感染松墨天牛的金龟子绿僵菌菌株的初步筛选

松墨天牛是松材线虫病的主要媒介昆虫,本文对分离自松墨天牛的6株金龟子绿僵菌,以及从光肩星天牛、大蜡螟、伊藤厚丝角叶蜂和土蝽上分离的各1个菌株,共10个菌株的产孢情况、孢子萌发率进行研究;在此基础上选出金龟子绿僵菌1291、1349和2049三个菌株及球孢白僵菌F-263菌株,采用成虫跗节接种法和幼虫浸渍法进行室内松墨天牛及大蜡螟的毒力测定比较。结果表明,供试绿僵菌菌株对松墨天牛幼虫在接种15天后的感染僵虫率为76.9％～93.1％(1×10⁷孢子/mL);成虫在接种20天后的僵虫率亦达57.9％～75.0％(6.5×10⁵～3.4×10⁶孢子/成虫);2049菌株表现尤其突出;对应的球孢白僵菌F-263对幼虫和成虫的僵虫率分别是96.3％(1×10⁷孢子/mL)和55％(9.7×10⁵孢子/成虫)。但这3个金龟子绿僵菌菌株对大蜡螟的毒力较低,存在较明显的寄主专化性。这3个菌株今后在防治松墨天牛方面具有较大开发应用价值。     

孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛幼虫的致病力的影响

【目的】球孢白僵菌Beauveria bassiana是一种被广泛用于害虫生物防治的生防菌。本研究探讨了孢悬液浓度和宿主体型大小对球孢白僵菌对松墨天牛Monochamus alternatus 幼虫的致病力的影响,旨在为松墨天牛的生物防治提供科学基础。【方法】分别用0.5% 吐温-80(CK)以及1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸和1×10⁹孢子/mL的球孢白僵菌孢悬液接种松墨天牛4龄幼虫,统计接种后15 d内幼虫死亡率和染菌率。同时用最佳浓度(1×10⁹孢子/mL)的球孢白僵菌孢悬液接种体型大小分别为100~150, 200~220, 300~320, 400~420, 500~520和600~650 mg/头的松墨天牛幼虫,测定接种后20 d内幼虫的死亡率和染菌率。【结果】接种1×10⁵~1×10⁹孢子/mL的球孢白僵菌后,松墨天牛4龄幼虫起初活动自如,后在头部出现烧灼状伤并且体色逐渐变红,最后周身长满菌丝。不同浓度下,随球孢白僵菌孢子浓度升高,松墨天牛4龄幼虫校正死亡率和校正染菌率增加。接种1×10⁶~1×10⁹孢子/mL球孢白僵菌孢悬液15 d的松墨天牛4龄幼虫累计校正死亡率均可达到100%,1×10⁷, 1×10⁸和1×10⁹孢子/mL浓度下,松墨天牛达到100%校正死亡率所需时间最少。接种0 (CK), 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸和1×10⁹孢子/mL球孢白僵菌孢悬液的松墨天牛4龄幼虫校正染菌率在第15天分别为0, 20.00%, 86.67%, 90.00%, 96.67%和100.00%,表现为白僵菌孢子浓度越高,染菌率越高。1×10⁹孢子/mL的接种浓度下,松墨天牛幼虫个体越大,天牛幼虫死亡率和染菌率越高。表现在第20天时,体型大小为100~150, 200~220, 300~320, 400~420, 500~520和600~650 mg/头的幼虫的死亡率分别为76.67%, 76.67%, 66.67%, 93.33%, 100.00%和100.00%,染菌率分别为60.00%, 63.33%, 60.00%, 86.67%, 96.67%和100.00%。【结论】球孢白僵菌悬浮液浓度对松墨天牛幼虫的死亡和侵染有显著影响,表现为随孢子浓度的增加而增加;同时,松墨天牛幼虫个体越大,死亡率和染菌率越高。研究结果对开展利用球孢白僵菌防治松墨天牛具重要借鉴和指导意义。     

