红火蚁病原真菌分离鉴定及致病性研究

红火蚁Solenopsis invicta是世界上最具破坏力和威胁性的入侵检疫害虫之一,对农林业生产安全、公共设施以及人体健康都造成了巨大影响。为筛选对红火蚁有致病性的病原真菌,本研究通过组织分离法对自然罹病的红火蚁进行致病菌分离,结合病原形态学及分子生物学的方法对病原真菌进行鉴定并采用喷雾法测定了病原真菌对红火蚁的致病力。结果表明：从罹病红火蚁上分离出3株病原真菌HHYSFJ01、HHYSFJ02和HHYSFJ05,分别为红绶曲霉Aspergillus nomius、淡紫拟青霉Purpureocillium lilacinum和球孢白僵菌Beauveria bassiana。致病性测定,这3株病原菌对红火蚁均具有较强的致病性,且3株病原菌致病性间差异显著。经1.0×10^8孢子/mL孢子悬浮液喷雾处理红火蚁10 d后,球孢白僵菌菌株HHYSFJ05的致病性最强,红火蚁工蚁的校正死亡率为84.71%,LT50为4.67 d;红绶曲霉菌株HHYSFJ01和淡紫拟青霉菌株HHYSFJ02次之,红火蚁工蚁的校正死亡率分别为78.82%和73.29%,LT50分别为4.99 d和5.20 d。     

淡紫紫孢菌PLF-1对感染尖刀镰孢菌百合种球的促生作用及其相关防御酶活性变化

为了探究淡紫紫孢菌(Purpureocillium lilacinum)PLF-1对百合种球的促生作用及对百合尖刀镰孢菌的防治效果,采用平板对峙法评估淡紫紫孢菌对尖孢镰刀菌的拮抗效果,以及淡紫紫孢菌对百合抗尖孢镰刀菌的抗性作用。同时监测百合种球中超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性变化情况。研究结果表明：浓度为4.34×10⁴ CFU/mL和4.34×10⁵ CFU/mL的淡紫紫孢菌孢子悬浮液对百合种球表现为促进作用,浓度为4.34×10⁴ CFU/mL时最高茎长达11 cm。平板拮抗实验中该淡紫紫孢菌菌株能有效抑制尖孢镰刀菌生长,抑制率高达72%。接种淡紫紫孢菌和病原菌的百合种球茎长会增长37.6%,根长会增长33%。该菌株能提高感染尖刀镰孢菌百合种球中的超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性,有效抑制尖刀镰孢菌的毒害作用,促进植株健康生长。     

红火蚁Toll受体家族免疫响应绿僵菌表达模式的研究

为探究红火蚁Solenopsis invicta Buren Toll受体家族基因如何免疫响应绿僵菌Metarhizium的侵染。本研究采用浸渍法测定了不同浓度下绿僵菌对红火蚁大中小3种不同大小工蚁的致病力,并用显微镜观察了绿僵菌侵染后红火蚁3种工蚁的表型变化。利用生物信息学筛选Toll受体基因,并对Toll受体家族基因的理化性质、结构域、染色体位置、系统进化进行分析鉴定。运用实时荧光定量PCR（RT-qPCR）检测了红火蚁大中小型工蚁中Toll受体家族的发育历期和绿僵菌侵染后的表达模式。结果表明,绿僵菌在96 h对红火蚁大中小型工蚁的致死中浓度（LC50）分别为5.8×10^7孢子/mL、3.1×10^7孢子/mL、1.5×10^7孢子/mL;绿僵菌侵染红火蚁第3天,显微镜下观察到红火蚁体壁细小的菌丝,虫体僵硬,寄主死亡后菌丝迅速蔓延并逐渐长成橄榄绿色分生孢子;RT-qPCR结果表明Toll受体基因在红火蚁不同发育阶段中的mRNA表达水平存在显著差异,且Toll受体基因在雌性生殖蚁表达水平显著高于雄性生殖蚁;RT-qPCR结果表明红火蚁大中小型工蚁Toll受体免疫响应绿僵菌不一样。在大型工蚁中,绿僵菌侵染后,Toll受体家族基因能显著上调表达,6 h是一个诱导高峰期,Toll2-1和LRR转录水平最高,响应绿僵菌最强;在中型工蚁中,绿僵菌不能刺激Toll2-2基因的表达,却能强烈诱导Toll1,Toll2-1,Toll6,Toll7和LRR基因的上调表达,Toll1和Toll2-1响应绿僵菌最强。在小型工蚁中,绿僵菌能显著诱导Toll受体家族基因基因的上调表达,在24 h时,LRR基因表达量最高,相比于对照,LRR基因表达提高25倍,LRR在6 h和24 h响应绿僵菌最强。以上研究表明Toll受体可以免疫响应入侵的绿僵菌,且不同的Toll对绿僵菌可能具有不同的响应机制。本研究为进一步阐明Toll受体的功能奠定基础,为进一步利用绿僵菌控制红火蚁提供技术指导。     

