Biodegradation of beta-cyfluthrin by fungi

Five fungal species, namely, Trichoderma viride strain 5-2, T. viride strain 2211, Aspergillus niger, A. terricola, and Phanerochaete chrysoporium were screened for degradation study of beta-cyfluthrin. Each fungal species was allowed to grow in Czapek dox medium containing beta-cyfluthrin (5 mg/mL) as the major carbon source of the medium. The highest degradation of beta-cyfluthrin was observed by T. viride 5-2 (T(1/2) = 7.07 days), followed by T.viride 2211 (T(1/2) = 10.66 days). The degradation of beta-cyfluthrin followed first-order kinetics with a fast degradation rate during first 7 days of growth of the fungi. In the case of T. viride strain 5-2, five degradation products were isolated after 20 days of growth of the fungi, out of which three products were identified as alpha-cyano-4-fluorobenzyl-3- (2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate, alpha-cyano-4-fluoro-3-phenoxy benzyl alcohol, and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanoic acid.     

降解玉米秸秆真菌复合菌系的构建及其降解效果评价

  【目的】  秸秆的木质纤维素含量丰富、结构复杂，在自然界中降解较慢，增加秸秆降解菌剂中菌株的多样性有利于提升还田秸秆的降解效果。探究菌株多样性水平和组成影响复合菌系秸秆降解的效果及原因，为复合菌系在秸秆降解中的应用提供理论支撑。  【方法】  通过富集驯化培养，从玉米秸秆还田土壤中筛选具有秸秆降解能力的真菌，从中挑选5株高效秸秆降解真菌进行基因间隔区序列 (ITS) 测定和物种鉴定，明确其分类地位。通过全组合构建菌株多样性为1～5的复合菌系，分别检测复合菌系的秸秆相对降解率及其滤纸酶、纤维素内切酶和木聚糖酶活性，利用方差分析和相关性分析等方法研究菌株多样性和组成对复合菌系玉米秸秆降解效果及其纤维素酶活性的影响。  【结果】  共筛选获得了15株具有秸秆降解能力的真菌，其中5株真菌的秸秆降解效果好、纤维素水解能力强。经ITS序列鉴定和系统发育分析，发现5株降解真菌的遗传差异较大，Z7-6、F7-5、F4-3、L1-1和J2-5分别与草酸青霉 (Z7-6: Penicillium oxalicum)、烟曲霉 (F7-5: Aspergillus fumigatus)、哈茨木霉 (F4-3: Trichoderma harzianum)、白囊耙齿菌 (L1-1: Irpex lacteus) 和木贼镰刀菌 (J2-5: Fusarium equiseti) 的ITS序列相似度均超过99.95%。全组合复配结果表明，复合菌系的秸秆降解能力和纤维素酶活力均高于各单一菌株，且随着菌株多样性水平的增加而提高。滤纸酶、纤维素内切酶和木聚糖酶的活力越强，复合菌系对玉米秸秆的降解效果越好，而其秸秆相对降解率主要取决于滤纸酶和纤维素内切酶的活性。抽样效应分析发现，不同菌株对复合菌系的秸秆降解效果、滤纸酶和纤维素内切酶活性的影响不同。不含菌株F7-5的复合菌系降解效果显著优于含有该菌株的组合，以Z7-6 (P. oxalicum)、F4-3 (T. harzianum)、L1-1 (I. lacteus) 和J2-5 (F. equiseti) 组合F1的玉米秸秆降解效果最佳、酶活性最高。  【结论】  秸秆降解复合菌系的构建过程需要同时考虑多样性效应和抽样效应，增加降解菌的多样性有助于增强秸秆的降解效果。本研究筛选获得的复合菌系F1在玉米秸秆降解中具有潜在的应用前景。     

