姜黄根茎内生真菌区系分析

目的了解姜黄植物根茎中内生真菌群落组成和生态分布规律。方法采用表面消毒法分别于春、冬季从姜黄植物根茎中分离内生真菌,通过形态特征和基于ITS序列的系统发育分析初步确定其分类地位。结果分离获得51株内生真菌(春季33株、冬季18株),初步鉴定其分别归于5个纲、7个目、8个科、8个属(Fusarium,Gibberel-la,Alternaria,Phomopsis,Diaporthe,Nectria,Botryosphaeria,Mucor),其中镰刀菌属(Fusarium,51.0%)和赤霉属(Gibberel-la,17.6%)为优势菌群;春季分离的内生真菌分属于7个属(分离率为82.5%),而冬季的内生真菌仅归入4个属(分离率为45.0%)。结论姜黄植物中内生真菌具有较丰富的物种和系统发育多样性,在类群组成和分布上存在季节性差异,某些内生真菌(Fusariumsp.)具有宿主偏好性。     

广西柳树桑寄生内生真菌的分离鉴定与抗肿瘤活性菌株筛选

该研究从广西钦州市采集健康的柳树桑寄生的根、茎和叶中分离并纯化内生真菌,对真菌进行形态学鉴定,提取内生真菌的DNA,采用真菌ITS序列对内生真菌进行分子鉴定。利用A549和H460细胞作为抗肿瘤活性指示细胞,采用MTT法测定真菌乙酸乙酯提取物的抗肿瘤活性。经过初步分离分析,从柳树桑寄生中纯化出27株内生真菌,经鉴定它们分别属于7个目9个属15个种。拟盘多毛孢属和间座壳属为优势属,其中拟盘多毛孢属全部定植于寄生根;其次为新壳梭孢属、拟茎点霉属和球座菌属,各分离到3株;其他包括青霉属、镰刀菌属、炭疽菌属和派伦霉属,各分离到1株。抗肿瘤活性研究表明,有一株与Pestalotiopsis protearum的ITS序列相似性达100%的拟盘多毛孢属菌株Gen24表现有抑制肿瘤细胞A549和H460生长的特性,在真菌乙酸乙酯浸提物浓度为800μg·mL~(-1)时,对A549细胞的抑制率达到了56.92%,对H460细胞的抑制率达到了70.11%。该研究结果表明广西柳树桑寄生内生真菌较丰富,在寄主中的分布表现了一定的组织特异性,而且还存在一些具有抗肿瘤活性的菌株及其活性物质可供进一步深入研究。     

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.     

Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini)

Endophyte microorganisms are organisms that live inside plants without causing any apparent damage to their hosts. Since all plants exhibit endophyte microorganisms, it is believed that mutual association is of great importance in nature. Luehea divaricata (Martius & Zuccarini), known popularly in Brazil as agoita-cavalo, is a big-sized tree with a wide distribution in the country that possesses medicinal qualities for: dysentery, leucorrhea, rheumatism, blennorrhoea, tumors, bronchitis, and depuration. This research aims at isolating and molecularly characterizing fungi isolates from L. divaricata by sequence analysis of ITS1-5.8S-ITS2 rDNA. Further, the colonization of endophyte in the host plant by Light and Scanning Electron Microscopy will also be investigated. Whereas, genera Alternaria, Cochliobolus, Diaporthe, Epicoccum, Guignardia, Phoma, and Phomopsis, were identified; rDNA sequence analysis revealed intra-species variability among endophyte isolates of the genus Phomopsis sp. Light and Scanning Electron Microscopy techniques showed the presence of endophyte fungi inside L. divaricata leaves, inhabiting inter- and intra-cellular spaces. These types of extensive colonization and dissemination were reported throughout all the leaf parts in palisade parenchyma, esclerenchyma, spongy parenchyma, adaxial epidermis, and vascular bundle indicating colonization of endophytes in múltiple structural sub-niches in the host plant.     

