桃小食心虫病原真菌
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摘要
桃小食心虫(简称桃小)是危害严重的果树食心虫类害虫。可应用病原真菌在土壤中对越冬老熟幼虫感染致病进行生物防治。由于病原真菌能在土壤中长期宿存,被感染的幼虫又作为新的感染源,从而可对桃小的种群数量起到持续控制作用。本文采用从自然染病的桃小越冬虫尸上分离病原真菌的方法,分离筛选出对桃小致病力高的一株病原真菌,对其进行形态学与分子鉴定,确定种属地位。系统地研究了该菌株对桃小幼虫的致病力,侵染桃小的致病过程以及桃小感染后的组织病理学特征和生理生化防御反应。并对该菌株的生物学特性与菌粉的制备,野外防治效果,菌株在自然环境中的宿存情况以及与常用化学杀虫剂的相容性进行了研究,为应用该菌株对桃小进行生物防治提供理论和应用依据。
研究结果如下：
1.从桃小的越冬虫尸上分离并筛选出一株对桃小致病力高的病原真菌TST05。通过回接实验证实其为桃小的病原真菌。该菌株在PDA培养基上的培养菌落初为白色毛绒状,后产生浓密的黄色粉状孢子。具有白僵菌属典型的特征,产孢轴末端“之”字型弯曲且孢子着生在小齿突上。产孢细胞多浓密簇生,显微镜下可见球状密集的孢子头。分生孢子卵圆形或近球形。应用rDNAITS序列分析方法对其进行分子鉴定。结合形态学和分子鉴定结果确定TST05菌株为白僵菌属Beauveria的球孢白僵菌Beauveria bassiana。
2.测定了TST05菌株对桃小老熟幼虫的致病力。结果显示,107-109孢子/mL的孢子悬浮液感染幼虫,9d的累积校正致死率可达到68.57%～97.50%。并且TST05菌株的致死速度较快,染菌2d后幼虫开始死亡,死亡高峰集中于处理后4-7d。随孢子浓度增大,幼虫的死亡高峰时间也随之提前并缩短。致死中时LTso随孢子浓度增加由6.57d缩短至4.31d。致死中浓度LC50从4d的2.16×109孢子/mL降低到6d的4.36×106孢子/mL。研究结果表明,TST05菌株对桃小幼虫有较高的致病力,且致死速度较快,可作为防治桃小的优良菌株。该菌株已保藏在中国普通微生物菌种保藏管理中心,编号为CGMCC4526。
3.采用光学显微镜、扫描电镜、透射电镜技术,系统地观察研究了TST05菌株孢子在桃小幼虫体表的附着、萌发、菌丝穿透体壁的过程和对内部组织的侵染及其引起的组织病理学变化。研究发现,桃小幼虫头壳硬而光滑,孢子附着很少,但在触角的基部和口器的皱褶处孢子附着较多。在幼虫胸部和腹部,体表着生密集的棘刺和稀疏的长刚毛,孢子主要附着在棘刺基部的体表上。在气门附近和光滑的刚毛上也发现有孢子的附着。孢子萌发后入侵的途径是穿透幼虫体壁。在染菌24-36h,孢子萌发出芽管直接穿透体表或在菌丝前端生成侵入钉侵入表皮。真菌的入侵可破坏原表皮层中的蛋白—几丁质片层结构。在真菌入侵过程中,幼虫表皮中出现黑斑、血淋巴中出现黑化体,显示幼虫体内免疫防御系统作出抵抗反应。真菌进入幼虫血腔后,产生大量芽生孢子并侵入幼虫内部组织器官。观察到脂肪体、消化道、马氏管、肌肉组织的感染症状,以及真菌对幼虫丝腺腔中的丝胶层和液体丝素的侵染和占据过程。最终,菌丝冲破虫尸的体壁,从虫尸内长出并产生新的分生孢子作为新的感染源感染其它寄主。研究结果直观地揭示了TST05菌株对桃小幼虫的感染过程。
4.桃小幼虫对TST05菌株感染的生理生化反应试验表明,在受到真菌侵染后,桃小幼虫体内的营养物质海藻糖和蛋白质的含量明显下降,葡萄糖含量先升后降。同时,感染后虫体内海藻糖酶活性升高,5d时达到了对照组的1.91倍。感染后,幼虫体内起免疫防御及保护作用的多酚氧化酶(PO)、谷胱甘肽-S-转移酶(GSTs)、羧酸酯酶(CarE)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和谷胱甘肽过氧化物酶(GSH-Px)的活性都表现出先升后降的趋势。其中GSTs和GSH-Px受感染影响变化最大,在染菌3d后分别达到对照组的3.84倍和3.95倍。SOD和POD酶活力在感染后3d比对照组高出0.61倍和0.53倍。PO和CarE的活性在感染5d后达到了最高值,分别达到对照组的1.48和1.50倍。表明TST05菌株的侵染,对桃小幼虫的营养代谢产生影响,并诱发了其生理防御反应。
染菌后虫体内的过氧化氢酶(CAT)活性始终都低于对照组,最低时仅为对照组的56.78%,显示TST05菌株的入侵对CAT酶有较强的抑制或破坏作用。同时发现,感染TST05菌株可导致桃小幼虫体内乙酰胆碱酯酶(AChE)的酶活性升高,显示该菌株的感染或其代谢毒素可能对幼虫的神经传导有影响。
5.测定不同培养基、不同温度和湿度对该菌株的菌丝生长、孢子产量、及孢子萌发的影响。发现该菌株在PDA、PPDA、SDAY、SMAY4种培养基上均生长良好,菌落厚而致密,产孢量均大于3.95×107孢子/mL；菌株适应的温度和湿度范围较宽,15～30℃之间,相对湿度(RH)30%-100%之间孢子均可萌发、生长和产孢。随着温度接近25℃、湿度增大,孢子的萌发率、菌落的生长速率和产孢量都显著增加。15℃、RH100%时产孢量为1.32×107孢子/mL；25℃、RH30%时,产孢量也能达到1.37×107/mL；25℃、RH100%时产孢量达到6.19×107孢子/mL。15℃、RH100%孢子萌发率为52.28%；25℃、RH>80%时,孢子萌发率都能达到90%以上。表明TST05菌株易于培养,能适应北方干旱低温条件,可开发成为防治桃小的生物制剂。
