红树林泥样中真菌筛选的培养基选择与样品处理方法

【背景】 海洋微生物是天然药物资源宝库，海洋环境中的微生物能够产生很多区别于陆生微生物的天然产物。红树林生长于陆地与海洋交界带的滩涂浅滩，是陆地向海洋过渡的特殊生态系统，可能蕴含着丰富的微生物资源和潜在的大量结构新颖的代谢产物。 【目的】 以厦门杏林湾红树林根际海泥和海水为研究对象，探索其中可培养海洋真菌的多样性及其发酵提取物的抗细菌和抗真菌活性，以期为发现新的药物先导化合物提供菌株资源。 【方法】 样品预处理后涂布于GPY、PDA、Martin和MEA这4种培养基，28 ℃倒置培养，待培养基上长出合适大小的菌落后，挑取单菌落接种于PDA培养基纯化。利用纸片扩散法对发酵提取物进行抗菌活性筛选，通过高效液相色谱分析提取物的化学多样性，并采用形态观察结合rDNA ITS序列分析对活性较好的4株真菌进行菌种鉴定。 【结果】 共分离出71株真菌，其中49株真菌对金黄色葡萄球菌有抗性，6株真菌对白色念珠菌有抗性，2株真菌对大肠杆菌有抗性，菌株HS5-MEA-4 and HS6-MEA-10初步鉴定为土曲霉，菌株HS5-GPY-7 and HS6-GPY-15分别为棘孢曲霉和 Aspergillus templicola 。 【结论】 初步认知了厦门杏林湾红树林环境真菌多样性及其发酵提取物的活性和化学多样性特征，为后续海洋真菌次级代谢产物研究提供了依据。

自雅浦海沟4个站位的12个沉积物样品中分离得到38株真菌菌株，基于形态观察和ITS序列信息对所得菌株进行分类鉴定，发现它们分别属于青霉属（ Penicillium ，17株）、曲霉属（ Aspergillus ，7株）、篮状菌属（ Talaromyces ，1株）、枝孢属（ Cladosporium ，6株）、赭霉属（ Ochroconis ，1株）、 Meyerozyma 属（1株）、梅拉菌属（ Meira ，1株）、 Cystobasidium 属（2株）、 Wallemia 属（1株）和红酵母属（ Rhodotorula ，1株）等10个属.其中青霉属、曲霉属和枝孢属菌株为优势菌株，分别占分离菌株总数的44.7%、18.4%和15.8%；而 Wallemia 属（节担菌纲）菌株是首次从深海环境中分离到.利用PCR技术在分离的深海真菌菌株中检测 nirK 和 P450nor 两种反硝化关键酶基因，结果显示 nirK 基因可在黄曲霉（ Aspergillus flavus ）、黑曲霉（ Aspergillus niger ）、杂色曲霉（ Aspergillus versicolor ）、枝孢菌（ Cladosporium sp.）、枝状枝孢（ Cladosporium cladosporioides ）和产黄青霉（ Penicillium chrysogenum ）等6种真菌菌株中检测到，而 P450nor 基因仅在黄曲霉菌株中检测到.同时还通过富集培养方法从上述沉积物样品中分离获得两株反硝化枝孢菌.结果表明，雅浦海沟深海沉积物环境中存在着丰富的真菌资源，它们在深海环境氮循环中起着一定作用，这将增强人们对深海环境可培养真菌多样性及其生态作用的认识.

对中国第29次南极科学考察获得的南极普里兹湾海域23个站位的深海沉积物样品进行了真菌分离培养，共获得了11个属的21株真菌，分别是：青霉属（ Penicillium ，9株）、枝孢属（ Cladosporium ，3株）、支顶孢属（ Acremonium ，1株）、短梗霉属（ Aureobasidium ，1株）、孢盘菌属（ Botryotinia ，1株）、散囊菌属（ Eurotiales ，1株）、外瓶霉属（ Exophiala ，1株）、镰孢菌属（ Fusarium ，1株）、丛赤壳属（ Nectria ，1株）、茎点霉属（ Phoma ，1株）、帚枝霉属（ Sarocladium ，1株）.南极普里兹湾海域的真菌多样性较高，其中青霉属菌株最多，占总菌株比例为42.85%，是普里兹湾真菌中的优势属，其次为枝孢属菌株，占总菌株比例为14.29%.分别用尼罗红和氯化三苯基四氮唑（TTC）对这21株菌进行产油筛选，得到8株产油菌，分属于 Penicillium （2株）、 Aureobasidium （1株）、 Cladosporium （1株）、 Eurotiales （1株）、 Exophiala （1株）、 Fusarium （1株）、 Sarocladium （1株）等7个属，这些产油菌产油量大致在20.1%到52.0%之间.对这8株菌的产油量和胞内脂肪酸成分进行分析，结果表明：这些产油菌所含的基本为短链脂肪酸，且不饱和脂肪酸含量较高，适合作为生物柴油的生产菌株.

