黑粉虫幼虫肠道细菌的研究

从黑粉虫幼虫肠道环境中分离、纯化、培养,获得5个细菌菌株,对其培养性状、染色反应、菌体形态、生理生化反应等进行了系统研究.鉴定结果表明：上述5个细菌菌株分别属于金杆菌属（Aureobacterium）、李斯特菌属（Listeria）、微杆菌属（Microbacterium）、莫拉菌属（Moraxella）、短小杆菌属（Curtobacterium）,其中金杆菌属、微杆菌属、短小杆菌属菌也发现于黄粉虫幼虫肠道环境中.通过研究,既可为黑粉虫生产所需微生态制剂提供微生态理论依据,又发掘了微生物资源,丰富了微生物资源库.     

窗胸萤肠道细菌分离及鉴定研究

从窗胸萤的5龄幼虫中分离纯化获得10个细菌菌株,并对其菌体形态、染色反应、培养性状、生理生化性状进行了系统研究。鉴定结果表明,上述10个细菌菌株分别属于埃希氏菌属(Escherichia)、金色单胞菌属(Chryseomonas)、放线杆菌属(Actinobacillus)、摩根氏菌属(Morganella)、李斯特菌属(Listeria)、金杆菌属(Aureobacterium)、微杆菌属(Microbacterium)、地杆菌属(Terrabacter)、芽孢杆菌属(Bacillus)和黄杆菌属(Flavibacterium)。     

栎黄掌舟蛾幼虫肠道好氧菌群分析及产纤维素酶菌的筛选

栎黄掌舟蛾Phalera assimilis是柞树主要叶部害虫之一。本研究以栎黄掌舟蛾幼虫为材料,从幼虫肠道中分离出好氧细菌23株,通过对16S r DNA扩增产物ARDRA分析后,代表性菌株测序结果表明,23株肠道好氧细菌分别属于葡萄球菌属Staphylococcus sp.、短小杆菌属Curtobacterium sp.、赖氨酸芽孢杆菌属Lysinibacillus sp.、肠球菌属Enterococcus sp.和阿特拉津降解菌属Arthrobacter sp.5个属的细菌,其中,以葡萄球菌属及短小杆菌属细菌种类最多。通过筛选培养基从23株菌中筛选出产纤维素酶菌6株。本研究可为深入了解栎黄掌舟蛾肠道菌群结构、寻找产纤维素酶菌及新的微生物资源等奠定了理论基础。     

中华通草蛉越冬成虫肠道可培养好氧细菌多样性分析

本研究以昆虫微生态学原理为指导探究中华通草蛉Chrysoperla sinica(Tjeder)的肠道菌群构成,为中华通草蛉的营养生理研究及人工繁殖利用提供理论依据和技术基础。采用无菌解剖及纯培养分离检测技术,从雌、雄成虫肠道环境中分离、纯化、培养,获得10个细菌菌株,对菌体形态、染色反应、培养形状、生理生化反应进行系统研究。结果表明,上述10个菌株分别属于摩根氏菌属(Morganella)、短小芽孢杆菌(Bacillus pumilus)、地杆菌属(Terrabacter)、爱德华氏菌属(Edwardsiella)、色杆菌属(Chromobacterium)、热黄杆菌(Flavobacterium thoupophilum)、放线杆菌属(Actinobacillus)、棍状杆菌属(Clavibacter)、邻单胞菌属(Plesiomonas)、预研菌属(Yokenella);越冬期雌、雄成虫肠道细菌的种类和数量存在较大差异,可能与雌、雄成虫不同的营养和生理需求有关,两者关系还需进一步研究。     

窗胸萤生物学特性及其幼虫肠道细菌种类的研究

对一种陆栖萤火虫窗胸萤(Pyrocoelis analis Fabricius)的生物学特性及其幼虫的肠道细菌进行了研究。结果表明:窗胸萤在24℃下从卵发育到成虫平均(349.50±8.15)d,其卵期、幼虫期、蛹期及成虫期分别为(117.22±0.90)d、(184.92±18.56)d、(12.65±1.09)d和(15.60±3.85)d;在其5龄幼虫中分离纯化出埃希氏菌属(Escherichia)、金色单胞菌属(Chryseomonas)、放线杆菌属(Actinobacillus)、摩根氏菌属(Morganella)、李斯特菌属(Listeria)、金杆菌属(Aureobacterium)、微杆菌属(Microbacterium)、地杆菌属(Terrabacter)、芽孢杆菌属(Bacillus)和黄杆菌属(Flavibacterium)共10个细菌菌株。     

