蚧虫受蜡蚧轮枝菌感染的组织病理学研究
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摘要
蚧虫是世界农林业的重要害虫,以刺吸式口器刺入植物组织,吸取汁液为生。蚧虫体表具有多种蜡腺,能分泌大量蜡质,对虫体起保护作用,使化学防治很难奏效。采用病原菌防治蚧虫是生物防治的主要方面,蚧虫在自然界中记录最重要的病原菌是蜡蚧轮枝菌Lecanicillium lecanii(Zimmermann)Gams & Zare。本文以日本龟蜡蚧Ceroplastes japonicus Green和褐软蚧Coccus hesperidum L.为例,研究蜡蚧轮枝菌对蚧虫的致病机理,为利用病原菌防治蚧虫提供直接的理论依据和实践思路,开展蚧虫生物防治新途径。
本文通过使用光学显微镜、扫描电镜和透射电镜技术相结合的方法,研究了日本龟蜡蚧消化和排泄系统的显微和超微结构,为蜡蚧轮枝菌对蚧虫的致病机理研究提供基础。重点研究了蜡蚧轮枝菌对日本龟蜡蚧和褐软蚧的侵染过程、侵染部位和蚧虫被蜡蚧轮枝菌感染后的的组织病理变化。研究结果如下:
1.日本龟蜡蚧的消化和排泄系统
日本龟蜡蚧的消化系统由口器、前肠、中肠、后肠和肛门组成。口器是消化道前端的开口,有四个尖细的口针。肠道白色膜质。前肠较细,又分为骨化的咽和食道两部分。前肠的中部附着一对发达的唾液腺,后端与中肠相通。中肠粗、长,形成一个大的中肠环。中肠的肠壁细胞大,向肠腔突起。后肠分为细的回肠和较粗的直肠。后肠的肠壁细胞小,肠腔大。直肠的末端终止于肛门。一个发达的滤室包被在直肠的前端。马氏管是蚧虫的排泄器官,位于中肠和后肠的交界处,为两条黄色念珠状的管。这两条管通过一个公共管联系在一起与肠道相连。马氏管的内部看似蜂窝状,内含大量的结晶,大约占据了细胞质体积的一半。
2.蚧虫感染蜡蚧轮枝菌后的组织病理变化
蜡蚧轮枝菌对不同蚧虫的致病性不同,研究发现,蜡蚧轮枝菌菌株V3.4505对日本龟蜡蚧的致病性比V3.4504的强;而V3.4504对褐软蚧的致病性比V3.4505强。
蜡蚧轮枝菌主要通过穿透蚧虫体壁侵染虫体,肛门和阴门也是菌丝侵入虫体的途径。虫体腹面的体缘区、口器和足的基部周围,气门路上以及阴门区以及背面的肛板区是容易侵染的部位,而覆盖在虫体背面的蜡壳抑制蜡蚧轮枝菌的侵染。蜡蚧轮枝菌对蚧虫的入侵过程以及蚧虫的组织病理变化如下:接菌24h后,分生孢子附着在虫体表面,在适宜的温度和湿度条件下,分生孢子萌发形成芽管,芽管伸长形成菌丝:菌丝沿寄主体表生长,在其前端或菌丝上侧向分支,特化成侵入钉,向体壁入侵。体壁的穿透在接菌后72h内发生,真菌入侵的部位虫体表面出现凹陷,入侵点处体壁颜色加深,真菌的入侵使表皮层被破坏,表皮层和真皮层分离,虫体表皮出现裂缝,菌丝通过这些裂缝进入虫体。接菌72h后,附着在虫体表面的菌丝分支,形成大量的菌丝体,覆盖在虫体表面;进入蚧虫体腔的菌丝,形成芽生孢子。芽生孢子在血腔中迅速繁殖,并侵入血细胞,消耗细胞质的营养,然后侵入细胞核,使细胞核破坏和染色质凝聚。在血腔被真菌占领的同时,内部的组织器官也受到侵染,例如,气管、肌肉束、马氏管内等等,都出现芽生孢子。随着真菌的侵入,蜡壳和虫体颜色也发生变化,日本龟蜡蚧的分别由白色和红色变成了黄色,褐软蚧的颜色由于营养物质的消耗变成黑色。菌丝的入侵使虫体内部的组织器官被完全分解,虫体死亡。在死亡虫体表面新的分生孢子形成,作为新的传染源释放到外界。
Scale insects are one group of the important pests in fruit orchards, forests, horticulture and ornamental plants, which feed on the plant juice with their piercing and sucking mouthpart piercing into the plant tissues. The most distinctive characteristic of the scale insects is their body with many types of wax glands that secrete a mountain of wax substances to form wax covering over the surface of body. The wax covering limits the efficacy of insecticide sprays so that it is difficult to control scale insects. The availability of biological control agents such as entomopathogenic fungi would be highly desirable, of which, Lecanicillium lecanii (Zimmermann) Gams & Zare is probably the most important one for the scale insects reported in the nature. In order to develop entomopathogenic fungi into an effective bio-insecticide, it is significant to investigate the histopathology of scale insects that infected by entomopathogenic fungi. In this thesis, the two species of soft scale, Japanese wax scale, Ceroplastes japonicus Green and brown soft scale, Coccus hesperidum L. (Hemiptera: Coccoidea:Coccidae) and the pathogenic fungus, L. lecanii were studied using light microscopy, scanning electron microscopy and transmission electron microscopy.