食用菌栽培相关木霉种的鉴定

从木霉污染的食用菌菌筒和子实体中分离纯化了49株木霉菌株。采用形态学方法以及ITS/5.8S测序分析,对这些木霉菌进行了分类鉴定。结果表明,中国福建、浙江等省食用菌栽培相关木霉种以哈茨木霉Trichoderma harzianum Rifai和长枝木霉T. longibrachiatum Rifai为主,少量为深绿木霉 T. atroviride Karsten和棘孢木霉T. asperellum Samuels;木霉污染菌的种类与采集地点、食用菌的种类有一定相关,如在浙江庆元香菇Lentinus edodes (Berk.)sing菌筒中分离的木霉主要是哈茨木霉,在广州刺芹侧耳（杏鲍菇）Pleurotus enyngii菌筒中分离的木霉主要是棘孢木霉,而从福建浦城食用菌污染袋中分离的木霉主要是深绿木霉。两种分类方法对木霉鉴定结果基本一致,因此形态学分类方法结合分子生物学方法使食用菌栽培相关木霉种的分类鉴定更准确和可靠。     

哈茨木霉A25-2纤维二糖水解酶I基因的克隆及其编码催化功能域的序列在绿色木酶HP35-3中的表达

本研究从哈茨木霉（rrichoderma harzianMm）A25—2总RNA中利用RT—PCR的方法扩增到其纤维二糖水解酶I基因的cDNA序列,并对该基因编码的氨基酸序列进行分析,得到cbhI基因中编码催化功能域的序列。将催化功能域编码序列克隆到表达载体pCP—GH中,用PEG—CaCl2介导的原生质体转化方法将重组质粒转化到绿色木霉（Trichoderma viride）HP35—3中,筛选得到12个转化子。以P-NPC为底物,测定了该12个转化子的酶活力,获得比活力最高的转化子Tv／CDHI-CBM-5,其纤维二糖水解酶活力是HP35—3的3．8倍。SDS—PAGE分析表明,绿色木霉表达了导入的含A25—2纤维二糖水解酶I催化功能域的编码序列。     

Trichoderma brevicompactum complex: Rich source of novel and recurrent plant-protective polypeptide antibiotics (peptaibiotics)

Three strains of Trichoderma brevicompactum and another four that are closely related to that species (Trichoderma cf. brevicompactum) were analyzed for the formation of polypeptide antibiotics (peptaibiotics) by LC/ESI-MS(n). These isolates were selected because of an antagonistic potential against Eutypa dieback and Esca disease of grapevine and have not yet been investigated for the production of peptide antibiotics. Fully grown cultures on potato dextrose agar were extracted with CH2Cl2/MeOH, and this extract was subjected to SPE using C18 cartridges. The methanolic eluates were analyzed by LC/ESI-MS(n). All strains were found to produce membrane-active alamethicins F30. In addition to that, novel peptaibiotics were detected, namely, 14 12-residue trichocryptins B, 12 11-residue trichocryptins A, 19 11-residue trichobrevins A and B, 6 10-residue trichoferins, and 17 8-residue trichocompactins. These compounds may partially be responsible for the plant-protective action of the producers. Chemotaxonomic considerations also indicated the necessity to introduce another new species that is closely related to T. brevicompactum.     

纤维素降解真菌A25-2的分离、鉴定及其产纤维素酶的酶学特性

本研究以Avicel-刚果红选择培养基为初筛培养基,从云南哀牢山国家级自然保护区和广西猫儿山国家级自然保护区的土壤样品中分离筛选得到4200株真菌,从中筛选出透明圈与菌落直径比较大、透明程度较为清晰的12个菌株。通过液体培养发酵,测定其上清液中的羧甲基纤维素酶活力、滤纸酶活力和Avicel酶活力,最终筛选出一株产该三种酶且其活力均最高的真菌菌株A25-2。通过对菌株A25-2形态学观察和其内转录间隔区（internal transcribed spacer,ITS）序列同源性比对分析,将菌株A25-2鉴定为哈茨木霉（Hypocrea lixii）。酶活测定结果表明菌株A25-2产纤维素酶的酶活力较高,在最适作用pH4.5和最适作用温度55℃下,其羧甲基纤维素酶活力为2.26IU/mL,滤纸酶活力为0.58IU/mL,Avicel酶活力为0.39IU/mL。薄层层析实验表明A25-2具有完整的纤维素酶系统。因此,真菌A25-2可作为饲料加工等生产和纤维素酶相关研究的备选菌株。     

