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。     

生姜共生真菌的分离及其体外抑菌效应初探

为筛选得到优良植物病害生防菌,对广西生姜Zingiber officinale种植区健康生姜根系和叶片中的共生真菌进行了组织分离,以生姜茎腐病菌群结腐霉Pythium myriotylum和香蕉枯萎病菌尖孢镰刀菌古巴专化型4号生理小种Fusarium oxysporum f. sp. cubense race 4为指示菌,通过平板对峙培养法和发酵液菌落直径法试验进行筛选评价,并结合形态学观察及ITS序列分析对筛选出的生防效果最好的共生真菌进行了鉴定。结果表明,从生姜植株共分离得到34株共生真菌,其中根系分离22株,叶片分离12株;对峙培养发现有6株共生真菌对生姜茎腐病菌和香蕉枯萎病菌均有抑制作用;其中菌株SBM-11拮抗作用最强,对生姜茎腐病菌抑制率达到93%,对香蕉枯萎病菌抑制率达到82%;SBM-11的发酵液对生姜茎腐病菌和香蕉枯萎病菌抑制率分别为82%、73%,与其他菌株发酵液抑制效果相比差异明显;结合形态和分子鉴定结果表明SBM-11菌株为绿色木霉Trichoderma viride,极具生防潜力。     

Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Morphological and molecular characterization of a new autochthonous Trichoderma sp. isolate and its biocontrol efficacy against Alternaria sp.

The development of agriculture requires the use of microorganisms in the management of phytopathogens as a way to compensate for the use of chemical pesticides, in order to produce healthy crops. The objective of this study was to characterize a new isolate of Trichoderma sp. based on morphological and molecular features, and its potential ability to control the pathogen Alternaria sp. The antagonistic isolate was isolated from soil samples of potato fields in Guasave Sinaloa, Mexico, whereas the pathogen was collected from infected apple leaves in the orchard “La Escondida” in Guerrero County, Chihuahua, Mexico. For morphological characterization both fungi were grown on solid PDA medium. DNA of Trichoderma sp. was isolated using the CTAB method and PCR analyses were done using ITS1, ITS4 primers resulting in amplified products of 600 bp. These were sequenced, submitted to Genbank (acc. no. MN950427) and used for further phylogenetic analysis through Bayesian inference approach. Five clades were identified and the polytome topography recovered from clade 4 indicates a high genetic similarity with T. asperellum. A BLAST examination of the resulting sequence in GenBank showed 98.11% similarity with T. asperellum. This result together with the morphological and the phylogenetic analyses indicates that the isolate belongs to Trichoderma asperellum Samuels, Lieckfeldt & Nirenberg. Biocontrol tests of this isolate showed inhibition of Alternaria sp. between 50% and 93%. These results are essential for biodiversity research and give some new possibilities for pest management.     

A fungal endophyte of black spruce (Picea mariana) needles is also an aquatic hyphomycete

An aquatic hyphomycete, Dwayaangam sp., was isolated from superficially sterilized black spruce (Picea mariana) needles submerged in aerated water in a small glass chamber (microcosm). The internal transcribed spacer (ITS) sequence of this fungus and of a commonly encountered foliar endophyte isolated from P. mariana showed a high degree of similarity. When sporulation was induced in the microcosm, both the aquatic hyphomycete and the endophyte isolate produced similar aquatic conidia after 30 days, which is longer than previously documented in similar studies. Without the use of molecular tools, the link between the aquatic and endophytic phases of the fungus would have gone unnoticed. This is the first time that a fungal endophyte of conifer needles has been shown to have an aquatic phase. Its presence both as a foliar endophyte and a sporulating aquatic fungus suggests an alternating life cycle between the two environments.     