采用PCBs移除实验及其降解机理，分析研究从胶州湾海洋沉积物筛选的4株青霉真菌的好氧-厌氧降解的可能性，比较不同pH值、不同培养基条件对真菌生长的影响，获取真菌在好氧、厌氧条件下的生理学特性。结果表明，4株真菌更适合在海水MMM培养基上生长。pH值为6~8时，均能正常生长；在偏碱性pH值下，更适合形成菌落。通过测定细胞数目建立的真菌生长曲线表明4株菌株均可在好氧、厌氧条件下形成菌落，在好氧条件下生长比厌氧条件下生长更好，这为进一步深入研究PCBs降解奠定了实验依据和技术基础。4株海洋真菌在不同母液的MMM培养基上的生长状况、其对不同pH值的适应能力以及在好氧一厌氧条件下的生长曲线均反映了菌株海洋来源的特性。

研究了南极罗斯海地区沉积物中可培养微生物多样性 , 并检测了菌株胞外酶活性 , 为开发利用南极资源奠定基础。采用 Zobell 2216E 培养基、土豆培养基 (PDA) 及稀释平板划线法分离并获得纯培养的细菌和真菌 , 分别通过 16S rDNA 、 18S/ITS 序列鉴定分析了可培养微生物的多样性和系统发育。从南极罗斯海沉积物样品中获得 78 株细菌和 54 株真菌 , 细菌、真菌的优势菌株分别为嗜冷杆菌属 ( Psychrobacter ) 、枝孢属 ( Cladosporium ); 78 株细菌菌株中有 2 株与模式菌株的 16S rDNA 基因序列相似性小于 97%, 可能是潜在的细菌新物种。细菌 API20NE 生理生化和真菌产胞外酶活性结果表明 , 获得的细菌 and 真菌大多数可检测到胞外水解酶活性。本研究丰富了南极可培养微生物资源 , 为南极罗斯海地区可培养微生物的多样性研究以及南极微生物资源的应用研究奠定了基础。

Fungi play important roles, as both decomposers and plant symbionts in mangrove ecosystems. Their ability to survive extreme environmental conditions makes them potential rich sources for recovery of novel bioactive compounds. In this study, biotechnological potential of fungi recovered from the rhizospheres of four mangrove species (Sonneratia alba, Rhizophora mucronata, Ceriops tagal and Avicennia marina) was explored. Rhizospheric sediment samples of the mangroves were collected using standard protocols and different media used for isolation of fungi. A total of 33 fungal isolates were recovered and characterized based on morphological, physiological and Internal transcribed spacer (ITS) gene region analysis. The disk diffusion method was used to evaluate the antibacterial activity of the isolates. Morphologically, the isolates showed different characteristics with regard to color, margin and elevation. Physiologically, all the isolates were able to grow at different pH ranges, ranging from pH 4 to pH 12 and at different temperatures from 20 to 30°C and tolerated sodium chloride up to 7%. Phylogenetic analysis grouped the isolates into two phyla: Ascomycota and Basidiomycota affiliated to different genera; Penicillium, Aspergillus, Talaromyces, Diatrypella, Thielavia, Hypocreales, Paracremonium, Geosmithia, Peniophora, Massarina and Fulvifomes. A total of 17 representative isolates produced inhibition zones against three pathogenic bacterial strains. The findings demonstrate that the tropical mangroves rhizospheres are a rich source of fungi with antibacterial compounds and traits relevant for biotechnological application.   Key words: Antimicrobial activity, tropical marine fungi, Mangrove sediments, Internal transcribed spacer (ITS).

Abstract Objective To isolate and identify of marine fungal metabolites against clinical bacterial pathogens. To optimize the production medium for isolated fungus. Method Marine fungus isolated from water and sediment samples from different places of Sundarbans mangrove, Muttukadu (Chennai) and Parangipettai in India. Antimicrobial substance from marine fungi was produced by agar plate method. The potent fungal were inoculated on production medium and extracted was done. The extracted compound was checked for anti bacterial activity. Suitable production medium were optimized. Result Totally 30 fungal isolates were recovered and morphologically 10 different strains were belongs to the fungal genera such as Fusarium , Aspergillus , Mucor and Penicillium . Preliminary screening results showed 3 fungal isolates showed promising activity. After production of potent fungal SS2 crude extracts showed highest inhibition against the bacterial pathogens out of 3 fungal isolates. The results showed maximum zone in 20mm against E. coli and minimum 10 mm against Vibrio sp. Conclusions The present study identified Fusarium sp isolated from Sundarbans mangrove water as a potential source for bioactive compounds. Further isolation of active compound from potential fungal isolates will leads to the discovery of effective antimicrobials.