Laboratory prescreening of Bradyrhizobium japonicum for low pH,Al and Mn tolerance can be used to predict their survival in acid soils

We examined whether strains of Bradyrhizobium japonicum selected for growth on acid media in vitro would also survive and grow better in acid soils. Four agar screening media for acid-tolerant rhizobia, which differed in the number of acid soil stresses imposed (pH, low calcium (Ca) and phosphorus (P), high aluminum (Al) and manganese (Mn)), were assessed for their effects on the survival of 14 Indonesian strains and two commercial strains of B. japonicum. Survival of B. japonicum in the agar media was compared with that in two acid soils. A repeat stab inoculation method which provided a declining range of inoculum cell number to 10³ cells per stab was used to assess the daily growth of the strains on the screening media at 5 pH levels (3.8, 4.2, 4.5, 5.0, and 6.8). The growth and survival of the 16 strains were then measured at days 1, 8, 18, and 28 after inoculation in two acid soils (pH 4.24 and 4.35) sterilized using γ-irradiation at 5.0 Mrad. Selectivity of the agar media improved as more acid stress factors were incorporated in the media. Those strains of Bradyrhizobium identified as acid, Al and Mn-tolerant in acidic agar media, also had better survival in the low pH soils. There was no relationship between acid or alkali production on agar media and acid tolerance on agar or in soil. There was no apparent relationship between symbiotic performance and acid tolerance, and one acid-tolerant strain was as effective as the commercial inoculant strain CB1809. The most acid-tolerant strain was also the most ineffective.     

矿区植物根内嗜鱼外瓶霉对重金属的耐性和超积累作用

从云南会泽铅-锌矿区自然生长植物密序野古草(Arundinella bengalensis(Spreng.)Druce)的健康根内分离得到一株深色有隔内生真菌(dark septate endophytes,DSE)。据其形态学特征和ITS1-5.8S-rDNA-ITS2序列分析,鉴定为一株嗜鱼外瓶霉(Exophiala pisciphila)。纯培养条件下研究了其对Pb2+、Zn2+、Cd2+的耐性和超积累作用。结果表明,此菌株在液体合成培养基上可以分别耐受2、3和0.5 g/L的Pb2+、Zn2+和Cd2+;在含Pb2+、Cd2+培养基中生长其菌丝可以富集超过其干重25%以上的Pb2+和5%以上的Cd2+;显示此菌株属于极端耐性并具有超积累作用的真菌。此真菌在植物根系的定殖或许可以通过菌丝体对金属离子的固持作用缓解植物根围土壤微环境中超量金属离子对植物的毒害作用。     

Vineyard Compost Supplemented with Trichoderma Harzianum T78 Improve Saline Soil Quality

Understanding soil degradation is especially important in the Mediterranean Region where desertification is a serious problem, and soil salinization is one of the causes. Salinity reduces soil quality, limits crop productivity and brings on long term soil degradation. Therefore the restoration of degraded soils is necessary to reduce land degradation, improve soil fertility and achieve a sustainable food production. The addition of compost supplemented with the beneficial microorganism Trichoderma harzianum isolate T78 to saline soils (NaCl) was studied to determine the impact on soil microbiology, which is the key to restore and rehabilitate degraded soils. The selected Trichoderma harzianum isolate T78 showed high salt tolerance despite the low osmotolerance of the genus Trichoderma . Increasing salt concentration reduced Trichoderma sp colony‐forming units (CFU) from natural soil and adversely affected soil microbial biomass C as well as dehydrogenase, β‐glucosidase, phosphatase and urease activities. Simultaneous amendment of the saline soil with compost and inoculation with T. harzianum T78 improved the soil microbiological quality; the number of T. harzianum T78 CFU did not decrease as NaCl increased. As T. harzianum strain T78 is salt tolerant, increasing the relative abundance of this specific strain would contribute to the rehabilitation of saline soils. Vineyard composts supplemented with T. harzianum T78 represent a promising approach for the treatment and improvement of saline soil properties. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of enrichment and plating media for isolating Listeria monocytogenes