茄病镰刀菌性角膜炎树鼩模型的建立

目的采用临床分离的茄病镰刀菌感染树鼩角膜,建立茄病镰刀菌性角膜炎树鼩模型。方法茄病镰刀菌接种到沙保氏培养基,26℃培养箱培养7 d,收集真菌混悬液,血细胞计数板调整孢子数量为1×10~(10)CFU/m L。清洁级树鼩40只随机分为实验组(n=30)、对照组(n=10)。实验组用胰岛素针头(29 G)将真菌孢子混悬液50μL注入角膜基中央,对照组注入生理盐水50μL。通过前段照相、共聚焦显微镜检查、病理组织学变化、感染角膜组织培养对模型进行评价。结果真菌浸润范围、角膜上皮细胞和内皮细胞水肿程度、菌丝数量均与时间呈正相关;炎性细胞浸润数量造模后第7天达到高峰,以中性粒细胞为主;实验各时间点均可见菌丝平行于基质纤维生长;感染后角膜组织培养可见茄病镰刀菌生长;造模成功率为86%。结论采用基质注射茄病镰刀菌孢子的方法首次成功建立茄病镰刀菌性角膜炎树鼩模型。     

绿僵菌MaFZ-13对橙带蓝尺蛾幼虫致病力及林间防治效果

【目的】橙带蓝尺蛾Milionia basalis近年来在我国南方地区严重危害罗汉松Podocarpusmacrophyllus和竹柏Podocarpus nagi,扩散蔓延速度快,造成了很大的损失。本研究旨在评估金龟子绿僵菌Metarhizium anisopliae MaFZ-13菌株对橙带蓝尺蛾幼虫的致病力,探寻橙带蓝尺蛾生物防治新资源。【方法】通过绿僵菌MaFZ-13不同浓度(1.0×10⁵~1.0×10⁹孢子/mL)孢子悬浮液接种橙带蓝尺蛾幼虫,运用时间-剂量-死亡率(TDM)模型对生物测定结果进行分析,同时通过林间喷施1.0×10⁸孢子/mL绿僵菌孢子悬浮液进行林间防治效果试验。【结果】橙带蓝尺蛾幼虫累计死亡率随着绿僵菌MaFZ-13孢子悬浮液孢子浓度的增加和接种时间的延长而增大。接种1.0×10⁹孢子/mL孢子悬浮液8 d后以及接种1.0×10⁸孢子/mL孢子悬浮液10 d后,幼虫死亡率均达到100%;接种1.0×10⁷孢子/mL孢子悬浮液15 d后幼虫死亡率为95.6%。应用TDM模型对生测数据进行分析,结果表明所建模型通过Hosmer-Lemeshow拟合异质性检验,并由模型估算出了该菌株对橙带蓝尺蛾幼虫的致死剂量和致死时间。绿僵菌MaFZ-13菌株接种4, 5, 6和7 d后的半致死剂量(LC₅₀)对数估计值分别为7.99, 7.12, 6.46和5.83。以1.0×10⁷, 1.0×10⁸和1.0×10⁹孢子/mL的孢子浓度接种时,绿僵菌MaFZ-13菌株对橙带蓝尺蛾幼虫的LT₅₀值分别为5.19,3.99和2.81 d。林间用1.0×10⁸孢子/mL绿僵菌孢子悬浮液喷洒9 d后橙带蓝尺蛾幼虫死亡率可达到85.8%,防治效果为85.05%。【结论】绿僵菌MaFZ-13对橙带蓝尺蛾幼虫具有较强的致病力,林间防治效果良好,具有较好的应用潜力。     

利用大蜡螟模型研究白念珠菌SUMO编码基因SMT3在致病性调控中的作用CSCD

目的探讨白念珠菌SMT3基因在致病性调控中的作用。方法利用瞬时CRISPR/Cas9系统构建SMT3纯合子缺失菌株,并检测其侵入生长和生物被膜形成能力;利用大蜡螟感染模型检测SMT3缺失株致病能力,通过糖原染色观察组织内白念珠菌菌体形态,并进一步检测SMT3缺失株对大蜡螟幼虫血细胞的破坏作用。结果SMT3缺失促进了侵入生长和生物被膜形成,其感染的大蜡螟幼虫平均存活期为3.5 d,致病性较野生型菌株(平均存活期为8.5 d)显著增强;SMT3缺失株感染的大蜡螟幼虫血细胞数为1.4×10^(7)/mL,较野生型组(2.9×10^(7)/mL)显著降低,仅为野生型组的48%。结论白念珠菌SMT3基因在致病性调控中发挥重要作用。     