香山黄栌叶片内生细菌的分离与鉴定

通过微生物培养方法对北京香山黄栌叶片内生细菌进行分离及筛选,共得到81株内生细菌,经形态学观察、16S rDNA扩增及系统发育分析,将其归为变形菌门(Proteobacteria)中的欧文氏菌属(Erwinia)和志贺氏菌属(Shigella);厚壁菌门(Firmicutes)中的芽孢杆菌属(Bacillus)、柯恩氏菌属(Cohnella)、类芽孢杆菌属(Paenibacillus)和葡萄球菌属(Staphylococcus);放线菌门(Actinobacteria)中的短小杆菌属(Curtobacterium)。这是国内外首次利用培养方法针对黄栌叶片内生细菌进行研究的报道。     

小龙虾肠道产木聚糖酶细菌的分离与鉴定

【背景】小龙虾肠道微生物是小龙虾降解纤维素和半纤维素的主要驱动力。【目的】研究肠道内细菌的相对丰度,为揭示肠道微生物在小龙虾纤维素降解过程中的作用提供理论支撑。【方法】采用纯培养法从小龙虾肠道筛选产木聚糖酶细菌,并且对小龙虾肠道细菌进行16S高通量测序。【结果】形态学和16SrRNA基因分子鉴定表明,筛选到的4株产木聚糖酶细菌均属于芽孢杆菌科芽孢杆菌属;结合进一步的生理生化特征鉴定,结果为:菌株Z-3为枯草芽孢杆菌(Bacillus subtilis),菌株Z-4为贝莱斯芽孢杆菌(Bacillus velezensis),菌株Z-29为蜡状芽孢杆菌(Bacillus cereus),菌株Z-30为高地芽孢杆菌(Bacillus altitudinis);16S rRNA基因高通量测序结果表明:在属水平上,小龙虾肠道细菌主要是Candidatus Bacilloplasma、拟杆菌属、弧菌属、不动杆菌属、Dysgonomonas、Tyzzerella3、气单胞菌属和希瓦氏菌属细菌。【结论】小龙虾肠道内细菌资源丰富,且芽孢杆菌属细菌在木质纤维素降解过程中发挥一定功能。     

黄瓜初花期与结瓜期叶片可培养内生细菌多样性研究

【目的】研究黄瓜初花和结瓜两个生长期叶片可培养内生细菌的多样性。【方法】采用叶片表面消毒、菌种分离、16S rDNA序列扩增和系统发育分析进行了系统研究。【结果】两时期黄瓜内生细菌的种类、数量及优势菌的种类都有明显差异。初花期叶片含菌量为(2.6±0.18)106CFU/g鲜重,分离出的38株内生细菌分别属于短小杆菌属(Curtobacterium sp.)、节杆菌属(Arthrobacter sp.)、微杆菌属(Microbacterium sp.)等14个已知属,其中优势种类为短小杆菌属菌株;结瓜期叶片含菌量为(5.2±0.42)105CFU/g鲜重,分离出的43株内生菌分别属于泛菌属(Pantoea sp.)、假单胞菌属(Pseudomonas sp.)、芽孢杆菌属(Bacillus sp.)等11个已知属,其中优势种类为泛菌属菌株。【结论】初花期内生菌含量是结瓜期的5倍,两时期内生菌的种类表现出很强的差异,体现出黄瓜不同生育期可培养内生菌数量和种类的多样性,相关研究为黄瓜促生内生细菌的理论探索和生产应用提供一定的研究基础。     

六斑异瓢虫成虫肠道细菌分离及鉴定研究

目的通过对肠道菌群的研究，探讨其营养生理，进而改善和研制人工饲料，提高六斑异瓢虫的抗逆力和成活率。方法按照传统的培养分离方法从六斑异瓢虫雌成虫肠道消化道内分离纯化获得5个不同菌株，分别对其培养性状、菌体形态、染色反应、生理生化性状进行了系统研究。结果上述5个细菌菌株分别属于地杆菌属（Terrabacter）、李斯特菌属（Listeria）、短小杆菌属（Curtobacterium）、纤维单胞菌属（Cellulomonas）和皮杆菌属（Demabacter）。结论六斑异瓢虫的菌群种类与其生活习性有关，为进一步研究适合六斑异瓢虫生长和繁殖的人工饲料提供数据参考。     

