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.     

Molecular phylogeny,diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae)

We investigated the endophytic fungal community associated with the Amazonian medicinal plant Carapa guianensis and its potential for providing bioactive compounds. A total of 162 endophytic fungal isolates were obtained and identified by molecular methods. These isolates were classified into 35 different taxa in the genera Aspergilllus, Beltrania, Botryosphaeria, Colletotrichum, Diaporthe, Endomelanconiopsis, Fusarium, Guignardia, Pestalotiopsis, Phomopsis, Pilidiella, Trichoderma, and Xylaria. The most frequent colonisers recovered of C. guianensis were Colletotrichum sp. 1, Diaporthe cf. mayteni, and Pestalotiopsis sp. 1. The fungal community had a moderate richness but high diversity and evenness indices. Colletotrichum sp. and Pilidiella wangiensis displayed selective antibacterial activity; Diaporthe cf. mayteni and Endomelanconiopsis endophytica showed high activity against amastigote forms of Trypanosoma cruzi; and Colletrotrichum sp. Guignardia mangiferae, Pestalotiopsis sp., and Diaporthe melonis were able to inhibit yellow fever virus proliferation. Our results suggest that the plants living in the tropical forest, such as the Amazonian hotspot region, can live in symbiosis with hidden and underestimated rich communities of endophytic fungi, which deserve protocols and/or specific laws to keep its future conservation. The capability of these endophytic fungi to produce bioactive compounds may be part of their chemical defense and adaptive response to survive and colonizing the plant host in wild environment. Consequently, these fungal communities may provide a source of bioactive molecules, including those able to inhibit or control neglected tropical diseases.     

The role of leaf cutting and fire on extrafloral nectaries and nectar production in Stryphnodendron adstringens (Fabaceae,Mimosoideae) plants

Herbivory pressure is an important ecological aspect to determine quantitative variation in plant defenses, such as the number of extrafloral nectaries (EFNs) and their nectar amount and quality. Extrafloral nectaries can attract ants, which can be considered a type of induced plant defense. Besides, plants tend to invest more in defense when they are more vulnerable to herbivores. Therefore, we aimed to elucidate if Stryphnodendron adstringens (Fabaceae), a common Cerrado tree species, when subjected to damage (by manual leaf cutting and experimental fire) would produce a greater number of EFNs and changes its nectar quality on newly produced leaves in comparison with plants not subjected to these treatments. Leaf damage was performed artificially directly on the plant branches and at the entire plant canopy (by means of scissors or fire events). Extrafloral nectary density was higher in new leaves produced after the treatment application (artificial herbivory and fire) in comparison with plants under control treatment. The amount of nectar was also higher under treatments in comparison with control, with a significant change on nectar quality in plants subjected to the treatments of artificial herbivory. The results provided support for the hypothesis that EFNs are an inducible defensive strategy in S. adstringens, confirming the existence of phenotypic plasticity given environmental pressures.     

Evaluation of antimicrobial activity of endophytic fungi from Camptotheca acuminata (Nyssaceae)

Agricultural research of plant-derived endophytic fungi has grown in recent decades. We isolated 26 endophytic fungi from the leaves, stems and fruits of the tree of life, Camptotheca acuminata, and tested them for antimicrobial activities based on growth inhibition measurements in a modified agar diffusion method. Fermentation broths from most of the isolates exhibited antifungal activity and 50% exhibited antibacterial activity; some of them also exhibited strong broad-spectrum antimicrobial activity. The strongest antimicrobial activity was exhibited by strains XSY10 and XSY15 against Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfectum, with 75% and 67% inhibition, respectively. Strain XSJ01 gave strong activity against pathogenic bacteria, with inhibition zones more than 20 mm in diameter. The isolates were identified by molecular methods as belonging to nine taxa: Nigrospora, Diaporthe, Alternaria, Colletotrichum, Pestalotiopsis, Sordariomycete, Guignardiai, Penicillium, and Zythia. Based on these results, we conclude that the endophytic fungi of C. acuminata are promising sources of novel bioactive compounds.     

