一株链霉菌21-1的分离鉴定及其对禾谷镰刀菌的抑菌活性

【背景】由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重威胁我国的小麦生产。【目的】筛选对禾谷镰刀菌具有拮抗能力的链霉菌菌株,为生防菌剂开发提供理论基础。【方法】利用平板对峙法筛选对禾谷镰刀菌具有拮抗能力的链霉菌;通过形态特征、生理生化特征和16S rRNA基因序列分析对其进行鉴定;通过病原菌菌丝生长、孢子产生及萌发抑制试验分析其发酵液的抑菌活性;利用人工接种试验测定该菌株发酵液的防病效果。【结果】筛选到一株对禾谷镰刀菌具有较强拮抗活性的链霉菌21-1,抑菌率为59.5%。依据形态特征、生理生化特性和16S rRNA基因序列分析,将该菌株鉴定为黄三素链霉菌(Streptomycesflavotricini)。菌株21-1发酵液能够抑制禾谷镰刀菌的菌丝生长、孢子产生及萌发过程,而且可以降低禾谷镰刀菌菌丝中可溶性蛋白质的含量,并增加丙二醛的含量。菌株21-1可以产生蛋白酶及纤维素酶。菌株21-1菌液10倍稀释液对小麦赤霉病的防效最佳,为70.1%。此外,菌株21-1发酵液对其他8种植物病原菌均有较好的抑制作用。【结论】菌株21-1对禾谷镰刀菌有较好的抑菌活性,具...     

绿僵菌无纺布菌条的制作(Ⅰ)液体种子培养条件

研究了金龟子绿僵菌菌株Ma83液体培养最佳营养及环境条件,为无纺布培养提供良好的液体种子.以生物量为指标,通过液体摇瓶试验确定了绿僵菌Ma83菌株液体种子培养参数,对不同的氮源、碳源及其浓度和初始pH进行单因素分析.结果表明,绿僵菌Ma83菌株液体种子培养最佳营养条件为4%黄豆粉,2%白砂糖,0.5%MgSO4·7H2O,0.05g/LKCl,0.1g/L FeSO4·7H2O,1.0g/L KH2PO4,pH6.3～6.5.70L发酵罐培养,干生物量第三天可达35g/L,显著优于筛选前培养基(SDA,28g/L,a=0.05).     

小麦内生细菌的分离及其对小麦纹枯菌的拮抗作用

利用涂布平板法从小麦根系中分离出8株内生细菌,从中筛选出1株对小麦纹枯菌(Rhizoctonia cerealis)具有拮抗作用的内生菌。室内测定该菌株培养液对小麦纹枯病菌的抑制作用,结果发现,小麦纹枯病菌在培养液中生长缓慢,培养6d后菌丝量与对照相比下降了89％,同时发现病菌菌丝生长畸形,出现断裂和细胞壁瓦解。双抗标记法测定该拮抗菌在小麦根系中的定殖情况,发现该菌能够在根系中长期定殖。初步的鉴定结果表明该菌为蜡样芽孢杆菌。     

小麦内生细菌的分离及其对小麦纹枯菌的拮抗作用*

利用涂布平板法从小麦根系中分离出8株内生细菌,从中筛选出1株对小麦纹枯菌(Rhizoctoniacerealis)具有拮抗作用的内生菌。室内测定该菌株培养液对小麦纹枯病菌的抑制作用,结果发现,小麦纹枯病菌在培养液中生长缓慢,培养6d后菌丝量与对照相比下降了89%,同时发现病菌菌丝生长畸形,出现断裂和细胞壁瓦解。双抗标记法测定该拮抗菌在小麦根系中的定殖情况,发现该菌能够在根系中长期定殖。初步的鉴定结果表明该菌为蜡样芽孢杆菌。     

