绿僵菌属的修订

本文报道绿僵菌属的修订和修订后本属区分种的主要标准。这对于自然界本属复杂类群的研究将起到新的推动和助益。     

绿僵菌属血清学的初步研究

本文采用凝集试验和免疫电泳的方法对不同来源的七株绿僵菌属真菌进行了免疫学对比研究。抗原是孢子悬液和菌丝体清液,通过对家兔接种抗原而获得抗血清。每一种抗原与其同源抗血清和异源抗血清进行交叉试验,并对凝集试验的结果以及免疫电泳反应产生的沉淀弧数目和免疫电泳图谱加以对比分析,试验结果表明:不同菌株间存在着明显的抗原类似性,同时各菌株间也表现出一定的抗原专一性。根据试验数据对供试菌株进行血清学分型的结果与形态学分类相符合。     

一株绿僵菌的分离研究

利用马铃薯葡萄糖琼脂培养基,对一株绿僵菌进行了分离培养。对不同生长发育天数的菌落特征即颜色、大小、孢子堆的形成等特点进行了描述。在光学显微镜下观察了菌丝的形态,成熟孢子及分生孢子梗的形状、颜色及连接状况。利用扫描电镜观察了分生孢子和孢子梗的超微形态。经培养特征和形态学鉴定,确定该昆虫病原真菌是金龟子绿僵菌。     

绿僵菌对小麦纹枯病菌的抑制作用研究

在实验室条件下,研究了金龟子绿僵菌（Metarhizium anisopliae）对小麦纹枯病菌（Rhizoctonia cerealis）的拮抗作用及其机理。结果表明,金龟子绿僵菌与小麦纹枯病菌对峙培养以及在培养基中加入金龟子绿僵菌孢子悬浮液,对小麦纹枯病菌菌丝生长均有较好的抑制作用。测定了培养不同天数的金龟子绿僵菌Ma55发酵液对小麦纹枯病菌菌丝生长、菌核产生量及菌核萌发率的影响。结果表明,液体振荡培养25 d的金龟子绿僵菌Ma55发酵液对小麦纹枯病菌的菌丝生长、菌核产生量及菌核萌发具有显著的抑制作用,且Ma55发酵液中的抑菌活性物质具有较好的热稳定性。在光学显微镜下,未观察到Ma55对小麦纹枯病菌的重寄生现象,但发现金龟子绿僵菌与小麦纹枯病菌对峙培养处小麦纹枯病菌营养菌丝的细胞质变稀薄、菌丝部分消解或断裂。上述结果显示,金龟子绿僵菌对小麦纹枯病菌的拮抗机制主要是营养竞争、空间竞争及抗生作用等。     

柱孢绿僵菌产抗氧化活性成分培养条件的研究

通过液体摇瓶培养,以自由基清除率为指标,对一株柱孢绿僵菌(Metarhizium cylindrosporae)在不同液体培养条件下的抗氧化活性进行了研究,确定了其产抗氧化活性物质的最佳培养条件。结果表明最佳培养条件为:碳源为葡萄糖和麦芽糖组成的组合碳源;氮源为蚕蛹粉;无机盐及生长因子为MgSO4.7H2O、柠檬酸铵和VB;起始pH值为6.5;装液量为40mL/100mL;接种量为5%~7.5%;摇床转速为160~180r/min;培养时间为8d。正交实验结果表明最佳培养基配方为麦芽糖10g/L、蚕蛹粉10g/L、MgSO4.7H2O0.1g/L、柠檬酸铵0.4g/L、VB0.3g/L时,其次生代谢产物的抗氧化活性最强,可达82.16%。     

绿僵菌属血清学的初步研究

本文采用凝集试验和免疫电泳的方法对不同来源的七株绿僵菌属真菌进行了免疫学对比研究。抗原是孢子悬液和菌丝体清液,通过对角兔接种抗原而获得抗血清。每一种抗原与其同源抗血清和异源抗血清进行交叉试验,并对凝集试验的结果以及免疫电泳反应产生的沉淀弧数目和免疫电泳图谱加以对比分析,试验结果表明：不同菌株间存在明显的抗原类似性,同时各菌株间也表现出一定的抗原专一性。根据试验数据对供试菌株进行血清学分型的结果与形     

绿僵菌属的一个新种

本文报导从贵州省贵阳罹病梨虎Rhynchites Coreanus Kono幼虫虫尸分离的一种绿僵菌新种的鉴定结果,该菌与绿僵菌属模式种金龟子绿僵菌Metarhizium anisopliae (Metsch) s-orkin 有明显区别,根据菌落颜色、分生孢子团块大小、连接紧密和牢固程度、孢子链连接方式和分生孢子形态等,我们鉴定为新种——翠绿绿僵菌Metarhizium iadini chen、Guo et zhou sp.nov.     