Fungal endophytes of turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.) and their biocontrol potential against pathogens <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> and <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Endophytic fungi have been isolated from the healthy turmeric (Curcuma longa L.) rhizomes from South India. Thirty-one endophytes were identified based on morphological and ITS–rDNA sequence analysis. The isolated endophytes were screened for antagonistic activity against Pythium aphanidermatum (Edson) Fitzp., and Rhizoctonia solani Kuhn., causing rhizome rot and leaf blight diseases in turmeric respectively. Results revealed that only six endophytes showed >?70% suppression of test pathogens in antagonistic dual culture assays. The endophyte T. harzianum TharDOB-31 showed significant in vitro mycelial growth inhibition of P. aphanidermatum (76.0%) and R. solani (76.9%) when tested by dual culture method. The SEM studies of interaction zone showed morphological abnormalities like parasitism, shriveling, breakage and lysis of hyphae of the pathogens by endophyte TharDOB-31. Selected endophytic isolates recorded multiple plant growth promoting traits in in vitro studies. The rhizome bacterization followed by soil application of endophyte TharDOB-31 showed lowest Percent Disease Incidence of rhizome rot and leaf blight, 13.8 and 11.6% respectively. The treatment of TharDOB-31 exhibited significant increase in plant height (85 cm) and fresh rhizome yield/plant (425 g) in comparison with untreated control under greenhouse condition. The confocal microscopy validates the colonization of the TharDOB-31 in turmeric rhizomes. The secondary metabolites in ethyl acetate extract of TharDOB-31 were found to contain higher number of antifungal compounds by high resolution liquid chromatograph mass spectrometer analysis. Thereby, endophyte T. harzianum isolate can be exploited as a potential biocontrol agent for suppressing rhizome rot and leaf blight diseases in turmeric.     

Endophytic fungal DNA, the source of contamination in spruce needle DNA

DNA isolated and amplified from higher plants may originate from symbiotic microbes occupying plant tissues. A recent report on the phylogeny of Picea contained sequence data that upon later analysis proved to originate from filamentous ascomycetes. Isolates of endophytic fungi from Picea foliage collected from the same location as the original samples were examined to identify the source of the contaminating DNA. The ITS region of isolates was screened by Southern blotting using an oligonucleotide probe homologous to a unique portion of the reported 'spruce' sequences. This study identifies a DNA sequence originally attributed to Picea engelmannii (Engelmann spruce) as Hormonema dematioides , a ubiquitous foliar endophyte of conifers. Infections of plants by endophytic fungi are common and their presence is not revealed by external symptoms. Plant molecular researchers should be aware of the potential for this type of DNA contamination.     

千层塔内生真菌的分离与鉴定

目的：为从千层塔中分离具有药用价值的内生真菌奠定基础。方法：新鲜千层塔茎段,经酒精和升汞消毒后,接种于PDA平板培养基上进行内生真菌的分离、纯化;根据菌落形态和孢子等形态特征,结合核糖体基因居间序列（ITS序列）进行菌株鉴定。结果：从千层塔的茎中分离出4株内生真菌。内生真菌I菌落形态和孢子特征与枝状枝孢霉属的特征相符合,ITS序列与GenBank中多条属于枝状枝孢霉的ITS序列相似,鉴定该菌株属于枝状枝孢霉;内生真菌II菌落形态和孢子特征与黄青霉的特征相符合,鉴定该菌株属于黄青霉;内生真菌III菌落形态和孢子特征与尖孢镰刀菌的特征相符合,ITS序列与GenBank中多条属于尖孢镰刀菌的ITS序列相似程度高,鉴定该菌株属于尖孢镰刀菌;内生真菌Ⅳ菌落形态与盾壳霉相似,ITS序列与GenBank中6条属于盾壳霉的ITS序列具有较高的相似性,鉴定该菌株属于盾壳霉。结论：从千层塔中分离和鉴定出4株内生真菌,分别属于枝状枝孢霉、黄青霉、尖孢镰刀茵和盾壳霉。     

