松褐天牛球孢白僵菌高毒力航天诱变菌株的筛选

【目的】松褐天牛Monochamus alternatus是我国松材线虫病Bursaphelenchus xylophilus的主要媒介昆虫。为了更好地开发利用松褐天牛病原微生物球孢白僵菌Beauveria bassiana, 本研究通过航天搭载诱变及室内筛选, 获得球孢白僵菌高毒力诱变菌株。【方法】将经神舟八号飞船航天搭载诱变后的孢子稀释液涂布在PDA平板上培养,获得单菌落菌株,进而筛选获得高毒力诱变菌株。观察所获9个航天诱变菌株的菌落形态、菌落生长速度、产孢量、孢子萌发率及抗高温胁迫能力等生物学特性, 在此基础上筛选出生物学性状优良的菌株B159, B252和B305, 并进一步对松褐天牛4龄幼虫进行生物测定。再通过撒菌粉和注射菌液方法, 检验B252和B305对松木段内松褐天牛幼虫的杀虫效果。【结果】球孢白僵菌航天诱变菌株的生物学特性与野生型菌株cfcc81357存在分化。9个航天诱变菌株的菌落形态发生了不同程度的改变,6个菌落生长速率出现负向变异,仅诱变菌株B159, B252和B305能产生分生孢子。航天诱变菌株B252和B305在浓度为1.0×107 cfu/mL时对松褐天牛4龄幼虫的校正死亡率均为100%, 半致死中时(LT₅₀)分别为8.08和8.56 d, 明显优于野生型菌株, 显示出对松褐天牛的极强毒力。使用撒菌粉和孢子液体注射方法, 诱变菌株B252和B305对松木段内松褐天牛幼虫死亡率比野生型菌株高。【结论】诱变菌株B252和B305可能是优良菌株, 对生物防治松褐天牛方面有潜在的实用价值。     

美国白蛾高毒力球孢白僵菌菌株筛选

【目的】美国白蛾Hyphantria cunea(Drury)是世界性检疫害虫,目前已经入侵到我国长江下游地区(从辽宁丹东至安徽芜湖)。作者在前期研究中,在芜湖地区成功获得了被球孢白僵菌Beauveria bassiana侵染的美国白蛾僵虫。本研究拟在前期已获取菌株的基础上,筛选生物学性状良好且对美国白蛾幼虫具有高致病力的球孢白僵菌菌株,以期为我国长江下游地区利用昆虫病原真菌防治美国白蛾奠定基础。【方法】对采集自美国白蛾僵虫的7株球孢白僵菌菌株(Bb01-1,Bb08-5,Bb08-6,Bb08-11,Bb22-2,Bb22-3和Bb22-5)进行生物学特性研究,以候选菌株菌落形态、生长速率、产孢量、萌发率和抗紫外能力等参数确定优良菌株;并将生物学性状良好的球孢白僵菌菌株配制成5个浓度(5×10~6,1×10~7,5×10~7,1×10~8和5×10~8孢子/m L)的孢悬液,采用浸渍法处理美国白蛾4龄幼虫,以幼虫死亡率、侵染率等指标评价菌株的致病力。【结果】供试菌株Bb22-2,Bb22-3和Bb22-5为粉状菌落,其菌落直径大,产孢早且孢子层厚;同时,这3个菌株在菌落生长速率和产孢量以及孢子萌发率、紫外照射处理后的萌发率也均显著优于其他4个菌株。毒力测定结果表明,这3个菌株对美国白蛾4龄幼虫表现出很强的致病力,各菌株处理11 d后美国白蛾幼虫校正死亡率为80%~90%。这3个菌株对美国白蛾幼虫的侵染率均高(侵染率53%~87%),并且呈现随浓度升高侵染率上升的趋势。其中,菌株Bb22-2侵染率均要高于其他2个菌株。【结论】本研究成功筛选获得了生物学性状良好且对美国白蛾高毒力的球孢白僵菌菌株。鉴于中国长江下游地区气候特点与美国白蛾生物学习性,可以预期采用昆虫病原真菌防治美国白蛾在该地区将具有很好的应用前景,但其实际应用价值尚需进一步的田间试验进行确认。     