茄病镰刀菌致ICR小鼠局部皮肤感染的实验研究

目的建立茄病镰刀菌感染皮肤的ICR小鼠模型。方法将茄病镰刀菌三个不同浓度的菌悬液分别接种于正常和免疫抑制ICR小鼠受损及正常未受损的皮肤,于接种后第7、14、21、28天处死动物,取皮疹和各脏器进行真菌镜检、培养和组织病理学检查。结果正常和免疫抑制组皮肤受损的小鼠均出现皮疹,而免疫抑制组皮疹程度重,持续时间长,且真菌镜检、培养及组织病理学大都可见菌丝。皮肤未受损的小鼠不发生皮疹。实验小鼠均未出现系统播散。结论接种109CFU/mL或1010CFU/mL茄病镰刀菌的孢子悬液于免疫抑制ICR小鼠擦伤的皮肤,可有效造成ICR小鼠的茄病镰刀菌皮肤感染。     

茄病镰刀菌致小腿慢性溃疡1例报告及病原真菌超微结构的研究

报道1例55岁男性农民,因右小腿溃疡4个月余就诊。取其溃疡组织作真菌培养和病理检查,真菌培养在含氯霉素、放线菌酮和不含氯霉素、放线菌酮的沙堡弱培养基中27℃都长出白色棉絮状菌落,而在不含氯霉素、放线菌酮条件下生长更好;在马玲薯培养基中长出镰刀状大分生孢子,菌种鉴定为茄病镰刀菌。8d后从溃疡处再取标本培养仍为茄病镰刀菌生长。病理切片经银染色后发现念珠状分隔菌丝,用透射电镜对病变组织内的菌体形态进行了超微结构研究。予伊曲康唑口服治疗,但患者未复诊而失访。     

分离自1例慢性阻塞性肺疾病患者的Aspergillus lentulus在蜡螟模型上的毒力初探

目的建立Aspergillus lentulus(A.lentulus或A.L)感染动物模型,借动物模型初步探究A.lentulus的毒力。方法将125只蜡螟随机分成5组,以Aspergillus lentulus临床株、Aspergillus lentulus标准株作为实验组,烟曲霉、白念珠菌为对照组,PBS为空白对照组。实验组及对照组菌株分别制成10~6 CFU/mL孢子悬液,感染各组蜡螟。记录72 h内蜡螟的生存情况并制作生存曲线,24 h后提取蜡螟肠道组织,HE染色组织切片观察肠道组织损伤情况,用组织匀浆法,测定蜡螟肠道内真菌载量及真菌逆培养阳性率,用真菌荧光染色法观察肠道培养真菌镜下形态。结果 A.lentulus临床株和A.lentulus标准株的蜡螟存活数与对照组之间差异有统计学意义(P0.05);结果显示A.lentulus临床株和A.lentulus标准株肠壁结构大致正常,局部可见水肿少量菌丝、孢子及炎症细胞浸润,对照组肠道结构破坏严重,可见菌丝、孢子及大量炎症细胞浸润;不同菌种感染蜡螟幼虫各组肠道载菌量及真菌逆培养阳性率比较,差异具有统计学意义(P0.05);真菌荧光显微镜观察,A.lentulus临床株和标准株菌丝多、孢子少,白念珠菌和烟曲霉组孢子多。结论与烟曲霉和白念珠菌相比,A.lentulus菌株对蜡螟毒力和肠道损伤能力较弱且致死率低。     

莱氏绿僵菌FJMR2对草地贪夜蛾不同虫态的致病性研究

【目的】为明确莱氏绿僵菌Metarhizium rileyi FJMR2对草地贪夜蛾Spodoptera frugiperda的生物防治潜力。【方法】在室内条件下,采用浸渍法测定了莱氏绿僵菌FJMR2对草地贪夜蛾卵、幼虫和蛹的致病性。【结果】当孢子浓度为2×10⁸孢子/m L时,草地贪夜蛾1-5龄幼虫和蛹的校正死亡率分别为100%、95.78%、87.15%、64.67%、38.00%和93.93%;草地贪夜蛾1-5龄幼虫和蛹的LC_(50)分别为3.12×108孢子/m L时,草地贪夜蛾1-5龄幼虫和蛹的校正死亡率分别为100%、95.78%、87.15%、64.67%、38.00%和93.93%;草地贪夜蛾1-5龄幼虫和蛹的LC_(50)分别为3.12×10⁴、7.94×104、7.94×10⁴、2.24×104、2.24×10⁶、2.13×106、2.13×10⁷、7.46×107、7.46×10⁸和1.25×108和1.25×10⁵孢子/m L。LT_(50)随孢子浓度的增大而缩短、随幼虫虫龄的增长而延长,当孢子浓度为2×105孢子/m L。LT_(50)随孢子浓度的增大而缩短、随幼虫虫龄的增长而延长,当孢子浓度为2×10⁸孢子/m L时,草地贪夜蛾1-5龄幼虫和蛹的LT_(50)分别为3.93、4.32、5.62、7.18、9.40和4.45 d。卵期为0 h和24 h的草地贪夜蛾卵经莱氏绿僵菌FJMR2浸渍处理后,孵化率分别为84.53%和89.77%,初孵化3 d的幼虫存活率分别为1.81%和3.02%,均显著低于对照组。【结论】莱氏绿僵菌FJMR2对草地贪夜蛾1-4龄幼虫和蛹具有较强侵染致病能力,同时显著降低卵的孵化率和初孵幼虫存活率。     

