产漆酶半知真菌Myrothecium verrucaria NF-08菌株的分离及产酶研究

【目的】采集凉水国家级自然保护区原始森林土壤样品,分离产漆酶真菌并优化产酶条件,旨在开发产漆酶半知真菌种质资源,提高漆酶产量,为微生物漆酶扩大生产提供菌种资源和条件参数。【方法】以木质素磺酸钠为唯一碳源的无机盐培养基为富集培养基,在愈创木酚-PDA选择性平板上分离产漆酶真菌。利用ABTS[2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸)]和SGZ[4-羟基-3,5-二甲氧基-苯甲醛连氮,即丁香醛联氮]显色反应初筛和摇瓶发酵复筛。确定供试菌株后,利用形态观察结合r DNA-ITS序列分析技术,将菌株鉴定至种。以ABTS法测定供试菌发酵液漆酶活性,以Lowry法测定发酵液总蛋白含量。在考察供试菌株生长、产酶及胞外蛋白总量动态变化基础上,通过单因素试验,研究碳源种类及浓度、氮源种类及浓度、培养基起始p H、装液量、接种量及底物诱导对菌株产漆酶的影响,获得菌株产酶最适培养基组分和培养条件参数。【结果】获得1株发酵周期短、初始酶活高的产酶菌株NF-08。结合形态学特征与r DNA-ITS序列分析结果,鉴定菌株NF-08属于为半知菌亚门疣孢漆斑菌。该菌株在液体发酵培养基中产漆酶活性与菌丝生长和胞外蛋白总量基本同步,发酵第6天达到酶活峰值9.28 U·m L-1。疣孢漆斑菌NF-08产酶最佳碳、氮源分别为4.0%葡萄糖和3.5%蛋白胨,培养基初始p H7.0。最适装液量60 m L·(250 m L)-1,最适接种量4%,最适培养温度和摇床转速分别为30℃和140 r·min-1。没食子酸、阿魏酸和单宁酸显著诱导疣孢漆斑菌NF-08漆酶活性的产生,以没食子酸效果最好。经过培养基组分和培养条件优化以及底物诱导,疣孢漆斑菌NF-08产酶水平达到16.82 U·m L-1,比优化前提高了81.25%。【结论】建立环境样品中产漆酶真菌的分离、纯化及筛选方法,获得了一株发酵周期短、产漆酶活力高的半知真菌疣孢漆斑菌NF-08。该菌株经单因素试验产酶条件优化效果明显,酶活水平提高显著,可为微生物发酵产漆酶提供新的菌株资源,在微生物发酵漆酶工业生产中具有应用潜力。后续对疣孢漆斑菌NF-08漆酶产生、性质及基因表达调控的深入研究,将有助于了解漆酶在半知真菌生活史中的作用及生态学功能,同时拓展半知真菌漆酶的应用领域。

Bioherbicidal Efficacy of a Myrothecium verrucaria-Sector on Several Plant Species

Comparative studies were conducted on mycelial preparations of the bioherbicide, Myrothecium verrucaria (MV) strain IMI 361690 and a recently discovered sector (MV-Sector BSH) of this fungus. The whitish sector was discovered, isolated, grown in pure culture on PDA and found to be a stable, non-spore producing mutant when cultured over several months under conditions that cause circadian sporulation during growth of its MV parent. Application of MV and MV-Sector BSH mycelial preparations to intact plants (hemp sesbania and sicklepod) and leaf discs (kudzu and glyphosate-resistant Palmer amaranth) showed that the sector efficacy was generally equal to, or slightly lower than MV. Bioassays of MV and this sector on seed germination and early growth of sicklepod and hemp sesbania seeds demonstrated that hemp sesbania seeds were slightly more sensitive to the fungus than sicklepod seeds and that the sector bioherbicidal activity was slightly less than that of MV. SDS-PAGE protein profiles of cellular extracts of MV and the sector and their respective culture supernatants showed several differences with respect to quantity and number of certain protein bands. Overall results showed that the isolate was a non-spore producing mutant with phytotoxicity to several weeds (including weeds tolerant or resistant to glyphosate), and that the phytotoxic effects were generally equivalent to those caused by MV treatment. Results of this first report of a non-sporulating MV mutant that suggest additional studies on protein analysis, and an extended weed host range under greenhouse and field conditions are needed in order to further evaluate its possible bioherbicidal potential.