不同发酵茶中优势真菌的分离鉴定及产消化酶活性比较

目的 分离鉴定云南普洱茶、广州小青柑、广西六堡茶和湖南熙茯茶中的主要真菌,并探讨其产消化酶活性。方法 采用平板涂布法分离不同发酵茶中的真菌,通过其ITS序列进行分子鉴定;利用平板透明圈法测定不同真菌的产淀粉酶、蛋白酶、纤维素酶和脂肪酶活性。结果 从4种发酵茶中共分离出8种真菌,其中,在云南普洱茶中分离出2种真菌,分别为篮状菌（Talaromyces. sp.）和T. variabilis;广州小青柑中分离出3种真菌,分别为阿曲霉（Aspergillus amstelodami）、烟曲霉（A. fumigatus）和绳状篮状菌（T. funiculosus）;广西六堡茶中分离出2种真菌,分别为黑曲霉（A. niger）和冠突曲霉（A. cristatus）;湖南熙茯茶中仅分离出谢瓦曲霉（A.chevalieri）。其中Talaromyces. sp.、T. variabilis、A. niger、A.amstelodami、A. fumigatus、T. funiculosus有产纤维素酶活性,Talaromyces. sp.、T. variabilis、A. cristatus、A. fumigatus、T. funiculosus有产淀粉酶活性,Talaromyces. sp.、T. variabilis、A. niger、A. fumigatus、T. funiculosus有产蛋白酶活性,A. niger有产脂肪酶活性。在8种真菌中,T. variabilis、烟曲霉和绳状篮状菌可同时产纤维素酶、淀粉酶和蛋白酶;黑曲霉能同时产纤维素酶、蛋白酶和脂肪酶;冠突曲霉仅有产淀粉酶活性;阿曲霉仅有产纤维素酶活性。通过比较不同真菌的产酶活性表明,烟曲霉的产蛋白酶活性能力最强;Talaromyces sp.的产纤维酶活性能力最强;Talaromyces. sp.和绳状篮状菌的产淀粉酶活性最强。结论 发酵茶的真菌以曲霉菌属Aspergillus、散囊菌属Eurotium和篮状菌属Talaromyces为主,多数真菌具有产消化酶活性,这些酶可能在茶叶发酵过程中起重要作用,并影响发酵茶叶的品质。     

宁夏贺兰山青海云杉根系共生真菌的分离与鉴定

[背景]青海云杉(Picea crassifolia)是中国特有植物,具有极高的生态价值,是西北地区重要的森林更新树种和荒山造林树种,其生长发育及其抗逆性与根系共生真菌多样性密切相关.[目的]从青海云杉根系分离并鉴定定殖的可培养共生真菌,阐明青海云杉根系可培养共生真菌的种类组成,为共生真菌在青海云杉育苗、造林及生态恢复...     

丛生盔形珊瑚共附生可培养真菌多样性分析

旨在研究珊瑚共附生可培养真菌多样性。运用稀释平板法和基于ITS-rDNA基因序列的系统发育分析对珊瑚共附生可培养真菌多样性进行研究, 将所得到的基因序列与NCBI数据库GenBank中的序列进行相似性比较并构建系统发育树。实验中从丛生盔形珊瑚表面和内部共分离得到19株菌落形态各异的真菌。ITS-rDNA序列分析及形态学鉴定表明, 丛生盔形珊瑚共附生真菌主要包括曲霉菌Aspergillus sp.、枝孢霉菌Cladosporium sp.、炭角菌Xylariales sp.、青霉菌Penicillium sp.、葡萄穗霉菌Stachybotrys sp.、赤霉菌Gibberella moniliformis、镰刀霉菌Fusarium sp.等。分离得到的菌株中, 4-13与GenBank中已报道的基因序列的相似性仅为89%。结果表明, 与丛生盔形珊瑚共附生的可培养真菌较为丰富, 是潜在的新的微生物菌种资源, 具有进一步研究的价值。     

Acetyl esterase production by Termitomyces clypeatus

Production of acetyl esterase by Termitomyces clypeatus was stimulated by xylan, cellulose, arabinose and arabinose-containing polysaccharides in the growth medium. The culture filtrate was equally active with p-nitrophenyl acetate and acetyl xylan. Acetyl xylan was completely deacetylated by the enzyme. Activity was optimum at pH 6.5 and at 50¡C. The Km values for p-nitrophenyl acetate and acetyl xylan were 0.83 mM and 0.38% (w/v) with Vm of 48 and 55 mmole acetate produced/min.mg protein, respectively.     