响应面法优化爪哇虫草菌的培养条件及其对斜纹夜蛾的毒力和保护酶活性影响

【目的】优化爪哇虫草菌Bd01的固态发酵培养条件,测定分生孢子对斜纹夜蛾3龄幼虫的毒力,研究被爪哇虫草菌侵染后寄主体内的保护酶活性变化。【方法】采用单因素试验确定爪哇虫草菌Bd01最佳的固态培养基及培养条件,利用Box-Behnken响应面法优化发酵参数,采用浸渍法测定分生孢子对斜纹夜蛾3龄幼虫的毒力,同时利用分光光度计法测定斜纹夜蛾3龄幼虫体内酶活性变化。【结果】以产孢量为指标,通过响应曲面法优化的爪哇虫草菌Bd01最佳产孢条件为：培养基营养成分含量为30.24g/L,pH值为7.55,光照时长为12.06h,在该条件下,培养基的产孢量为2.78×10⁸孢子/mL。浓度为1×10⁸孢子/mL的爪哇虫草菌孢子液对斜纹夜蛾3龄幼虫具有一定毒力,处理7 d时致死中浓度(LT₅₀)为3.11 d,致死中时(LC₅₀)为4.68×10⁵孢子/mL,校正死亡率为88.68%。处理后未死亡的斜纹夜蛾幼虫体内保护酶活性与对照组相比发生显著变化。【结论】优化后的培养基能够显著增加爪哇虫草菌的产...     

Tn5-induced Xenorhabdus bovienii lecithinase mutants demonstrate reduced virulence for Galleria mellonella larvae

A derivative of Tn5 was used to construct a variety of stable insertion mutations in the entomopathogenic bacterium, Xenorhabdus bovienii T228/1. Mutants which had altered expression of Congo Red binding ability, ampicillin resistance, haemolytic activity and lecithinase were isolated. Isolates with altered lecithinase activity had either lost ability to produce this enzyme or showed reduced expression. The role of lecithinase in pathogenesis of X. bovienii T228/1 for Galleria mellonella larvae was examined by LD₅₀ analysis. Maximum killing of G. mellonella was observed at 72 h post infection for both the wild-type parent strain and a lecithinase mutant 34(45). However, the LD₅₀ value for the wild-type parent strain (8·7 cells per larva) was significantly less than that calculated for the lecithinase mutant (35·5 cells per larva). The data suggested that although lecithinase is a virulence factor produced by X. bovienii , lecithinase activity alone is not sufficient for killing of G. mellonella larvae.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

构建噬菌体展示抗体库过程中电穿孔法的条件优化

目的：确定稳定且高效的电转化实验方案以达到构建高库容噬菌体展示抗体库的目的。方法：探索电压、脉冲时间、噬菌粒DNA质量与浓度、TG1大肠杆菌的生长周期、重悬洗涤缓冲液及培养基优化等方面对TG1大肠杆菌电转化效率的影响。结果：当电转杯电极间距为2mm时,设定电转仪参数为3kV、25μF、5ms、200Ω;外源DNA经纯化后加入感受态菌液中使终浓度为1ng/μl;培养基中加入20mmol/L的MgCl₂,并将TG1的生长阶段调控在OD₆₀₀=0.8,用无菌超纯水重悬及洗涤细胞,将感受态细胞浓度调整为4×10¹⁰个/ml。在上述条件下,电转化效率可达到4.9×10⁹CFU/μg DNA。结论：通过多种条件优化,提高了电转化效率,为构建高库容噬菌体展示抗体库建立了基础。     

Pathogenicity of Bacteria Symbiotically Associated with Insect Pathogenic Nematodes against the Greater Wax Moth,Galleria Mellonella (L.)