产杀虫抗菌单端孢霉烯族毒素海绵真菌Myrothecium verrucaria Hmp-F73的筛选和鉴定

从98株分离自大连海域繁茂膜海绵Hymeniacidon perleve共附生的真菌中,筛选得到具有较强杀虫抗菌活性的丝状真菌Hmp-F73。96孔板卤虫生物检测(brine shrimp assay,BSA)结果显示,该菌株发酵滤液甲醇提取物浓度为0.25μg.mL-1时,卤虫校正死亡率为100%;甲醇提取物经硅胶柱层析洗脱得到7个馏分,其中馏分3浓度为400μg.mL-1时对甜菜夜蛾具明显抑制取食活性,拒食率达到85.25%;1mg.mL-1的馏分6对玉米纹枯病菌Rhizoctonia solani抑菌圈直径达9.25mm。经色谱柱层析分离纯化得到4个杀虫抗菌活性化合物,氢核磁数据显示这些化合物均属于12,13-环氧单端孢霉烯族化合物。通过形态、培养特征及ITS1-5.8S-ITS2序列分析,将该菌株鉴定为疣孢漆斑菌Myrothecium verrucaria。

疣孢漆斑菌(Myrothecium Verrucaria)J-1胆红素氧化酶的纯化及性质初步研究

从中国土样中筛选到一株能产生胆红素氧化酶的微生物(Myrothecium Verrucaria)J-1,培养后,分离纯化,最后经QAE—Sephadex A50柱层析,得到胆红素氧化酶比活为207.65 U/A 280nm,总产率为22.3％。纯酶紫外吸收峰为278 nm,凝胶电泳为单一色带。分子量估计为52000。它能迅速、特异地氧化胆红素为胆绿素,并进一步氧化成目前还不清楚的紫色化合物。最佳作用pH为7.0,最佳作用温度为40℃。

大豆孢囊线虫生防菌株Myrothecium verrucaria ZW-2发酵条件优化及活性物质分析

疣孢漆斑菌(Myrothecium verrucaria)ZW-2菌株对大豆孢囊线虫具有(Heterodera glycines)明显的杀线虫活性,为了确定该菌株的最优发酵条件以及代谢产物,采用4因素3水平的正交试验进行了发酵条件优化以及代谢组学分析。结果表明:菌株ZW-2以Czapek培养基为基础培养基,最佳培养温度为28℃,摇床转速为180 r/min,装瓶量为100 mL,培养基pH为8,最佳碳源和氮源为蔗糖和硝酸钠。经代谢组学分析,根据代谢物质荷比、精确分子量及一级碎片离子得分等信息共筛选出20种差异代谢物,包括酰胺类化合物和脂肪族化合物。菌株ZW-2上述试验结果为进一步开发利用该菌株提供重要的理论和应用价值。

疣孢漆斑菌Myrothecium verrucaria GH-01漆酶的纯化和酶学性质研究

疣孢漆斑菌具有生长周期短,分泌漆酶酶活高等特点。利用已分离得到的疣孢漆斑菌GH-01(Myrothecium verrucaria GH-01)发酵产生粗酶液。进而通过分级沉淀、透析和层析的方法对漆酶粗酶液进行纯化。SDS-PAGE和Native-PAGE结果表明纯化可获得具有漆酶活性的单体蛋白。酶学性质研究表明,该漆酶催化最适温度为40℃,在底物ABTS存在条件下的最适p H值为4.0,且在低温及碱性条件具有较好的稳定性。此外通过漆酶对4大类染料脱色能力的研究,发现该漆酶对偶氮类的橙黄Ⅰ和蒽醌类的茜素红脱色有较好的脱色效果,反应1 h脱色率达80%以上;对三苯甲烷类的碱性品红脱色能力较弱,脱色率只能达到20%左右;脱色率最差的是杂环类的亚甲基蓝。在含10 U漆酶的体系中,对50 mg/L的染料降解效果相对最佳。