Caracterización morfológica y molecular del antagonismo entre el endofito Diaporthe sp. aislado de frailejón (Espeletia sp.) y el fitopatógeno Phytophthora infestans

Endophytic fungi produce a great variety of secondary metabolites both in vivo and in vitro. In this study, we characterized the ability of a sterile-mycelium endophytic fungus isolated from Espeletia sp. to control the growth of Phytophthora infestans in Petri dishes. Sequence from the ITS regions (internal transcribed spacer) of the endophyte showed 94% similarity to Diaporthe phaseolorum's. The antagonistic interaction between Diaporthe sp. and P. infestans was evaluated in three different culture media. Diaporthe sp. showed an antagonistic effect towards P. infestans, with some variation depending on which medium was used. In an attempt to identify possible genes involved in this antagonism, we detected a gene from the endophyte encoding an amylase, which was differentially expressed during this biotic interaction.     

Bradyrhizobium iriomotense sp. nov., isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan

A polyphasic study was performed to determine the taxonomic position of strain EK05(T) isolated from a root-outgrowth of Entada koshunensis, a legume available in Okinawa, Japan. Phylogenetic analysis of the 16S rRNA gene showed that the strain belongs to the genus Bradyrhizobium. Subsequent multilocus sequence analysis with ITS, glnII, recA, gyrB, and atpD sequences revealed that the isolate represents a distinct evolutionary lineage within the genus Bradyrhizobium. DNA-DNA hybridization indicated that strain EK05(T) shares <61% DNA relatedness with the type strains of all six recognized species of Bradyrhizobium, confirming that this strain is a novel species within the genus. Phylogenetic trees based on symbiotic loci, nifH and nodC, also placed strain EK05(T) clearly in a novel branch. On the basis of its phylogenetic distinctiveness, we propose Bradyrhizobium iriomotense sp. nov. for strain EK05(T). The type strain is EK05(T) (= NBRC 102520(T) = LMG 24129(T)).     

一株无花果内生真菌及其次生代谢产物

从无花果Ficus carica 叶中分离获得1株具有抗菌活性的内生真菌ZJWCF255,通过形态学和ITS rDNA序列分析将菌种鉴定为炭角菌属Xylaria sp.,其发酵液经活性跟踪分离,采用硅胶柱层析及HPLC等方法获得4个化合物。根据波谱数据,化合物1–4分别鉴定为cytochalasin C、D、Q、R。Cytochalasin Q（3）对11种植物病原真菌均具有较强的抑制活性,对蔓枯病菌的抑制率最强,EC50值为0.04μg/mL。该化合物对3种肿瘤细胞SMMC-772、MCF-7、MGC80-3具有较强体外抑制活性,其IC50值分别为（17.24±2.55）、（7.75±1.37）、（10.30±1.34）μg/mL。首次报道了cytochalasin Q（3）的抗菌活性及对3种肿瘤细胞的体外抑制活性,植物内生真菌是获得抗菌与抗肿瘤活性先导化合物的重要来源。     

培菌白蚁与鸡枞菌研究进展

培菌白蚁起源于非洲,蚁巢内具有复杂的社会分工.培菌白蚁依靠独特的蚁巢结构维持内部稳态和气体循环.菌圃是白蚁培育鸡枞菌的场所.鸡枞菌隶属于担子菌亚门,但其传播方式和生活史具有区别于其它担子菌的特点.鸡枞菌协助白蚁进行植物纤维的消化,白蚁则为鸡枞菌提供合适的生长环境,并控制鸡枞菌的遗传结构.培菌白蚁和鸡枞菌形成紧密的共生关...     