The bacterial symbionts isolated from the entomopathogenic nematodes were compared for their pathogenicity to last instar larvae of G. mellonella at both Phases I and II. Most bacterial symbionts at Phase I cause 100% mortality within 2-3 days post-injection with 1 times; 10 3 cells/larva. The pathogenicity of Phase I decreased in the following order: Xenorhabdus nematopbilus, Flavimonas oryzihabitans, Photorhabdus luminescens and Xenorhabdus bovienii with LD 50 values of 40, 55, 70 and 170 cells/larva. The injection of Phase II of the bacterial symbionts did not give 100% mortality even after 4 days post-injection. The time mortality response of G. mellonella larvae to both phases of the bacterial symbiont was significantly different usually at the two highest concentrations tested. The significancy in case of Phase I was in the following order from lowest to highest, F . oryzihabitans , X . nematophilus , P. luminescens and X. bovienii . It was 20.57, 23.96, 23.99 and 53.76 h, respectively. Also, F. oryzihabitans gave the lowest LT 50 value for its Phase II form. It was 36.85 h, and this is followed by X. bovienii , X. nematophilus , and P. luminescens , the LT 50 values of which were 69.29, 74.08 and 74.49 h, respectively. The results suggest that there is a direct correlation between toxin concentration and rate of killing the larvae. On the other hand, there is an inverse correlation between the LT 50 values and the injected concentration.     

昆虫病原真菌长孢蜡蚧菌TF-2菌株对豌豆蚜的侵染过程和致病力

【目的】豌豆蚜Acyrthosiphon pisum是世界性的重要农业害虫,给豆类作物造成巨大的经济损失。本研究在前期筛选已获得豌豆蚜致病菌长孢蜡蚧菌Lecanicillium longisporum TF-2菌株的基础上,检测了该致病菌分生孢子对豌豆蚜的毒力效用及侵染方式,以期为利用昆虫病原真菌防治豌豆蚜提供理论基础。【方法】采用离体叶片饲养法检测在不同浓度长孢蜡蚧菌TF-2菌株孢子悬浮液(1.0×10~3～1.0×10~7孢子/mL)中浸渍后对豌豆蚜成虫的致病力;应用扫描电镜和体视显微镜观察豌豆蚜成虫接种TF-2菌株(1.0×10~7孢子/mL)后长孢蜡蚧菌TF-2菌株侵染症状和侵染过程。【结果】不同浓度长孢蜡蚧菌TF-2菌株分生孢子浸染豌豆蚜成虫致病力随分生孢子浓度升高而逐渐增强,侵染后6 d对豌豆蚜成虫的半致死浓度(LC_(50))为3.26×10~4孢子/mL,最高浓度分生孢子(1.0×10~7孢子/mL)对豌豆蚜成虫的半致死时间(LT_(50))为2.92 d。扫描电镜观察结果表明,接种后24 h,TF-2菌株分生孢子在豌豆蚜成虫体表皮萌发形成芽管和附着胞;接种后48 h,芽管伸长在复眼、触角基部、足基节、腹末生殖节等部位形成网络结构;接种后96 h,菌丝布满整个虫体,新的分生孢子产生。【结论】长孢蜡蚧菌TF-2菌株对豌豆蚜成虫具有较强的致病力,扫描电镜观察揭示了其分生孢子在其虫体上的侵染过程,结果为进一步应用昆虫病原真菌进行生物防治提供理论基础和参考依据。     

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.     