… a substrate by Penicillium fellutanum isolated from coastal mangrove sediment. The biosynthesis was … The present work highlighted the possibility of using the marine fungal strain of P. …

… fungi requires special methods and equipments. A variety of culture media is used to cultivate marine fungi… Hence, results obtained using such methods for marine fungi should be …

Despite the rapid development of molecular techniques relevant for natural product research, culture isolates remain the primary source from which natural products chemists discover and obtain new molecules from microbial sources. Techniques for obtaining and identifying microbial isolates (such as filamentous fungi) are thus of crucial importance for a successful natural products’ discovery program. This review is presented as a “best-practices guide” to the collection and isolation of marine fungi for natural products research. Many of these practices are proven techniques used by mycologists for the isolation of a broad diversity of fungi, while others, such as the construction of marine baiting stations and the collection and processing of sea foam using dilution to extinction plating techniques, are methodological adaptations for specialized use in marine/aquatic environments. To this day, marine fungi, Sensu stricto, remain one of the few underexplored resources of natural products. Cultivability is one of the main limitations hindering the discovery of natural products from marine fungi. Through encouraged collaboration with marine mycologists and the sharing of historically proven mycological practices for the isolation of marine fungi, our goal is to provide natural products chemists with the necessary tools to explore this resource in-depth and discover new and potentially novel natural products.

… In order to investigate the barotolerance of marine fungi and to elucidate their ecological … sensitive growth determination methods are used. All marine yeasts cultivated under simulated …

Polythene is the most widely used plastic around the globe. Among the total plastic waste generated, polythene contributes the maximum share (64%). Various strategies/methods are being utilized to deal with the increasing rate of plastic waste, but among all the methods, bioremediation is regarded as the ecofriendly and widely accepted method. In the current investigation, we have attempted to discover the elite polythene deteriorating fungi (isolated from the rhizosphere soil of Avicennia marina). From 12 different eco-geographical locations along the West Coast of India, total 109 fungal isolates were recorded. The polythene deteriorating fungi were screened at varied pH (3.5, 7 and 9.5) based on changes in weight and tensile strength of the treated polythene at ambient temperature with continuous shaking for 60 days. BAYF5 isolate (pH 7) results in maximum reduction in weight (58.51 ± 8.14) whereas PNPF15 (pH 3.5) recorded highest reduction in tensile strength (94.44 ± 2.40). Surprisingly, we have also reported weight gain, with highest percent weight gain (28.41 ± 6.99) with MANGF13 at pH 9.5. To test the reproducibility of the results, the elite polythene degrading fungal isolates based on weight loss and reduction in tensile strength were only used for repetition experiment and the results based on the reduction in tensile strength were found only reproducible. Polythene biodegradation was further confirmed using Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) analysis. The most efficient polythene deteriorating fungal isolates were identified as Aspergillus terreus strain MANGF1/WL and Aspergillus sydowii strain PNPF15/TS using both morphological keys and molecular tools.

Background Fungi play a crucial role in ecosystems, and they have been widely considered a promising source for natural compounds that are crucial for drug discovery. Fungi have a high diversity, but about 95% of them remain unknown to science. The description rate of fungi is very low, mainly due to the inability of most fungi to grow in artificial media, which could not provide a sufficiently similar environment to their natural habitats. Moreover, many species in nature are in a state of low metabolic activity which cannot readily proliferate without proper resuscitation. Previously developed culturomics techniques are mostly designed and applicable for bacteria, with few attempts for fungal isolation because of their significantly larger cell size and hyphal growth properties. Results This study attempted to isolate previously uncultured and rare fungi from mangrove sediments using newly developed fungal enrichment culture method (FECM) and fungal isolation chips (FiChips). Comparison of fungal community composition at different enrichment stages showed that FECM had great influence on fungal community composition, with rare taxa increased significantly, thus improving the isolation efficiency of previously uncultured fungi. Similarly, in situ cultivation using FiChips has a significant advantage in detecting and culturing rare fungi, as compared to the conventional dilution plate method (DPM). In addition, based on morphological comparisons and phylogenetic analyses, we described and proposed 38 new ascomycetous taxa, including three new families, eight new genera, 25 new species, and two new combinations (presented in additional file 1). Conclusions Our study demonstrated that mangrove sediments harbor a high diversity of fungi, and our new isolation approaches (FECM and FiChips) presented a high efficiency in isolating hitherto uncultured fungi, which is potentially usable for fungal isolation in other similar environments. Video Abstract

