Application of terminal restriction fragment length polymorphism (T-RFLP) analysis to monitor effect of biocontrol agents on rhizosphere microbial community of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi related T-RFs significantly decreased.     

Pod and Seed Mycoflora on Transgenic and Conventional Soybean [Glycine max (L.) Merrill] cultivars in Mississippi

A 2-year (1999-2000) study was conducted at Starkville and Stoneville, MS to determine if the occurrence of the mycoflora varied on Roundup Ready (transgenic) compared to conventional soybean (Glycine max) cultivars. A total of 7,658 fungal isolates were identified from the pod and seed tissues of four cultivars compared at growth stages R6 and R8. Ninety-nine percent of all fungi isolated were mitosporic fungi and ascomycetes. In both years, total fungal isolates from the two locations were greater from the pod (65%) than from seed (33%) tissues. Isolation frequency from conventional cultivars was 54% compared to 46% for the transgenic cultivars. The most common fungi identified that are reported pathogens of soybean included Alternaria, Cercospora, Cladosporium, Diaporthe, Fusarium and Verticillium spp. When main effects and interactions were compared among the frequency data for the fungal genera, significant differences occurred, but consistent trends were not noted. Isolation frequencies of Diaporthe spp. during the R6 growth stage, were significantly greater on the conventional than on the transgenic cultivars in both years of the study, but only at Starkville. Isolation frequencies from samples taken during the R8 growth stage were similar at both locations in 1999 and 2000. Fusarium spp. isolated at R6 and R8 growth stages from pod and seed tissues were significantly greater on conventional than on transgenic cultivars in 2000. Even though frequencies were often significantly different between the transgenic and conventional cultivars, the data was not consistent between locations, pod and seed tissues, or growth stages. The pod and seed mycoflora of transgenic and conventional soybean cultivars was, therefore, similar in Mississippi.     

Molecular Identification of Melanised Non-Sporulating Moulds: A Useful Tool for Studying the Epidemiology of Phaeohyphomycosis

Subcutaneous infections caused by melanised fungi have been increasingly reported among transplant patients, and these infections have the potential for blood and visceral dissemination. Some moulds, such as Mycelia sterilia, cannot grow and sporulate on different media, making their identification impossible by conventional methods. The fast and accurate identification of melanised fungi at the species level is important because species may have tropism to different organs and different susceptibilities to antifungal agents. Molecular tools have been reported to be helpful for the species identification of non-sporulating moulds. Our goal was to identify the species of M. sterilia isolates obtained from clinical samples of transplant patients using sequences of ITS and the D1/D2 regions of rDNA. Clinical samples were obtained from eight kidney transplant recipients who developed subcutaneous fungal infections. The diagnosis was confirmed by histopathology and conventional culture. Histopathology showed septated, melanised hyphae, and the cultures identified non-sporulating fungi. Therefore, the DNA from the M. sterilia isolates was subjected to PCR amplification and sequencing of the ITS and D1/D2 regions. Genus/species identification was obtained by comparison with gene banks. We obtained the following identifications: Alternaria sp. (2), Cochliobolus lunatus/Curvularia lunata (2), Cochliobolus hawaiiensis/Bipolaris hawaiiensis (1), Ochroconis sp. (1), Medicocopsis romeroi/Pyrenochaeta romeroi (1) and Nigrograna mackinnonii/Pyrenochaeta mackinnonii (1).     

Determination of microbial diversity in meju,fermented cooked soya beans,using nested PCR‐denaturing gradient gel electrophoresis

Aims: To identify the microbiota in meju, fermented cooked soya beans, that may directly affect the microbial communities of Korean fermented soya bean foods. Methods and Results: Using conventional bacterial 16S rDNA, bacilli‐specific 16S rDNA or fungi 18S rDNA‐specific primers, PCR products were amplified through a series of PCRs using the DNA extracted from ten meju samples. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE), which showed that Enterococcus durans was commonly detected in nine of ten meju samples. Bacillus subtilis was shown to be the major strain of bacilli in the samples tested. Based on the DGGE analysis of fungi in meju, we determined that Absidia corymbifera, Aspergillus sp. and Candida rugosa were the main fungi in the tested samples. Conclusions: A variety of bacterial and fungal micro‐organisms were identified in meju samples, in addition to the micro‐organisms already known to be present. Significance and Impact of the Study: This is the first report showing the differences and similarities in the populations of micro‐organisms in meju samples using nested PCR‐DGGE, a culture‐independent method. The results may be applicable to the development of improved meju, in which the indigenous micro‐organisms required for fermentation can be standardized.     

Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.     