Endophytic fungi from the root tubers of medicinal plant Stephania dielsiana and their antimicrobial activity

The adverse effects of chemical synthetic fungicides on agricultural fields and the environment are driving a need to search for safer and less environmentally harmful plant protectants to move toward more sustainable development of agriculture. The endophytic fungal community associated with the medicinal plant Stephania dielsiana, and its potential for providing antimicrobial secondary metabolites were investigated. A total of 26 isolates of endophytic fungi were obtained, and 21 isolates were identified and classified into eight different genera, including Briansuttonomyces, Glomerella, Pleosporales, Diaporthe, Phoma, Penicillium, Periconia and Colletotrichum, and the most frequent endophytic species obtained were Diaporthe phaseolorum, Penicillium sp., Periconia igniari and Colletotrichum sp. The ethyl acetate (EtOAc) extract of the endophytic fungus Diaporthe phaseolorum Stdif6 displayed the most significant antifungal activity against all tested phytopathogens, with EC₅₀ values ranging from 0.0138 to 0.3103 mg/mL. While the EtOAc extract of the endophytic fungus Penicillium sp. Stdif9 exhibited greater potential for antibacterial activity, with the minimum inhibitory concentration (MIC) values against seven bacteria ranging from 1.25 to 6 mg/mL. The remarkable antimicrobial activity of fungal endophytes suggests that fungal endophytes harbored inside the root tubers of S. dielsiana hold great promise as biocontrol agents against a broad spectrum of economically significant pathogens.     

Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches' Broom Disease

The basidiomycete fungus Crinipellis perniciosa (Stahel) Singer is the causal agent of Witches' Broom Disease of Cacao (Theobromacacao L.) which is the main factor limiting cacao production in the Americas. Pod losses of up to 90% are experienced in affected areas as evidenced by the 50% drop in production in Bahia province, Brazil following the arrival of the C. perniciosa in the area in 1989. The disease has proven particularly difficult to control and many farmers in affected areas have given up cacao cultivation. In order to evaluate the potential of endophytes as a biological control agent of this phytopathogen, the endophytic fungal community of resistant and susceptible cacao plants as well as affected branches was studied between 2001 and 2002. The fungal community was identified by morphological traits and rDNA sequencing as belonging to the genera Acremonium, Blastomyces, Botryosphaeria, Cladosporium, Colletotrichum, Cordyceps, Diaporthe, Fusarium, Geotrichum, Gibberella, Gliocladium, Lasiodiplodia, Monilochoetes, Nectria, Pestalotiopsis, Phomopsis, Pleurotus, Pseudofusarium, Rhizopycnis, Syncephalastrum, Trichoderma, Verticillium and Xylaria. These fungi were evaluated both in vitro and in vivo by their ability to inhibit C. perniciosa. Among these, some were identified as potential antagonists, but only one fungus (Gliocladium catenulatum) reduced the incidence of Witches' Broom Disease in cacao seedlings to 70%.     

Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.     

Obligate biotrophic pathogens of the genus Albugo are widespread as asymptomatic endophytes in natural populations of Brassicaceae

Mutualistic interactions of plants with true fungi are a well‐known and widespread phenomenon, which includes mycorrhiza and non‐mycorrhizal endophytes like species of Epichloë. Despite the fact that these organisms intrude into plants, neither strong defence reactions nor the onset of symptoms of disease can be observed in most or even all infested plants, in contrast to endophytic pathogens. Oomycetes are fungal‐like organisms belonging to the kingdom Straminipila, which includes diatoms and seaweeds. Although having evolved many convergent traits with true fungi and occupying similar evolutionary niches, widespread oomycete endophytes are not known to date, although more than 500 endophytic pathogens, including species of the obligate biotrophic genus Albugo, have been described. Here, we report that oomycetes of the genus Albugo are widespread in siliques of natural host populations. A total of 759 plants, encompassing four genera with rare reports of white blister incidents and one with common incidents, were collected from 25 sites in Germany. Nested PCR with species‐specific primers revealed that 5–27% of the hosts with rare disease incidence carried asymptomatic Albugo in their siliques, although only on a single plant of 583 individuals, an isolated pustule on a single leaf could be observed. Control experiments confirmed that these results were not because of attached spores, but because of endophytic mycelium. Vertical inheritance of oomycete infections has been reported for several plant pathogens, and it seems likely that in nature this way of transmission plays an important role in the persistence of asymptomatic endophytic Albugo species.     