绿僵菌与3种杀虫剂混配对樟巢螟的协同作用

采用定量喷雾法测定了绿僵菌、白僵菌以及3种杀虫剂对樟巢螟Orthaga achatina低龄幼虫的室内毒力,并测定了金龟子绿僵菌Ma09与3种杀虫剂混配后的协同作用。结果显示：4种昆虫病原菌菌株均对樟巢螟2~3龄幼虫具有较好的致死效果,用绿僵菌和白僵菌孢悬液处理后,死亡高峰期在3~4 d;白僵菌GDLY9的防治效果最好,处理浓度为1×109孢子/mL下LT50和LT90分别为3.01、3.95 d,金龟子绿僵菌Ma09、3297效果相当,黄绿绿僵菌Mf985的LT50相对低一些,但是LT90优于金龟子绿僵菌Ma09和3297。金龟子绿僵菌Ma09与苯氧威、除虫脲和短稳杆菌混配后均表现出增效作用,协同指数分别为20.43、27.53和33.13。     

一株马铃薯干腐病拮抗菌的筛选、鉴定及其生物防效

【背景】马铃薯干腐病是一种由镰刀菌引起的田间和储藏期都普遍发生的病害,主要引起块茎腐烂,致使马铃薯品质和产量降低,严重影响其食用价值和经济价值。【目的】发掘有效的生防菌株以控制马铃薯干腐病,并探究其抑菌作用。【方法】从甘肃定西地区马铃薯根际土壤中分离到109株细菌,以硫色镰刀菌(Fusarium sulphureum)为靶标菌,采用平板对峙法筛选拮抗菌,并通过形态学、生理生化特征及16S r RNA基因序列分析对拮抗菌株进行鉴定。检测拮抗菌无菌发酵液对F.sulphureum菌丝生长、孢子萌发、马铃薯块茎损伤接种病斑直径、干腐病发病率及对绿豆种子发芽的影响。【结果】筛选到一株对马铃薯干腐病有较强抑制作用的菌株YL11,经鉴定其为假单胞菌属(Pseudomonas sp.)菌株。YL11菌株无菌发酵液对F.sulphureum菌丝生长、孢子萌发、马铃薯块茎病斑扩展、干腐病发病率、毒素活性均有显著抑制作用。20%无菌发酵液对F.sulphureum菌落生长的抑制率达到87.3%;75%无菌发酵液可完全抑制孢子萌发;无菌发酵液浸泡能有效抑制马铃薯干腐病病斑的扩展,14 d时对病斑扩展的抑制率达到67.1%;90 d后干腐病的发生率降低了68.4%;同时降低了F.sulphureum毒素的活性。【结论】拮抗菌株YL11能显著抑制F.sulphureum的生长,对马铃薯干腐病有较强的生物防治效果,具有潜在的应用价值。     

金龟子绿僵菌固态培养生物变量优化研究

金龟子绿僵菌属于真菌类生物杀虫剂,本文研究了接种量、种龄、菌种代数等生物因素对金龟子绿缰菌生长及产孢量的影响,并对液-固两步发酵工艺进行了研究.经过优化,得到金龟子绿僵菌产孢的最佳生物参数是:接种量为2.5g/100g、种龄为5～6天;采用固体二代种,培养6天,孢子产量可达1.531×1010孢子/g干培养基.     

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。     

一株高效拮抗向日葵菌核菌的细菌菌株YC16的筛选及其作用效果研究

[目的]筛选高效拮抗向日葵菌核菌的细菌菌株,为开发防治菌核菌病害、提高向日葵产量的生物菌剂提供菌种资源。[方法]以羧甲基纤维素钠(CMC)、小麦秸秆纤维素为唯一碳源的无机盐培养基,分离高效降解纤维素的细菌菌株;采用纤维素降解菌与菌核菌的平板对峙方法,进一步筛选拮抗菌核菌的菌株;利用16S rDNA序列鉴定菌株、PDYA平板对峙实验检验上述所选拮抗菌株的抑菌谱;采用离体向日葵新鲜叶片、草炭土基质盆栽实验,观察拮抗菌菌株抑制菌核菌生长的能力;温室盆栽和田间试验条件下,研究其防治向日葵菌核菌病害、促进生长和提高产量的效果。[结果]筛选了一株高效抑制菌核菌的细菌YC16,经过16S rDNA序列分析,鉴定为解淀粉芽孢杆菌。YC16菌株能够抑制8种病原真菌生长,包括齐整小核菌、腐皮镰孢菌、尖孢镰刀菌、稻梨孢、辣椒疫霉、镰刀菌、尖镰孢黄瓜专化型和向日葵菌核菌;抑制菌核菌感染叶片,抑制率达到了80.42%;抑制盆栽基质中菌核菌的菌丝生长,基质表面菌丝密度比对照减少了50%以上。盆栽接种YC16的向日葵生物量比对照提高54.9%,田间向日葵接种YC16菌剂对菌核菌引发的盘腐病防治效果达39%-100%,产量提高24.4%-30.2%。[结论]YC16生物菌剂施用于土壤,能够有效防治向日葵的茎腐病和盘腐病,展现了防治向日葵菌核病和提高产量的双重效果,是一株具有良好应用前景的高效菌种资源。     