6.试验制备了TST05菌株的菌粉制剂。采用楔形瓶和两端插入玻璃通气管的塑料袋作为TST05菌株的固体培养容器。结果显示,该菌株在两种培养容器中都能旺盛生长,培养基表面都已经长满白色浓密的菌丝。通过检测产孢量和胞外蛋白酶活性,证实培养20d后塑料袋中菌粉产孢量为12.50x109孢子/g,蛋白酶活性达到了770.34U/mg pr,分别是楔形瓶中菌粉的1.42倍和1.54倍。表明通气效果好更有利于TST05菌株的生长和产孢。
7.进行了果园防治应用试验。在9月份,当钻蛀在枣果内的桃小幼虫老熟脱果入土期,用制备的菌粉制剂与土壤按1：200的比例拌匀后均匀撒施于枣树下,将桃小老熟幼虫撒在土上由其自然入土,试验入土幼虫的感染死亡率。结果显示,菌粉制剂对桃小幼虫的野外校正致死率达到75.29%,具有较好的应用前景。
8.试验了TST05菌株菌株在土壤中的存活能力。如上述果园防治试验的方法,在9月份,将菌粉制剂撒于果园枣树下的表土层中,4个月后采样,在实验室培养测得成菌落数为1.09×104CFU/g,比初始的108.67×104CFU/g下降了两个数量级,但随后几个月中,成菌落数一直较为稳定,甚至略有回升。6个月和8个月后从土壤中直接分离的菌株的产孢量也较稳定,达到106孢子/mL。说明该菌株在野外土壤中具有高的存活能力,有利于对桃小种群数量的持续控制。
9.研究了TST05菌株菌株与7种常用化学杀虫剂的相容性。采用高效氯氰菊酯、高氯甲维盐、辛硫磷、高效氯氟氰菊酯、阿维菌素、阿维杀虫单、虫酰肼,试验它们对TST05菌株的生长、孢子萌发、产孢的影响。结果表明,在林间常用浓度下,7种杀虫剂对该菌株的菌丝生长抑制率在27.93%～38.74%之间。在虫酰肼处理组,孢子萌发率为33.19%,几乎不形成菌落,产孢量仅为0.05×106孢子/mL；而在其余6种杀虫剂处理组,孢子萌发率在50.34%～70.62%之间,能形成较致密的菌落,产孢量为2.51～3.58×106孢子/mL。杀虫剂对该菌株的抑制作用随着浓度的降低而减小,林间浓度再稀释5倍后,各处理组的孢子萌发率升高到72.08%～84.92%：产孢量为3.01～9.61×106孢子/mL。显示,TST05菌株与除虫酰肼之外的其余6种杀虫剂的相容性较好,并且相容性随着杀虫剂浓度的降低而提高。
Carposina sasakii (Matsmura) is an important fruit borer pest. The entomogenous fungi for biological control can be applid in the soil to infect C. sasakii overwintering larvae. Applying entomogenous fungi can sustainable control on the population of the pest because the fungi can survival in a long time in the soil and the infected larvae can be a new source of infection. In the thesis, one higher virulent strain TST05of entomopathogenic fungus was obtained by isolating from the naturally disease larvae of Carposina sasakii. Based on morphological characteristics and ITS gene sequence analysis, the taxonomy position of the strain was identified. The pathogenicity and virulence of strain TST05against C. sasakii larvae was studied. The histopathological characteristics and physiological and biochemical reaction of C. sasakii larvae infected by the strain were investigated. A series of experiment were conducted on the strain in biological characteristics, fungal powder preparation, application in the field condition, the survival ability in natural environment, and the compatibility with common chemicals pesticides. The results may provide a base in theory and application for biological control on C. sasakii.
