红树林内生真菌研究进展

红树林作为一种特殊的植物群落具有丰富的内生真菌资源,目前已分离鉴定的红树林真菌超过200种,成为海洋真菌的第二大类群,已报道的红树林内生真菌主要类群是链格孢霉(Alternaria)、曲霉(Aspergillus)、芽枝霉(Cladosporium)、炭疽菌(Colletotrichum)、镰孢霉(Fusarium)、拟青霉(Paecilomyces)、拟盘多毛孢(Pestalotiopsis)、青霉(Penicillium)、茎点霉(Phoma)、拟茎点霉(Phomopsis)、叶点霉(Phyllosticta)和木霉(Trichoderma)等.大部分红树林内生真菌具有较宽的宿主范围,极少数只有单一的宿主,不同红树林植物的内生真菌区系及优势种群有很大差异.红树林内生真菌的定殖因宿主植物不同部位、植株的年龄及季节和环境的变化明显不同.红树林内生真菌能产生多种代谢产物,具有抗菌、抗肿瘤等药用价值.红树林植物内生真菌的研究和开发具有重要意义.本文综述了红树林内生真菌的生物多样性及其分布、生物学功能和次生代谢产物等方面的研究进展.     

天目山山胡椒不同部位内生真菌组成及多样性分析

采用表面消毒法,从浙江天目山野生山胡椒[ Lindera glauca( Sieb.et Zucc.) Blume]的茎、叶和树皮中分离出内生真菌,基于ITS序列分析进行分类鉴定;并以内生真菌的分离率、定殖率、分离频率、多样性指数(H')及相似性系数为指标,分析了山胡椒内生真菌的菌群组成及多样性.结果显示:在26株山胡椒样株的728块组织块中共分离得到328株内生真菌(茎、叶和树皮中分别有161、40和127株);共鉴定出44个分类单元(茎、叶和树皮中各有19、18和28个),其中25个分类单元鉴定到种、17个鉴定到属、2个鉴定到科,ITS序列的GenBank登录号从JF502420至JF502462.在44个分类单元中,有40个分类单元属于子囊菌(310株),存在于山胡椒的各个部位;仅有4个分类单元属于担子菌(18株),且仅存在于茎和树皮中.山胡椒茎、树皮和叶中内生真菌的定殖率分别为65％、60％和15％,分离率分别为0.77、0.61和0.19;叶和树皮中内生真菌的多样性指数均为2.63,远大于茎(H’=1.83).山胡椒内生真菌的优势属为Phomopsis、Paraconiothyrium、Phoma和Colletotrichum,大量存在于叶、茎和树皮中.山胡椒茎与树皮、茎与叶及叶与树皮间内生真菌的相似性系数分别为0.27、0.19和0.18,显示树皮和叶之间以及树皮和茎之间内生真菌的组成极不相似.研究结果表明:山胡椒体内存在大量的内生真菌,其茎、叶和树皮的内生真菌菌群组成具有一定程度的多样性和差异性,且内生真菌的分布具有组织特异性.     

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.     

Endophytic fungal diversity of 2 sand dune wild legumes from the southwest coast of India

Endophytic fungi of 3 age classes (seeds, seedlings, and mature plants) and 5 tissue classes (cotyledons, seed coats, roots, stems, and leaves) of coastal sand dune legumes Canavalia cathartica and Canavalia maritima were assessed by plating surface-sterilized segments on malt extract agar. Forty-six fungal taxa comprising 6 ascomycetes, 33 mitosporic fungi, 2 zygomycetes, and 5 sterile morphospecies were recovered. There was no significant difference in the colonization frequency of endophytes between plant species (p = 0.4098, Student's t test). Among the age classes, endophytic fungi colonized over 90% of seedlings and mature plants. Similarly, among tissue classes, endophytic fungi colonized over 90% of root, stem, and leaf segments. Diversity and richness of endophytic fungi were higher in C. cathartica than in C. maritima. Rarefaction curves revealed a "higher expected number of species" in mature plants of C. cathartica and seedlings of C. maritima, whereas it was highest in leaves of both plant species. The most dominant endophyte, Chaetomium globosum, colonized over 50% of the root, stem, and leaf segments of C. maritima and over 50% of the root segments of C. cathartica. The colonization frequency of C. globosum was found to be 5%-12.5% in seeds and increased up to 40%-64.4% in seedlings or mature plants. Halosarpheia sp. was the only marine fungus recovered among the endophytes.     