柑橘黄龙病寄主长春花内生细菌的分离及功能鉴定

【目的】感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构及功能对柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】利用兼性厌氧可培养技术以及植物内生菌功能特性分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。29种内生细菌进行功能分析,其中6株内生细菌至少具有4种功能特性,分属于马胃葡萄球菌、苏云金芽孢杆菌、巨大芽孢杆菌、短小杆菌属、摩氏摩根菌(Morganella morganii)及溶杆菌属(Lysobacter sp.)5个属。【结论】感病与健康长春花植株中均含有丰富的内生细菌且差异较大,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的菌种。     

Further evidence for the phylogenetic coherence of actinomycetes with Group B-peptidoglycan and evidence for the phylogenetic intermixing of the genera Microbacterium and Aureobacterium as determined by 16S rDNA analysis

Abstract A 16S rDNA study was performed on at least two species, including the type strain of the type species, of each genus described to possess peptidoglycan of Group B. Analyses confirm that these actinomycetes form a phylogenetically coherent cluster within the arthrobacteria subline of descent. While members of the genera Agromyces, Clavibacter, Curtobacterium and Rathayibacter appear to be phylogenetically coherent, members of Microbacterium and Aureobacterium do not cluster according to their taxonomic affiliation but form one large cluster in which species of both genera are intermixed. Species with no taxonomic standing, i.e. "Corynebacterium aquaticum" , and "Brevibacterium helvolum" form three separate sublines of descent and can be considered nuclei of future novel genera.     

Isoprenoid quinones in the classification of coryneform and related bacteria.

Menaquinones were the only isoprenoid quinones found in 85 of the 95 coryneform bacteria examined. Dihydromenaquinones having nine isoprene units were the main components isolated from Corynebacterium bovis, from other glutamic acid-producing strains, and from Arthrobacter globiformis and related species. Dihydromenaquinones with eight isoprene units were found in Brevibacterium linens, the remaining Corynebacterium species and strains probably belonging to the genus Rhodococcus. Tetrahydromenaquinones with eight isoprene units were found in Arthrobacter simplex and Arthrobacter tumescens, and with nine isoprene units in Cellulomonas and Oerskovia. Kurthia and Curtobacterium were characterized by menaquinones with seven and nine isoprene units, respectively, and Microbacterium lacticum and Corynebacterium aquaticum had comparable amounts of menaquinones with 10 and 11 isoprene units. Strains received as Brevibacterium leucinophagum, Corynebacterium autotrophicum, Corynebacterium nephridii, Mycobacterium flavum, Mycoplana rubra and Protaminobacter ruber contained uniquinones as their sole isoprenoid quinones. The isoprenoid quinone data correlate well with major trends in coryneform taxonomy and are of value in the classification of coryneform and related bacteria.     

Development of the Predatory Stinkbug Brontocoris tabidus Signoret Heteroptera: Pentatomidae on Different Proportions of an Artificial Diet and Pupae of Tenebrio molitor L. Coleoptera: Tenebrionidae

The development of the Neotropical predatory pentatomid Brontocoris tabidus on an artificial diet based on beef meat and liver was evaluated. The predator showed significantly longer nymphal development and lower adult weights on this diet than when reared on pupae of the mealworm Tenebrio molitor. The survival of nymphs fed exclusively on the artificial diet was somewhat lower compared with feeding on T. molitor pupae. When B. tabidus was bred on this artificial diet during part of its nymphal period i.e. during the second; second and third; and second, third and fourth instars , and was subsequently returned to T. molitor pupae, the predator nymphs completed the nymphal stage with a developmental rate similar to that of nymphs fed on live prey throughout. The adults attained after switching from the artificial diet to live prey from the third and fourth instar onwards had similar weights to those in the control. Considering the relatively good results obtained with B. tabidus and other members of the pentatomid subfamily Asopinae, this meat-based diet may be a valuable alternative for use in the mass production of predatory pentatomids.     