The contents are as follows:
1. The strain TST05was isolated from the natural cadaver in winter-cocoons of Carposina sasakii. It was proved that the strain TST05was a pathogen of C. sasakii larvae by re-inoculation test on the host insect. Cultured on potato dextrose agar (PDA) media, the colony of the strain was white and villous in early cultivation and produced thick conidia in yellow and powdery-like in later cultivation. The upper of conidiogenous cell extended into a "Z"-bending conidiogenous axis. Conidia located on the small pectina which were born on the axis. Many conidiogenous cells densely clustered on vegetal mycelia. And many spherical conidiogenous cells group were observed under microscope. Conidia were oval or near spherical. Based on the morphological identification and alignment analysis of rDNA ITS region, the strain TST05was identified to be Beauveria bassiana.
2. The virulence of strain TST05to C. sasakii larvae was tested. The results showed that by innoculation with107-109conidia/mL of strain TST05, adjusted accumulative mortality rate of the larvae was68.57%97.50%during9d after treatment. The larvae began to die at2d after treatment, and the peak time of death appeared in4-7d. The death peak appeared earlier and quicker with conidia concentration increasing in treatment. The median lethal time (LT50) decreased from6.57d to4.31d with conidia concentration changing from1x109to1x107conidia/mL in the treatment. The median lethal concentration (LC50) was2.16x109conidia/mL at4d but it was4.36x106conidia/mL at6d. Strain TST05displayed a higher virulence to C. sasakii larvae and a rapid action in killing host. The strain can be used as a new strain for biology control on C. sasakii and was preserved in China General Microbiological Culture Collection, No.4526.
3. The conidial adhesion and germination on C. sasakii larvae surface, the integument penetration of hyphae on the larvae, the internal tissues infection, defense response of the larvae, and the host pathological changes were investigated by using light, scanning electron microscopy and transmission electron microscopy. The result showed that the head capsule of the larva was so strong and smooth that very few conidia attached to there. But there were many conidia in the basal area around the mouthparts and antennae. On the thorax and abdomen of the larvae, dense acanthae were distributed over the cuticle and in which few long seta scattered. Conidia adhered mainly to the area around the acanthae. Some conidia were observed in the peritreme socket of the spiracles and on the smooth seta.
After conidia germinated, the fungus invaded the larvae mainly by penetrating the integument. The conidia germinated during a24-36h period after inoculation, and the germ tubes might directly penetrated the cuticle or produced a infection peg at the hyphal tips to penetrate into the cuticle. The fungal infection destroyed the regular protein-chitin parallel lamellar structure in the procuticle. The defensive response of the larvae to the fungal attack was indicated by some dark spots appeared on the cuticle and some melanization appeared in the hemocoel. After overcoming the host's defense system, the fungus grew and reproduced many blastospores in the hemocoel, then infected the internal tissues and organs. Infection symptoms were observed in the fat body, alimentary canal, the Malpighian tubules and the muscle tissue. The fungus attacked and colonized the sericin layer and the liquid fibroin in the silk gland lumen. Finally, the fungus emerged through the cuticle of the dead insect and released conidiophores that could act as new pathogens to infect other larvae. The results clearly revealed the process of strain TST05infected C. sasakii larvae.