几种土壤真菌对烟粉虱的生物活性研究

B型烟粉虱Bemisia tabaci是重大入侵害虫,发展生物防治技术控制其危害具有重要意义。本研究采用浸渍法测定了9种20株土壤真菌对B型烟粉虱的室内活性,结果表明,供试菌株对B型烟粉虱都有一定致病活性,1.0±10^8 孢子/mL的分生孢子悬浮液处理10 d后,烟粉虱2龄若虫的累计校正死亡率为53%~93%,其中淡紫紫孢菌Purpureocillium lilacinum PITS01、PlMS-12和PlMS-15菌株,以及刀孢蜡蚧菌Lecanicillium psalliotae LpTS01菌株引起B型烟粉虱91%以上的死亡率。进一步测定了PITS01和LpTS01菌株对烟粉虱的毒力,结果表明PITS01和LpTS01对烟粉虱2龄若虫在处理第4天的LC50分别为2.8×10^7 孢子/mL和3.6×10^7 孢子/mL,第6天分别为1.7×10^6 孢子/mL和3.6×10^5 孢子/mL;当孢子浓度为1.0×10^7 孢子/mL时,PITS01和LpTS01菌株LT50值分别为4.4 d和4.2 d。菌株PITS01和LpTS01对烟粉虱防治具有一定的应用潜力。     

广西南宁市柑橘木虱虫生真菌种类调查

自柑橘木虱Diaphorina citri体上分离虫生真菌,通过形态特征和ITS序列分析,鉴定虫生真菌的种类;通过致病性测定,明确昆虫病原真菌种类。结果发现,自死体柑橘木虱上分离到18个虫生真菌菌株,隶属4种真菌;自活体上分离到985个虫生真菌菌株,隶属25种真菌。致病性测定结果发现,仅4种真菌对柑橘木虱成虫有致病性,包括刀孢蜡蚧菌Lecanicillium psalliotae、球孢白僵菌Beauveria bassiana、爪哇虫草Cordyceps javanica和淡紫紫孢菌Purpureocillium lilacinum,该4种真菌的分生孢子悬浮液（浓度5×10^7个孢子/mL）接种柑橘木虱成虫后10 d的累计死亡率分别为100%、100%、98.89%和43.33%。其中爪哇虫草仅自死体柑橘木虱上分离到,刀孢蜡蚧菌仅在活体上分离到,球孢白僵菌和淡紫紫孢菌在死体和活体上均被分离到。可见,柑橘木虱活体上的虫生真菌种类丰富。生产上应加强保护和利用昆虫病原真菌,提高其对柑橘木虱的自然抑制力。     