不同耕作措施对土壤真菌群落结构与生态特征的影响

以传统耕作（T）为对照,研究了免耕秸秆覆盖（NTS）、免耕（NT）、秸秆还田（TS）3种保护性耕作措施对黄土高原西部雨养农业区耕层土壤真菌数量、垂直分布及群落结构的影响。结果表明：不同耕作措施对土壤真菌数量有一定影响,耕层土壤真菌总数．TS〉T,NTS〉NT,T〉NT;耕作措施对真菌群落结构的影响比较明显,3种保护性耕作处理其土壤均以绿僵菌属（Metarrhizium）、腐霉属（Phythium）、曲霉属（Aspergillus）占绝对优势,B-P优势度指数三者之和都在0．6左右;而T处理占绝对优势的菌属是绿僵菌属（Metarrhizium）和镰刀菌属（Fusarium）,B—P优势度指数二者之和在0．7左右;土壤真菌群落的多样性指数和均匀性指数均呈现NTS〉TS〉T〉NT。因此,在黄土高原西部雨养农业区实施NTS和TS耕作措施,既有利于土壤有益真菌数量的增加,提高养分转化率,又有利于促进该区农业的可持续发展。     

Occurrence of fungi in water used at a haemodialysis centre

Aims: The aim of this study was to identify and determine the diversity, occurrence and distribution of fungi in water used at a haemodialysis centre. Methods and Results: Samples in the hydraulic circuit for the distribution of the water, dialysate samples and samples of sterilization solution from dialysers were collected over a 3‐month period, and 500 ml of each sample was filtered through membranes. All together 116 isolates of fungi were recovered from 89% of all water samples collected inside the haemodialysis unit, with prevalence of moulds in tap water samples and of yeasts in dialysate samples. Fusarium spp. was the most abundant genus found, whereas Candida parapsilosis was the predominant yeast species. Conclusions: This study demonstrated that various fungi were present in the water system. These data suggest the inclusion of the detection and quantification of fungi in the water of haemodialysis. Significance and Impact of the Study: The recovery of fungi from aqueous haemodialysis environments implies a potential risk for haemodialysis patients and indicates the need for continuous maintenance and monitoring. Further studies on fungi in haemodialysis water systems are required to investigate the organism ability to persist, their role in biofilm formation and their clinical significance.     

Ecological and physiological studies on fungi associated with human hair

Thirty-seven species attributed to 19 genera of keratinophilic fungi were recovered from 100 human hair samples collected from the Assiut governorate. The genera Aspergillus followed by Penicillium and Chrysosporium were frequently isolated from 65, 43 and 30% of the samples respectively. Fifteen species and 13 genera of thermophilic and thermotolerant fungi (recovered at 45 degrees C) were identified. The thermotolerant Aspergillus fumigatus was frequently encountered and emerged from 82% of the samples. Thirteen isolates of keratinophilic and 20 isolates of thermophilic fungi were tested for lipolytic and proteolytic activities. All the keratinophilic fungi showed lipolytic and proteolytic activities while 100 and 85% of the thermophilic fungi showed lipolytic and proteolytic activities. Using the paper-disc plate method, 12 types of shampoos and oils were tested for their antifungal activities on 42 strains of keratinophilic and thermophilic or thermotolerant fungi. Three out of four types of shampoo proved to be highly effective against all the test fungi.     

湘西地区蛇足石杉内生真菌的分离

采集不同生境的药用植物蛇足石杉,运用不同灭菌方法处理外植体,并用PDA平板培养基及孟加拉红培养基进行内生真菌的分离纯化.经过几种灭菌方法的摸索和比较研究,发现蛇足石杉外植体的灭菌十分困难(尤其是其茎部),难以在保持植物鲜活状态下达到一次彻底灭菌,有必要二次灭菌.通过二次灭菌分离得到的9株内生真菌中,来自蛇足石杉茎部的6株,来自叶片的3株.蛇足石杉的9株内生真菌在平板培养时生长极其缓慢,仅一株可在培养时产生孢子.     

不同产地杜仲树皮可培养内生真菌群落组成和多样性

以湖南、四川、贵州、河南、安徽、江西和陕西7个产地的杜仲Eucommia ulmoides为研究对象,采用可培养法对树皮内生真菌进行分离培养,结合形态学和分子生物学进行鉴定,并分析不同产地杜仲内生真菌群落组成和多样性差异。结果表明,不同产地杜仲树皮中共分离到真菌545株,隶属于7纲、15目、27科、43属,其中间座壳属Diaporthe为7个产地的共有属,各地优势属不同。在属水平上,四川样本中真菌群落多样性最高,河南样本真菌群落多样性最低。Margalef丰富度指数及Pielou均匀度指数表明,在河南样本中的内生真菌分布最均匀,丰富度最低;贵州样本内生真菌丰富度最高,但最不均匀。相似性分析显示不同产地杜仲树皮内生真菌组成和多样性不同。这些结果揭示了杜仲树皮中内生真菌菌群具有丰富的多样性,7个不同产地的杜仲树皮内生真菌群落组成、相对多度及优势类群皆存在差异。     