4. The physiological and biochemical reaction of C. sasakii larvae were studied when they were infected by the strain TST05. The results showed that after infected, the content of trehalose and protein in the larvae decreased obviously, the trend of changes of glucose content was increased at first and then decreased, the activity of trehalase rose to1.91times as that of control groups at5d after inoculation.
During the initial infection stage, the activities of PO, GSTs, CarE, SOD, POD and GSH-Px that play a role in immune and protection for the host, increased quickly, while during the later stage, their activities decreased at different degrees. The change of GSTs and GSH-Px activities were biggest in those enzymes. Their activities were2.84times and2.95times respectively as those in control groups at3d after inoculation. The activities of SOD and POD were1.61times and1.53at3d, the activeties of PO and CarE was1.48times and1.50times as that of control groups at5d, respectively. The results indicated that the infection of strain TST05affected nutrient metabolism and induced the physiological defense reaction of C. sasakii larvae.
The activity of CAT in infected larvae always lower than that in control larvae, and the lowest activity is56.78%as control activity. It indicted that the infection of the strain TST05can inhibited and destroyed CAT. As the same time, the increase of the AChE activity in infection period indicted the infection or the toxin of the strain can effect the nerve conduction of larvae.
5. The effect of culture media, temperature, and humidity on the mycelium growth, conidia yield, and conidia germination of the strain TST05were investigated. The results showed that the strain could grow well in the four culture media, PDA, PPDA, SDAY and SMAY. The fungal colonies were sick and dense, and the conidia yield exceeded3.95x107conidia/mL in these culture media.
The TST05strain was adapt to a wide range of temperature and humidity. The conidia could germinate, and the mycelia could grow and sporulate in15-30℃and RH30%-100%. With the temperature closing to25℃and the humidity increasing, the rate of conidia germination and mycelium growth and the conidia yield were all significantly increased. The conidia yield was1.32x107conidia/mL under15℃, RH100%,1.37x107conidia/mL under25℃, RH30%, and6.19x107conidia/mL under25℃, RH100%. The conidia germination rate was52.28%under15℃, RH100%, and90%or more under25℃, RH>80%. The results indicted that the strain TST05was easy to culture and adapt to low temperature and humidity in north. The strain could become a biological agent to control C. sasakii.
6. The powder preparation of strain TST05was preparated by cultured in wedge-shaped bottle and plastic bag with glass cavity tube in the both ends. The results showed that the strain grew vigorously in the two culture vessels. The white dense mycelia overgrow in the solid media. The conidia yield and the protease activity of powder preparation were12.50x109/g and770.34U/mg pr respectively at20d in the plastic bag, which were1.42times and1.54times respectively as that in wedge-shaped bottle. The results indicted the good ventilation effect is more conducive to the growth and sporulation of strain TST05.
7. The control efficiency of powder preparation to C. sasakii mature larvae in an orchard was determined. In September, when the mature larvae of C. sasakii bored out jujube and dropped in the soil for overwintering, the fungal powder preparation was mixed with soil in the proportion of1:200and then was applied in the upper soil layer under the tree. The larvae were laid on the surface of the soil and naturally bored into the soil. The mortality rate of powder preparation to C. sasakii larvae was determined. The adjusted mortality rate of C. sasakii larvae reached75.29%by the fungus infecting in the soil.
8. The survival ability of the strain TST05in soil was tested. The fungal powder preparation was scattered into soil by using the method as above mentioned. The CFUs (colony forming unit) of the strain TST05that survived in soil were determined. The results showed that the CFUs of the strain were1.09x104CFU/g after4months, while the value at the start was108.67x104CFU/g. But in the following months, the CFUs were relatively stable. The conidia yield of the strain isolated from the mixed soil can reach106conidia/mL after6and8months. The results indicated that the strain TST05possessed higher survive ability in the field condition which was propitious to sustainable control on the population of C. sasakii.
9. The compatibility of the strain TST05was studied with seven common chemicals pesticides including beta cypermethrin, cypermethrin-emamectin benzoate, phoxim, cyhalothrin, abamectin, Avi monosultap and tebufenozide. The effect of the chemicals pesticides on mycelium growth, conidia germination and conidia yield of the strain were tested.