不同土壤类型和含水量下金龟子绿僵菌对红火蚁的致病力

[背景]红火蚁是一种危险性入侵生物,虫生真菌对其防治效果会受到外界环境因子的影响。[方法]应用致病力测定的方法研究了不同剂量金龟子绿僵菌M09对红火蚁的毒力,同时研究了含水量和土壤类型对绿僵菌毒力的影响。[结果]红火蚁的死亡率与金龟子绿僵菌的剂量呈正相关,处理4d后LG50为0．37g。金龟子绿僵菌在砂土、壤土和粘土中对红火蚁的致死率均与对照差异显著,其中在砂土中的毒力最强。此外,在不同含水量的土壤中,金龟子绿僵菌的致死率也不相同（P〈0．01）。[结论与意义]土壤类型和土壤湿度会显著影响金龟子绿僵菌M09对红火蚁的防治效果。选择高湿和砂土类型的土壤施用金龟子绿僵菌M09可以达到较好的效果。     

Effects of validamycin A on the morphology, growth and sporulation of Rhizoctonia cerealis, Fusarium culmorum and other fungi

All Basidiomycotina screened were sensitive to validamycin A, whereas most Ascomycotina and all Mucorales and Oomycetes were insensitive. Studies with Rhizoctonia cerealis and Fusarium culmorum showed that, in semi-solid culture, the antibiotic caused a decrease in colony radial growth rate and that this was associated with a decrease in mean hyphal extension rate and an increase in hyphal branching. However, the antibiotic did not alter the morphology of R. cerealis grown in liquid culture (shaken or stationary). Validamycin A caused a reduction in the number and viability of conidia produced by F. culmorum.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

两株椰心叶甲分离物的鉴定及其系统发育分析

采用形态学方法对2株从自然罹病死亡的椰心叶甲虫尸上分离到的致病菌株Dz01和Ma4进行了鉴定,发现2个菌株在菌丝、瓶梗和分生孢子等形态特征上与金龟子绿僵菌小孢变种基本一致,可将2个菌株鉴定为金龟子绿僵菌小孢变种。基于Dz01和Ma4菌株和其它31个代表绿僵菌主要种或变种菌株rDNA上ITS1-5.8S-ITS2区序列构建的最大简约树显示,Dz01和Ma4菌株均聚在金龟子绿僵菌小孢变种所构成的分支中,这为2个菌株形态学鉴定结果提供了分子依据。     

Impact of moisture on survival of Aedes aegypti eggs and ovicidal activity of Metarhizium anisopliae under laboratory conditions

The effect of relative humidity (43%, 75%, 86% and > 98%) on Aedes aegypti eggs treated with Metarhizium anisopliae or water only was tested for up to a six months exposure at 25 degrees C. Survival of larvae inside eggs was clearly affected by the lowest humidity (43%) tested, and eclosion diminished at all humidities after increasing periods of exposure. M. anisopliae showed to have a strong ovicidal activity only at humidity close to saturation. No difference of activity was found between conidia and hyphal bodies tested. This fungus affected larvae inside eggs and has potential as a control agent of this important vector in breeding sites with high moisture.     