室内条件下莱氏猛叩甲对松墨天牛和黄粉虫的捕食能力

【目的】本研究旨在明确莱氏猛叩甲Tetrigus lewisi老熟幼虫分别对黄粉虫Tenebrio molitor和松墨天牛Monochamus alternatus末龄幼虫和蛹的捕食量的差异,评估莱氏猛叩甲的捕食能力,为松墨天牛M.alteratus的有效治理找到潜在的高效生物防治因子。【方法】于2014年11月在浙江富阳昌东村马鞍山砍下松墨天牛虫害木,通过林间野外调查,明确松墨天牛和莱氏猛叩甲的发生情况;将野外采集到的莱氏猛叩甲幼虫带回室内,分别饲喂黄粉虫和松墨天牛的末龄幼虫和蛹,每天记录捕食量,确定莱氏猛叩甲的捕食能力。【结果】野外调查中,我们在松墨天牛幼虫蛀食的坑道中发现了莱氏猛叩甲幼虫和松墨天牛幼虫被取食后的残体;在采集到1 094头松墨天牛幼虫的危害木内,共采得莱氏猛叩甲末龄幼虫36头,约为松墨天牛幼虫数量的3%。室内捕食实验表明,5 d内莱氏猛叩甲捕食黄粉虫幼虫总量为3.2头,平均每天捕食约0.6头,而捕食松墨天牛幼虫总量为8.0头,平均每天捕食约1.6头,莱氏猛叩甲对松墨天牛的总捕食量显著高于对黄粉虫幼虫的总捕食量(P0.0001);4 d内莱氏猛叩甲捕食黄粉虫蛹总量为5.6头,平均每天捕食约1.4头,而捕食松墨天牛蛹总量为7.8头,平均每天捕食约2.0头,莱氏猛叩甲对松墨天牛蛹的捕食量显著高于黄粉虫蛹(P=0.028)。回归分析还表明,莱氏猛叩甲幼虫的捕食量不受叩甲个体大小的影响。【结论】莱氏猛叩甲普遍分布在松墨天牛发生区,同时发现松墨天牛被取食后的残体,表明莱氏猛叩甲是松墨天牛的捕食性天敌。莱氏猛叩甲均能捕食黄粉虫和松墨天牛的幼虫和蛹;相比于捕食黄粉虫,莱氏猛叩甲对松墨天牛的幼虫和蛹具有更好的捕食效率,具有很好的生物防治前景,是未来松墨天牛治理潜在高效生物防治因子。     

Nucleic acid hybridization studies on Microbacterium, Curtobacterium, Agromyces and related taxa

Thirty strains of Agromyces, Arthrobacter, Curtobacterium, Brevibacterium, Corynebacterium and Microbacterium, exhibiting the rare peptidoglycan of group B, were subjected to extensive nucleic acid hybridization studies. The DNA homology values indicate that Corynebacterium insidiosum DSM 20157 is genetically identical with Corynebacterium michiganense DSM 20134. Corynebacterium sepedonicum NCPPB 378 and Corynebacterium nebraskense DSM 20400 are closely related to Corynebacterium michiganense DSM 20134. Corynebacterium betae DSM 20141, Corynebacterium oortii ATCC 25283 and Corynebacterium poinsettiae ATCC 9682 are genetically identical with Corynebacterium flaccumfaciens DSM 20129. In addition, Curtobacterium citreum ATCC 15828, Curtobacterium luteum ATCC 15830 and Curtobacterium pusillum ATCC 19096 share a high degree of relatedness to Corynebacterium flaccumfaciens DSM 20129. All other described species are more distantly related to each other. DNa-rRNA cistron similarity studies reveal that all corynebacterium with a peptidoglycan group B are members of one homogeneous cluster for which the rank of a genus is suggested.     

