Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family

Aspergilli are filamentous, cosmopolitan and ubiquitous fungi which have significant impact on human, animal and plant welfare worldwide. Due to their extraordinary metabolic diversity, Aspergillus species are used in biotechnology for the production of a vast array of biomolecules. However, little is known about Aspergillus species that are able to adapt an endophytic lifestyle in Cupressaceae plant family and are capable of producing cytotoxic, antifungal and antibacterial metabolites. In this work, we report a possible ecological niche for pathogenic fungi such as Aspergillus fumigatus and Aspergillus flavus. Indeed, our findings indicate that A. fumigatus, A. flavus, Aspergillus niger var. niger and A. niger var. awamori adapt an endophytic lifestyle inside the Cupressaceous plants including Cupressus arizonica, Cupressus sempervirens var. fastigiata, Cupressus semipervirens var. cereiformis, and Thuja orientalis. In addition, we found that extracts of endophytic Aspergilli showed significant growth inhibition and cytotoxicity against the model fungus Pyricularia oryzae and bacteria such as Bacillus sp., Erwinia amylovora and Pseudomonas syringae. These endophytic Aspergilli also showed in vitro antifungal effects on the cypress fungal phytopathogens including Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. In conclusion, our findings clearly support the endophytic association of Aspergilli with Cupressaceae plants and their possible role in protection of host plants against biotic stresses. Observed bioactivities of such endophytic Aspergilli may represent a significant potential for bioindustry and biocontrol applications.     

Bioactivity of endophytic Trichoderma fungal species from the plant family Cupressaceae

Trichoderma fungal species are universal soil residents that are also isolated from decaying wood, vegetables, infected mushroom and immunocompromised patients. Trichoderma species usually biosynthesize a plethora of secondary metabolites. In an attempt to explore endophytic fungi from healthy foliar tissues of the plant family Cuppressaceae, we explored Cupressus arizonica, C. sempervirens var. cereiformis, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, Juniperus excelsa, Juniperus sp. and Thuja orientalis plants and recovered several endophytic Trichoderma fungal strains from Trichoderma atroviride and Trichoderma koningii species. We found that the host plant species and biogeographical location of sampling affected the biodiversity and bioactivity of endophytic Trichoderma species. Furthermore, the bioactivity of Trichoderma isolates and the methanol extracts of their intra- and extra-cellular metabolites were assessed against a panel of pathogenic fungi and bacteria. Fungal growth inhibition, conidial cytotoxicity, minimum inhibitory concentration and minimum bactericidal concentration were evaluated and analyzed by statistical methods. Our data showed that both intra- and extracellular secondary metabolites from all endophytic isolates had significant cytotoxic and antifungal effects against the model target fungus Pyricularia oryzae and the cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. Further research indicated their significant antimicrobial bioactivity against the model phytopathogenic bacteria Pseudomonas syringae, Erwinia amylovora and Bacillus sp., as well. Altogether, the above findings show for the first time the presence of T. atroviride and T. koningii as endophytic fungi in Cupressaceae plants and more importantly, the Trichoderma isolates demonstrate significant bioactivity that could be used in future for agrochemical/drug discovery and pathogen biocontrol.     

Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots

 The most widespread type of mycorrhiza is the so-called vesicular-arbuscular mycorrhiza. In this endomycorrhiza, fungal hyphae penetrate plant cell walls in the root cortex. There they form densely branched arbuscules. Fungus and plant plasma membrane are separated by a common interfacial apoplast. The pH of the compartment between the symbionts is of pivotal importance for nutrient transfer. Histochemical experiments were conducted to check for an acidic nature of the interface in the model system Glomus versiforme (Karst.) Berch-Allium porrum L. Two chemically different acidotropic dyes (neutral red and LysoSensor Green DND-189) stained the arbuscules intensely. The staining of arbuscules could be eliminated by addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) or treatments leading to membrane rupture. Therefore, the staining of the arbuscules was based on the ion-trap mechanism, which indicates acidic, membrane-bound compartments. Microscopic examination of stained arbuscules at high optical resolution revealed a peripheral accumulation of the dye. Since plasmolysis rapidly destained the arbuscules, it is concluded that the dyes accumulate in the arbuscular interface, indicating the highly acidic nature of this compartment. The findings are discussed with respect to their relevance for the nutrient transfer in mycorrhizas. In addition, evidence for a discontinuity in the arbuscular interface between the stem and the branches of the arbuscule is given. Received: 15 September 1999 / Accepted: 20 February 2000     

