、葡萄座腔菌和桑砖红镰孢是两品种共有的优势种群种类,而且优势种群会随季节变化而变化.在两品种的内生真菌中,链格孢、葡萄座腔菌是最为常见的优势种群.     

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.     

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.     

Association of seed‐borne Fusarium species on Pinus ponderosa with germination and seedling viability in Argentina

Pinus ponderosa seedlots from eight seed orchards in Western Patagonia, Argentina, were evaluated for the presence of Fusarium species, and isolates obtained were subjected to pathogenicity tests. The following species were isolated: Fusariumoxysporum, Fusarium equiseti, Fusarium verticillioides, Fusarium incarnatum, Fusarium acuminatum, Fusarium graminearum and Fusarium proliferatum. With the exception of F. proliferatum and F. graminearum all Fusarium species were recovered from P. ponderosa seedlings with symptoms of damping‐off. Five Fusarium species were re‐isolated from necrotic roots of seedlings showing chlorosis (F. oxysporum, F. equiseti, F. incarnatum, F. acuminatum and F. verticillioides). High damping‐off losses occurred in Fusarium‐contaminated seed of P. ponderosa and low disease incidence occurred on less contaminated seed. The role of seed‐borne inoculum in disease spread within forest nurseries is discussed.     

Ophiostomatoid fungi associated with root‐feeding bark beetles on Scots pine in Poland

Ophiostomatoid fungi are known to be associated with various species of bark beetles. However, information about fungal associates of root‐feeding bark beetles in Europe is still fragmentary. For this reason, the fungal associates of Hylastes ater, H. opacus and Hylurgus ligniperda on Pinus sylvestris were isolated and identified. A total of 743 fungal isolates were collected and separated into 10 morphological groups. Analyses of ITS rDNA and partial β‐tubulin gene sequences confirmed that these groups represented distinct species. The 10 species included a total of 13 associations between fungi and bark beetles that had not been recorded previously. All of the bark beetles examined were frequently associated with ophiostomatoid fungi. The fungal diversity and relative abundance of species were very similar in the three species of root‐feeding bark beetles. The most commonly encountered associates of these beetles were Grosmannia radiaticola, Leptographium lundbergii, L. procerum and L. truncatum. Insect infestation data furthermore suggest that Hylastes spp. and Hg. ligniperda are also important vectors of the fungal pathogen Sphaeropsis sapinea.