黔西南薏苡内生真菌多样性及其抗菌活性研究

为进一步了解贵州黔西南薏苡(Coix lacryma-Jobi)可培养内生真菌的多样性组成及其抗菌活性。通过组织块分离法,选取薏苡根、叶和种仁为分离对象,进行内生真菌分离,并通过形态学观察,分子生物学特征对所分离内生真菌进行鉴定。采用平板菌块对峙法对薏苡内生真菌进行抗菌活性筛选。从薏苡根、叶和种仁中共分离纯化获得76株薏苡内生真菌,根据形态和分子生物学特征,它们属于10个目、14个科、26个属和1个不明属以及未知菌;以大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphyloccocus aureus)、枯草芽胞杆菌(Bacillus subtilis)、酿酒酵母(Saccharomyces cerevisiae)为指示菌株,对薏苡内生真菌进行抗菌活性筛选。其中有25株对大肠杆菌具有抗菌活性,占分离菌株的比例为32. 9%;有34株对金黄色葡萄球菌具有抗菌活性,其比例为44. 7%;有24株对枯草芽胞杆菌具有抗菌活性,比例是31. 6%;有23株对酿酒酵母具有抗菌活性,占比例为30. 3%;对4种标准菌株都有抗菌活性的有13株,占总株数的17. 1%。薏苡的不同组织结构中存在丰富的内生真菌资源,部分内生真菌具有抑制其他微生物生长的活性,具有产生天然活性产物成分的潜力,为工业应用奠定了资源基础,具有进一步发掘和研究的价值。     

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.     

植物叶片内生放线菌的分离、分类与拮抗活性

以四川采集的芍药和北京采集的三叶草的叶片为材料,经表面消毒程序后,分离出内生放线菌15株。通过表观特征比较和BOX-PCR基因指纹图谱分析的方法,将分离出的放线菌归并于12个不同的基因型,其中6个来自芍药,6个来自三叶草。结合16S rRNA基因序列分析可知,分离菌株除C4、C5属于假诺卡氏菌外,其余的13株都是链霉菌;菌株C12与最近模式种的16S rRNA基因序列相似性较低,为96.6%。对分离菌株的抗菌活性实验表明,有11株在一个或一个以上的测试中呈阳性;其中6株对植物致病菌立枯丝核菌有明显抗性,占阳性菌株的55%。     

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.     

Testing the optimal defense hypothesis in Stryphnodendron adstringens (Fabaceae,Mimosoideae) leaves: the role of structure,number, position and nectar composition of extrafloral nectaries

Stryphnodendron adstringens is a common Cerrado tree that possesses extrafloral nectaries (EFNs) on its leaves, which are located at the base and apex of the rachis and along the secondary veins. The position of EFNs and their nectar production can be affected by defense strategies because plant organs possess different values and herbivory vulnerability. Here we aimed to elucidate anatomy, histochemistry, nectar composition and EFN number on leaves of S. adstringens in the light of the optimal defense hypothesis. We found a convergence on anatomy and histochemical characterization because the three studied types of EFNs have epidermis, secretory parenchyma and vascular tissue, showing phenolic compounds and polysaccharides in the secretory parenchyma cells. The nectar contained glucose, fructose and sucrose, which attract ants of the Camponotus and Cephalotes genus. We found differences in the number of EFNs along the secondary veins and in the nectar composition between EFNs located at the base and apex of the rachis of the leaf. The number of EFNs on the secondary veins increases from the base to the apex, suggesting a strategy to induce ant patrolling over the entire leaf region. EFNs at the base secreted more nectar, which should be related to the protection of the leaf base, which is the part most vulnerable to herbivore attack and the most valued organ. We concluded that EFNs of S. adstringens are anti‐herbivore defenses whose pattern matches the predictions of the optimal defense hypothesis.     

Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae)

The diversity and antimicrobial activity of endophytic fungi associated with the Brazilian medicinal plant Solanum cernuum Vell. were studied during summer and winter seasons. A total of 246 fungal isolates were obtained, including 225 filamentous fungi and 21 yeasts. They were identified by morphological, physiological, and molecular methods. Fifty-five different taxa represented by the phyla Ascomycota (33 taxa), Basidiomycota (21 taxa), and Zygomycota (one taxon) were identified. The most abundant taxa were closely related to Arthrobotrys foliicola , Colletotrichum gloeosporioides , Coprinellus radians , Glomerella acutata , Diatrypella frostii , Phoma glomerata , Mucor sp., Phlebia subserialis , Phoma moricola , Phanerochaete sordida , and Colletotrichum sp. A total of 265 fungal extracts were screened and 64 (26.01%) displayed antimicrobial activities. Among these extracts, 18 (28.12%) presented antibacterial and antifungal activities, 42 (65.62%) displayed selective antibacterial activity, and four (6.25%) exhibited only antifungal activity. The best values of minimum inhibitory concentration were obtained from extracts of Cryptococcus rajasthanensis , Glomerella acutata, Leptosphaeria sp., and Phoma glomerata ranging from 7.8 to 15.62?μg/mL. This study is the first survey of the endophytic fungi community associated with S. cernuum, and our results show that they can represent a promising source of bioactive compounds.     

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.     

High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by cultivation-independent approach

Heterosmilax japonica Kunth is well recognized for its diuretic effects in China. However, little is known about its endophytic fungi. In this study, microbial communities inhabiting the stems of H. japonica in spring and summer were investigated by light microscopy and cultivation-independent approaches, such as RFLP analysis and sequencing of rDNA ITS library. Molecular phylogenetic analysis showed that a broad spectrum of fungi, including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia, is able to colonize the plants internally. Particularly, several rDNA sequences determined in this study like YJ4-61 are not specifically affiliated with any currently documented fungal sequences in the public database. Several sequence types, such as YJ4-9 and YJ4-70, are significantly similar to some uncultured environmental samples. Furthermore, our result also showed that the samples collected in spring harbored more abundant endophytic populations than that in summer, implying a seasonal fluctuation for the endophytes in H. japonica.     

Coniochaeta ligniaria: antifungal activity of the cryptic endophytic fungus associated with autotrophic tissue cultures of the medicinal plant Smallanthus sonchifolius (Asteraceae)

Few studies have addressed the presence and bioactivity of endophytic fungi living in plantlets growing under in vitro conditions. After unfruitful attempts to grow axenic shoot cultures of the medicinal plant Smallanthus sonchifolius (yacon) were made, healthy shoots grew on half strength Murashigue and Skoog media supplemented with 2.2 μM benzylaminopurine without sucrose. We isolated a fungus UM109 from these autotrophic tissue cultures and it was identified as Coniochaeta ligniaria using molecular, physiological and morphological methods. Dichloromethane extracts from C. ligniaria and its host S. sonchifolius exhibited antifungal activity against phytopathogenic fungi Colletotrichum acutatum, C. fragariae and C. gloeosporioides. Both extracts of C. ligniaria and S. sonchifolius were subjected to antifungal bioassay-directed fractionation using NMR spectroscopy and GC-FID analysis. Twelve antifungal fatty acids were identified and 8 out of the total were produced by the fungus and the plant including caproic, caprylic, myristic, palmitic, heptadecanoic, stearic, oleic and linoleic acids. Additionally, caproic, caprylic and palmitic acids were produced at high concentrations by the endophytic fungus and its host. The detection of these antifungal fatty acids produced by both C. ligniaria and S. sonchifolius suggests that these bioactive compounds may be partially responsible for the high resistance of S. sonchifolius to phytopathogenic fungal attacks. This finding also indicates the existence of an interesting chemical symbiosis between an endophytic fungus and its host. Furthermore, the isolation of C. ligniaria from tissue culture of S. sonchifolius demonstrates that plantlets growing in vitro as autotrophic cultures can shelter specific endophytic fungal communities. The use of autotrophic tissue cultures may become an important tool for studies on the taxonomy, ecology, evolution and biotechnological application of endophytes.     