The Endophytic Mycoflora of Bark,Leaf, and Stem Tissues of <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss (Neem) from Varanasi (India)

A systematic study was made of the endophytes of Azadirachta indica A. Juss (the neem tree) growing in several of its natural habitats in India. A total of 233 isolates of endophytic fungi representing 18 fungal taxa were obtained from segments of bark, stem, and leaves of this tree. Hyphomycetes (62.2%) were the most prevalent followed by the Coelomycetes (27.4%) and Mycelia Sterilia (7.7%). As mathematically determined, the maximum species richness and frequency of colonization of endophytes appeared in leaf segments rather than stem and bark tissues from each location. Endophytic colonization frequency was also greater in leaves (45.5%) than bark (31.5%). The leaf samples from all locations were nearly constant in their endophytic composition, whereas bark samples showed maximum diversity at different locations. Inter-site comparisons for endophytic diversity, however, were not significantly different with Loc1 and Loc2 having a maximum of 66.67% J _c. The smallest similarity was between Loc2 and Loc3 of 54.17% J _c. The dominant endophytic fungi isolated were Phomopsis oblonga, Cladosporium cladosporioides, Pestalotiopsis sp., Trichoderma sp, and Aspergillus sp. Genera such as Periconia, Stenella, and Drechslera are reported here for the first time as endophytes from this host plant. This report illustrates the value of sampling different tissues of a given plant in several locations to obtain the greatest species diversity of endophytes. The rich and sizeable collection of endophytic fungi from this specific plant may represent a unique source of one or more of the interesting and useful bioactive compounds normally associated with A. indica such as the azadirachtins and related tetranortriterpenoids.     

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.     

Seasonal and tissue age influences on endophytic fungi of Pinus tabulaeformis (Pinaceae) in the Dongling Mountains, Beijing

Endophytic fungi associated with Pinus tabulaeformis Carr.were investigated in the Dongling Mountains of Beijing.A total of 16200 tissue segments of P.tabulaeformis from four sample collections were processed,and 10659 fungal isolates were recovered.The overall colonization and isolation rates of endophytic fungi from high to low were spring>winter>autumn>summer and in different tissues were bark>needle>xylem,irrespective of sampling seasons.The colonization rates of endophytic fungi of needles increased with age in the four sample collections.There were no significant differences of the colonization rates of endophytic fungi among 1-,2-,and 3-year-old bark and xylem,except for significantly lower colonization rates in 1-year-old bark than in 2- and 3-year-old bark in summer.A similar trend of the isolation rates of endophytic fungi occurred.A total of 24 fungal taxa were recorded,of these five taxa Alternaria alternata,Leptostroma sp.,Pestalotiopsis besseyi,Phoma lingam,and Phomopsis archeri,were consistently isolated as the common fungi in each sample collection.Our results suggest that some fungi show a certain degree of tissue recurrence or specificity,and the composition of endophytic assemblages is not conspicuously influenced by the seasonal factor.     

Diversity and ecological distribution of endophytic fungi associated with medicinal plants

A total of 973 isolates of endophytic fungi were recovered from 1144 tissue fragments of the six me-dicinal plant species belonging to 4 families collected in the Beijing Botanical Garden. Of these isolates 778 sporulated and were identified into 21 taxa by morphological characteristics. Among the taxa 11 belonged to Coelomycetes, 6 to Ascomycetes, and 4 to Hyphomycetes. Various numbers of endophytic fungi (5―8 taxa) were obtained from each plant. Alternaria alternata was the dominant species in the 6 plants, and Microsphaeropsis conielloides was also dominant in Eucommia ulmoides. There were high colonization rates (47.9%―63.1%) and isolation rates (0.7―0.93) of endophytic fungi, and they were conspicuously higher in twigs than those in leaves in the 6 plants examined. The colonization and isolation rates of endophytic fungi increased with the twig age. The results based on the analyses of cluster and Sorenson's similarity coefficients indicated that some endophytic fungi showed a certain degree of host and tissue preference.     

Diversity and ecological distribution of endophytic fungi associated with medicinal plants

A total of 973 isolates of endophytic fungi were recovered from 1144 tissue fragments of the six medicinal plant species belonging to 4 families collected in the Beijing Botanical Garden. Of these isolates 778 sporulated and were identified into 21 taxa by morphological characteristics. Among the taxa 11 belonged to Coelomycetes, 6 to Ascomycetes, and 4 to Hyphomycetes. Various numbers of endophytic fungi (5–8 taxa) were obtained from each plant. Alternaria alternata was the dominant species in the 6 plants, and Microsphaeropsis conielloides was also dominant in Eucommia ulmoides. There were high colonization rates (47.9%–63.1%) and isolation rates (0.7–0.93) of endophytic fungi, and they were conspicuously higher in twigs than those in leaves in the 6 plants examined. The colonization and isolation rates of endophytic fungi increased with the twig age. The results based on the analyses of cluster and Sorenson’s similarity coefficients indicated that some endophytic fungi showed a certain degree of host and tissue preference.     