绿僵菌产海藻糖水解酶培养条件研究

丝状真菌绿僵菌能产生一系列二糖水解酶,其中包括海藻糖水解酶。这些酶在绿僵菌对昆虫的致病过程中起着重要的作用。本文研究了不同碳源、氮源对金龟子绿僵菌Metarhizium anisopliae var. acridum菌株CQMa102产生与分解昆虫血淋巴中海藻糖等二糖相关的海藻糖水解酶活性的影响。结果表明:分别以葡萄糖、麦芽糖、蔗糖、山梨醇和可溶性淀粉为碳源,金龟子绿僵菌均可产生海藻糖水解酶,但最佳碳源是可溶性淀粉,因为由其诱导产生的海藻糖水解酶具有最高的总活性和比活性以及更多的同工酶,山梨醇次之。硝态氮(NaNO₃)作为唯一氮源时,几乎检测不出海藻糖水解酶活性,而铵态氮((NH₄)₂SO₄)或NaNO₃和有机氮(蛋白胨和酵母浸膏)混合氮源作氮源时,海藻糖水解酶活性都很高。在绿僵菌菌丝提取液和滤液的海藻糖水解酶活性比较中发现:CQMa102在多数碳源的培养基中产生的海藻糖水解酶主要分泌到培养基中,仅有少数结合在细胞壁上。     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

新疆地区绿僵菌菌株筛选及抗逆性研究

本文对新疆地区土壤中分离得到的17株绿僵菌Metarhizium菌株以东亚飞蝗Locusta migratoria manilensis为供试昆虫进行毒力测定,筛选获得高毒力菌株,并对筛选后的高毒力绿僵菌菌株进行耐短时高温能力、抗紫外线能力和耐干旱能力的测试,分析高毒力绿僵菌菌株的抗逆性,以期获得致病力高且抗逆性好的菌株,为下一步绿僵菌生物农药的开发提供依据。研究发现,M1-17、M1-13、M1-09、M1-16、M1-05五株菌株为对东亚飞蝗高致病力的菌株,平均僵虫率在80.00%~96.67%之间,LT50在2.92~3.65之间。对高温的抗性效果较好的菌株为M1-17和M1-05;对紫外线的抗性效果较好的菌株为M1-17和M1-16;而菌株M1-09、M1-17和M1-16抗旱能力较好。菌株M1-17较其它菌株具有更好的抗逆性,具有很好的开发利用价值。     

Effect of formulation on the viability of Metarhizium anisopliae conidia

A slide agglutination test using antibody-sensitized latex particles was developed for the specific detection in the early infection of the flacherie virus of the silkworm, Bombyx mori. With this test, 0.63 μg/ml of virus protein could be detected. The tests was completed within 5 min. Extracts from flacherie virus-infected silkworm larvae agglutinated latex particles specifically, while there was no agglutination by extracts of normal and nuclear polyhedrosis virus-infected silkworm larvae. The results showed that the sensitivity and simplicity of this technique for the detection of flacherie virus were greater than those of conventional serological techniques such as the immunofluorescence test and the immunodiffusion test.     

Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

Repeated in vitro subculturing alters spore surface properties and virulence of Metarhizium anisopliae

Repeated subculturing caused rapid changes in the spore surface properties and virulence of Metarhizium anisopliae. Of the two strains evaluated, M. anisopliae V245 attenuated more rapidly than V275. Electrophoretic mobility and Radial Flow Chamber assays were used for the first time to generate qualitative and quantitative information on the adhesive forces of M. anisopliae conidia. Independent of strain, adhesion, hydrophobicity and spore-bound Pr1 declined after the first subculture; however, spore surface charge decline was erratic. Adhesion and hydrophobicity stabilized after the third subculture, whereas spore-bound Pr1 continues to decline following repeated subculturing. Decline in spore bound Pr1 was directly correlated with decline in virulence, however, such correlation with adhesion, hydrophobicity or surface charge could not be established. Because spore-bound Pr1 activities were directly correlated with M. anisopliae virulence; it could be used as a quality-control marker to monitor changes in virulence.     

MTT比色法测定促肝细胞生长物质对肝细胞生长的刺激活性

本实验建立了用简便的ＭＴＴ比色法对促肝细胞生长物质的促肝细胞增殖作用的测定方法,确定了实验的最适条件。与传统的３Ｈ ＴｄＲ掺入法进行比较的结果显示,ＭＴＴ比色法与３Ｈ ＴｄＲ掺入法测定结果基本相符,灵敏度相近,但消除了同位素的污染,是一个测定促肝细胞生长物质刺激肝细胞增殖活性的简便方法。     

Rapid colorimetric assay for intestinal peptide hydrolases

A simple two-step method is described for quantitating the release of free l-phenylalanine, l-leucine, l-methionine, or l-isoleucine from di- or polypeptides. The colorimetric assay is based on the ability of l-amino acid oxidase to catalyze the oxidation of free l-amino acid, but not of peptides. The potent metal chelator 1,10-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.     

A colorimetric assay for determination of cell viability in algal cultures

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.     

Termite pathogens: Transfer of the entomopathogen Metarhizium anisopliae between Reticulitermes sp. termites

Subterranean termites (Reticulitermes sp.) exposed to whole cultures of Metarhizium anisopliae for 4, 8, 12, or 48 hr transfer disease to previously healthy termites. Healthy termites concentrate grooming activity on diseased individuals and thereby become infected. Termites which have been killed by the fungus are avoided by healthy individuals and are less effective in spreading disease than are exposed living termites.     

Rapid continuous colorimetric enzyme assay for penicillin G acylase

A rapid, continuous, colorimetric enzyme assay for penicillin G acylase has been developed. The assay measures the formation of the acidic products of penicillin G hydrolysis by following the decrease in pH using Phenol Red as an indicator. The activity measured is directly proportional to the amount of enzyme added to the assay, having a linear relationship with an R ² value of 0.9994.