昆虫病原真菌紫外线诱变育种研究进展

昆虫病原真菌是控制害虫发生与危害的重要生物防治因子之一,优良的菌种是生物防治的关键。紫外线诱变育种是昆虫病原真菌菌种选育中最常用的方法之一,具有易操作、效果好、作用时间长等优点。本文以白僵菌、绿僵菌、玫烟色棒束孢等三种常用的昆虫病原真菌为例,从诱变育种的材料、方法和应用等方面综述了昆虫病原真菌紫外线诱变育种的研究现状,以期为昆虫病原真菌的生物防治研究提供参考。     

86A—2真菌制剂防治山楂叶螨,棉叶螨的研究

从田间山楂叶螨尸体上分离到一株真菌８６Ａ—２菌株。经形态观察和生物学性状研究,该菌株与顶头孢霉菌（Ｃｅｐｈａｌｏｓｐｏｒｉｕｍａｃｒｅｍｏｎｉｕｍｃｏｒｄａ）近似［１］。经证病试验表明该菌株是叶螨属昆虫的病原真菌,有很强的致病力,田间试验表明对山楂叶螨、棉叶螨具有较好的生物防治效果。     

航天对MT工程菌株的影响研究

为了探讨航天与常规诱变技术对MT工程菌株的影响,进行了HNO2.UV、γ-射线、LiCl及γ-射线 LiCl分别与航天共诱变对MT工程菌株活性的影响试验,并且就航天对MT工程菌株的致死作用及质粒的稳定性进行了研究。结果表明:MT工程菌株经航天处理后,活菌数降低,死亡率为34.59%,航天对MT工程菌株具有一定的致死作用;MT工程菌株经UV、γ-射线、LiCl及γ-射线 LiCl分别与航天共诱变处理后的生长活性没有改变,MT工程菌株经HNO2与航天共诱变处理后的生长速率减慢;在航天处理的作用下,MT工程菌株的质粒不易丢失,能够维持很好的稳定性,丢失率仅为8.26%,而置于地面的MT工程菌株的质粒易丢失,丢失率为83.51%。     

航天搭载对小桐子种子萌发及幼苗生长的影响

研究了航天搭载对两个种源地小桐子种子萌发及幼苗生长的影响.结果表明:航天搭载降低了种子的活力;在幼苗生长方面,航天搭载对小桐子幼苗的成苗率没有显著影响,航天搭载扩大了版纳种源当代幼苗株高及地径的变异范围,对元阳种当代幼苗的株高及地径影响不明显.这说兑叫不同种源地的植物对太空环境的反应有所不同.本研究为航天技术有效的运用于小桐子诱变育种提供依据.     

温莪术内生真菌Gibberella moniliformis EZG0807诱变及其诱变株遗传稳定性研究

利用大梯度超导磁体(JMT-16T50F)模拟失重和超重环境对温莪术内生真菌Gibberella moniliformis EZG0807进行诱变,以期得到代谢产物活性高、遗传稳定性好的菌株。诱变24 h、48 h和72 h后,通过稀释涂布平板法得到139株诱变菌株;经滤纸片抑菌法初筛和MTT法抗肿瘤细胞活性实验复筛,筛选出高活性诱变菌株M7226。采用群体传代的方法考察菌株M7226十代以内菌株的生长状况和次级代谢产物抗菌抗肿瘤活性的能力。结果显示活性内生真菌EZG0807经大梯度超导磁体诱变,筛选得到一株代谢产物活性高、遗传稳定性好的诱变菌株M7226,为后续次级代谢产物的分离纯化奠定基础,同时此法为真菌诱变育种提供了一种新的可供选择的方法。     

诱变育种新途径——航天搭载太空诱变育种

1 航天搭载,太空(空间)诱变 航天搭载,空间诱变可以简称为航天诱变育种,亦称太空育种.它是将农作物种子或供试诱变材料搭乘返回式卫星或宇宙飞船,送到距地球200～400km的太空,利用空间宇宙射线的强辐射在高真空、微重力和交变磁场等特殊环境中进行诱变处理,使供试的农作物种子和材料产生变异,返回地面后筛选、培育出符合要求的新品种.     