Endophytic fungi assemblages from 10 <Emphasis Type="Italic">Dendrobium</Emphasis> medicinal plants (Orchidaceae)

Dendrobium is the largest genus of tropical epiphytic orchid, some of which are traditional Chinese medicinal plants. The therapeutic components varied significantly among species. Endophytic microbes (fungi) hidden in medicinal plants may play an important effect on the overall quality of herb. Investigation of fungal composition in host plants is the first step toward elucidating the relationship endophyte-therapeutic content of herbal medicine. In this study, 401 culturable fungal endophytes were isolated and identified from 10 species of medicinal Dendrobium based on morphological and molecular techniques. The results showed that endophytic fungi from Dendrobium plants exhibited high biodiversity (37 genera, about 80 species). Acremonium, Alternaria, Ampelomyces, Bionectria, Cladosporium, Colletotrichum, Fusarium, Verticillium and Xylaria were the dominant fungal endophytes. Tropical epiphytic orchids appear to vary in degree of host specificity in their endophytic fungi.     

四绺孢球腔菌科真菌地理分布及其生态学功能

根据ITS、LSU、rpb2、tef1和tub2多基因系统发育分析,将云南禾本科植物格孢腔菌目的7株内生真菌鉴定归属于格孢腔菌目Pleosporales四绺孢球腔菌科Tetraplosphaeriaceae的四绺孢属Tetraploa和假四绺孢属Pseudotetraploa以及该目下的一个未定属genera incertae sedis。羧甲基纤维素钠培养基和愈创木酚培养基筛选发现四绺孢球腔菌科的2个菌株具有较强的纤维素酶和漆酶活性,而这个未定属的菌株仅具有较弱的纤维素酶活性、无漆酶活性,表明格孢腔菌目的2个内生真菌类群的纤维素和木质素降解能力不同。多重对应分析发现四绺孢球腔菌科真菌的属与寄主、分离来源和地理位置有关联,其中四绺孢属和假四绺孢属可在活的健康植物作为内生真菌存活,并在植物凋落物和土壤中分离得到,推测四绺孢属和假四绺孢属两属为内生和腐生双生态位真菌。因此,进一步深入探究四绺孢球腔菌科内生真菌参与的禾本科植物凋落物的分解将深化我们对禾本科植物内生真菌多样性和生态学功能的认识。     

Differential Growth Inhibition of Diaporthe and Phomopsis Isolates by the Metabolic Activity of Five Actinomycetes

Fifty-five cultures derived from Diaporthe perithecia and Phomopsis pycnidia found on diverse host plant species collected at different times and sites in Vojvodina, Yugoslavia, showed distinguishing quantitative reactions to the fungistatic activity of five actinomycetes obtained as fortuitous laboratory contaminants coming from field material. Streptomyces albidoflavus , S. albus , S. diastaticus , Streptomyces sp., and Streptoverticillium sp. could be ranked by their growth-inhibitory potential, with S. albus showing the strongest, and Streptomyces sp. the lowest. The responses of the fungi varied depending on the tested actinomycetes, but two major groups could be distinguished: A, which encompased the isolates that were less affected by the proximity of the actinomycetes; and B, with those which exhibited high sensitivity in all the experiments. Group A was typically represented by Diaporthe arctii , Phomopsis longicolla, and the Phomopsis type-1 cultures from Xanthium italicum; group B was typically represented by Diaporthe/Phomopsis helianthi, Phomopsis type-2 cultures from X. italicum , and isolates from Lactuca serriola . The results obtained underscore the dissimilarities between D. arctii and D. helianthi , and corroborate the value of the physiological aspects of congeneric isolates in considering taxonomic problems in the coelomicete genus Phomopsis.     

Diversity of the endophytic fungi associated with the ancient and narrowly endemic neotropical plant Vellozia gigantea from the endangered Brazilian rupestrian grasslands

