Screening of endophytic bacteria against fungal plant pathogens

Bacterial endophytes were found from 6 plant leaves among 35 plant leaves screened. Two of the isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1 showed the clear inihibition against plant pathogens, Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. Supernatant of the bacterial culture also showed the clear inhibition against the fungal growth on the plate and the antibiotic substance was identified as iturin A by HPLC analysis. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. Very thin hyphae of R. solani was miccroscopically observed when the fungus was co-cultivated with KL1.     

Isolation of antifungal bacteria from Japanese fermented soybeans, natto

An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.     

Isolation of antifungal bacteria from Japanese fermented soybeans,natto

An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.     

Aqueous and organic extracts of Trigonella foenum-graecum L. inhibit the mycelia growth of fungi

Aqueous extracts from various plant parts of fenugreek (3%) (aerial parts: leaves and stems (LS), roots (R), ground seeds (GS) and not ground seeds (NGS)) and petroleum ether, ethyl acetate and methanolic fractions of the aerial parts were assayed to determine their antifungal potential against Botrytis cinerea, Fusarium graminearum, Alternaria sp., Pythium aphanidermatum, and Rhizoctinia solani. All fenugreek plant parts showed antifungal potential and the magnitude of their inhibitory e ects was species and plant parts dependent. R extract was shown less toxic (30.38%), whereas NGS extract expressed the strongest inhibition, with an average of 71.44%, followed by GS (58.56%) and LS (57.1%). Screening indicated that P. aphanidermatum was the most resistant species, with an average inhibition of 34.5%. F. graminearum, Alternaria sp. and R. solani were the most sensitive species, and were similarly inhibited (63.5%). The stability test indicated that the aqueous extracts of all plant parts lost approximately 50% of their relative activity after one month of storage at 4°C, whilst they lost 60%–90% of their activity when stored at ambient temperature for one month. The antifungal activity resided mainly in the methanol fraction and the minimum inhibitory concentration (MIC) of methanol fraction witch caused total inhibition of R. solani and Alternaria sp. was 60 g/ml. Results of current study suggested that the constituents of Trigonella foenum-graecum have potential against harmful pathogenic fungi. Therefore, fenugreek could be an important source of biologically active compounds useful for developing better new antifungal drugs.     

Reduced Chitinase Activities in Ant Plants of the Genus Macaranga

Many plant species have evolved mutualistic associations with ants, protecting their host against detrimental influences such as herbivorous insects. Letourneau (1998) reported in the case of Piper that ants defend their plants principally against stem-boring insects and also reduce fungal infections on inflorescences. Macaranga plants that were experimentally deprived of their symbiotic Crematogaster ants suffered heavily from shoot borers and pathogenic fungi (Heil 1998). Here we report that ants seem to reduce fungal infections actively in the obligate myrmecophyte Macaranga triloba (Euphorbiaceae), while ant-free plants can be easily infected. We also found extremely low chitinase activity in Macaranga plants. The plants' own biochemical defense seems to be reduced, and low chitinase activity perhaps may represent a predisposition for the evolution of myrmecophytism. These plants are therefore highly dependent on their ants, which obviously function not only as an antiherbivore defense but also as an effective agent against fungal pathogens. Received: 5 August 1998 / Accepted in revised form: 18 November 1998     

Impact of biocide treatments on the bacterial communities of the Lascaux Cave

The Lascaux Cave contains a remarkable set of paintings from the Upper Palaeolithic. Shortly after discovery in 1940, the cave was modified for public viewing and, in 2001, was invaded by a Fusarium solani species complex. Benzalkonium chloride was used from 2001 to 2004 to eliminate the fungal outbreak. In this study, we carried out a sampling in most of the cave halls and galleries. Sequence analysis and isolation methods detected that the most abundant genera of bacteria were Ralstonia and Pseudomonas. We suggest that, as a result of years of benzalkonium chloride treatments, the indigenous microbial community has been replaced by microbial populations selected by biocide application.     