三株金龟子绿僵菌对红火蚁的致病力测定

本研究在实验室条件下测定了金龟子绿僵菌M09、CQMa117和CQMa128三个菌株对红火蚁的侵染能力。结果表明在1.0×10~8孢子/mL浓度下M09、CQMa128和CQMa117菌株处理后10 d红火蚁的工蚁累计死亡率分别为73.3%、14.6%和55.5%;M09菌株对工蚁的LC50为3.50×106孢子/mL,8.0×107孢子/mL浓度处理LT50为4.35 d。对M09菌株侵染红火蚁幼虫和蛹的能力测定结果显示,1.0×10~8孢子/mL浓度处理后10 d该菌株对幼虫和蛹的累计侵染率为98.9%和100%,对幼虫和蛹LC50分别为6.34×10~4孢子/mL、1.01×10~4孢子/mL。以上研究证实金龟子绿僵菌M09菌株对红火蚁具较强的致病力,致死速度较快,可作为该蚁生物防治的候选菌株。     

白僵菌、绿僵菌对雷公藤丽长角巢蛾的侵染力测定

经室内侵染力测定,球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae皆能侵染雷公藤丽长角巢蛾Xyrosaria lichneuta幼虫,不同菌株的侵染力存在显著差异。3株白僵菌中,Bb27菌株毒力最强,菌液为1×10^5～1×10^9个·mL^-1五个不同浓度梯度的孢子悬浮液时,害虫15d校正死亡率在58．3％-100％之间;LT50分别为9．36d,6．26d,3．89d、2．54d．2．10d;LC50为4．87×10^4个·ML^-1。3株绿僵菌中,MaZPTP01菌株毒力最强,5种孢子浓度15d校正死亡率分别为52．4％、69．0％、76．2、92．9％、95．2％;LT50分别为11．47d、6．89d、4．36d、2．68d、2．13d;LC50为7．39×10^4个·mL^-1。Bb27和MaZFIT01菌株可应用于林间作进一步防治试验。     

一株白僵菌对红火蚁的毒力及对免疫酶活性与基因表达的影响

白僵菌是最具防治害虫潜力的一类昆虫病原真菌。本研究测定了球孢白僵菌Beauveria bassiana 237菌株对红火蚁Solenopsis invicta的感染毒力,并探究了白僵菌侵染对红火蚁免疫相关酶活性及相关基因表达的影响。结果显示,该白僵菌菌株对红火蚁的毒力较强,在孢子悬浮液108孢子/mL浓度下,对红火蚁的致死中时间LT50为5.288±0.2014 d;在10^4~10^8孢子/mL的不同浓度处理下,红火蚁死亡率随孢子浓度的增加而显著增加。经计算,第4~10天致死中浓度LC50值由8.82×10^6孢子/mL降低到8.95×10^5孢子/mL。红火蚁被球孢白僵菌感染后,体内保护酶和解毒酶发生了不同程度的改变。其中保护酶类酚氧化酶（PO）的活性在白僵菌处理后的第12 h已出现抑制,而在第24 h、48 h、72 h时均显著高于对照;超氧化物歧化酶（SOD）活性在12 h时与对照无显著差异,但在24~48 h阶段酶活显著上升,之后下降;过氧化物酶（POD）在较早时段保持与对照无显著差异水平,至中后期48~72 h出现持续升高。解毒酶类混合功能氧化酶系（MFO）的活性在整个检测时间段内表现为抑制-上升-抑制-上升的波动状态;谷胱甘肽S-转移酶（GSTs）活性的变化与SOD相似,只在24~48 h阶段出现上升。白僵菌还导致红火蚁免疫信号通路Toll途径相关基因表达量变化。在处理后12 h,识别因子GNBP1、Spaetzle即被激活,维持上调-回调波动趋势;而信号传递因子Myd88、pelle在检测的12~72 h内基本处于被抑制状态,只有Myd88在48 h时表达量上升;转录因子Dorsal以及抗菌肽Defensin在12~24 h都已被显著激活,而在后续48~72 h被抑制。综上所述,球孢白僵菌237菌株通过调节红火蚁酶活以及免疫相关基因的表达量实现成功侵染和致病作用,具有很高的生防应用价值。     