Interaction of the Bioherbicide <i>Myrothecium verrucaria</i>with Technical-Grade Glyphosate on Glyphosate-Susceptible and -Resistant Palmer Amaranth

Previously we found that a strain of Myrothecium verrucaria (MV) exhibited bioherbicidal activity against several important weeds, and that some commercial formulations of glyphosate applied with MV resulted in synergistic interactions that improved weed control efficacy. We also found that MV had bioherbicidal activity against glyphosate-resistant Palmer amaranth. We have also reported that some commercial formulations are inhibitory to MV. Our objectives were to test the effect of unformulated glyphosate (high purity, technical-grade glyphosate) alone and in combination with MV for bioherbicidal activity on glyphosate-susceptible and -resistant Palmer amaranth biotypes under greenhouse conditions and to examine technical-grade glyphosate on the growth of this bioherbicide. High purity glyphosate (without adjuvants/surfactants) was not toxic to MV growth and sporulation at concentrations up to 2.0 mM when grown on agar supplemented with the herbicide. Both biotypes were injured by MV and MV plus glyphosate treatments as early as 19 h after application (3 h after a dew period of 16 h). These injury effects increased and were more evident through the 6-day time course, when after 120 h the MV plus glyphosate treatment had killed all glyphosate-susceptible and -resistant plants. The interaction of glyphosate plus MV was synergistic toward the control of Palmer amaranth. Data strongly suggest that the active ingredient is responsible for the synergy previously found when this bioherbicide was combined with some commercial formulations of glyphosate. Results demonstrated that MV can control both glyphosate-resistant and -susceptible Palmer amaranth seedlings and act synergistically with high-purity glyphosate to provide improved weed control.

Effects of <i>Myrothecium verrucaria</i>on Ultrastructural Integrity of Kudzu (<i>Pueraria montana var. lobata</i>) and Phytotoxin Implications

The fungus Myrothecium verrucaria (Alb. & Schwein.) (MV), originally isolated from diseased sicklepod (Senna obtusifolia L.), has bioherbicial activity against kudzu and several other weeds when applied with low concentrations of the surfactant Silwet L-77. To more fully understand the initial events of MV infection or disease progression, and to improve knowledge related to its mechanism of action, the effects of MV and its product (roridin A) on kudzu seedlings were examined at the ultrastructural level. Ultrastructural analysis of MV effects on kudzu seedlings revealed a rapid (~1 h after treatment) detachment of the protoplast from the cell wall and plasmodesmata appeared to be broken off and retained in the wall. These symptoms occurred well in advance of the appearance of any fungal growth structures. Some fungal growth was observed after severe tissue degeneration (24 to 48 h after treatment), but this occurred primarily at the extra-cellular location with respect to the kudzu tissues. Kudzu seedlings treated with roridin A, a trichothecene produced by the fungus, exhibited some symptoms similar to those induced by the fungus applied in spore formulations with surfactant. The overall results are the first to report the ultrastructural effects of this bioherbicide on plants and suggest that penetration of a phytotoxic substance(s) in the fungal formulation was facilitated by the surfactant, and that roridin A exerts phytotoxicity toward kudzu.

Interaction of the Bioherbicide Myrothecium verrucaria and Glyphosate for Kudzu Control