铜尾矿白羊草重金属含量对叶际和根际真菌群落的影响

植物的叶际和根际微生物与宿主植物之间存在复杂的相互关系,能促进植物的生长,增强宿主植物对逆境胁迫的耐受性.本实验选择铜矿区主要恢复植被白羊草为研究对象,采用高通量测序的方法,分析白羊草叶际和根际真菌群落结构与多样性差异,探究白羊草叶片和根部富集的重金属含量对叶际和根际真菌群落特征的影响.结果表明,在白羊草的根际和叶际区域,子囊菌门（Ascomycota）和担子菌门（Basidiomycota）均为优势菌门.白羊草根际真菌群落的多样性和丰富度显著高于叶际真菌.白羊草植株叶际和根际真菌多样性受到不同重金属的影响,白羊草叶际多样性主要受到叶片Zn和Cu含量的影响,而根Pb含量是影响根际真菌群落多样性的关键因子.此外,叶际子囊菌门的孢菌科与叶片Cd含量有极显著正相关关系,根际丛赤壳科与根Zn含量显著正相关,可作为重金属污染区域生态恢复的指示菌种.本研究为铜尾矿生态恢复过程中发掘和利用叶际或根际真菌资源提供科学依据,也为筛选重金属污染区生态修复的植物-微生物共生体提供基础.     

合肥市夏季大气颗粒物中微生物群落的高通量测序分析

大气颗粒物的组分、来源和时空变化特征等方面已进行了广泛的研究,但对占大气颗粒物25%的生物气溶胶中细菌和真菌等微生物群落的研究较少.本文使用电感耦合等离子体质谱仪（ICP-MS）和离子色谱仪（IC）分别测定了大气颗粒物中痕量元素和水溶性离子的含量,并结合高通量测序和荧光定量PCR分析了合肥市7~9月大气颗粒物PM_1.0、PM_2.5和PM₁₀中微生物群落组成及其来源.结果表明,不同粒径下细菌群落多样性无显著差异（ANOVA,P>0.05）,雨天细菌和真菌群落多样性均高于晴天,所有样品中细菌群落多样性均显著高于真菌群落多样性（ANOVA,P<0.01）;大气中细菌的优势菌门为变形菌门（46.19%）、厚壁菌门（33.42%）、拟杆菌门（10.99%）、蓝藻门（3.33%）和放线菌门（2.11%）,真菌的优势菌门为子囊菌门（73.23%）、担子菌门（5.78%）、被孢霉菌门（3.41%）和毛霉门（0.10%）;通过分析潜在源环境的指示物种,发现土壤、植物叶片和动物粪便是合肥市大气中细菌群落的主要来源,真菌群落的主要来源是植物叶片和土壤;细菌群落主要受K、Pb、Al、Fe、Mg、Ca、Na⁺、NO₂^-和风速（WS）影响,而真菌群落的主要影响因素是V、Mn、Sr、NO₂^-、NO₃^-、Na⁺、Cl^-、空气质量指数（AQI）和PM₁₀;此外,在细菌和真菌群落分析中鉴定到了不动杆菌属、链球菌属、肠杆菌属、假单胞菌属、代夫特菌属、沙雷氏菌属、木霉属、链格孢属和曲霉属等9种致病菌,它们可以导致人类和其它生物的多种疾病.本文的研究结果有助于揭示大气微生物的各种特性及其影响因素,以及对人类健康的影响,对后续研究和政府相应政策的制定具有重要的参考价值.     