Microbial community analysis in the termite gut and fungus comb of Odontotermes formosanus: the implication of Bacillus as mutualists

The microbial communities harbored in the gut and fungus comb of the fungus-growing termite Odontotermes formosanus were analyzed by both culture-dependent and culture-independent methods to better understand the community structure of their microflora. The microorganisms detected by denaturing gradient gel electrophoresis (DGGE), clonal selection, and culture-dependent methods were hypothesized to contribute to cellulose-hemicellulose hydrolysis, gut fermentation, nutrient production, the breakdown of the fungus comb and the initiation of the growth of the symbiotic fungus Termitomyces. The predominant bacterial cultivars isolated by the cultural approach belonged to the genus Bacillus (Phylum Firmicutes). Apart from their function in lignocellulosic degradation, the Bacillus isolates suppressed the growth of the microfungus Trichoderma harzianum (genus Hypocrea), which grew voraciously on the fungus comb in the absence of termites but grew in harmony with the symbiotic fungus Termitomyces. The in vitro studies suggested that the Bacillus sp. may function as mutualists in the termite-gut-fungus-comb microbial ecosystem.     

Phylogenetic relationships in Termitomyces (Family Agaricaceae) based on the nucleotide sequence of ITS: a first approach to elucidate the evolutionary history of the symbiosis between fungus-growing termites and their fungi

Termitomyces constitutes a very poorly known genus of fungi whose essential characteristic is that all representatives of the genus are cultivated by termites (Macrotermitinae) in their nest and that all the fungi cultivated by termites belong to this genus. For the first time, the phylogenetic relationships of several African Termitomyces species was studied by the sequencing of their internal transcriber spacer region (ITS1--5.8S--ITS2). It appeared that this group is clearly monophyletic and belongs to the Tricholomataceae family. The total homology of the ITS zone of several Termitomyces symbionts of different termite genera indicated that the specific diversity of this group is in fact less important than previously supposed. Finally, the comparison between the Termitomyces phylogenetic tree and the taxonomic tree of Macrotermitinae showed that if for certain genera the hypothesis of termite/fungus coevolution is acceptable, it should not be applied for all symbiosis.     

凉山州龙肘山锈红杜鹃与薄叶马银花根部真菌分子检测

【目的】为检验直接分子检测法用于揭示杜鹃花属(Rhododendron)植物根部真菌(Root-associated fungi,RAF)组成的有效性。【方法】采用不依赖于培养的分子检测技术直接从锈红杜鹃(R.bureavii)与薄叶马银花(R.leptothrium)的发根(Hair root)提取DNA,用真菌特异性引物扩增r DNA-ITS区、经克隆后测序,对获得的ITS序列进行分析;通过收集NCBI中与本研究的RAF相似性97%以上的所有序列对应的真菌来源(土壤或根系的身份)数据,分析真菌的生态学特性,并用FUNGuild软件提供的方法划分真菌的生态类型。【结果】从两种杜鹃花根部共检测到15种真菌,其中担子菌门(Basdiomycota)真菌3种,子囊菌门(Ascomycota)真菌12种。柔膜菌目(Helotiales)真菌在两种杜鹃花RAF群落中占据优势,并且在两种杜鹃花根系中均检测到该类真菌。此外,两种杜鹃花根部有多种生态类型的RAF共存,包括曾被频繁报道的杜鹃花类菌根菌Oidiodendron sp.和Rhizoscyphus sp.、内生真菌Phialocephala fortinii、共生一致病过渡型真菌Pezoloma ericae、外生菌根共生菌Meliniomyces sp.,以及腐生型真菌Myceana sp.、Lachnum virgineum、Herpotrichia sp.。【结论】直接分子检测法从两种杜鹃花属植物根部检测到的真菌谱系多样性较高、生态类型复杂,这一方法能较为全面地反映杜鹃花属植物RAF多样性。