野生五唇兰根部内生真菌多样性研究

兰科植物根部的内生真菌在兰科植物的整个生活史中起着重要的作用, 为了解不同生境不同类型的兰科植物内生真菌菌群的多样性, 作者于2004年7月至2005年10月, 以海南岛霸王岭自然保护区内的野生五唇兰(Doritis pulcherrima)作为实验材料, 对不同生境、不同形态的五唇兰植株根部的内生真菌群落多样性进行了研究。从附生于石上及生于杂木林或灌丛中、叶背绿色及叶背紫红色的五唇兰植株新鲜营养根段中共分离出83株内生真菌, 鉴定为19个属, 其中包括培养基筛选实验中分离出的30株14属。镰刀菌属(Fusarium)(24.1%)和丝核菌属 (Rhizocto-nia)(14.5%)为优势属。两种叶色的五唇兰内生真菌群落丰富度较为一致; 而两种不同生境中的五唇兰内生真菌群落丰富度则表现出较大的差异: 附生于灌木的五唇兰内生真菌群落Shannon多样性指数远高于附生岩石的。研究结果表明五唇兰内生真菌多样性更多地受生境的影响而不是受植株形态类型的影响。     

Biological activities of extracts from endophytic fungi isolated from Garcinia plants

Sixty-five crude extracts from 51 selected endophytic fungi isolated from Garcinia species were tested for various bioactivities. Eighty per cent of the fungal extracts from fermentation broths and mycelia displayed bioactivities: antimycobacterial (76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1% against NCI-H187 and 12.7% against KB cells), and cytotoxicity to Vero cells (40.0%). Based on internal transcribed spacer rRNA sequence analysis, 15 bioactive isolates were identified as Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis spp. One isolate (N24) was matched with an unidentified fungal endophyte. These results indicate that endophytic fungi isolated from Garcinia plants in Thailand are potential sources of various bioactive natural products.     

Diversity of endophytic fungal community associated with Piper hispidum (Piperaceae) leaves

Tropical and subtropical plants are rich in endophytic community diversity. Endophytes, mainly fungi and bacteria, inhabit the healthy plant tissues without causing any damage to the hosts. These fungi can be useful for biological control of pathogens and plant growth promotion. Some plants of the genus Piper are hosts of endophytic microorganisms; however, there is little information about endophytes on Piper hispidum, a medicinal shrub used as an insecticide, astringent, diuretic, stimulant, liver treatment, and for stopping hemorrhages. We isolated the fungal endophyte community associated with P. hispidum leaves from plants in a Brazilian forest remnant. The endophytic diversity was examined based on sequencing of the ITS1-5.8S-ITS2 region of rDNA. A high colonization frequency was obtained, as expected for tropical angiosperms. Isolated endophytes were divided into 66 morphogroups, demonstrating considerable diversity. We identified 21 isolates, belonging to 11 genera (Alternaria, Bipolaris, Colletotrichum, Glomerella, Guignardia, Lasiodiplodia, Marasmius, Phlebia, Phoma, Phomopsis, and Schizophyllum); one isolate was identified only to the order level (Diaporthales). Bipolaris was the most frequent genus among the identified endophytes. Phylogenetic analysis confirmed the molecular identification of some isolates to genus level while for others it was confirmed at the species level.     

Assessment of diversity,distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant <Emphasis Type="Italic">Adenocalymma alliaceum</Emphasis> Miers

A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds.     

Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India

Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15.0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0.473 for Jaccard’s and 0.642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56.25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55.87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries.     

The foliar endophytic fungal community composition in Cirsium arvense is affected by mycorrhizal colonization and soil nutrient content

Foliar fungal endophytes are ubiquitous, but understudied symbionts of most plant species; relatively little is known about the factors affecting their occurrence, diversity and abundance. We tested the effects of soil nutrient content and arbuscular mycorrhizal (AM) colonization on the occurrence of foliar endophytic fungi in Cirsium arvense in two field studies. In the first study, we assessed relationships between soil moisture, organic matter, carbon and nitrogen content and plant water, nitrogen and carbon content and AM colonization and the occurrence of foliar endophytic fungal species. In the second study, we manipulated soil nutrient content and AM colonization of potted seedlings and identified differences in endophytic fungal species composition of the leaves and stems. The results reveal that endophytes can occur either more or less frequently, depending on soil nutrient and plant water content and AM colonization. We propose that these patterns were the result of differences in fungal growth responses to nutrient availability in the leaves, which can be affected by resources obtained from the soil or symbiotic fungi in the roots.     