Phylogenetic diversity of endophytic leaf fungus isolates from the medicinal tree Trichilia elegans (Meliaceae)

Various types of organisms, mainly fungi and bacteria, live within vegetal organs and tissues, without causing damage to the plant. These microorganisms, which are called endophytes, can be useful for biological control and plant growth promotion; bioactive compounds from these organisms may have medical and pharmaceutical applications. Trichilia elegans (Meliaceae) is a native tree that grows abundantly in several regions of Brazil. Preparations using the leaves, seeds, bark, and roots of many species of the Meliaceae family have been widely used in traditional medicine, and some members of the Trichilia genus are used in Brazilian popular medicine. We assessed the diversity of endophytic fungi from two wild specimens of T. elegans, collected from a forest remnant, by sequencing ITS1-5.8S-ITS2 of rDNA of the isolates. The fungi were isolated and purified; 97 endophytic fungi were found; they were separated into 17 morpho-groups. Of the 97 endophytic fungi, four genera (Phomopsis, Diaporthe, Dothideomycete, and Cordyceps) with 11 morpho-groups were identified. Phomopsis was the most frequent genus among the identified endophytes. Phylogenetic analysis showed two major clades: Sordariomycetes, which includes three genera, Phomopsis, Diaporthe, and Cordyceps, and the clade Dothideomycetes, which was represented by the order Pleosporales.     

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.     

一株芦荟抗菌内生细菌的分离鉴定及生物学性质研究

从药用植物中华芦荟组织中分离出一株抗菌内生细菌A11#,该菌产生广谱抗菌活性物质,对金黄色葡萄球菌、枯草芽孢杆菌等革兰氏阳性细菌和植物病原真菌均有较强的抑制作用;通过形态学观察及生理生化特征测定,初步鉴定归属于芽孢杆菌属(Bacillus)。该菌在初始pH值为5．0～10．0的PDA培养基上生长旺盛,生长最适pH为5～7,培养液初始pH为6～10时发酵产生大量粘性物质;生长温度范围在10～45℃,最适温度28～37oc;在初始pH5．0和30．5℃条件下发酵,其发酵液抑菌作用最强;该菌所产抗菌物质能耐121℃处理30min而不影响其活性。在阿须贝无氮培养基上生长良好,在不加葡萄糖、蔗糖或淀粉的LB和牛肉膏蛋白胨培养基上不生长;同时还产生对高岭土有絮凝活性的物质。     

Relationship between plant development, tannin concentration and insects associated with Copaifera langsdorffii (Fabaceae)

Plant development is the main factor that determines the insect-ontogeny interaction, since it leads to variations in resource quality and availability. The aim of this study was to test the hypothesis that plant development and varying tannin concentration leads to changes in species richness, abundance and composition of ants, free-feeding herbivores and galling insects associated with Copaifera langsdorffii (Fabaceae). The plant ontogeny and tannin concentration effects on insects were tested on 60 individuals with height varying from 0.9 to 11.0 m. A positive correlation was observed for tree height and species richness and abundance of ants, free-feeding and galling insects. In contrast, we did not find a significant relation between leaf tannin concentration and plant height, or richness and abundance of the different insect guilds. The assemblage of ants (composition of species) did not change between saplings and adults of C. langsdorffii. However, the assemblage of free-feeding herbivores and galling insects varied between the two development stages studied. The present study reveals an ontogenetic succession pattern for herbivore insects along the C. langsdorffii growth, probably due to both indirect and direct benefits from the host plant architecture and quality. Those plants with more complex architectures should support a wider diversity of insects, since they present higher number of sites for egg laying, housing, feeding and better environmental conditions. This is the first work to investigate the host plant ontogeny effect on insects in Cerrado “Savanna” vegetation. The pattern described, along with other previous studies, suggests a vast occurrence of ontogenetic succession in tropical areas.