Occurrence and diversity of entomopathogenic fungi in cultivated and uncultivated soils in Pakistan

The distribution of entomopathogenic fungi in various geographical areas of Punjab, Pakistan, is poorly understood. The present study was planned to explore the occurrence and diversity of entomopathogenic fungi from soils collected from cultivated and non‐cultivated habitats. The detailed survey of different habitats (crop fields, fruits, vegetables, forests) was conducted to collect soil samples and the associated fungi were isolated using Galleria bait method. Among 210 soil samples, 168 fungal isolates were recovered and identified, with 98 from forests, 32 from vegetables, 30 from field crops and 8 from fruits. The major entomopathogenic fungi recovered from these samples were Beauveria bassiana, Metarhizium anisopliae, Paecilomyces lilacinus, B. brongniartii, P. chlamydosporia and Lecanicillium attenuatum. The diversity of entomopathogenic fungi was greater in soil samples from forests compared to crop fields, vegetables and fruits, respectively. The geographical attributes (altitude, longitude, latitude) greatly influenced the occurrence of entomopathogenic fungi with the highest number of isolates found from >600 m altitude, 33°–34′N latitude, and 73°74′E longitude from soil samples. The results of the surveys clearly indicated that the entomopathogenic fungi are distributed in the soils which may be used in successful IPM programs in future.     

Diversity of keratinophilic mycoflora in the soil of Agra (India)

Diversity of keratinophilic mycoflora in the soil of Agra was under observation for 1 year (July 2001-June 2002) and isolation of keratinophilic fungi was followed by the hair-baiting method. The frequency of occurrence of keratinophilic fungi in 284 soil samples collected from various hospitals, cattle yards, poultry farms, crop fields and playgrounds was determined, 204 samples (72%) having been found to be positive. A total of 33 species classified into 11 genera (Acremonium, Aspergillus, Chrysosporium, Emmonsia, Geomyces, Keratinophyton, Microsporum, Myceliophthora, Penicillium, Sporotrichum, Trichophyton) were encountered from the soil samples. Sporotrichum spp. were found to be the most dominant species followed by Trichophyton simii. The parameter of keratinophilic fungi found in the samples studied ranged from 62 to 80% where playgrounds yielded the maximum number of species (80%) while the least dominating soil was hospital soil (62%). Among all the baits used maximum fungi occurred on human hairs (82%) followed by chicken feather (74%), wool (61%) and the least on horns (45%). The spectrum of keratinophilic fungi isolated from different sites differed considerably according to the frequency of use by humans.     

城市供水系统对水中真菌数量和群落结构的影响

【背景】对于饮用水中的微生物污染,绝大多数研究集中在细菌、病毒及原虫和蠕虫。由于真菌中包含多种潜在致病菌,应当引起人们的关注。【目的】研究城市供水系统中真菌数量、群落组成变化及可能存在的潜在致病真菌。【方法】利用MEA和RB两种培养基,对供水系统中的原水和两个水厂净水工艺过程及不同供水模式用户龙头水样进行培养法计数统计。提取上述样本总DNA,并应用Illumina MiSeq平台进行ITS1区高通量测序。【结果】从15个样本中共获得有效序列579 470条,1 260个OTU,包含8个门26个纲67个目228个属的真菌。门水平上子囊菌门真菌为供水系统优势真菌,属水平上不同样本存在差异,但曲霉属和枝顶孢属真菌在所有样本中均存在;培养法和高通量测序结果共同显示,活性炭过滤出水真菌数量和物种丰富度均较前一工艺有所上升;氯化消毒对真菌数量、物种多样性及物种组成影响显著;经过供水管网输配和二次供水设施后,用户龙头水样本真菌数量和物种丰富度明显高于出厂水。【结论】供水系统中的优势真菌为子囊菌门(Ascomycota),该门真菌可穿透净水工艺过程的多级屏障;水厂净水工艺能有效去除水中可培养真菌,生物活性炭过滤工艺能够影响真菌数目及物种多样性;供水管网及二次供水设施是末端饮用水中真菌污染的重要来源;供水系统中存在潜在致病真菌。     