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。     

黄绿绿僵菌Mf82菌株对不同虫态褐飞虱的毒力

对水稻重要害虫褐飞虱Nilaparvata lugens目前市场上尚无一种理想的微生物杀虫剂。昆虫病原真菌具有从体壁侵入的能力因而对刺吸性害虫的防治具有优势。为此, 本研究选用不同原寄主和来源地的3种昆虫病原真菌（金龟子绿僵菌Metarhizium anisopliae、 黄绿绿僵菌Metarhizium flavoviride和球孢白僵菌Beauveria bassiana）的12个不同菌株, 以1 100孢子/mm²孢子悬浮液进行室内毒力测定。结果表明： 在参试的不同菌种12个菌株中, 黄绿绿僵菌Mf82菌株对褐飞虱成虫致病力最高, 10 d累计校正死亡率为83.5%, 致死中时（LT₅₀）为4.6 d。其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力, 毒力大小顺序为成虫>高龄若虫>低龄若虫。黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、 卵粒均有侵染作用, 10 d侵染率分别为66.7%和51.2%, 卵龄越低, 侵染效果越好, 卵龄为0.5 d时侵染率最高。本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、 若虫和卵均有较强的致病性, 是一株极具应用潜力的生防真菌。     

泡盛曲霉抗FOA突变株的筛选与转化

根据泡盛曲霉SG1菌株分生孢子的紫外线致死曲线,选择死亡率为85%～90%的诱变时间诱变分生孢子,然后将其涂布于含FOA(5-flourooroticacid)和尿嘧啶核苷的基本培养基上,选择抗FOA的突变株。经过纯化和回复突变检测后,获得了5株需要尿嘧啶或尿嘧啶核苷才能在基本培养基上生长的稳定突变株。进一步分析鉴定结果表明,这些突变株的URA3基因发生了突变。Northern杂交及RTPCR方法证明这些突变株中URA3基因突变均发生在转录水平上。选择突变株SA5作为受体菌,用含来自黑曲霉的野生型URA3基因的质粒转化该受体菌,结果获得了稳定的转化子。Southern杂交证明野生型URA3基因取代了突变株的ura3基因。     

绿僵菌M189菌株特异性SCAR标记的建立及田间监测应用

为了监测生防真菌绿僵菌菌株在田间释放后的回收,有必要建立菌株DNA分子标记,以此将应用的菌株与其他菌株或田间的土著分离株鉴别开来。作者采用16条随机引物扩增了51株绿僵菌菌株的基因组DNA,得到81个多态性位点。其中M189菌株多态性位点30个,分析得到1个特异性位点,并将该位点的DNA片段测序后转化成为特异性SCAR标记。检验确定了该标记的敏感性,可以从供试的51株菌株中准确鉴定出目的菌株M189。并用该标记检测了从田间回收的3个分离株,确定其中1个与应用菌株M189一致。     

草地贪夜蛾高致病力绿僵菌菌株的筛选与鉴定

为获取对草地贪夜蛾Spodoptera frugiperda具有高致病力的生防真菌,从福建省不同地区分离得到8株寄主为鳞翅目和半翅目幼虫僵虫的绿僵菌Metarhizium,采用浸渍法测定了其对草地贪夜蛾2龄幼虫和蛹的致病力,并根据形态学和分子生物学方法对高致病力菌株进行种类鉴定。结果表明,8株绿僵菌菌株对草地贪夜蛾2龄幼虫和蛹均表现出不同程度的致病力,其中菌株FJMR2和FJXY7表现出较强的致病力。在5×107个/mL孢子浓度下,FJMR2和FJXY7对草地贪夜蛾2龄幼虫的致死率分别为88.76%和82.13%,对蛹的致死率分别为86.57%和84.00%;对草地贪夜蛾2龄幼虫的LT50分别为4.81 d和4.93 d,对蛹的LT50分别为4.94 d和4.83 d。经鉴定菌株FJMR2和FJXY7均为莱氏绿僵菌Metarhizium rileyi。本研究获得2株对草地贪夜蛾2龄幼虫和蛹具有高致病力的莱氏绿僵菌菌株,在草地贪夜蛾的生物防治中具有较大应用潜力。     

Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate.     