Hidden diversity of endophytic fungi in an invasive plant

Fungal endophytes are important in plant ecology and common in plants. We attempted to test cointroduction and host-jumping hypotheses on a community basis by comparing endophytes isolated from invasive spotted knapweed (Centaurea stoebe, Asteraceae) in its native and invaded ranges. Of 92 combined, sequence-based haplotypes representing eight classes of Fungi, 78 occurred in only one of the two ranges. In the native range of C. stoebe, one haplotype of Alternaria alternata was clearly dominant, whereas in the invaded range, no haplotype was dominant. Many haplotypes were closely related to one another and novel. For example, six putative, new species of Botrytis were discovered as endophytes of C. stoebe, which has never been reported to have Botrytis spp.. Apparent differences between the two communities of endophytes were significant according to an analysis of similarity, but phylogenetic community structure did not differ significantly between the ranges. Both host-jumping and cointroduction of fungal endophytes likely took place during the spotted knapweed invasion.     

Birch family and environmental conditions affect endophytic fungi in leaves

We investigated whether variation in foliar endophyte frequency among mountain birch trees from different maternal families was due in part to genetic differences among trees. The effect of different environmental conditions on the susceptibility of these mountain birch families to foliar endophytes was tested. The 3-year study was conducted in two tree gardens (altitudinal difference 180 m) with ten families of mountain birch. The frequency of the commonest endophytic fungus in mountain birch leaves, Fusicladium sp., was significantly affected by mountain birch family, with the ranking of families relative to Fusicladium sp. frequency being consistent from environment to environment. Variation in the frequency of Melanconium sp. was difficult to interpret because of significant family × garden × year interaction. Fusicladium sp. and Melanconium sp. endophytes were most frequent in different tree individuals, families and gardens. We conclude that mountain birch trees show heritable variation in their foliar endophyte frequency, and thus one of the conditions necessary for foliar endophytes of mountain birch trees to be able to affect the evolution of their host plant is fulfilled. However, the effect of mountain birch family on the frequency of endophytic fungi varies depending on the endophytic genera in question and partially also on environmental conditions. Received: 28 March 1998 / Accepted: 14 October 1998     

Assessment of root endophytic aquatic hyphomycetous fungi on plant growth

The role of root endophytic aquatic hyphomycetous fungi in plant health was studied by employing pot experiments. Three aquatic hyphomycetous fungi Heliscus lugdunensis, Tetrachaetum elegans and Tetracladium nainitalense isolated as root endophytes of riparian plants were artificially inoculated into two test plants viz., Solanum melongena and Hibiscus esculentus. T. elegans and H. lugdunensis showed significant effects (fresh weight, dry weight and length of shoots and roots) on both test plants, whereas T. nainitalense had no effect.     