For well understanding pathogenic mechanism of L. lecanii to the scale insects, the micro- and ultra-structure of digestive and excretory system of C. japonicus were studied at first showed in chapter 2 of the thesis. The infection processes and the histopathological changes of the two scale insects infected with L. lecanii were showed in chapter 3 and 4, respectively. The results of these studies are showed as follows.
1. Digestive and excretory system of the adult female Japanese wax scale, C. japonicus
The digestive system of C. japonicus consisted of mouthpart, foregut, midgut, hindgut and anus. Ceroplastes japonicus possessed piercing and sucking mouthpart. The mouthpart was the anterior open of the alimentary canal and had four sharp stylets, a pair of maxillary stylets and mandibular stylets. On the inner base of each stylet, there was a lever for muscle attachment. Within the stylets were located the food and salivary canals. The former was placed dorsally and was connected with the pharyngeal duct, while the latter was ventral and joins with the salivary pump. The extremity of each stylet was sharply pointed for penetrating the plant tissue. The gut was white membraneous. The foregut was narrow and subdivided into a sclerotised pharynx and an oesophagus. Its middle part was attached by a pair of salivary glands and its posterior communicated with the midgut. The midgut was thick, long and loop-shaped with big epithelia protruded to the enteric cavity. The midgut was divided into segments by constrictions. Gastric caecum was a blind-ending lateral divericulum of the midgut with some tracheae attached on its surface. The filter chamber was formed from loops of the anterior midgut and part of the ileum enclosed within an invaginated cavity developed from the enlarged anterior part of rectum. This envelope enclosed most of the gut, isolating the filter chamber from the haemocoel. Hindgut was divided into a narrow ileum and a relative broad rectum. The surface of the ileum was characterized with a layer of longitudinal muscle. The epithelia of ileum loose arranged and protruded to the enteric cavity, while the epithelia of the rectum were relatively small in the enteric cavity that made the enteric cavity of rectum appeared very large. The inner surface of intestinal wall was rough with small promontories. The posterior rectum ended at anus. The anal apparatus lay at the inner end of an internal anal tube and consisted of an anus surrounded by a sclerotized anal ring with 6 anal ring setae and covered by a pair of anal plates. Two brownish-yellow moniliform malpighian tubules located at the junction of midgut and hindgut. The two tubules communicate to the gut by a common tubule. Malpighian tubules were composed of basement membrane, basal fold, cytoplasm and microvilli. Its inner appeared honeycombed with many crystals possessed most volume of the cytoplasm.
2. Histopathological changes of the scale insects infected by L. lecanii
L. lecanii showed different infectivity to the scale insects. It was observed that the infectivity of I. lecanii strain V3.4505 to C. japonicus was stronger than that of strain V3.4504; while the infectivity was contrary to Coccus hesperidum, that strain V3.4504 was stronger.