Kudzu is an exotic invasive weed in the southeastern U.S. that is difficult to control with current commercial herbicides. Some success for its control has been achieved using a bioherbicidal agent, Myrothecium verrucaria (MV). Spore and mycelial formulations of MV were tested alone and in combination with glyphosate for control of kudzu (Pueraria lobata) under greenhouse and field conditions in naturally-infested areas. In greenhouse and field experiments, kudzu control increased as the concentration of spores or mycelia increased. Glyphosate alone provided 10%, 35%, 50% and 60% control in field experiments at 0.25, 0.50, 0.75 and 1.0X rates, respectively and MV alone spores provided 15%, 50%, 65% and 85% control at 0.25, 0.50, 0.75 and 1.0X rates, respectively. However, when MV spores were combined with glyphosate, significantly higher control occurred than that caused by either component alone. Similar levels of control were observed for MV mycelial formulations applied alone or with glyphosate at equivalent concentrations of the fungus. The rate of disease progression was more rapid and severe at all fungal spore or mycelial formulations and herbicide rates when these propagules were applied in combination with glyphosate. In field tests, 24 h after application, only 20% of kudzu plants were severely damaged by MV alone (0.25X), whereas 80% were severely diseased when MV spores and glyphosate were mixed and applied at 0.25X rates each. A similar trend occurred with the MV mycelial formulation applied at these rates. Synergist interactions on kudzu control were observed, especially when lower levels of MV (spores or mycelia) and glyphosate were combined and applied to kudzu in the greenhouse or in the field. These results suggest that it may be possible to incorporate glyphosate to improve the bioherbicidal control potential and reduce herbicide and inoculum requirements of M. verrucaria spores or mycelium for controlling kudzu.

Extending the Shelf-Life of <i>Myrothecium verrucaria</i>, a Bioherbicide

The shelf-life of a bioherbicide product is an important factor with regard to its commercial potential. The bioherbicidal efficacy of freshly fermented Myrothecium verrucaria (strain IMI 368023) (MV) mycelia formulations and MV mycelia preparations that had been freeze-dried and then stored at -20&deg;C for 8 years was compared. Two concentrations of each formulation (1.0x and 0.5x) were tested, utilizing bioassays on seedlings of the weed, hemp sesbania (Sesbania exaltata) under greenhouse conditions or in darkness utilizing hydroponically grown seedlings. Freeze drying of freshly prepared MV mycelium produced a light, brownish-colored powder. Efficacy tests of this reconstituted 8-year-old dried material showed that some bioherbicidal activity was lost during long-term storage, i.e., ~20% and ~60% seedling dry weight reduction at the 1.0x and 0.5x rate, respectively. Although plant mortality was greater in the fresh mycelial preparations treatments versus the freeze-dried and stored samples at all time points in the time-course, the stored material still caused >80% mortality, 15 days after treatment. Comparative disease progression ratings also showed a similar trend. Overall results show that freeze-drying MV is a useful method to reduce the bulk and cumbersomeness of storing heavy liquid fermentation product, while retaining bioherbicidal activity. These findings increase the utility of this bioherbicide and offer the potential to use the dried material in soil treatments or in a more concentrated form than attainable via the fermented product.

Interaction of a <i>Myrothecium verrucaria</i>Mycelial Preparation and a Glyphosate Product for Controlling Redvine (<i>Brunnichia ovata</i>) and Trumpet Creeper (<i>Campsis radicans</i>)

A mycelial formulation of the bioherbicidal fungus Myrothecium verrucaria (Alb. & Schwein.) Ditmar: Fr. (MV) was tested alone and in combination with a commercially available glyphosate [N-(phosphonomethyl)glycine] (GLY) product for controlling the invasive vines, redvine [Brunnichia ovata (Walt.) Shinners], and trumpet creeper [Campsis radicans (L.) Seem. ex Bureau] in field experiments conducted near Stoneville, MS. Several application timing regimens were evaluated (Fall, Spring, Fall + Spring, and Spring + Fall). We found that a Fall + Spring application of MV + GLY controlled redvine and trumpet creeper by 95%, 12 days after the second treatment, through a synergistic interaction of the fungus and glyphosate. Disease symptomatology was characterized by rapid necrosis of leaf and stem tissues, with mortality occurring within 72 h. Neither glyphosate alone, nor MV alone, effectively controlled either weed species under any application timing regimen. No visual disease or herbicide damage occurred on glyphosate-resistant soybean plants in the treated test plots. These results suggest that some formulations of glyphosate, mixed with the bioherbicide MV, can effectively control redvine and trumpet creeper, two of the most troublesome weeds in the row crops of the Mississippi Delta region in the mid-southern U.S.