植物内生真菌次级代谢产物抗菌机制分析

正细菌是世界上已知分布最广的生命体,可引起人体疾病,临床主要采用抗生素进行治疗,与此同时,抗生素的广泛应用也导致细菌产生耐药性,进而阻滞对其引起的感染性疾病的治疗。因此,人们渴望获得更多新型、疗效更为显著的抗菌药物。植物内生真菌是一个特殊的真菌类群,据估计约有一百万至数百万种内生真菌存活于维管植物细胞内或细胞间[1]。研究表明,内生真菌代谢产物类型与其宿主植物息息相关,     

鱼藤属植物内生真菌的分离及杀虫活性研究

本文是以毛鱼藤、白花鱼藤、蜜花鱼藤3种鱼藤属植物为试验材料,分离筛选代谢物具有生物活性的内生真菌菌株。通过组织分离法,从3种鱼藤属植物中分离得到4株代谢表现旺盛的内生真菌菌株。杀虫活性实验表明:4种内生真菌的代谢产物对松材线虫都具有一定的毒杀作用,其中青霉菌的代谢产物活性最高,48 h校正死亡率达90%以上。4种内生真菌中,只有青霉菌的代谢产物对家蚕4龄幼虫具有触杀活性,1.5 mg/头剂量处理时,24 h校正死亡率即达到100%。     

具有乙酰胆碱酯酶抑制活性的蛇足石杉内生真菌分子鉴定

在马铃薯葡萄糖液体培养基(PDB)、沙氏液体培养基(SDB)和查氏液体培养基(CDB)中发酵培养蛇足石杉内生真菌菌株SF88和LF52,乙酰胆碱酯酶(AChE)抑制活性检测结果表明,菌株SF88和LF52在PDB、SDB和CDB发酵后菌体总生物碱对AChE活性的抑制率分别为45.6%和56.7%、56.3%和68.5%、40.8%和48.3%。对这两株不产孢内生真菌的DNA ITS、18S rDNA、28S rDNA、TUB和RPB进行了测序,系统发育分析表明,SF88和LF52为Phaeosphaeriaceae科中一未知种,为开发生物碱类乙酰胆碱酯酶抑制剂(AChEI)药物提供了新的潜在菌种资源。     

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.     

茶树内生真菌混合培养增强对植物病原真菌的拮抗作用

采用分离自健康茶树叶片组织中的2株内生真菌,长枝木霉CSN-18和曲霉CSN-3,研究了单独培养和混合培养条件下培养原液与培养原液乙酸乙酯萃取液对植物病原真菌的抑制作用。与2菌株单独培养相比,混合培养对稻瘟病菌Magnaporthe grisea、茶花褐斑病菌Phyllosticta camelliaecola和茶云纹叶枯病菌Guignardia camellia等8种植物病原真菌的抑制作用明显增强。混合培养原液的乙酸乙酯萃取液对茶花褐斑病菌、稻瘟病菌和茶云纹叶枯病菌的抑制率分别为79.48%±1.46%、76.99%±0.91%和71.51%±4.93%;对茶花褐斑病菌、茶云纹叶枯病菌和稻瘟病菌孢子萌发的抑制率分别为100.00%、90.90%±2.59%和84.00%±5.29%。混合培养原液对茶花褐斑病菌菌丝生长的抑制作用也比两菌株的单独培养液有明显增强;在低浓度下,混合培养原液对茶花褐斑病菌的孢子萌发有促进作用;但浓度超过40%,则明显抑制茶花褐斑病菌孢子的萌发。     

喜树内生真菌抗水稻纹枯病菌活性的研究

从喜树健康组织叶、茎以及果实中经过分离、纯化共得到26株内生菌株。利用生长速率法测定这26株菌株对植物病原菌——水稻纹枯病病原菌的抑制作用。抑菌试验结果显示：26株菌株中有22株喜树内生真菌的发酵液对植物病原菌菌丝生长均有不同程度的抑制作用;其中,菌株XSY10的发酵液抑菌效果最强,达到了74.97%。水稻盆栽试验结果表明：喷施菌株XSY10次生代谢产物7d后,对水稻纹枯病的防治效果为34.77%。以上试验结果说明喜树内生真菌XSY10对植物病害的防治具有一定的功效。     

Enzymatic activity of fungi endophytic on five medicinal plant species of the pristine sacred forests of Meghalaya,India

Fungal species that establish an endophytic role inside the tissues of medicinal plants are known to produce a wide range of biologically active metabolites and enzymes. In the present study, the most dominant and representative endophytic fungal species of five ethno-medicinal plants prevalent in the pristine sacred forests of Meghalaya, were screened for their ability to produce amylase, cellulase, protease, lipase, and xylanase. Each of endophytic fungal isolates showed a wide range of enzyme activity. Mycelial biomass generation and root colonization, in addition to the enzyme activity of the endophytic fungal isolates, provided insights into their probable origin and ecological roles within the plant host.     