The results showed that the inhibition rate of the mycelium growth was27.93%-38.74%under the conventional concentrations of the pesticides. In the group treated with tebufenozide, the conidia germination rate was33.19%, and the conidia yield was only0.05×106conidial/mL, while in other groups treated with other6pesticides, the conidia germination rate ranged from50.34%to70.62%, and the conidia yield was2.51-3.58x106conidial/mL. The inhibition action of the pesticides to the strain decreased with the concentration decreasing. When the pesticides were diluted5times of the conventional concentrations, the conidia germination rate was up to72.08%-84.92%, and the conidia yield was3.01-9.61x106conidia/ml. The results showed that the compatibility of the strain TST05with the six pesticides was relatively good. And the compatibility was increased with the concentration of pesticides decreasing.
研究结果如下：
1.从桃小的越冬虫尸上分离并筛选出一株对桃小致病力高的病原真菌TST05。通过回接实验证实其为桃小的病原真菌。该菌株在PDA培养基上的培养菌落初为白色毛绒状,后产生浓密的黄色粉状孢子。具有白僵菌属典型的特征,产孢轴末端“之”字型弯曲且孢子着生在小齿突上。产孢细胞多浓密簇生,显微镜下可见球状密集的孢子头。分生孢子卵圆形或近球形。应用rDNAITS序列分析方法对其进行分子鉴定。结合形态学和分子鉴定结果确定TST05菌株为白僵菌属Beauveria的球孢白僵菌Beauveria bassiana。
2.测定了TST05菌株对桃小老熟幼虫的致病力。结果显示,107-109孢子/mL的孢子悬浮液感染幼虫,9d的累积校正致死率可达到68.57%～97.50%。并且TST05菌株的致死速度较快,染菌2d后幼虫开始死亡,死亡高峰集中于处理后4-7d。随孢子浓度增大,幼虫的死亡高峰时间也随之提前并缩短。致死中时LTso随孢子浓度增加由6.57d缩短至4.31d。致死中浓度LC50从4d的2.16×109孢子/mL降低到6d的4.36×106孢子/mL。研究结果表明,TST05菌株对桃小幼虫有较高的致病力,且致死速度较快,可作为防治桃小的优良菌株。该菌株已保藏在中国普通微生物菌种保藏管理中心,编号为CGMCC4526。
3.采用光学显微镜、扫描电镜、透射电镜技术,系统地观察研究了TST05菌株孢子在桃小幼虫体表的附着、萌发、菌丝穿透体壁的过程和对内部组织的侵染及其引起的组织病理学变化。研究发现,桃小幼虫头壳硬而光滑,孢子附着很少,但在触角的基部和口器的皱褶处孢子附着较多。在幼虫胸部和腹部,体表着生密集的棘刺和稀疏的长刚毛,孢子主要附着在棘刺基部的体表上。在气门附近和光滑的刚毛上也发现有孢子的附着。孢子萌发后入侵的途径是穿透幼虫体壁。在染菌24-36h,孢子萌发出芽管直接穿透体表或在菌丝前端生成侵入钉侵入表皮。真菌的入侵可破坏原表皮层中的蛋白—几丁质片层结构。在真菌入侵过程中,幼虫表皮中出现黑斑、血淋巴中出现黑化体,显示幼虫体内免疫防御系统作出抵抗反应。真菌进入幼虫血腔后,产生大量芽生孢子并侵入幼虫内部组织器官。观察到脂肪体、消化道、马氏管、肌肉组织的感染症状,以及真菌对幼虫丝腺腔中的丝胶层和液体丝素的侵染和占据过程。最终,菌丝冲破虫尸的体壁,从虫尸内长出并产生新的分生孢子作为新的感染源感染其它寄主。研究结果直观地揭示了TST05菌株对桃小幼虫的感染过程。
4.桃小幼虫对TST05菌株感染的生理生化反应试验表明,在受到真菌侵染后,桃小幼虫体内的营养物质海藻糖和蛋白质的含量明显下降,葡萄糖含量先升后降。同时,感染后虫体内海藻糖酶活性升高,5d时达到了对照组的1.91倍。感染后,幼虫体内起免疫防御及保护作用的多酚氧化酶(PO)、谷胱甘肽-S-转移酶(GSTs)、羧酸酯酶(CarE)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和谷胱甘肽过氧化物酶(GSH-Px)的活性都表现出先升后降的趋势。其中GSTs和GSH-Px受感染影响变化最大,在染菌3d后分别达到对照组的3.84倍和3.95倍。SOD和POD酶活力在感染后3d比对照组高出0.61倍和0.53倍。PO和CarE的活性在感染5d后达到了最高值,分别达到对照组的1.48和1.50倍。表明TST05菌株的侵染,对桃小幼虫的营养代谢产生影响,并诱发了其生理防御反应。
染菌后虫体内的过氧化氢酶(CAT)活性始终都低于对照组,最低时仅为对照组的56.78%,显示TST05菌株的入侵对CAT酶有较强的抑制或破坏作用。同时发现,感染TST05菌株可导致桃小幼虫体内乙酰胆碱酯酶(AChE)的酶活性升高,显示该菌株的感染或其代谢毒素可能对幼虫的神经传导有影响。