Extraction and quantification of "condensed tannins" as a measure of plant anti-herbivore defence? Revisiting an old problem

Contents of phenolic compounds in leaf extracts often serve as a measure of plant anti-herbivore defence. This method suffers from the multifunctionality of phenolics and from problems with their colorimetric quantification. Here we present further evidence for the pertinence of these problems. Contents of condensed tannins (CCT) were spectrophotometrically quantified in leaf extracts of 11 closely related mimosoid species, and Spodoptera littoralis caterpillars were reared on artificial diet containing these extracts. The relationship of CCT with caterpillar growth differed considerably among plant species, since both positive and negative correlations were detected. There was, however, a negative correlation of CCT with fungal spore germination, indicating a role of these compounds in resistance to fungi. Detailed knowledge on the structure and biological function of defensive compounds and on the overall composition of leaves is required to estimate a plant's defensive efficacy against a particular group of enemies.     

攀枝花矿区糙野青茅根际耐铬放线菌筛选及促生能力评价

从攀枝花矿区糙野青茅根际土中分离放线菌,筛选获得对重金属铬具有强耐受性、抗菌促生和产胞外酶能力的放线菌,为微生物-植物联合修复重金属污染提供菌种资源.采用稀释涂布法进行放线菌的分离,通过放线菌在不同铬浓度高氏一号培养基上的生长状况判断其对重金属铬的耐受性,进一步采用原子吸收分光光度法分析菌株对铬耐受性强的菌株耐铬及去铬能力;通过对菌株产铁载体、溶磷、产吲哚乙酸(IAA)、产纤维素酶、产淀粉酶、产木糖酶、产酪蛋白酶能力及抗菌活性的筛选,综合评价菌株抗菌促生能力.结果显示,分离获得的27株根际菌株,光学显微形态及扫描电镜结果均显示为放线菌,其中有14株能在含铬的培养基上生长,具有耐铬能力.16S rRNA基因测序结果显示,14株放线菌分属于链霉菌属(streptomyces)、小单孢菌属(Micromonospora)和冢村氏菌属(Tsukamurella).菌株SCAU9002和SCAU9008在铬浓度为300mg·L~(-1)的处理下培养6 d,去铬率分别达到81.17%和78.41%;菌株SCAU9002、SCAU9006、SCAU9008、SCAU9009、SCAU9013同时具有3种促生功能,盆栽实验结果进一步表明,在重金属胁迫下,经菌株处理后的玉米植株更高,生物量更大,根系更发达,促生效果较好.此外,菌株SCAU9006对6种病原菌具有拮抗作用,5株菌同时具有3种及以上产胞外水解酶的能力,4株菌能在7%的NaCl浓度下生长,13株菌在pH5.0~8.0的范围内均可生长.结果表明,根际放线菌在促进矿区植物生长和铬尾矿污染土壤的生物修复研究中具有很大潜力.     

三江平原不同退化阶段小叶章湿地土壤真菌群落结构组成变化

为了了解三江平原小叶章湿地真菌群落结构及多样性变化,探讨湿地水分下降对真菌群落结构的影响.本研究采用高通量测序技术,分析和比较了黑龙江省科学院自然与生态研究所三江平原野外试验研究站内3个不同退化阶段小叶章生态类型湿地真菌群落结构多样性,探讨真菌群落改变的原因.结果表明,从小叶章沼泽湿地(w0)→小叶章沼泽化草甸湿地(w1)→小叶章草甸湿地(w2)的变化,土壤Shannon-Wiener指数(H)呈现出逐步上升的规律,即:w0w1w2.序列比对结果显示,不同小叶章湿地土壤真菌分别属于子囊菌(Ascomycota)、担子菌(Basidiomycota)、壶菌(Chytridiomycota)、未知菌(Fungi_unclassified)、接合菌(Zygomycota)这5个真菌类群,优势菌门为未知菌门(75.12%)、子囊菌门(56.56%)、担子菌门(72.65%).而且Heatmap分析也显示沼泽化草甸湿地和草甸湿地的真菌群落结构和组成比较接近.真菌群落结构受到了土壤养分以及植物组成等影响,其中土壤养分解释贡献率达88.62%,植物组成解释贡献率达到9.85%.三江平原小叶章湿地不同退化阶段湿地真菌群落结构存在较大差异,土壤水分因子对真菌多样性影响较大.这为研究三江平原湿地退化后的真菌结构多样性及空间异质性提供了科学依据.     