… isolation and characterization of fungi from different ecosystems like forests, mangroves and … The mangroves soil substrata are generally firm to soft mud with aerial roots known as …

Fusarium genus comprises important saprophytic and phytopathogenic fungi and is widespread in nature. The present study reports the occurrence of Fusarium spp. in soils from two mangrove forests in northern Peninsular Malaysia and analyzed physico-chemical properties of the mangrove soil. Based on TEF-1α sequences, nine Fusarium species were identified: Fusarium solani species complex (FSSC) (n = 77), Fusarium verticillioides (n = 20), Fusarium incarnatum (n = 10), Fusarium proliferatum (n = 7), Fusarium lateritium (n = 4), Fusarium oxysporum (n = 3), Fusarium rigidiuscula (n = 2), Fusarium chlamydosporum (n = 1), and Fusarium camptoceras (n = 1); FSSC isolates were the most prevalent. Phylogenetic analysis of the combined TEF-1α and ITS sequences revealed diverse phylogenetic affinities among the FSSC isolates and potentially new phylogenetic clades of FSSC. Soil analysis showed varied carbon content, pH, soil moisture, and salinity, but not nitrogen content, between sampling locations. Regardless of the physico-chemical properties, various Fusarium species were recovered from the mangrove soils. These were likely saprophytes; however, some were well-known plant pathogens and opportunistic human pathogens. Thus, mangrove soils might serve as inoculum sources for plant and human pathogenic Fusarium species. The present study demonstrates the occurrence of various Fusarium species in the extreme environment of mangrove soil, thereby contributing to the knowledge on species diversity in Fusarium.

… Previous research on bacterial isolation from mangrove soils indicated that microbial isolates have various potentials.Some fungi and bacteria isolated from mangrove soil in Cardoso …

… fungi using different culturing techniques and culture media … in underestimating fungal diversity in deep-sea sediments in … (2007) also reported low fungal diversity of uncultured fungal …

Abstract A fungal strain of Aspergillus niger was recovered from sediments collected in the Northeast coast of Brazil (Pecém’s offshore port terminal). Cultivation in different growth media yielded a new ester furan derivative, 1, along with malformin A1, malformin C, cyclo (trans-4-hydroxy-L-Pro-L-Leu), cyclo (trans-4-hydroxy-L-Pro-L-Phe), cyclo (L-Pro-L-Leu), cyclo (L-Pro-L-Phe), pseurotin D, pseurotin A, chlovalicin, cyclo (L-Pro-L-Tyr) and cyclo (L-Pro-L-Val). Compound 1 was cytotoxic against HCT-116 cell line, showing IC50 = 2.9 μg/mL (CI 95% from 1.8 to 4.7 μg/mL).

… the cultivable fungi present in deep marine sediments of the Antarctic Ocean and evaluated … Despite the variety of culture media used in the present study, there was a low frequency of …

Abstract In recent years, the Arctic has become a hot spot for microbial research. However, fungal diversity in the Chukchi Sea (adjacent to the Arctic Ocean) is little known. The purpose of this study was to explore fungal diversity in sediments of the Chukchi Sea and the temperature adaptability of the cultured fungi. A total of 74 fungal strains were isolated from nine marine sediment samples collected from the Chukchi Sea. These fungi were identified based on morphological characteristics and sequence comparison of the internal transcribed spacers of the ribosomal DNA (ITS-rDNA) with the GenBank reference sequence database. Amongst these fungi, 61 strains belonged to the Ascomycota and 13 strains were affiliated with the Basidiomycota. The dominant genus was Penicillium. These fungal strains were cultured at 4°C, 10°C, and 25°C to determine their temperature adaptability. Most strains were able to grow at 4°C, and Thelebolus ellipsoideus was identified as a psychrophilic fungus. Our results revealed a high fungal diversity in marine sediments of the Chukchi Sea and found that some fungal species (Penicillium chrysogenum, Eutypella scoparia, Alternaria alternata, Cladosporium tenuissimum, C. perangustums and Thelebolus ellipsoideus) isolated from these sediments were able to grow at 10°C and 4°C.

… This also applies to the presence of a large number of sterile and ambiguous cultures [27]. We discovered 32 species of fungi using morphological-cultural and molecular methods. The …

… baiting dilute nutrient medium with sediments and incubating … technique yielded fungi that showed good growth under low … detect one such fungus directly in the deep-sea sediments, a …

… Fungi were then isolated from sediments by a modified dilution plate technique from 1:10 up to 1:100. Fungal … increased knowledge of fungi from marine and dredged sediments in six …