Endophytic Phomopsis sp. colonization in Oryza sativa was found to result in plant growth promotion and piperine production

Endophytic fungi have been reported to have the acquired ability to synthesize host plant specific medicinal natural products. Many fungi with such properties have been characterized and optimized for the conditions which favor maximal production of desired products. However, the inherent plant colonization property of promising endophytic fungi is least studied. Exploiting the transgenome functioning of these fungi have immense applications to add beneficial features to nonhost plants. In the present study, the endophytic fungus Phomopsis sp. isolated from Piper nigrum was confirmed for piperine production by HPLC and LCMS/MS. Further, the fungal isolate was studied for its colonization ability in Oryza sativa. Interestingly, the fungi treated plants were found to have significant plant growth enhancement when compared to the control. Further screening of extract from treated plants by HPLC and LCMS/MS resulted in the confirmation of presence of piperine. The observed result is extremely significant as it opens up novel applications of endophytic fungal colonization in taxonomically diverse plants.     

Measuring diversity of endophytic fungi in leaf fragments: does size matter?

Endophytic fungi inhabit living plant tissues without causing disease symptoms. Although abundant, the extent of their contribution to fungal biodiversity remains unclear. Since endophytic fungi are poorly known, especially in the tropics, current estimates of fungal species are probably conservative. Here we tested strategies for sampling endophytic fungi in tropical plants. We compared the number of fungi isolated from 400 mm2 leaf pieces that were divided into increasingly small fragments. Leaf pieces were surface-sterilized, cut into fragments and plated on culture media. For a given area, cutting leaf pieces into smaller fragments significantly increased the number of fungal morphospecies recovered. There was a strong linear relationship between size of fragments and number of fungi isolated. By extrapolation, an estimated 16 +/- 3 fungi could be recovered from a 2 x 2 cm leaf piece, using infinitely small fragments. This represents a large part of the fungal diversity estimated to exist in leaf endophytes in a population. We conclude that reducing the size and increasing the number of leaf fragments will increase the number of fungal species isolated. This strategy will help to estimate real values of endophytic fungal diversity.     

Biodiversity and distribution of endophytic fungi associated with <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cultivated in a forest reserve

Endophytic fungi residing in Panax quinquefolium (American ginseng) have not been well characterized. We collected American ginseng of one-, two-, three- and four-year-old plants cultivated in a forest reserve and identified the endophytic fungal isolates using traditional methods. The colonization frequency and the dominant endophytic fungal species were investigated. Endophytic fungal diversity indices and similarity coefficient were also assessed and all age groups of American ginseng studied were colonized by endophytic fungal assemblages; 134 fungal isolates were assigned to 27 taxa. The infection frequencies varied with the host age and tissue. The dominant endophytic fungi were recorded for each tissue and age of host. The roots of two- and four-year-old American ginseng exhibited the highest and the lowest Shannon-Wiener index respectively. Four-year-old American ginseng had a low similarity coefficient when compared with each of the other three ages classes. The possible role of endophytic fungi in relation to American ginseng cultivation is discussed.     

Seasonality of arbuscular mycorrhizal symbiosis and dark septate endophytes in a grassland site in southwest China

Arbuscular mycorrhizal and dark septate endophytic fungal colonization in a grassland in Kunming, southwest China, was investigated monthly over one year. All plant roots surveyed were co-colonized by arbuscular mycorrhizal and dark septate endophytic fungi in this grassland. Both arbuscular mycorrhizal and dark septate endophytic fungal colonization fluctuated significantly throughout the year, and their seasonal patterns were different in each plant species. The relationships between environmental (climatic and edaphic) factors and fungal colonization were also studied. Correlation analysis demonstrated that arbuscular mycorrhizal colonization was significantly correlative with environmental factors (rainfall, sunlight hours, soil P, etc.), but dark septate endophytic fungal colonization was only correlative with relative humidity and sunlight hours.     

茶树品种、叶片生育期和茶叶化学成份对内生真菌的影响

通过茶树品种、叶片龄期及叶片化学成份对茶树内生真菌分布影响的研究,发现:(1)在供试的4个茶树品种中,黄旦的内生真菌分离频率达100%,而福云六号只有7.5%,表明茶树品种不同或遗传背景不同,其叶片中内生真菌的发生存在明显差异。(2)随着茶树叶片的成熟,叶片中内生真菌的种类与数量逐渐增多,与叶片龄期呈正相关,叶片龄期影响茶树内生真菌的种群结构。(3)不同龄期叶片水浸提液对Colletotrichum、Pestalotiopsis、Guignardia三种茶树上的主要内生真菌的生长产生不同的影响;在PDA培养基中添加15%的茶多酚,对此3种内生真菌均有一定的抑制作用,但抑制程度有所不同,表明茶树叶片的化学成份是影响内生真菌分布的重要因素之一。