嘉陵江滨岸带不同土地利用类型土壤真菌群落结构与功能多样性

通过明确嘉陵江滨岸带不同土地利用类型土壤真菌群落多样性的差异,为嘉陵江生态环境保护修复提供理论依据.选择嘉陵江中下游滨岸带的人工湿地、天然湿地、林地和农田这4种典型土地利用类型作为研究样地,利用高通量测序技术对土壤真菌群落进行测序,分析不同土地利用类型对土壤真菌群落多样性、结构和功能的影响.结果表明,林地与天然湿地土壤真菌的Chao1指数显著高于其他两种土地利用类型（P<0.05）,林地土壤真菌的Shannon指数显著高于农田和人工湿地两种土地利用类型（P<0.05）;嘉陵江流域滨岸带土壤真菌被划分为15个菌门,在门分类水平上的优势真菌群落（相对丰度>0.01）分别为：子囊菌门（Ascomycota）、担子菌门（Basidiomycota）、油壶菌门（Olpidiomycota）、罗兹菌门（Rozellomycota）、被孢霉门（Mortierellomycota）和壶菌门（Chytridiomycota）;相比之下,罗兹菌门偏好选择林地生境,而油壶菌门和被孢霉门偏好选择农田,担子菌门在人工湿地具有显著优势;天然湿地中的优势功能类群为植物病原菌,人工湿地中的优势功能类群为粪腐菌,农田中优势功能类群为动物病原菌-真菌寄生菌;冗余分析表明,土壤含水率（MC）、全氮（TN）、有机碳（TOC）和碱解氮（AN）是影响真菌群落变化的主要环境因子.由此可见,林地是嘉陵江流域真菌多样性最高和功能类群最均衡的土地利用类型,其次为天然湿地,人为干扰导致嘉陵江滨岸带土壤真菌群落多样性水平降低.     

小麦与不同作物多样化轮作对土壤真菌群落的影响

为明确华北平原冬小麦与不同作物多样化轮作下的土壤真菌群落差异,为生态可持续种植制度的构建和优化提供理论依据,采用实时荧光定量PCR和高通量测序技术研究了连续冬小麦-夏玉米M、冬小麦-夏花生(夏玉米)PM和冬小麦-夏大豆(夏玉米)SM轮作处理的土壤真菌群落丰度、组成和多样性.结果表明,与连续冬小麦-夏玉米处理相比,轮作花生PM2和大豆SM2处理显著降低了土壤真菌ITS序列拷贝数,轮作花生或大豆处理增加了土壤真菌群落丰富度和多样性.非度量多维尺度分析(NMDS)结果显示,不同轮作茬口之间土壤真菌群落存在明显分离,轮作作物对土壤真菌群落结构的影响达显著水平.华北平原砂壤质潮土中不同作物轮作处理的土壤真菌群落均以Ascomycota为优势菌门,以Sordariomycetes和Eurotiomycetes为优势菌纲.不同轮作茬口土壤真菌群落组成存在差异显著的类群,Neocosmospora、Plectosphaerella和Gibellulopsis等潜在病原真菌在冬小麦-夏花生(夏玉米)轮作处理中显著富集,而Penicillium和Zopfiella等潜在有益真菌在冬小麦-夏大豆(夏玉米)轮...     