Myrothecium verrucaria NF-08产漆酶条件的响应面法优化及粗酶染料脱色研究

以半知菌属疣孢漆斑菌Myrothecium verrucaria NF-08为供试菌,采用响应面方法优化菌株产漆酶条件,并以其漆酶粗酶进行3种结构类型6种染料的脱色试验,考察了粗酶浓度、温度和p H值对脱色率的影响.两水平析因设计、中心组合设计和验证试验结果表明:葡萄糖浓度为15.4 g·L^(-1)、蛋白胨浓度为43.3 g·L^(-1)和培养基初始p H值为6.68时,漆酶活性达到最大值(25.58±1.60)U·m L^(-1),是单因素试验(16.82 U·m L^(-1))的1.52倍,提高了52.1%.以上述发酵条件获得的M.verrucaria NF-08漆酶粗酶对3种结构类型染料均有脱色效果,排序为偶氮类优于芳甲烷类优于蒽醌类.脱色率随粗酶浓度增大(2~8 U·m L^(-1))、反应温度升高(15~35℃)而上升,随p H值上升(4~10)而下降.粗酶浓度为5 U·m L^(-1),反应温度为25℃,p H值为4时,偶氮类染料铬黑T和芳甲烷类染料碱性品红即可几乎完全脱色,脱色率分别为99.2%和94.6%.

真菌电化学修复除草剂污染土壤:降解动力学探索

【目的】微生物燃料电池(microbial fuel cell,MFC)在去除污染物的同时产出电能,是一种颇有前景的生态修复手段。构建真菌强化MFC装置,比较电动力(EK)、真菌、MFC修复除草剂污染土壤效果及优缺点,探索MFC在有机污染物修复中的应用潜力。【方法】设计了一种添加真菌进行生物强化的MFC,并用EK、真菌、MFC三种方法修复两种除草剂污染的灭菌土壤。经筛选和驯化的疣孢漆斑菌和踝节菌菌株用于后两种方法,研究真菌强化对MFC去除除草剂的影响。测量土壤pH、电导率、除草剂去除率,MFC产电性能,用气相色谱-质谱鉴定两种除草剂的降解产物。【结果】EK修复中,添加模拟电解液、碳纤维条、加电10 V的处理组7 d后氯氟吡啶酯(F)和高效氟吡甲禾灵(H)去除率分别为71%和38%。真菌、MFC处理F的最大去除率达到100%。对比踝节菌,疣孢漆斑菌对两种除草剂的降解性能更好,疣孢漆斑菌、踝节菌单菌构建的MFC对H的去除率分别为62.5%和24.1%。F降解产物为氟氯吡啶酸,H降解产物为乙酸大茴香酯,推测了降解路径和降解动力学。三种方法降解F以及EK降解H均符合动力学一级反应,而真菌和MFC降解H符合二级反应。【结论】对比EK、真菌修复,MFC修复效果更好,该方法可以较快地修复土壤又无需额外供电,是一种经济有效的自持式修复策略。

一种真菌对人参皂苷Rg_3的转化

【目的】筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份。【方法】从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定。【结果】从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性。【结论】阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法。

胆红素氧化酶产酶菌株的分离及最佳产酶条件的研究

胆红素是铁卟啉化合物在人体内的代谢产物之一,主要来源于衰老红细胞崩解后的血红蛋白,随胆汁排泄。凡能引起胆红素的生成过多或使肝细胞对胆红紊的摄取、结合、排泄过程发生障碍的因素,都可使血中胆红素浓度增高,出现高胆红素血症。因此,测定血中胆红素水平对临床诊断肝胆疾病,溶血等有着重要的意义。