This study focused on the taxonomy and diversity of the endophytic fungi associated with Vellozia gigantea, an endemic, ancient, and endangered plant species that occurs only in the rupestrian grasslands of Brazil. A total of 285 fungal isolates were recovered from leaves and roots of the V. gigantea, which were identified in 27 genera and 87 different taxa using molecular taxonomy methods. Xylaria berteri, Diaporthe sp. 1, Nigrospora oryzae, Muscodor sp. 1, Colletotrichum aeschynomene, and Trichoderma viride occurred in the highest frequency in both the leaf and root. Diaporthe was the most abundant genus, with 70 endophytic isolates recovered from the leaves and roots. Among all the taxa identified, 62 occurred as singlets, including those of the genera Clonostachys, Coccomyces, Crucellisporiopsis, Daldinia, Myxotrichum, Pallidocercospora, Pezicula, Peyronellaea, and Pseudocercospora. The diversity indices displayed high values, showing that V. gigantea shelters a diverse and rich mycobiota. Our results indicate that V. gigantea shelters in its tissues a highly diverse and cryptic mycobiota, including several rare species previously unreported as endophytes, but that are reported to have different ecological functions, which might be an important biological component contributing to the fitness of the plants living in the rupestrian grassland.     

湖北烟草内生真菌生物多样性和种群结构分析

【目的】研究传统药用植物烟草(Nicotiana tabacum L.)内生真菌的丰富度,揭示其种群多样性和群落结构,为烟草内生真菌资源的有效利用奠定基础。【方法】采用组织分离法进行烟草内生真菌的分离,通过形态学和分子生物学相结合的方法进行菌株分类鉴定,以香农多样性指数及相对分离频率反映内生真菌物种多样性及分布规律。【结果】从不同组织部位、不同生长时期、不同海拔样地的健康烟草中共分离获得539株内生真菌,根据r DNA-ITS系统发育分析鉴定为31属73种,香农多样性指数为2.78,曲霉属Aspergillus和镰孢属Fusarium为优势菌群,其相对分离频率分别为22.63%和12.99%。其分布规律表现为茎部内生真菌的多样性高于叶部和根部;随着生育期的延长,内生真菌多样性逐步增多;随着海拔高度升高,内生真菌的种类和数量呈现降低的趋势。【结论】烟草内生真菌具有丰富的生物多样性,其分布表现出组织、生长时期、海拔高度专化性。阐明内生真菌在烟草中的分布规律,可以为烟草内生真菌在农业生产领域的开发应用提供科学依据。     

云南、浙江、内蒙古禾本科植物内生真菌多样性研究

从云南、浙江和内蒙古的30属禾本科植物分离到1821株内生真菌,根据ITSrDNA系统发育分析和ITS预测真菌的阈值,将这些菌株鉴定为3门10纲34目216属,其中子囊菌门5纲26目192属,担子菌门3纲6目21属,毛霉门2纲2目3属.粪壳菌纲和座囊菌纲为主要优势纲,相对频率分别为54.8％和30.9％;座囊菌纲的格孢...     

假橐吾内生菌分离及其拮抗菌的筛选与鉴定

从菊科植物假橐吾(Ligulariopsis shichuana Y.L.Chen)中分离得到110株内生真菌和26株内生放线菌,依照形态培养特征对菌株进行初步分类鉴定,采用琼脂块法进行拮抗菌株筛选,并对拮抗性最好的2株内生放线菌进行分子鉴定.结果表明:(1)经初步鉴定,将分离到的假橐吾内生真菌合并为86种,分为10个属,其中交链孢霉属和丝核菌属占优势(分别为41.9%和16.3%);内生放线菌分为3个属,其中链霉菌属占优势(92.3%).(2)抗菌活性试验表明,有31株内生真菌分别对细菌、植物病原真菌具有显著抑制作用,全部内生放线菌对靶标菌有抗性,其中菌株wz57和wz01拮抗性最强.(3)根据16S rDNA序列比对结果,菌株wz57和wz01与链霉菌属(Streptomyces griseoplanus)相似性最高,分别达到99.3%和99.5%,初步归为Streptomyces griseoplanus.     