Antimicrobial activity in cultures of endophytic fungi isolated from Garcinia species

The aim of the present study was to screen for antimicrobial activity in endophytic fungi isolated from surface sterilized leaves and branches of five Garcinia plants, G. atroviridis, G. dulcis, G. mangostana, G. nigrolineata and G. scortechinii, found in southern Thailand. Fermentation broths from 377 isolated fungi were tested for antimicrobial activity by the agar diffusion method. Minimum inhibitory concentrations (MICs) were obtained for crude ethyl acetate extracts. Seventy isolates (18.6%) displayed antimicrobial activity against at least one pathogenic microorganism, such as Staphylococcus aureus, a clinical isolate of methicillin-resistant S. aureus, Candida albicans and Cryptococcus neoformans. The results revealed that 6-10%, 1-2% and 18% of the crude ethyl acetate extracts inhibited both strains of S. aureus (MIC 32-512 microg mL(-1)), Ca. albicans and Cr. neoformans (MIC 64-200 microg mL(-1)), and Microsporum gypseum (MIC 2-64 microg mL(-1)), respectively. Isolates D15 and M76 displayed the strongest antibacterial activity against both strains of S. aureus. Isolates M76 and N24 displayed strong antifungal activity against M. gypseum. Fungal molecular identification based on internal transcribed spacer rRNA gene sequence analysis demonstrated that isolates D15 (DQ480353), M76 (DQ480360) and N24 (DQ480361) represented Phomopsis sp., Botryosphaeria sp. and an unidentified fungal endophyte, respectively. These results indicate that some endophytic fungi from Garcinia plants are a potential source of antimicrobial agents.     

Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants

Endophytic filamentous fungi from coffee plants (Coffea arabica and C. robusta) deposited in the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI) were characterized taxonomically by using molecular tools and investigated concerning their antimicrobial activity against different human pathogenic bacteria. Thirty-seven out of 39 CBMAI strains investigated were identified to at least at genus level by ITS and rDNA D1/D2 sequencing and phylogenetic analyses. Bioactivity screening of fungal extracts against Salmonella choleraesuis (CBMAI 484), Staphylococcus aureus (CBMAI 485), Pseudomonas aeruginosa (CBMAI 489) and against four different Escherichia coli serotypes showed that 17 fungi inhibited at least one of the bacteria studied. The endophytic fungi Trichoderma harzianum (CBMAI 43), Guignardia sp. (CBMAI 69) and Phomopsis sp. (CBMAI 164) inhibited from four to five bacterial species, while five fungi were active against all pathogenic bacteria tested and were identified as Aspergillus versicolor (CBMAI 46), Fusarium oxysporum (CBMAI 53), Glomerella sp. (CBMAI 63) and Cladosporium spp. (CBMAI 64 and CBMAI 66). The Minimal Inhibitory Concentration (MIC) for the fungus extracts varied from 0.025 to 1.0 mg ml⁻¹, demonstrating antimicrobial potential of some of these fungi.     