5.测定不同培养基、不同温度和湿度对该菌株的菌丝生长、孢子产量、及孢子萌发的影响。发现该菌株在PDA、PPDA、SDAY、SMAY4种培养基上均生长良好,菌落厚而致密,产孢量均大于3.95×107孢子/mL；菌株适应的温度和湿度范围较宽,15～30℃之间,相对湿度(RH)30%-100%之间孢子均可萌发、生长和产孢。随着温度接近25℃、湿度增大,孢子的萌发率、菌落的生长速率和产孢量都显著增加。15℃、RH100%时产孢量为1.32×107孢子/mL；25℃、RH30%时,产孢量也能达到1.37×107/mL；25℃、RH100%时产孢量达到6.19×107孢子/mL。15℃、RH100%孢子萌发率为52.28%；25℃、RH>80%时,孢子萌发率都能达到90%以上。表明TST05菌株易于培养,能适应北方干旱低温条件,可开发成为防治桃小的生物制剂。
6.试验制备了TST05菌株的菌粉制剂。采用楔形瓶和两端插入玻璃通气管的塑料袋作为TST05菌株的固体培养容器。结果显示,该菌株在两种培养容器中都能旺盛生长,培养基表面都已经长满白色浓密的菌丝。通过检测产孢量和胞外蛋白酶活性,证实培养20d后塑料袋中菌粉产孢量为12.50x109孢子/g,蛋白酶活性达到了770.34U/mg pr,分别是楔形瓶中菌粉的1.42倍和1.54倍。表明通气效果好更有利于TST05菌株的生长和产孢。
7.进行了果园防治应用试验。在9月份,当钻蛀在枣果内的桃小幼虫老熟脱果入土期,用制备的菌粉制剂与土壤按1：200的比例拌匀后均匀撒施于枣树下,将桃小老熟幼虫撒在土上由其自然入土,试验入土幼虫的感染死亡率。结果显示,菌粉制剂对桃小幼虫的野外校正致死率达到75.29%,具有较好的应用前景。
8.试验了TST05菌株菌株在土壤中的存活能力。如上述果园防治试验的方法,在9月份,将菌粉制剂撒于果园枣树下的表土层中,4个月后采样,在实验室培养测得成菌落数为1.09×104CFU/g,比初始的108.67×104CFU/g下降了两个数量级,但随后几个月中,成菌落数一直较为稳定,甚至略有回升。6个月和8个月后从土壤中直接分离的菌株的产孢量也较稳定,达到106孢子/mL。说明该菌株在野外土壤中具有高的存活能力,有利于对桃小种群数量的持续控制。
9.研究了TST05菌株菌株与7种常用化学杀虫剂的相容性。采用高效氯氰菊酯、高氯甲维盐、辛硫磷、高效氯氟氰菊酯、阿维菌素、阿维杀虫单、虫酰肼,试验它们对TST05菌株的生长、孢子萌发、产孢的影响。结果表明,在林间常用浓度下,7种杀虫剂对该菌株的菌丝生长抑制率在27.93%～38.74%之间。在虫酰肼处理组,孢子萌发率为33.19%,几乎不形成菌落,产孢量仅为0.05×106孢子/mL；而在其余6种杀虫剂处理组,孢子萌发率在50.34%～70.62%之间,能形成较致密的菌落,产孢量为2.51～3.58×106孢子/mL。杀虫剂对该菌株的抑制作用随着浓度的降低而减小,林间浓度再稀释5倍后,各处理组的孢子萌发率升高到72.08%～84.92%：产孢量为3.01～9.61×106孢子/mL。显示,TST05菌株与除虫酰肼之外的其余6种杀虫剂的相容性较好,并且相容性随着杀虫剂浓度的降低而提高。
Carposina sasakii (Matsmura) is an important fruit borer pest. The entomogenous fungi for biological control can be applid in the soil to infect C. sasakii overwintering larvae. Applying entomogenous fungi can sustainable control on the population of the pest because the fungi can survival in a long time in the soil and the infected larvae can be a new source of infection. In the thesis, one higher virulent strain TST05of entomopathogenic fungus was obtained by isolating from the naturally disease larvae of Carposina sasakii. Based on morphological characteristics and ITS gene sequence analysis, the taxonomy position of the strain was identified. The pathogenicity and virulence of strain TST05against C. sasakii larvae was studied. The histopathological characteristics and physiological and biochemical reaction of C. sasakii larvae infected by the strain were investigated. A series of experiment were conducted on the strain in biological characteristics, fungal powder preparation, application in the field condition, the survival ability in natural environment, and the compatibility with common chemicals pesticides. The results may provide a base in theory and application for biological control on C. sasakii.