Spatial and temporal variation of fungal endophytic richness and diversity associated to the phyllosphere of olive cultivars

Fungal endophytes are micro-organisms that colonize healthy plant tissues without causing disease symptoms. They are described as plant growth and disease resistance promoters and have shown antimicrobial activity. The spatial-temporal distribution of endophytic communities in olive cultivars has been poorly explored. This study aims to investigate the richness and diversity of endophytic fungi in different seasons and sites, within the Alentejo region, Portugal. Additionally, and because the impact of some pathogenic fungi (e.g. Colletotrichum spp.) varies according to olive cultivars; three cultivars, Galega vulgar, Cobrançosa and Azeiteira, were sampled. 1868 fungal isolates were identified as belonging to 26 OTUs; 13 OTUs were identified to the genera level and 13 to species level. Cultivar Galega vulgar and season autumn showed significant higher values in terms of endophytic richness and diversity. At site level, Elvas showed the lowest fungal richness and diversity of fungal endophytes. This study reinforces the importance of exploring the combined spatio-temporal distribution of the endophytic biodiversity in different olive cultivars. Knowledge about endophytic communities may help to better understand their functions in plants hosts, such as their ecological dynamics with pathogenic fungi, which can be explored for their use as biocontrol agents.     

Insect pathology and fungal endophytes

Fungi that occur inside asymptomatic plant tissues are known as fungal endophytes. Different genera of fungal entomopathogens have been reported as naturally occurring fungal endophytes, and it has been shown that it is possible to inoculate plants with fungal entomopathogens, making them endophytic. Their mode of action against insects appears to be due to antibiosis or feeding deterrence. Research aimed at understanding the fungal ecology of entomopathogenic fungi, and their role as fungal endophytes, could lead to a new paradigm on how to successfully use these organisms in biological control programs.     

Cultural Growth Patterns and Incompatibility Reactions in Diaporthe and Phomopsis Populations

Comparative studies were carried out with cultures isolated from Phomopsis condiomata and/or Diaporthe ascomata found on naturally-infected living stems or on overwintered stem fragments of Arctium lappa L., Helianthus annuus L., Glycine max (L.) Merr., and Xanthium italicum Moretti, growing in or nearby the same fields in Vojvodina. Results underscored the heterogeneity of the Phomopsis anamorphs in the D. arctii (Lasch) Trav, complex, Intra-specific vegetative incompatibility groups were distinguished among Phomopsis and/or Diaporthe populations from A. lappa. More than one phytopathogenic Phomopsis and/or Diaporthe species were identified on X. italicum. P. longicolla Hobbs was isolated for the first time in Yugoslavia from soybean stem debris. The holomorph Diaporthe (Phomopsis) helianthi Muntanola- Cvetkovic, Mihaljcevic et Petrov showed distinguishing characteristics with respect to other congeneric fungi.     

青檀果内生真菌菌群的组成及多样性

为进一步了解我国古老特有的野生植物青檀内生真菌的组成特征,以安徽琅琊山自然保护区内的野生青檀果为研究对象,采用组织分离和形态学鉴定的方法对青檀果内生真菌进行了分离和鉴定.结果表明: 从500个组织块中共分离内生真菌428株,隶属于3目4科20属,未产孢的菌株暂归为无孢菌群(80株,18.7%).暗梗孢科和球壳孢科是青檀果内生真菌的优势科,分别占38.1%和32.9%,交链孢属和拟茎点霉属为其优势属,分别占27.1%和16.8%.青檀果不同部位内生真菌的组成存在差异,以种子中分离的内生真菌数量最多(19属181株,占42.3%),果翅中分离的内生真菌最少(7属88株,占20.6%).除了交链孢属为3个不同部位的共同优势属之外,拟茎点霉属和大茎点霉属分别是果柄和果翅的优势属.果柄和果翅内生真菌菌群的组成相似性最高(C_S=0.78),种子和果翅的相似性系数最低(C_S=0.50).分析显示,果柄与果翅在内生真菌菌群组成上无显著差异,而种子与果柄、种子与果翅在内生真菌菌群组成上均存在显著差异.     

云南大理烟区烟叶内生真菌多样性及分布特征

【目的】研究大理烟区烟叶内生真菌的多样性及分布特征。【方法】通过形态特征和ITS-rDNA序列分析对获得的菌株进行分类。【结果】从烟草叶片中共分离得到内生真菌1 568株,分别归于31个属,在属水平上的优势菌群为链格孢属(Alternaria)和镰孢属(Fusarium),分别占真菌总数的40.79%和18.73%。【结论】烟草内生真菌的数量、种类、多样性和分布分别在不同部位叶片间和不同地区间存在差异,在下部叶数量较多,而上部叶的种类较多,多样性更丰富。随着烟草的发育成熟,内生真菌数量、种类和多样性增加,在现蕾期和成熟期最大。