两种色型黄粉虫抗冻蛋白cDNA克隆、序列分析与表达分析

产生抗冻蛋白(antifreeze proteins, AFPs)是大多数昆虫抵抗低温的一个重要策略。为给黄粉虫Tenebrio molitotr耐寒机理研究提供参考, 本研究采用RT-PCR、 5′ RACE和3′ RACE法克隆了黄、 黑两种色型黄粉虫幼虫的抗冻蛋白基因Tm-afp, 分析了其基因序列及所编码的氨基酸序列, 并检测了其在这两种色型间的mRNA水平差异。结果表明： 从黄、 黑两种色型黄粉虫幼虫克隆出的Tm-afp cDNA全长分别为579 bp和588 bp, 它们包含一个20 bp的5′端非翻译区、 一个402 bp的开放阅读框和一个变异较大的3′ 端非翻译区, 其碱基序列一致性为95%。由于这两个抗冻蛋白基因编码的蛋白成熟肽存在两个氨基酸差异： 第35位（D→E）和130位（T→S）, 因此将其判定为黄粉虫抗冻蛋白基因Tm-afp的两个异构体, 并分别命名为Tm-afp-1和Tm-afp-2。Tm-afp-1和Tm-afp-2编码的抗冻蛋白异构体（分别为Tm-afp-1和Tm-afp-2 ）信号肽之间有3个氨基酸差异： 第2位（G→A）、 9位（S→T）和27位（Y→N）; 其成熟肽的第一个氨基酸为谷氨酰胺, 含8个高度保守的12 aa短串联重复序列, 每个重复序列的第2位和第8位为半胱氨酸。此外, Tm-afp-1和Tm-afp-2均属富含苏氨酸和半胱氨酸的昆虫抗冻蛋白, 其二级结构均由大量的β-折叠和无规则卷曲组成, 三级结构为8个特殊的右手β-螺旋, 每一圈螺旋由12个氨基酸组成; 在β-螺旋的一个侧面规则排列着由保守XCT形成的β折叠片层。同时, 低温可以诱导黄、 黑两种色型黄粉虫Tm-afp的表达, 但长时间低温诱导下黑色型黄粉虫幼虫的Tm-afp表达量显著高于黄色型黄粉虫幼虫Tm-afp表达量。结果提示, 黄粉虫种内不同色型间, 抗冻蛋白基因Tm-afp可能存在多种异构体, 并且黑色型黄粉虫Tm-afp比黄色型黄粉虫Tm-afp对低温的应答更强烈。这一研究结果为进一步探索黄粉虫的耐寒性机理提供了有益参考。     

Phylogenetic analysis of the genus Microbacterium based on 16S rRNA gene sequences

Abstract 16S rRNA gene (rDNA) studies of the six species of the genus Microbacterium, M. lacticum, M. laevaniformans, M. dextranolyticum, M. imperiale, M. arborescens and M. aurum , were performed and the primary structures were compared with those of 29 representative actinobacteria and related organisms. Phylogenetic analysis indicated that six species of the genus Microbacterium and representative four species of the genus Aureobacterium appear to be phylogenetically coherent as was suggested by Rainey et al., although the peptidoglycan types of these two genera are different (peptidoglycan type B1 or B2). Thus, the phylogenetical analyses revealed that members of actinobacteria with group B-peptidoglycan do not cluster according to their peptidoglycan types, but form compact cluster different from actinobacteria or actinomycetes with group A-peptidoglycan.     

发酵牛粪对黄粉虫幼虫生长发育的影响

为寻找大型养殖场牛粪资源化与产业化利用途径,本文探讨了杂食性黄粉虫转化与利用牛粪的可行性.将牛粪和黄粉虫常规饲料(65％麦麸、30％玉米面、5％豆粕)按梯度比例混合后,用有益微生物菌群(EM)发酵,筛选出FD1、FD2(牛粪含量分别为60％、80％)两组发酵饲料作为处理,以常规饲料为对照(CK)进行黄粉虫幼虫饲养试验,研究不同发酵料对黄粉虫幼虫生长曲线、死亡率、化蛹率、抗氧化系统等的影响,评价黄粉虫幼虫对发酵牛粪饲料的适应性.结果表明:与CK相比,虽然FD1处理黄粉虫幼虫生长周期延长了20 d,死亡率有所上升,但黄粉虫幼虫单体取食总量增加49％ (P＜0.01),末龄幼虫单体质量增加28％(P＜0.01),粗脂肪含量提高26％(P＜0.05),不饱和与饱和脂肪酸含量的比值比CK高32％(P＜0.05),抗氧化酶活性显著增强,对发酵饲料表现出良好的适应性;而FD2处理各项指标均比CK差,表现出较低的适应性.表明在FD1处理条件下,可利用黄粉虫3龄幼虫对牛粪进行资源化利用,具有较好的应用前景.