六株虫生真菌的生物学特性及其对油茶象甲的致病力

为了筛选防治油茶象甲的虫生真菌菌株,本研究测定了不同绿僵菌(Ma09、Ma25、Ma05、Mac985)和白僵菌(Bb01、Bb02)菌株的生长速度、产孢量以及孢子萌发率,采用孢悬液浸渍法测定了上述菌株对油茶象甲成虫和幼虫的致病力。结果表明,Ma09、Ma05、Bb01、Bb02菌株的菌落直径分别为63.13、62.55、61.99、61.90 mm(25℃、15 d),生长速度显著快于其余菌株(P0.05);Ma25、Bb01、Ma09菌株的产孢量分别为3.04×10~8、2.88×10~8、2.85×10~8孢子/cm~2,显著高于其余菌株(P0.05);Bb01和Ma09菌株的孢子萌发率较高,分别达到97.80%和96.40%。不同菌株对油茶象甲成虫和幼虫致病力差异显著,其中Bb01和Ma09在1×10~7孢子/mL时,对油茶象甲成虫的LT_(50)分别为4.64 d和4.93 d,处理后15 d累计校正死亡率均达到100.00%,僵虫率分别为76.00%和78.67%;对幼虫的LT_(50)分别为2.15 d和3.48 d,10 d累计校正死亡率均达到100.00%,僵虫率分别为81.00%和86.31%。本研究筛选的Bb01和Ma09菌株具有生长快、产孢量大、孢子萌发率高、对油茶象甲致病力强等特性,在油茶象甲的生物防治中具有潜在的应用价值。     

粗放经营茶园土壤昆虫病原真菌种群多样性及时间生态位

本文系统调查了粗放经营状态下黄山毛峰茶园根际土壤昆虫病原真菌的种群多样性及其时间生态位水平.于2012年8月至2013年6月,在安徽省黄山市汤口镇选择粗放经营茶园采集土壤样品140份,经稀释平板法分离共获得昆虫病原真菌1041株.结果表明： 昆虫病原真菌种群多样性较为丰富,经鉴定分为6属13种.其中,淡紫紫孢菌、球孢白僵菌和金龟子绿僵菌为优势种群,其菌株数分别为309、255和101株,占总数的29.7%、24.5%和9.7%.茶园土壤中淡紫紫孢菌的时间生态位宽度最大,球孢白僵菌最小.昆虫病原真菌之间以球孢白僵菌和布氏白僵菌的时间生态位重叠值最大,为1.965;而爪哇棒束孢和球孢白僵菌的时间生态位重叠值最小,仅为0.374.     

Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Asiatic citrus psylla, Diaphorina citri, Kuwayama (Hemiptera: Liviidae) is an economic pest of citrus groves and a vector of the bacterium, Candidatus Liberibacter spp., one of the causative agents of citrus greening. In order to estimate the infectivity of six different isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea, fungal bioassay was performed on the adults of D. citri. Adults of D. citri were treated individually with 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, 2 × 10⁸ spores/mL fungal concentrations by the immersion method. Subsequent to fungal bioassay, treated D. citri were used to determine the levels of esterase and glutathione S‐transferases (GST) enzymes over a period of 3–7 days. The mortality results suggest that I. fumosorosea isolates (If‐02) caused 82.2% mortality on the seventh day of treatment. However, B. bassiana isolate (Bb‐08) with lowest LC₅₀ (1.4 × 10⁷ spores/mL) proved to be highest potential isolate against D. citri. Biochemical determination of esterase and GST activity assay showed significant differences in activities after infection of fungi. Significantly high activity of esterase was observed by Bb‐01 (27.0 unit per mg protein) on the seventh day, while Ma‐11.1 and If‐2.3 (16.9 and 36.3 unit per mg protein) on the third day post treatment. However, maximum GST's activity was showen by isolates Bb‐08,Ma‐M₂ and If‐2.3 (37.6, 1.40 and 10.9 unit per mg protein) on the third day. The current investigation will help to explore the relations between the insect defense system and entomopathogenic fungi. Moreover, the determination of enzymatic activities will be useful for selecting the most pathogenic isolates.     