3种菊花病毒/类病毒实时荧光定量RT-PCR检测体系的构建与准确性评价

菊花容易受到病毒感染而造成品质下降,目前国内对菊花病毒的检测主要根据外观表现或者定性PCR检测,无法准确判定病毒载量。为构建一种可同时用于检测菊花B病毒(Chrysanthemum virus B,CVB)、番茄不孕病毒(Tomato aspermy virus,TAV)和菊花褪绿斑驳类病毒(Chrysanthemum chloritic mottle viroid,CChMVd)的实时荧光定量RT-PCR检测方法,本研究分别以保守区域作为靶标设计相应的引物探针,通过优化扩增体系中CVB、TAV、CChMVd 3种病毒/类病毒探针浓度、引物浓度、Mg²⁺浓度、dNTPs浓度,摸索扩增程序中反转录时间、退火温度和扩增循环数,构建了一种可同时用于CVB、TAV、CChMVd的3重实时荧光定量RT-PCR检测体系,优化后的扩扩增体系中CVB、TAV和CChMVd的探针浓度分别为100 nmol/L、120 nmol/L和80 nmol/L,引物浓度分别为200 nmol/L、240 nmol/L和160 nmol/L,Mg²⁺浓度为3.0 mmol/L;dNTPs浓度200μmol/L;最适反转录时间为25 min,退火温度为60℃,循环数为40。敏感性实验结果表明,该反应体系对3种病毒/类病毒的敏感性为1.0×¹⁰3拷贝/mL,敏感性好;定量线性范围为1.0×¹⁰3拷贝/mL~1.0×¹⁰10拷贝/mL,线性范围宽;特异性好,对菊花矮化类病毒、烟草花叶病毒和黄瓜花叶病毒核酸检测结果为阴性;对1.0×¹⁰4拷贝/mL的低浓度参考品平行检测10次,定量结果lg值偏差(CV%)为4.81%,重复性好。在南京农业大学"中国菊花种质资源保存中心"基地随机选择菊花20株进行本研究试剂检测,检出6例CVB病毒株和4例TAV病毒株,其病毒载量为2.5×¹⁰4拷贝/mL~5.5×¹⁰7拷贝/mL,随机选择1株CVB病毒株定量PCR,产物进行TA克隆后经测序与NCBI Blast比对,其与MH678704.1的同源性为100%。因此,本研究建立了一种能同时检测CVB、TAV、CChMVd 3种菊花常见病毒/类病毒的灵敏、快速、可定量的检测方法。     

昆虫病原线虫Heterorhabditis beicherriana LF品系与Bt HBF-18菌株混用对华北大黑鳃金龟幼虫的防治效果

【目的】明确昆虫病原线虫 Heterorhabditis beicherriana LF品系(LF)与苏云金芽孢杆菌 Bacillus thuringiensis HBF-18菌株(Bt HBF-18)混用后对华北大黑鳃金龟 Holotrichia oblita 幼虫的致病力的协同增效作用,为该害虫的防治提供新的技术措施。【方法】在室内测定了LF在不同使用剂量、不同环境温度及不同土壤湿度条件下对华北大黑鳃金龟7-10日龄幼虫的致病力;通过室内生测测定了Bt HBF-18对LF存活的影响,以及Bt HBF-18与LF两者混用后对7-10日龄华北大黑鳃金龟幼虫的防治效果;同时通过室外盆栽试验测定了两者混用对华北大黑鳃金龟幼虫的防治效果。【结果】华北大黑鳃金龟幼虫死亡率随LF施用剂量和处理时间的增加而升高,其中,侵染期线虫(infective juveniles, IJs)800 IJs/100 μL及以上剂量处理7 d后幼虫死亡率达到了100%;25℃为该线虫侵染的最适宜环境温度;适宜土壤湿度范围为14%~20%,湿度过低或过高都会显著影响其侵染效率。室内生测结果表明, Bt HBF-18处理9 d对华北大黑鳃金龟幼虫的致死中浓度(LC 50 )为 1.44× 10^8 CFU/g土,此浓度对LF的存活基本没有影响。另外,室内生测和室外盆栽试验结果均表明,将LF与Bt HBF-18混用能显著提高对华北大黑鳃金龟幼虫的防治效果,混用后具有不同程度的加成或协同增效作用。室内生测试验中LC 50 Bt+200 IJs/100 μL LF混用处理3 d后,较单独LF和Bt HBF-18处理幼虫死亡率分别提高了约43.07%和36.05%,具有显著的协同增效作用;室外盆栽试验中1/2 LC 50 Bt+1 000 IJs/mL LF, LC 50 Bt+1 000 IJs/mL LF和1/2 LC 50 Bt+1 500 IJs/mL LF均具有协同增效作用,其中1/2 LC 50 Bt+1 500 IJs/mL LF增效作用最佳,较单独LF和Bt HBF-18处理幼虫死亡率分别提高了约38.89%和80.55%。【结论】将昆虫病原线虫LF与Bt HBF-18混用对华北大黑鳃金龟幼虫的防治具有加成或协同增效作用。