We compared selective enrichment broths used by the U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service, for their efficiency in the quantitative recovery of Listeria monocytogenes from a naturally contaminated Brie cheese that was obtained as part of an epidemic investigation. Quantitative recovery of Listeria in FDA broth (greater than 2.4 x 10(5) colony forming units/mL) was significantly better than recovery in USDA broth (9.3 x 10(3) colony forming units/mL). When USDA broth was supplemented with D-glucose and Phytone (papaic digest of soy protein), its recovery efficiency improved but did not equal that of FDA broth for isolating L. monocytogenes from Brie cheese. A comparison of 4 selective plating media [modified McBride's agar, gum base nalidixic acid agar, lithium chloride-phenylethanol-moxalactam agar (LPM), and acriflavine-ceftazidime agar (AC)] showed that 3 L. monocytogenes strains belonging to serotype 1/2a were partially or completely inhibited on LPM and AC agars. One strain of serotype 1/2a formed microcolonies on modified McBride's agar after 48 h of incubation.     

根癌农杆菌介导的绿色木霉HP35-3转化及表达体系的构建

本研究通过根癌农杆菌介导转化法,构建丝状真菌绿色木霉HP35-3的遗传转化表达体系,并以红色荧光蛋白基因（DsRed2）为报告基因来验证本表达体系。首先构建含有潮霉素抗性基因（HygR）、磷酸甘油醛脱氢酶启动子（Pgpd）和红色荧光蛋白基因（DsRed2）的双元载体pCAMT-CPRFP。然后将该双元载体转化到根癌农杆菌EHA105中,并筛选阳性转化子。将该转化子和绿色木霉HP35-3分生孢子进行液体共培养,并在含有150μg/mL潮霉素抗性平板上筛选出绿色木霉HP35-3RFP阳性转化子。最后分别通过如下三种方法检测：提取HP35-3RFP的总DNA进行PCR验证;对菌丝体进行荧光显微镜观察;对菌丝体提取物进行荧光酶标仪检测。结果表明：获得的转化子能够稳定遗传,外源红色荧光蛋白基因整合到木霉HP35-3的基因组中并成功表达。     

Viridenepoxydiol, a new pentasubstituted oxiranyldecene produced by Trichoderma viride

A new pentasubstituted oxiranyldecene, named viridenepoxydiol, has been isolated (0.9 mg/L) from the culture filtrate of a strain of Trichoderma viride showing in vitro and in vivo antagonistic activity against Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridenepoxydiol was characterized as 3,5,9-trimethyl-2-oxiranyl-dec-8-ene-2,5-diol (3) using spectroscopic methods. It showed inhibitor effect on mycelial growth of S. rolfsii and its minimum inhibitory concentration (over 90% inhibition) was found to be 396 mug/mL. This is the first time that viridenepoxydiol was reported.     

Production of chlamydospores and conidia by Trichoderma spp in liquid and solid growth media

Isolates of Trichoderma spp grew and produced chlamydospores as well as conidia in molasses-corn steep liquor (M-CSL), sucrose nitrate (SN), and glucose tartrate (GT) media. In M-SCL, isolates of T. hamatum, T. viride, and T. harzianum formed 10.4, 5.9 and 1.1 × 10⁸ chlamydospores g^?1 dry weight of mycelium. Fewer chlamydospores formed in SN and GT. Although T. harzianum formed the least number of chlamydospores, it produced the highest number of conidia in all three media. Molasses-corn steep liquor was superior to SN or GT in supporting development of both spore types. Spore production was not influenced by initial pH of the media or by continuous maintenance of the media at pH 4 or 7. Equal numbers of chlamydospores were formed in liquid media incubated in shake or static culture. Conidia formation, however, was stimulated in static culture. Chlamydospores and conidia of several naturally occurring isolates and induced biotypes of Trichoderma spp were abundantly produced on a variety of solid substrates moistened with liquid nutrients or water, preferably at pH 4. Bran, cornmeal, and peanut hull meal were better than eight other solid substrates for production of chlamydospores and conidia. A u.v.-induced, benomyl-resistant biotype of T. viride (T-1-R9) formed 22 × 10⁷ and 18 × 10⁸ chlamydospores and conidia, respectively, g^?1 of bran-SN. The ratio of conidia to chlamydospores was always greater in solid than liquid media. In solid media, 10 times more conidia than chlamydospores were formed, whereas in liquid only two or three times more were formed. Chlamydospores from liquid and solid fermentations were viable (ca 80%) and fresh chlamydospores germinated well (ca 75%) on nutrient agar. Although dried chlamydospores were viable, as determined with tetrazolium bromide, their germination on agar was poor (ca 8%).     