疣孢漆斑菌发酵液的化学成分及其农药活性的研究

对一株羌活内生真菌—疣孢漆斑菌(Myrothecium verrucaria)的发酵液进行化学成分研究,采用乙酸乙酯萃取,硅胶柱色谱和制备型HPLC等色谱手段分离、纯化,从中分离得到14个单体化合物。通过理化性质与波谱数据分析,分别鉴定为:疣孢菌素B(1)、8-乙酰基漆斑菌素H(2)、疣孢菌素M(3)、疣孢菌素J(4)、漆斑菌素D(5)、乙酰基疣孢菌素L(6)、异漆斑菌素E(7)、漆斑菌素E(8)、漆斑菌素H(9)、漆斑菌素A(10)、乙酰基漆斑菌素E(11)、Isotrichoverrin A(12)、Isotrichoverrin B(13)、疣孢菌素A(14)。其中化合物2、6、7为首次从该种真菌中分离得到。并对14个化合物进行农药活性测试,结果表明化合物10 ppm对拟南芥有显著的生长抑制作用,32 ppm对早熟禾有生长抑制作用,1000 ppm对绿棉铃虫的致死率大于70%,具有良好的除草和杀虫活性。这为进一步开发疣孢漆斑菌作为生物除草剂和杀虫剂提供理论依据。

越南槐内生真菌GRPH-0的抗真菌活性及其鉴定

目的为新型抗生素的研究提供有价值的菌种资源。方法采用改良的琼脂扩散法从越南槐内生真菌中筛选拮抗白念珠菌的活性菌株并测定其对念珠菌属真菌的抑菌谱;以改良的微量肉汤稀释法测定其代谢产物的抗菌活性;根据形态和分子特征鉴定该菌株的分类学地位。结果菌株GRPH-0活菌块对白念珠菌的抑菌圈直径比阳性对照大,并且对参试念珠菌属真菌均具有不同程度的抑制作用,具有较广的抑菌谱;其代谢产物对白念珠菌及其多重耐药临床分离株的最小抑菌浓度均为4μg/m L,仅为阳性对照的16倍;初步鉴定该菌株为疣孢漆斑菌。结论菌株GRPH-0鉴定为疣孢漆斑菌,对参试病原真菌均显示强的抗真菌活性,具有较大的开发应用潜力。

疣孢漆斑菌发酵液正丁醇部位化学成分研究

目的:对疣孢漆斑菌发酵液正丁醇部位进行化学成分研究。方法:采用各种提取分离纯化技术进行化学成分分离,运用波谱技术进行结构鉴定。结果:从疣孢漆斑菌发酵液正丁醇部位分离鉴定了14个化合物,分别鉴定为:分别为cyclo-(Pro-Phe)(1)、cyclo-(4-OH-Pro-Phe)(2)、cyclo-(4-OH-Pro-Leu)(3)、cyclo-(Ala-Pro)(4)、cyclo-(4-methyl-Pro-9-propyl-Gly)(5)、cyclo-(Pro-Gly)(6)、cyclo-(Phe-Gly)(7)、cyclo-(Leu-Leu)(8)、N-乙酰色胺(9)、N-(2-苯乙基)乙酰胺(10)、N-(2-羟基苯乙基)乙酰胺(11)、N-(4-羟基苯乙基)乙酰胺(12)、尿嘧啶(13)、胸腺嘧啶(14)。结论:所有化合物均为首次从该真菌中分离得到。

胆红素氧化酶产生菌的选育及发酵工艺条件的研究

对胆红素氧化酶产生菌的菌和选育进行了研究。以科研室保藏菌种Mv9201为出发菌株，通过紫外线、硫酸二乙酯单独处理和复合诱变，从353株突变株中筛选出一株高产菌株MyrotheciumverrucariaMv9352，并对其产酶条件进行了研究。以20％的土豆汁（含0．25％的葡萄糖和蛋白胨）为发酵培养基，初始pH6～7，摇床转速为140～160r／min，24～26℃培养96h，酶活力可达4．0～4．5u／ml，比出发株的产酶能力提高近4．5倍。

平板培养结合宏测序法调查小麦田土壤真菌多样性

从河南省商丘市小麦田里于扬花期采集土样3份,通过ITS2宏测序方法结合平板培养法研究了小麦扬花期田间真菌种类,发现小麦田块物种多样性指数明显大于甜瓜地块,平均有1 630个OTUS。培养法获得的主要菌株为枝细枝孢(Cladosporium ramotenellum)、疣孢漆斑霉(Myrothecium verrucaria)和链格孢(Alternaria alternata),并对其形态进行详尽的描述。