Air- and dustborne mycoflora in houses free of water damage and fungal growth

Typically, studies on indoor fungal growth in buildings focus on structures with known or suspected water damage, moisture, and/or indoor fungal growth problems. Reference information on types of culturable fungi and total fungal levels are generally not available for buildings without these problems. This study assessed 50 detached single-family homes in metropolitan Atlanta, Ga., to establish a baseline of "normal and typical" types and concentrations of airborne and dustborne fungi in urban homes which were predetermined not to have noteworthy moisture problems or indoor fungal growth. Each home was visually examined, and samples of indoor and outdoor air and of indoor settled dust were taken in winter and summer. The results showed that rankings by prevalence and abundance of the types of airborne and dustborne fungi did not differ from winter to summer, nor did these rankings differ when air samples taken indoors were compared with those taken outdoors. Water indicator fungi were essentially absent from both air and dust samples. The air and dust data sets were also examined specifically for the proportions of colonies from ecological groupings such as leaf surface fungi and soil fungi. In the analysis of dust for culturable fungal colonies, leaf surface fungi constituted a considerable portion (>20%) of the total colonies in at least 85% of the samples. Thus, replicate dust samples with less than 20% of colonies from leaf surface fungi are unlikely to be from buildings free of moisture or mold growth problems.     

Fungal community analysis by large-scale sequencing of environmental samples

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized. All sequences were deposited in GenBank, with accession numbers AY 969316 to AY 970290 for the ITS sequences and AY 969135 to AY 969315 for the SSU sequences.     

Air- and Dustborne Mycoflora in Houses Free of Water Damage and Fungal Growth

Typically, studies on indoor fungal growth in buildings focus on structures with known or suspected water damage, moisture, and/or indoor fungal growth problems. Reference information on types of culturable fungi and total fungal levels are generally not available for buildings without these problems. This study assessed 50 detached single-family homes in metropolitan Atlanta, Ga., to establish a baseline of “normal and typical” types and concentrations of airborne and dustborne fungi in urban homes which were predetermined not to have noteworthy moisture problems or indoor fungal growth. Each home was visually examined, and samples of indoor and outdoor air and of indoor settled dust were taken in winter and summer. The results showed that rankings by prevalence and abundance of the types of airborne and dustborne fungi did not differ from winter to summer, nor did these rankings differ when air samples taken indoors were compared with those taken outdoors. Water indicator fungi were essentially absent from both air and dust samples. The air and dust data sets were also examined specifically for the proportions of colonies from ecological groupings such as leaf surface fungi and soil fungi. In the analysis of dust for culturable fungal colonies, leaf surface fungi constituted a considerable portion (>20%) of the total colonies in at least 85% of the samples. Thus, replicate dust samples with less than 20% of colonies from leaf surface fungi are unlikely to be from buildings free of moisture or mold growth problems.     

垃圾填埋场中厌氧真菌18SrDNA的PCR扩增及鉴定

采用机械破壁法直接从来自7个不同地区的垃圾填埋场滤液样本中提取真菌DNA,应用真菌通用引物NS1和NS8扩增18SrDNA（约1800bp),多聚酶链式反应（PCR）产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增;以PCR产物作为模板,采用厌氧真菌Chytridiomycetes科的专用引物Chyt-719和Chyt-1553进行二次PCR扩增（约857bp),该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中,且为Neocallimastix属。     

浙江省仓贮稻谷大米真菌区系的研究

在浙江省采集仓贮稻谷及大米样品,共分离到真菌17属48种(不包括酵母菌)。其中:曲霉属(Aspergillus)18种,青霉属(Penicillium)10种,镰孢霉属(Eusarium)4种。研究表明:在浙江省地区之间,贮粮真菌种类无明显差异。稻谷加工成大米后,籽粒表面带菌量及内部带菌率均明显下降,其真菌区系以贮藏真菌为主。稻谷在贮藏前以田间真菌为主。贮藏1—4个月的稻谷因贮藏真菌的种类、数量明显增长,而田间真菌的种类和数量仍保持在较高的水平上,其表面带菌量及谷粒带菌率处于高峰期;贮藏1年以上的稻谷,其主要带菌种类为贮藏真菌,带菌量及带菌率明显下降。早籼谷和晚粳谷在相同贮藏条件下其带菌种类和数量基本一致。     

Investigation of keratinophilic fungi from soils in Western Australia a preliminary survey

In order to determine which species of geophilic dermatophytes were present in Western Australian soils 299 samples were investigated. These samples were collected from a range of locations, 208 from towns throughout the state and 91 samples from the Perth Metropolitan area.Most samples were collected from areas frequented by people and animals, such as home gardens, parks and animal yards.Of the total 299 soils, 271 (90.6 %) yielded keratinophilic fungi. A total of 181 dermatophytes were isolated, and there were 205 isolations of other keratinophilic fungi. Microsporum gypseum (30.7 %) was the most prevalent dermatophyte recovered from soil followed byMicrosporum cookei (21.7 %) and thenTrichophyton ajelloi (8.0 %). No other dermatophytes were recovered.Chrysosporium indicum was the most common of all the keratinophilic fungi and was isolated from 50.1 % of the samples. Mixed growth was obtained from 33.5 % of the soil samples.