Involvement of pachybasin and emodin in self-regulation of Trichoderma harzianum mycoparasitic coiling

Our aim was to determine the effects of two secondary metabolites secreted by Trichoderma harzianum, pachybasin and emodin, on the mycoparasitic coiling behavior and cAMP content of T. harzianum. The number of T. harzianum coils around Nylon 66 fiber was increased in the presence of R. solani. The number of T. harzianum coils around R. solani hyphae and Nylon 66 fiber were significantly increased in the presence of pachybasin and emodin. The cAMP level in T. harzianum was significantly increased by close contact with R. solani and much higer cAMP level in the presence of exogenous pachybasin and emodin. A cAMP inhibitor diminished the effect of pachybasin and emodin on T. harzianum coiling around Nylon 66 fiber. The results suggest that pachybasin and emodin mediate the increase in the number of Trichoderma mycoparasitic coils via cAMP signaling. This is the first report to suggest that pachybasin and emodin play roles in the biocontrol mechanism of Trichoderma.     

Viridepyronone, a new antifungal 6-substituted 2H-pyran-2-one produced by Trichoderma viride

A new antifungal 6-substituted 2H-pyran-2-one, named viridepyronone, has been isolated from a cultural filtrate of a strain of Trichoderma viride showing antagonistic activity in vitro toward Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridepyronone was characterized as 6-(4-oxopentyl)-2H-pyran-2-one 2 with spectroscopic methods. Bioassays showed that viridepyronone had a good antifungal activity against S. rolfsii, and its minimum inhibitory concentration (over 90% inhibition) was found to be 196 microg/mL. This is the first report of viridepyronone produced by any species of fungi.     

Piperidinyl amides with insecticidal activity from the maritime plant Otanthus maritimus

Two new piperidine amides, N-[(2E,4E,8Z)-tetradecatrienoyl]piperidine (1) and N-[(2E,4E,8Z,11Z)-tetradecatetrenoyl]piperidine (2), along with the known metabolites N-[(2E,4E)-tetradecadienoyl]piperidine (3), N-isobutyl-(2E,4E,)-tetradecadienamide (4), N-isobutyl-(2E,4E,8Z)-tetradecatrienamide (5), N-isobutyl-(2E,4E,8Z,11Z)-tetradecatetraenamide (6), sesamine (7), pinoresinol (8), and espeletone (9), were isolated from the dichloromethane/methanol extracts of the plant Otanthus maritimus Hoffman & Link collected from coastal areas in Greece. Pinoresinol (8) and espeletone (9) are reported for the first time as metabolites of O. maritimus. The structures of the new natural products were elucidated by interpretation of their NMR and high-resolution mass spectral measurements. The insecticidal properties of the crude extracts, essential oil, and isolated metabolites 1-9 were evaluated on Crematogaster scutellaris (Olivier) ants and Reticulitermes balkanensis (Clement) termites, showing significant levels of activity.     