L. lecanii generally infected the wax scale by penetrating the integument. Besides, it also can invade the body of the scale insects from the anus and vulva. The anal area, the body margin, around the base of mouthparts and legs, over the stigmatic furrow and the area around the vulva were susceptible places, while the wax test which covered the dorsal surface had inhibitory effect to L. lecanii. The infection process and the histopathological changes of the scale insects infected by L. lecanii were shown as follows. 24h after inoculation, the conidia attached to the cuticle. The conidia germinated into germ tube and the germ tube developed into hyphae in proper temperature and relative humidity. The hyphae developed along the surface of the host or branched laterally with their tips differentiated into specialized infection pegs and began to invade the integument. Penetration of the cuticle occurred within 72h of inoculation. The fungal infection made the emergency of depression on the surface and the color darken of the infection place. The hyphae extended laterally in the cuticlar layer or penetrated across the cuticle which damaged the cuticular structure and separated the cuticle from the epidermis. The fungus also caused the insect cuticle to rupture and hyphae entered the insect body through these openings. 72h after inoculation, amounts of mycelia, which covered the surface of the host, were formed by the branching of the hyphae attached on the insect, while blastospores were produced from the hyphae invaded in the hemocoele of the scale insect. After 96h, blastospores were dispersed throughout the hemolymph and completely disrupted the hemocytes, resulting in damage of the cell nucleus and agglutination of chromatin. Concomitant to colonization of the hemolymph, the internal organs and tissues, e.g. in trachea, malpighian tubules and muscle fiber, etc. were also infected. As the infection progressed, the wax test and body of C. japonicus changed color from white and red, respectively, to yellowish. And the color of Coccus hesperidum changed to dark brown. Muscle fibers were separated from surrounding tissues. Malpighian tubule damage was characterized by disruption of the basal laminar and basal infold walls separating them from each other and many irregular cystals changed to vacuoles. The midgut was damaged with desquamation of the intima from the enterocytes, emergence of many vacuoles in the enterocyters, formation of vesicles in the endoplasmic reticulum and alternation of the nuclei in the enterocytes. After 144h, the internal tissue structure was totally compromised, the insects died. Many hyphae covered the whole body. New conidiophores produced on the surface of the cadavers and released as new pathogen.
本文通过使用光学显微镜、扫描电镜和透射电镜技术相结合的方法,研究了日本龟蜡蚧消化和排泄系统的显微和超微结构,为蜡蚧轮枝菌对蚧虫的致病机理研究提供基础。重点研究了蜡蚧轮枝菌对日本龟蜡蚧和褐软蚧的侵染过程、侵染部位和蚧虫被蜡蚧轮枝菌感染后的的组织病理变化。研究结果如下:
1.日本龟蜡蚧的消化和排泄系统
日本龟蜡蚧的消化系统由口器、前肠、中肠、后肠和肛门组成。口器是消化道前端的开口,有四个尖细的口针。肠道白色膜质。前肠较细,又分为骨化的咽和食道两部分。前肠的中部附着一对发达的唾液腺,后端与中肠相通。中肠粗、长,形成一个大的中肠环。中肠的肠壁细胞大,向肠腔突起。后肠分为细的回肠和较粗的直肠。后肠的肠壁细胞小,肠腔大。直肠的末端终止于肛门。一个发达的滤室包被在直肠的前端。马氏管是蚧虫的排泄器官,位于中肠和后肠的交界处,为两条黄色念珠状的管。这两条管通过一个公共管联系在一起与肠道相连。马氏管的内部看似蜂窝状,内含大量的结晶,大约占据了细胞质体积的一半。
2.蚧虫感染蜡蚧轮枝菌后的组织病理变化
蜡蚧轮枝菌对不同蚧虫的致病性不同,研究发现,蜡蚧轮枝菌菌株V3.4505对日本龟蜡蚧的致病性比V3.4504的强;而V3.4504对褐软蚧的致病性比V3.4505强。
蜡蚧轮枝菌主要通过穿透蚧虫体壁侵染虫体,肛门和阴门也是菌丝侵入虫体的途径。虫体腹面的体缘区、口器和足的基部周围,气门路上以及阴门区以及背面的肛板区是容易侵染的部位,而覆盖在虫体背面的蜡壳抑制蜡蚧轮枝菌的侵染。蜡蚧轮枝菌对蚧虫的入侵过程以及蚧虫的组织病理变化如下:接菌24h后,分生孢子附着在虫体表面,在适宜的温度和湿度条件下,分生孢子萌发形成芽管,芽管伸长形成菌丝:菌丝沿寄主体表生长,在其前端或菌丝上侧向分支,特化成侵入钉,向体壁入侵。体壁的穿透在接菌后72h内发生,真菌入侵的部位虫体表面出现凹陷,入侵点处体壁颜色加深,真菌的入侵使表皮层被破坏,表皮层和真皮层分离,虫体表皮出现裂缝,菌丝通过这些裂缝进入虫体。接菌72h后,附着在虫体表面的菌丝分支,形成大量的菌丝体,覆盖在虫体表面;进入蚧虫体腔的菌丝,形成芽生孢子。芽生孢子在血腔中迅速繁殖,并侵入血细胞,消耗细胞质的营养,然后侵入细胞核,使细胞核破坏和染色质凝聚。在血腔被真菌占领的同时,内部的组织器官也受到侵染,例如,气管、肌肉束、马氏管内等等,都出现芽生孢子。随着真菌的侵入,蜡壳和虫体颜色也发生变化,日本龟蜡蚧的分别由白色和红色变成了黄色,褐软蚧的颜色由于营养物质的消耗变成黑色。菌丝的入侵使虫体内部的组织器官被完全分解,虫体死亡。在死亡虫体表面新的分生孢子形成,作为新的传染源释放到外界。