Host-plant mediated interactions between two aphid species

Three isolates of Isaria fumosoroseus Wize (Hyphomycetes) were cultured on six media composed of differing amounts of chitin, carbon, and nitrogen. The effect of nutrition on growth and virulence was studied by measuring colony growth, spore yield, germination rate, spore-bound protease (Pr₁) and lipase activity, and virulence of inoculum produced by different media against second instars of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Chitin peptone nutrition media resulted in the highest colony growth but spore yields were lowest for this medium, whereas the osmotic stress medium resulted in the lowest colony growth with fewer spores. Highest lipase activity (4.14 µmol/ml/min) was observed for spores produced on high C/N medium for isolate IF28.2, whereas highest Pr1 activity (2.64 µmol/ml/min) was also observed on high C/N medium for isolate IF28.2. A higher rate of Pr1 and lipase activity for all three isolates was observed on low C/N, 2% peptone, and the osmotic stress media. Conidia from nutrient-poor media (2% peptone) proved to be the least virulent for all three isolates with median survival time values of 2.23, 2.05, and 1.79 days for IF32, IF28.2, and IF49, respectively. Median survival time values for the various nutrient media proved to be positively correlated with spore-bound Pr1 and lipase activity, having correlation coefficient values of 0.115 and 0.538, respectively.     

黑龙江黑木耳优良菌株筛选研究

在2012年春季初筛和秋季复筛基础上,对选出的4个野生黑木耳菌株和5个栽培品种,以及在多个木耳基地分离获得的7个菌株进行了进一步栽培比较试验。基于菌丝生长速度、长势、拮抗试验、抗菌试验、栽培农艺性状和产量等因素,最终从16个菌株中筛选出适合黑龙江栽培的优良菌株6个,其中野生菌株2个：A14和A15;栽培菌株4个：M23、M43、M44和M46。     

Coexistence of parentalBacillus brevis and its gramicidin S-negative mutant during spore germination stages

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared in separate as well as in mixed cultures with respect to germination of their spores in several media. Mixed-culture experiments were facilitated by the observation that colonies of wild and mutant cultures are distinctly different in appearance on nutrient agar. We found that there was complete coexistence in both strains throughout the outgrowth phase of germination, during which gramicidin S-induced suicide normally occurs in the wild-type prior to vegetative growth. Coexistence was also observed in media supporting germination but not growth, i.e., alanine-salts and alanine-water. The same was found when spores of the two strains were incubated in a soil suspension. We found that both strains become sensitive to starvation in a salts mixture only after development into vegetative cells, the mutant strain being more sensitive than the parent in this regard, but again coexistence was observed in mixed culture.     

A field experiment in Ghana on the tolerance of cocoa seedlings to cocoa swollen-shoot and cocoa mottle-leaf viruses

Seedings of Amelonado Cocoa and of two progenies obtained by crossing Iquitos (Upper Amazon) parents were infected with three strains of cocoa swollen-shoot virus (CSSV) and with cocoa mottle-leaf virus (CMLV). CSSV strain I A had the most severe effects on the growth and canopy condition of all varieties. All four virus isolates had more effect on the growth of Amelonado than on the Iquitos progenies, except that CSSV strain I W affected the growth of Amelonado and one of the Iquitos progenies equally, as did CMLV which caused the greatest decrease in the first crop of all varieties. The relative virulence of different CSSV strains in any one variety can be assessed from single criteria such as length of latent period or effect on growth; CMLV had a shorter latent period than CSSV Strain I A but less effect on growth.     

Characterization of a mutant strain of a filamentous fungus Cladosporium phlei for the mass production of the secondary metabolite phleichrome

UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.     

Mating System and Basidiospore Formation in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel⁻). The fruiting, nonfruiting, and Cel⁻ phenotypes differed from each other and from the parental wild-type strain in a variety of characteristics, including growth, conidiation, and evolution of ¹⁴CO₂ from ¹⁴C-side chain-labeled lignin, indicating that strain OCG101 is a heterokaryon.