The contents are as follows:
1. The strain TST05was isolated from the natural cadaver in winter-cocoons of Carposina sasakii. It was proved that the strain TST05was a pathogen of C. sasakii larvae by re-inoculation test on the host insect. Cultured on potato dextrose agar (PDA) media, the colony of the strain was white and villous in early cultivation and produced thick conidia in yellow and powdery-like in later cultivation. The upper of conidiogenous cell extended into a "Z"-bending conidiogenous axis. Conidia located on the small pectina which were born on the axis. Many conidiogenous cells densely clustered on vegetal mycelia. And many spherical conidiogenous cells group were observed under microscope. Conidia were oval or near spherical. Based on the morphological identification and alignment analysis of rDNA ITS region, the strain TST05was identified to be Beauveria bassiana.
2. The virulence of strain TST05to C. sasakii larvae was tested. The results showed that by innoculation with107-109conidia/mL of strain TST05, adjusted accumulative mortality rate of the larvae was68.57%97.50%during9d after treatment. The larvae began to die at2d after treatment, and the peak time of death appeared in4-7d. The death peak appeared earlier and quicker with conidia concentration increasing in treatment. The median lethal time (LT50) decreased from6.57d to4.31d with conidia concentration changing from1x109to1x107conidia/mL in the treatment. The median lethal concentration (LC50) was2.16x109conidia/mL at4d but it was4.36x106conidia/mL at6d. Strain TST05displayed a higher virulence to C. sasakii larvae and a rapid action in killing host. The strain can be used as a new strain for biology control on C. sasakii and was preserved in China General Microbiological Culture Collection, No.4526.
3. The conidial adhesion and germination on C. sasakii larvae surface, the integument penetration of hyphae on the larvae, the internal tissues infection, defense response of the larvae, and the host pathological changes were investigated by using light, scanning electron microscopy and transmission electron microscopy. The result showed that the head capsule of the larva was so strong and smooth that very few conidia attached to there. But there were many conidia in the basal area around the mouthparts and antennae. On the thorax and abdomen of the larvae, dense acanthae were distributed over the cuticle and in which few long seta scattered. Conidia adhered mainly to the area around the acanthae. Some conidia were observed in the peritreme socket of the spiracles and on the smooth seta.
After conidia germinated, the fungus invaded the larvae mainly by penetrating the integument. The conidia germinated during a24-36h period after inoculation, and the germ tubes might directly penetrated the cuticle or produced a infection peg at the hyphal tips to penetrate into the cuticle. The fungal infection destroyed the regular protein-chitin parallel lamellar structure in the procuticle. The defensive response of the larvae to the fungal attack was indicated by some dark spots appeared on the cuticle and some melanization appeared in the hemocoel. After overcoming the host's defense system, the fungus grew and reproduced many blastospores in the hemocoel, then infected the internal tissues and organs. Infection symptoms were observed in the fat body, alimentary canal, the Malpighian tubules and the muscle tissue. The fungus attacked and colonized the sericin layer and the liquid fibroin in the silk gland lumen. Finally, the fungus emerged through the cuticle of the dead insect and released conidiophores that could act as new pathogens to infect other larvae. The results clearly revealed the process of strain TST05infected C. sasakii larvae.
4. The physiological and biochemical reaction of C. sasakii larvae were studied when they were infected by the strain TST05. The results showed that after infected, the content of trehalose and protein in the larvae decreased obviously, the trend of changes of glucose content was increased at first and then decreased, the activity of trehalase rose to1.91times as that of control groups at5d after inoculation.