药用植物内生真菌的多样性及生物功能研究进展

药用植物内生真菌资源丰富,其代谢产物常具有抗肿瘤、抗氧化、抑菌等作用,能产生药用植物生长调节物质及与宿主相同或类似的次生代谢产物,从而成为近年来的研究热点。本文对药用植物内生真菌的分离鉴定、多样性、生物活性及生物学功能等方面进行综述,以期为今后筛选及利用有效的药用植物内生真菌奠定基础。     

Differential effects of foliar endophytic fungi on insect herbivores attacking a herbaceous plant

Foliar endophytic fungi appear to be ubiquitous in nature, occurring in a very wide range of herbaceous plants. However, their ecological role within forbs is very poorly known and interactions with foliar-feeding insects virtually unexplored. In this study, leaves of Cirsium arvense were infected with different combinations of endophyte fungi that had been previously isolated from this plant species. Two months later, leaf material was fed to larvae of a generalist insect, Mamestra brassicae, and adults of a specialist feeder, Cassida rubiginosa. Endophytes had different effects on the two insects; one species, Chaetomium cochliodes, reduced growth of M. brassicae but increased feeding by C. rubiginosa. Another species, Cladosporium cladosporioides, increased beetle feeding also, but had no effect on M. brassicae. Interactions were also seen between fungal species and dual infection with C. cladosporioides and Trichoderma viride greatly reduced beetle feeding. It is concluded that endophytes have significant effects on foliar feeding insects that differ with degree of specialism of the herbivore. We suggest that these effects are due to chemical changes in the host, brought about by fungal infection. These fungi have received remarkably little attention in the study of insect–plant interactions and yet could be important determinants of insect growth and even population dynamics.     

银杏内生真菌多样性研究(英文)

采用组织块分离法,从中国福建、江苏、贵州三省银杏Ginkgo biloba的根、茎、叶、树皮组织中分离内生真菌,利用形态学与ITS rDNA序列分析相结合的方法对所分离的菌株进行鉴定。结果表明从根、茎、叶和树皮分离出175株内生真菌,归为47类,每一类取代表菌株进行ITS测序及系统分析,分别属于子囊菌门的8个目,即Eurotiales、Hypocreales、Xylariales、Trichosphaeriales、Glomerellales、Diaporthales、Botryosphaeriales、Pleosporales,11科,16属。其中刺盘孢属Colletotrichum(19.75%)、链格孢属Alternaria(19.15%)、镰孢菌属Fusarium(10.64%)和拟茎点霉属Phomopsis(10.64%)为优势菌群;并且新丛赤壳属Neonectria和生赤壳属Bionectria为首次从银杏中分离出。Shannon-Wiener指数(H=2.4192)和Simpson指数(1-D=0.8856)的计算结果反应出所获得的银杏内生真菌菌群具有较高的多样性。     

Effect of different root endophytic fungi on plant community structure in experimental microcosms

Understanding the effects of root‐associated microbes in explaining plant community patterns represents a challenge in community ecology. Although typically overlooked, several lines of evidence point out that nonmycorrhizal, root endophytic fungi in the Ascomycota may have the potential to drive changes in plant community ecology given their ubiquitous presence, wide host ranges, and plant species‐specific fitness effects. Thus, we experimentally manipulated the presence of root endophytic fungal species in microcosms and measured its effects on plant communities. Specifically, we tested whether (1) three different root endophyte species can modify plant community structure; (2) those changes can also modified the way plant respond to different soil types; and (3) the effects are modified when all the fungi are present. As a model system, we used plant and fungal species that naturally co‐occur in a temperate grassland. Further, the soil types used in our experiment reflected a strong gradient in soil texture that has been shown to drive changes in plant and fungal community structure in the field. Results showed that each plant species responded differently to infection, resulting in distinct patterns of plant community structure depending on the identity of the fungus present. Those effects depended on the soil type. For example, large positive effects due to presence of the fungi were able to compensate for less nutrients levels in one soil type. Further, host responses when all three fungi were present were different from the ones observed in single fungal inoculations, suggesting that endophyte–endophyte interactions may be important in structuring plant communities. Overall, these results indicate that plant responses to changes in the species identity of nonmycorrhizal fungal community species and their interactions can modify plant community structure.