Mortality of Delia floralis, Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi

Abstract:  The susceptibility of Delia floralis eggs, neonates and larvae and the susceptibility of Galleria mellonella and Mamestra brassicae larvae to seven different Norwegian isolates of the insect pathogenic, hyphomycetous fungi Tolypocladium cylindrosporum , Metarhizium anisopliae and Beauveria bassiana , were investigated. Metarhizium anisopliae isolate ARSEF 5520 was highly virulent to G. mellonella larvae and caused 100% mortality when tested at a concentration of 3.6 × 10⁶ conidia/ml. The same M. anisopliae isolate was not virulent to D. floralis larvae. Isolates of T.cylindrosporum , were equally virulent to G. mellonella and D. floralis causing up to 36.0% mortality of larvae. It is suspected, however, that the use of grated rutabaga as a food source in the D. floralis bioassay reduced the fungal virulence of both M. anisopliae and T. cylindrosporum to D. floralis . Among three T. cylindrosporum isolates tested at a concentration of 1.0 × 10⁷ conidia/ml against eggs of D. floralis none of them reduced the hatching percentage. One isolate, ARSEF 5525 did, however, significantly reduce the longevity of neonates. Beauveria bassiana isolates ARSEF 5510 and ARSEF 5370 tested at a concentration of 1.0 × 10⁷ conidia/ml resulted in M. brassicae mortality levels of 70.0 and 55.0%, respectively. The B. bassiana isolate ARSEF 5557, however, was not virulent to M. brassicae . Among the three isolates tested against M. brassicae the two virulent isolates produced a red pigment, probably oosporein, when cultured in Sabouraud dextrose agar.     

Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.     

实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础,本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列,Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani)AG1-IA融合群菌株GD118为对照,采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小,研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。     

Structure of the outer surfaces of sclerotia of certain fungi

Summary The surfaces of sclerotia of Rhizoctonia solani, Botrytis cinerea and Sclerotinia rolfsii were examined with the Stereoscan electron microscope.The periphery of the sclerotium of R. solani consists of a loose net-work of hyphae which are not sufficiently thickened to withstand the extreme desiccation that takes place when the material is coated with gold-palladium alloy.The surface of the sclerotium of B. cinerea has many closely packed hyphal tips which project outwards from the centre of the structure. The thickening of the walls of the hyphae enable them to retain their shapes. A film was observed on the most exposed areas and this may have been dried-up melanin pigment.The outer skin of the sclerotium of S. rolfsii is an almost continuous layer which is thrown into ridges and troughs to give a wavy appearance.     

绿僵菌侵染小菜蛾幼虫过程的透射电镜观察

利用透射电镜观察了绿僵菌Metarhizium anisopliae对小菜蛾Plutella xylostella幼虫的侵染过程。结果表明：接种后22 h最早观察到绿僵菌穿透到寄主表皮层中,在穿透表皮过程中伴随着机械力和酶的活动。菌体进入寄主血腔后,以菌丝出芽生殖、菌丝分隔及菌丝段分隔3种方式大量增殖,主要形成颗粒状的菌丝段在寄主体腔内扩散,也形成少量的丝状菌丝。     

New bioassay method to assess the pathogenicity of Colombian strains of Metarhizium anisopliae (Metsch.) Sorokin and Paecilomyces sp. (Deuteromycotina: Hyphomycetes) against the subterranean burrower bug Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae)

Using a new sand bioassay method, the pathogenicity of three Colombian strains of Metarhizium anisopliae (i.e., CIAT 224, 245, and 230) and one strain of Paecilomyces sp. (CIAT 308) were tested against fourth instars of the subterranean burrower bug Cyrtomenus bergi Froeschner (Hem.: Cydnidae) in the laboratory. The proposed bioassay simulates the habitat of C. bergi and at the same time enables the evaluation of a large number of insects in a short period of time and under controlled conditions. All tested fungal species/strains were pathogenic against C. bergi. However, only low levels of mortality were obtained, and never exceeded 55%. In the screening experiment, M. anisopliae strains CIAT 224 and CIAT 245 caused the highest levels of mortality. The use of the bioassay procedure described herein is discussed.