Scale insects are one group of the important pests in fruit orchards, forests, horticulture and ornamental plants, which feed on the plant juice with their piercing and sucking mouthpart piercing into the plant tissues. The most distinctive characteristic of the scale insects is their body with many types of wax glands that secrete a mountain of wax substances to form wax covering over the surface of body. The wax covering limits the efficacy of insecticide sprays so that it is difficult to control scale insects. The availability of biological control agents such as entomopathogenic fungi would be highly desirable, of which, Lecanicillium lecanii (Zimmermann) Gams & Zare is probably the most important one for the scale insects reported in the nature. In order to develop entomopathogenic fungi into an effective bio-insecticide, it is significant to investigate the histopathology of scale insects that infected by entomopathogenic fungi. In this thesis, the two species of soft scale, Japanese wax scale, Ceroplastes japonicus Green and brown soft scale, Coccus hesperidum L. (Hemiptera: Coccoidea:Coccidae) and the pathogenic fungus, L. lecanii were studied using light microscopy, scanning electron microscopy and transmission electron microscopy.
For well understanding pathogenic mechanism of L. lecanii to the scale insects, the micro- and ultra-structure of digestive and excretory system of C. japonicus were studied at first showed in chapter 2 of the thesis. The infection processes and the histopathological changes of the two scale insects infected with L. lecanii were showed in chapter 3 and 4, respectively. The results of these studies are showed as follows.
1. Digestive and excretory system of the adult female Japanese wax scale, C. japonicus
The digestive system of C. japonicus consisted of mouthpart, foregut, midgut, hindgut and anus. Ceroplastes japonicus possessed piercing and sucking mouthpart. The mouthpart was the anterior open of the alimentary canal and had four sharp stylets, a pair of maxillary stylets and mandibular stylets. On the inner base of each stylet, there was a lever for muscle attachment. Within the stylets were located the food and salivary canals. The former was placed dorsally and was connected with the pharyngeal duct, while the latter was ventral and joins with the salivary pump. The extremity of each stylet was sharply pointed for penetrating the plant tissue. The gut was white membraneous. The foregut was narrow and subdivided into a sclerotised pharynx and an oesophagus. Its middle part was attached by a pair of salivary glands and its posterior communicated with the midgut. The midgut was thick, long and loop-shaped with big epithelia protruded to the enteric cavity. The midgut was divided into segments by constrictions. Gastric caecum was a blind-ending lateral divericulum of the midgut with some tracheae attached on its surface. The filter chamber was formed from loops of the anterior midgut and part of the ileum enclosed within an invaginated cavity developed from the enlarged anterior part of rectum. This envelope enclosed most of the gut, isolating the filter chamber from the haemocoel. Hindgut was divided into a narrow ileum and a relative broad rectum. The surface of the ileum was characterized with a layer of longitudinal muscle. The epithelia of ileum loose arranged and protruded to the enteric cavity, while the epithelia of the rectum were relatively small in the enteric cavity that made the enteric cavity of rectum appeared very large. The inner surface of intestinal wall was rough with small promontories. The posterior rectum ended at anus. The anal apparatus lay at the inner end of an internal anal tube and consisted of an anus surrounded by a sclerotized anal ring with 6 anal ring setae and covered by a pair of anal plates. Two brownish-yellow moniliform malpighian tubules located at the junction of midgut and hindgut. The two tubules communicate to the gut by a common tubule. Malpighian tubules were composed of basement membrane, basal fold, cytoplasm and microvilli. Its inner appeared honeycombed with many crystals possessed most volume of the cytoplasm.