Beauveria bassiana as a pathogen of the Mexican fruit fly (Diptera: Tephritidae) under laboratory conditions

Bioassays were carried out under controlled conditions (27 +/- 2 degrees C, 80 +/- 5% RH, and a photoperiod of 12:12 [L:D] h) to evaluate the effect of eight strains of the entomopathogenic fungus Beauveria bassiana upon larvae, pupae, and adult females of the Mexican fruit fly, Anastrepha ludens (Loew). Mortality of the immature stages was low, 2-8% in larvae and 0% in pupae. However, very high levels of mortality were obtained for adult flies, with values of 100, 98, and 98% for the strains Bb16, Bb24, and Bb26, respectively. LC50 values for these three strains ranged from 3.12 x 10(6) to 9.07 x 10(6) conidia/ml. Lethal time 50 (LT50) was 2.8, 3.7, and 4.2 d for Bb16, Bb26, and Bb24 strains, respectively, with an average LT50 of 4.4 d across all strains. The fungal mycelium emerged through the soft parts of the exoskeleton, such as the wing bases, mouth, intersegmental regions of the legs, and membranous regions of the abdomen, coxae, and neck. Maximum percentage sporulation ranged from 66.4 to 74.7% for the three most virulent strains.     

Evaluation of Beauveria bassiana (Ascomycota: Hypocreales) as a control of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) emerging from fallen,infested coffee berries on the ground

The effects of Beauveria bassiana strains on coffee berry borers (CBB), which emerge from infested berries left on soil, and its impact on the infestation of coffee berries on tree branches were evaluated at two Experimental Stations (Naranjal-Caldas and Paraguaicito-Quindio) in the Colombian coffee zone. Using a completely randomized design with 10 repetitions, 50 coffee berries artificially infested with CBB were placed on the base of a coffee tree. Four treatments including B. bassiana strain Bb9205, a mixture of Cenicafé strains (Bb9001, Bb9024 and Bb9119), a commercial formulation of B. bassiana and a control (water) were sprayed with 1×10⁹ conidia per tree. After 30 days, all fungal strains lowered the infestation levels of the coffee berries on the trees at both locations. The mixture of Cenicafé strains decreased the tree infestation between 50 and 30% at both locations. In the berries dissected from each treated tree, insect mortality was about 40% at both locations compared to 15% in the control. B. bassiana strains also decreased the insect population inside the newly infested berries on the trees by 55–75%. The mixture of Cenicafé strains was the most effective for decreasing insect populations. B. bassiana significantly decreased CBB populations that emerged from fallen, infested, coffee berries and reduced future insect generations.     

Effect of entomopathogenic fungi upon adults of Stomoxys calcitrans and Musca domestica (Diptera: Muscidae)

The pathogenicity and virulence of 10 isolates of entomopathogenic fungi from the soil of lodging pens of dairy production units in Aguascalientes, Mexico, on adults of Stomoxys calcitrans and Musca domestica were determined. All isolates were pathogenic when exposed by aspersion to a concentration of 1×10⁸ conidia/ml, causing between 20.3 and 91.7% mortality in S. calcitrans and between 31 and 91.7% in M. domestica at 7 days post-exposure; in S. calcitrans isolates of Beauveria bassiana (Bb114) and Metarhizium anisopliae (Ma135), sensu lato, were the most noteworthy as mortality reached above 90% with an LC₅₀ of 3.5×10⁵ conidia/ml for Bb114, while for Ma135 reached 1.6×10⁴ conidia/ml. In M. domestica Ma134 and Ma135 showed mortality above 90% with an LC₅₀ of 4.3×10⁴ and 1.4×10⁵ conidia/ml, respectively.