抗福美双假单胞菌株的NTG化学诱变及分子探针鉴定

利用微生物的抗药性筛选抗福美双的假单胞菌株Ｔ４６，然后通过亚硝基胍（ＮＴＧ）化学诱变获得了对福美双敏感的三株突变株Ｔ４６Ｎ１０、Ｔ４６Ｎ７和Ｔ４６Ｎ１７。利用这三个突变株作受体，通过基因克隆的方法筛选到了可使三个突变株恢复抗性的克隆，抗性基因分别被定位在７ｋｂ、２．５ｋｂ和２０ｋｂ的ＥｃｏＲⅠ片段上。将这三个克隆分别用（３２）Ｐ标记后制成分子探针，然后分别与三个突变菌株的总ＤＮＡ进行ＤＮＡ－ＤＮＡ分子杂交，结果表明，各突变株均存在与菌株Ｔ４６的抗福美双基因克隆的高度同源性，经结合形态、生理生化等分析后证实这三株突变株均源自于出发菌株Ｔ４６。     

Solubilization of inorganic phosphates and growth emergence of wheat as affected by Azotobacter chroococcum mutants

 Phosphate-solubilizing strains of A. chroococcum isolated from the wheat rhizosphere were evaluated for their ability to solubilize tricalcium phosphate (TCP), Mussoorie rock phosphate (MRP) and also for indole-acetic-acid (IAA) production. Strains were selected on the basis of the clearance zone on solid agar media of Pikovskaya and Jensen's media containing TCP, and phosphate solubilization in Jensen's liquid culture medium containing both TCP and MRP. Mutants of the best phosphate-solubilizing (TCP 1.52 μg ml^–1 MRP 0.19 μg ml^–1), IAA-producing A. chroococum strain P-4, were developed and screened for P solubilization and phytohormone production. Five mutants solubilized more P (in the range of 1.5–1.7 μg/ml^–1 of TCP and 0.19–0.22 μg ml^–1 of MRP) than the parent strains. In vitro growth emergence studies of three wheat varieties, viz. C-306, WH-542 and HD-2009, showed better performance with phosphate-solubilizing mutants than with the parent strain. Received: 15 October 1997     

Increased emergence of spring wheat after inoculation with Pseudomonas chlororaphis isolate 2E3 under field and laboratory conditions

Inoculation at the time of planting with Pseudomonas chlororaphis strain 2E3 increased the emergence of spring wheat by 8% and 6% at two different sites in northern Utah. Isolate 2E3 strongly inhibited the growth of the wheat pathogen Fusarium culmorum on artificial media. A second isolate of P. chlororaphis (strain O6) also inhibited fungal growth on artifical media but did not increase emergence of the spring wheat at the same field sites. Inoculation of winter wheat by 2E3 did not promote emergence when planted into field soil sterilized by fumigation with methyl bromide. Under laboratory conditions, emergence of spring wheat in sterilized soils from both sites was at least 90%. In the soils that were not sterilized, emergence was below 25% in soil from one site and below 50% in soil from the other. Treating seeds with 2E3 significantly improved emergence in a sterile soil-containing matrix that had been inoculated with the wheat pathogen Fusarium culmorum. Consequently, we propose that increases in wheat emergence can be attributed to the suppression by 2E3 of pathogenic organisms present in the native field soils. A strain of Rhizoctonia solani, shown to the pathogenic on winter wheat, was isolated from one of these soils.     

Synthesis and biological evaluation of new analogues of the active fungal metabolites N-(2-methyl-3-oxodecanoyl)-2-pyrroline and N-(2-methyl-3-oxodec-8-enoyl)-2-pyrroline (II)

New analogues of the bioactive enamides isolated from P. brevicompactum (2 and 3) have been synthesized to improve the biological activities. Two different structural modifications have been introduced: substitution of the aliphatic side chain present in the natural products (1-4) by other groups frequently found in other active compounds and use of other nitrogen-containing five-membered rings with different degrees of oxidation. In this way, the insecticidal and fungicidal activities have been improved. Thus, compound 9, which possess a 3-pyrroline ring, exhibited important insecticidal activity against third-instar nymphs of Oncopeltus fasciatus Dallas (100% mortality at 7.5 microg/cm(2)). Remarkable fungicidal activity was also found, and preliminary structure-activity relationships could be established.