2. Histopathological changes of the scale insects infected by L. lecanii
L. lecanii showed different infectivity to the scale insects. It was observed that the infectivity of I. lecanii strain V3.4505 to C. japonicus was stronger than that of strain V3.4504; while the infectivity was contrary to Coccus hesperidum, that strain V3.4504 was stronger.
L. lecanii generally infected the wax scale by penetrating the integument. Besides, it also can invade the body of the scale insects from the anus and vulva. The anal area, the body margin, around the base of mouthparts and legs, over the stigmatic furrow and the area around the vulva were susceptible places, while the wax test which covered the dorsal surface had inhibitory effect to L. lecanii. The infection process and the histopathological changes of the scale insects infected by L. lecanii were shown as follows. 24h after inoculation, the conidia attached to the cuticle. The conidia germinated into germ tube and the germ tube developed into hyphae in proper temperature and relative humidity. The hyphae developed along the surface of the host or branched laterally with their tips differentiated into specialized infection pegs and began to invade the integument. Penetration of the cuticle occurred within 72h of inoculation. The fungal infection made the emergency of depression on the surface and the color darken of the infection place. The hyphae extended laterally in the cuticlar layer or penetrated across the cuticle which damaged the cuticular structure and separated the cuticle from the epidermis. The fungus also caused the insect cuticle to rupture and hyphae entered the insect body through these openings. 72h after inoculation, amounts of mycelia, which covered the surface of the host, were formed by the branching of the hyphae attached on the insect, while blastospores were produced from the hyphae invaded in the hemocoele of the scale insect. After 96h, blastospores were dispersed throughout the hemolymph and completely disrupted the hemocytes, resulting in damage of the cell nucleus and agglutination of chromatin. Concomitant to colonization of the hemolymph, the internal organs and tissues, e.g. in trachea, malpighian tubules and muscle fiber, etc. were also infected. As the infection progressed, the wax test and body of C. japonicus changed color from white and red, respectively, to yellowish. And the color of Coccus hesperidum changed to dark brown. Muscle fibers were separated from surrounding tissues. Malpighian tubule damage was characterized by disruption of the basal laminar and basal infold walls separating them from each other and many irregular cystals changed to vacuoles. The midgut was damaged with desquamation of the intima from the enterocytes, emergence of many vacuoles in the enterocyters, formation of vesicles in the endoplasmic reticulum and alternation of the nuclei in the enterocytes. After 144h, the internal tissue structure was totally compromised, the insects died. Many hyphae covered the whole body. New conidiophores produced on the surface of the cadavers and released as new pathogen.
引文
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[40]王音.绿僵菌LD65和LD68菌株生物学特性及对小菜蛾的致病机理.中国农业大学博士学位论文.2005,55-68.
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[44]殷凤鸣,陈权才,叶燕华等.蜡蚧轮枝菌防治湿地松粉蚧的技术研究.林业科技通讯,1996,5:15-18.
[45]殷凤鸣,秦生长.蜡蚧轮枝菌防治湿地松粉蚧的研究.广东林业科技,2000,16(1):41-44.
[46]袁盛勇,孔琼,张虹等.蜡蚧轮枝菌MZ041024菌株对温室白粉虱和吹棉蚧的室内毒力.西南师范大学学报(自然科学版),2007,32(1):111-114
[47]张仙红,李文英,贺运春等.菜青虫感染蜡蚧轮枝菌后的组织病理变化.昆虫学报,2003,46(5):655-659
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[24] Kanaoka, M., Isogai, A., Murakoshi S., Ichinoe, M., Suzuki, A., Tamura, S. Bassianolide, a new insecticidal cyclodepsipeptide from Beauveria bassiana and Verticillium lecanii. Agric Biol Chem, 1978,42 (3): 629-635.