岩性对喀斯特灌丛土壤固氮菌与丛枝菌根真菌群落结构及丰度的影响

运用末端限制性片段长度多态性(terminal restriction fragment length polymorphism,T-RFLP)和荧光定量PCR(real-time PCR)法,检测喀斯特灌丛生态系统中不同岩性条件下土壤固氮菌与丛枝菌根(arbuscular mycorrhizal,AM)真菌群落结构与丰度的变化,揭示岩性对灌丛生态系统土壤中固氮菌与AM真菌群落结构与丰度的影响.结果表明不同岩性条件下,土壤固氮菌与AM真菌丰度存在显著差异,其中,固氮菌与AM真菌丰度在石灰岩土壤中最大,白云岩土壤中最小,石灰岩-白云岩夹层土壤介于两者之间;同样,不同岩性土壤中固氮菌与AM真菌群落结构存在显著性差异.土壤Olsen-P、有机碳、黏粒含量与固氮菌丰度存在显著正相关关系,而土壤全氮、黏粒含量与AM真菌丰度存在显著正相关.RDA分析表明,植物均匀度影响固氮菌群落组成结构,而植物均匀度、香农多样性指数及丰富度指数影响AM真菌群落组成结构.以上的研究结果表明:岩性主要是通过影响植物与土壤养分来影响土壤固氮菌与AM真菌群落组成结构及丰度.     

黄土丘陵区退耕还林对土壤真菌群落的影响

退耕还林显著影响了黄土高原土壤细菌群落结构与组成特征.然而,作为土壤微生物群落的重要组成部分,黄土高原土壤真菌落结构与组成特征如何响应退耕还林并不十分清楚.以山西省右玉县贾家窑黄土高原缓坡丘陵为研究区域,以耕地(玉米地和糜子地)和人工林地(沙棘林和柠条林)土壤为研究对象,通过18S rRNA高通量测序技术,研究了退耕还林对土壤真菌群落结构与组成特征的影响.结果发现,黄土高原丘陵区耕地和林地土壤中子囊菌门(Ascomycota)占绝对优势(61.03%～78.06%),毛霉门(Mucoromycota)次之(12.11%～17.13%),担子菌门(Basidiomycota)再次之(6.15%～18.42%),壶菌门(Chytridiomycota)等其余真菌门的相对丰度较低.退耕还林显著降低了子囊菌门的相对丰度而增加了担子菌门的相对丰度.属分类水平下,退耕还林显著影响了毛壳菌属(Chaetomium)、被孢霉属(Mortierella)、镰刀菌属(Fusarium)和地舌菌属(Geoglossum)等的丰度.镰刀菌属(Fusarium)等植物病原性真菌在耕地土壤中被富集.非度量多维尺度...     

Interactive effects of ozone and powdery mildew (Sphaerotheca fuliginea) on bottle gourd (Lagenaria siceraria)

Interacting effects of ozone at 0.05, 0.1 and 0.2 ppm and powdery mildew infection were studied with respect to the growth, flowering and fruit-setting of bottle gourd, and ozone injury, fungal colonization, conidia size and germination. Intermittent exposure in closed-top chambers to ozone at 0.1 or 0.2 ppm, and infection by Sphaerotheca fulginea, each caused significant suppressions of plant growth, flowering and fruit-setting. Fungal colonization was increased by exposures to 0.05 ppm ozone, but decreased by 0.2 ppm ozone. Less browning and necrosis (symptoms of ozone phytotoxicity) appeared on the leaves of mildew-inoculated plants exposed to ozone at 0.1 and 0.2 ppm. Conidia collected from plants exposed to 0.1 and 0.2 ppm were smaller, contained fewer fibrosin bodies and showed poor germination in-vitro. Fumigations with ozone at 0.05 ppm increased the germination of conidia collected from the exposed plants or of the conidia directly exposed to ozone on glass slides in microgas exposure cabinets. Distortion and wall rupturing of the conidia were induced by exposure to ozone at 0.2 ppm for 12 h. Ozone at 0.05 ppm and S. fuliginea jointly suppressed the root and shoot growth of bottle gourd significantly more than the sum of individual effects (synergistic interaction). The fungus infection partially protected the plants from injury by ozone at 0.2 ppm, and exposure to ozone at 0.2 ppm inhibited fungal development. Antagonistic reductions of damage to bottle gourd growth by simultaneous mildew infection and ozone at 0.2 ppm were observed. The study revealed that powdery mildew infection may become severe on bottle gourd grown in areas polluted with mild level of ozone (0.05 ppm) whereas, at 0.1 ppm O₃ or more, the disease will be suppressed.     