During the initial infection stage, the activities of PO, GSTs, CarE, SOD, POD and GSH-Px that play a role in immune and protection for the host, increased quickly, while during the later stage, their activities decreased at different degrees. The change of GSTs and GSH-Px activities were biggest in those enzymes. Their activities were2.84times and2.95times respectively as those in control groups at3d after inoculation. The activities of SOD and POD were1.61times and1.53at3d, the activeties of PO and CarE was1.48times and1.50times as that of control groups at5d, respectively. The results indicated that the infection of strain TST05affected nutrient metabolism and induced the physiological defense reaction of C. sasakii larvae.
The activity of CAT in infected larvae always lower than that in control larvae, and the lowest activity is56.78%as control activity. It indicted that the infection of the strain TST05can inhibited and destroyed CAT. As the same time, the increase of the AChE activity in infection period indicted the infection or the toxin of the strain can effect the nerve conduction of larvae.
5. The effect of culture media, temperature, and humidity on the mycelium growth, conidia yield, and conidia germination of the strain TST05were investigated. The results showed that the strain could grow well in the four culture media, PDA, PPDA, SDAY and SMAY. The fungal colonies were sick and dense, and the conidia yield exceeded3.95x107conidia/mL in these culture media.
The TST05strain was adapt to a wide range of temperature and humidity. The conidia could germinate, and the mycelia could grow and sporulate in15-30℃and RH30%-100%. With the temperature closing to25℃and the humidity increasing, the rate of conidia germination and mycelium growth and the conidia yield were all significantly increased. The conidia yield was1.32x107conidia/mL under15℃, RH100%,1.37x107conidia/mL under25℃, RH30%, and6.19x107conidia/mL under25℃, RH100%. The conidia germination rate was52.28%under15℃, RH100%, and90%or more under25℃, RH>80%. The results indicted that the strain TST05was easy to culture and adapt to low temperature and humidity in north. The strain could become a biological agent to control C. sasakii.
6. The powder preparation of strain TST05was preparated by cultured in wedge-shaped bottle and plastic bag with glass cavity tube in the both ends. The results showed that the strain grew vigorously in the two culture vessels. The white dense mycelia overgrow in the solid media. The conidia yield and the protease activity of powder preparation were12.50x109/g and770.34U/mg pr respectively at20d in the plastic bag, which were1.42times and1.54times respectively as that in wedge-shaped bottle. The results indicted the good ventilation effect is more conducive to the growth and sporulation of strain TST05.
7. The control efficiency of powder preparation to C. sasakii mature larvae in an orchard was determined. In September, when the mature larvae of C. sasakii bored out jujube and dropped in the soil for overwintering, the fungal powder preparation was mixed with soil in the proportion of1:200and then was applied in the upper soil layer under the tree. The larvae were laid on the surface of the soil and naturally bored into the soil. The mortality rate of powder preparation to C. sasakii larvae was determined. The adjusted mortality rate of C. sasakii larvae reached75.29%by the fungus infecting in the soil.
8. The survival ability of the strain TST05in soil was tested. The fungal powder preparation was scattered into soil by using the method as above mentioned. The CFUs (colony forming unit) of the strain TST05that survived in soil were determined. The results showed that the CFUs of the strain were1.09x104CFU/g after4months, while the value at the start was108.67x104CFU/g. But in the following months, the CFUs were relatively stable. The conidia yield of the strain isolated from the mixed soil can reach106conidia/mL after6and8months. The results indicated that the strain TST05possessed higher survive ability in the field condition which was propitious to sustainable control on the population of C. sasakii.
9. The compatibility of the strain TST05was studied with seven common chemicals pesticides including beta cypermethrin, cypermethrin-emamectin benzoate, phoxim, cyhalothrin, abamectin, Avi monosultap and tebufenozide. The effect of the chemicals pesticides on mycelium growth, conidia germination and conidia yield of the strain were tested.
The results showed that the inhibition rate of the mycelium growth was27.93%-38.74%under the conventional concentrations of the pesticides. In the group treated with tebufenozide, the conidia germination rate was33.19%, and the conidia yield was only0.05×106conidial/mL, while in other groups treated with other6pesticides, the conidia germination rate ranged from50.34%to70.62%, and the conidia yield was2.51-3.58x106conidial/mL. The inhibition action of the pesticides to the strain decreased with the concentration decreasing. When the pesticides were diluted5times of the conventional concentrations, the conidia germination rate was up to72.08%-84.92%, and the conidia yield was3.01-9.61x106conidia/ml. The results showed that the compatibility of the strain TST05with the six pesticides was relatively good. And the compatibility was increased with the concentration of pesticides decreasing.
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