[25] Kim, J. J. Influence of Lecanicillium attenuatum on the development and reproduction of the cotton aphid, Aphis gossypii. BioControl, 2007, 52 (6): 789-799.
[26] Kosztarab, M. Why study the scale insects? (Homoptera: Coccinea). In: Proceedings (Part II) of the sixth international symposium of scale insect studies. Cracow, Poland, 1990, pp. 7-10.
[27] Lo, P. L., Chapman, R. B. The role of parasitoids and entomopathogenic fungi in mortality of third-instar and adult Ceroplastes destructor and C. sinensis (Hemiptera: Coccidae: Ceroplastinae) on citrus in New Zealand. Biocontrol Science and Technology, 1998, 8 (4): 573-582.
[28] Marzo, L.D. and Marotta, S. Anatomy of the alimentary canal in female Coccoidea: an iconographic review. In: Bollettino di Zoologia Agraria e di Bachicoltura. Serie II, 2001, 33(3): 77-83.
[29] Pena, J.E. & McMillan, R.T. Verticillium lecanii, a new fungal parasite of the scale Philephidra tuberculosa n.sp. (Homoptera: Coccidae) in Florida. Florida Entomologist, 1986,69(2): 416-417.
[30] Pesson, P. Contribution a l' Etude morphologique et functionnelle de la Tele, de l' appareil buccal et du tube digestif des femelles de cowides. Monographic Centre National de Recherches Agronomiques, Service de Documentation, Versailles, 1944, pp. 266.
[31]St.Leger,R.J.,Joshi,L.,Bidochka,M.J.,Rizzo,N.W.,and Roberts,D.W.Characterization and ultrastructural-localization of chitinases from Metarhizium anisopliae,M.flavoviride,and Beauveria bassiana during fungal invasion of host (Manduca sexta) cuticle.Appl.Envir.Microbiol,1996,62(3):907-912.
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[33]Velimirovic,V.Influence of Verticillium lecanii Viegas on Coccus peudomagnoloarum Kuwana.Zastita Bilja,Beogra,1994,45:187-193.
[34]程云吉,牟吉元.山东农业大学学报(自然科学版),1998,29(2),144-150.
[35]高明媛.昆虫表皮碳氢化合物在昆虫分类中的应用.昆虫学报,2001,44(1):119-122.
[36]南开大学,中山大学,北京大学等.昆虫学(第二卷).北京,人民教育出版社,1980,12-13.
[37]冉江洪,吴次彬.白蜡虫Ericerus pe-la Chavannes唾液腺的研究.四川大学学报(自然科学版),1995,32(6):713-719.
[38]宋建.韭菜迟眼蕈蚊幼虫消化系统结构和消化酶的研究.山东农业大学硕士学位论文.2004,33-34
[39]王联德.蜡蚧轮枝菌毒素及其对烟粉虱种群控制作用的研究.福建农林大学博士学位论文.2003,114-139.
[40]王音.绿僵菌LD65和LD68菌株生物学特性及对小菜蛾的致病机理.中国农业大学博士学位论文.2005,55-68.
[41]谢映平.山西林果蚧虫.北京,中国林业出版社,1998,22-38.
[42]谢映平,薛皎亮,郑乐怡.蚧虫昆虫的蜡泌物:超微结构和化学成分.北京,中国林业出版社,2006,162-167.
[43]徐天瑞,刘晨光.白蜡虫马氏管的超微结构.昆虫学报,1997,40(3):283-287.
[44]殷凤鸣,陈权才,叶燕华等.蜡蚧轮枝菌防治湿地松粉蚧的技术研究.林业科技通讯,1996,5:15-18.
[45]殷凤鸣,秦生长.蜡蚧轮枝菌防治湿地松粉蚧的研究.广东林业科技,2000,16(1):41-44.
[46]袁盛勇,孔琼,张虹等.蜡蚧轮枝菌MZ041024菌株对温室白粉虱和吹棉蚧的室内毒力.西南师范大学学报(自然科学版),2007,32(1):111-114
[47]张仙红,李文英,贺运春等.菜青虫感染蜡蚧轮枝菌后的组织病理变化.昆虫学报,2003,46(5):655-659
[48]周燚,王中康,喻子牛.微生物农药研发与应用.北京,化学工业出版社,2006,105-115.