生物炭和秸秆还田对微咸水滴灌棉田土壤真菌群落结构多样性的影响

微咸水灌溉增加土壤盐分,改变土壤环境,进而影响土壤真菌的结构和多样性.在长期微咸水灌溉的基础上,分别添加生物炭和秸秆（采用等碳量设计,分别为3.7 t·hm^-2和6 t·hm^-2）,探究生物炭和秸秆对土壤理化性质和真菌群落结构多样性的影响.结果表明：与不施生物炭和秸秆（对照）相比,生物炭施用显著增加土壤的pH、全碳、速效钾和速效磷含量,但显著降低土壤电导率,降低幅度为20.71%;秸秆处理显著增加土壤的速效钾和速效磷含量,但显著降低土壤容重和电导率,降低幅度为4.17%和64.50%.生物炭和秸秆处理对真菌群落Chao1指数和ACE指数有增加趋势,对Shannon指数和Simpson指数有降低趋势.土壤优势真菌门类为子囊菌门、被孢霉门、担子菌门、壶菌门和球囊菌门;优势真菌属为毛壳菌属、赤霉菌属、镰刀菌属、Idriella和被孢霉属.施加生物炭和秸秆提高子囊菌门、被孢霉门、担子菌门、球囊菌门和毛壳菌属的相对丰度;但降低壶菌门、赤霉菌属和Idriella的相对丰度.LEfSe分析表明,施用生物炭和秸秆还田降低真菌群落潜在生物标志物数量.RDA结果显示,土壤真菌群落结构与EC_1：5和TN显著相关.微咸水灌溉给土壤带来了不利影响,其中EC_1：5和TN是驱动土壤真菌群落结构变化的主要因子,土壤真菌群落通过生物炭和秸秆对土壤的改良作用来适应盐胁迫环境.     

堆肥厂不同工作区空气真菌的多样性与群落结构

动物粪便堆肥厂是空气中真菌的重要来源.目前对其不同工作区逸散真菌的种类和丰度差异尚无相关报道,无法全面评估堆肥厂空气真菌的生态健康风险.针对这一问题,对动物粪便堆肥厂的堆肥区、包装区、办公区和下风向区空气样本进行采集,利用高通量测序技术对4个区域空气中真菌的生物多样性和群落结构进行分析,并进一步对办公区及下风向区空气真...     

Effect of methamidophos on soil fungi community in microcosms by plate count, DGGE and clone library analysis

Methamidophos was widely used a pesticide in northern China. The potential influences of methamidophos on soil fungal community in black soil were assessed by plate count, 28S rDNA-PCR-DGGE, and clone library analysis. Three methamidophos levels (50, 150, and 250 mg/kg) were tested in soil microcosms. Results from plate count during a 60-d microcosm experiment showed that high concentrations of methamidophos (250 mg/kg) could significantly stimulate fungal populations. DCGE (denaturing gradient gel electrophoresis) fingerprinting patterns showed a significant difference between the responses of culturable and total fungi communities under the stress of methamidophos. Shannon diversity indices calculated from DGGE profiles indicated that culturable fungi in all microcosms with methamidophos treatment increased after 1 week of incubation. However, the diversity indices of total fungi decreased in the first week, as compared to the stimulation of culturable fungi. At the 8th week, however, all the microcosms treated by methamidophos were similar to the control microcosms in community structure as suggested by the Shannon diversity indices for both culturable and total fungi. In contrast, after 1 week the fungal structure of culturable and unculturable both were disturbed to different extent under the stresses of methamidophos by clustering analysis. Clone sequencing analysis indicated the stimulation of pathogenic and unculturable fungal populations by methamidophos treatment, suggetsing potential risks of plant disease outbreak.