全 文 ：文章编号: 1007-0435( 2006) 02-0198-03 ·博士论文摘要·
虫生菌四脊裸胞壳 Dh菌株生理特性、
致病性和分子生物学研究
博士生: 朱建兰1　导师: 张自和1, 2
( 1. 甘肃农业大学, 兰州　730070; 2. 兰州大学农业部草地农业生态系统学重点开放实验室, 兰州　730020)
　　四脊裸胞壳(Emer icella quadr iclineata) Dh 菌株为虫
生真菌, 对沙枣尺蠖( Ap ocheima cinerarius)、苜蓿钩麦蛾
( Ap roaer ema anthy lidella)等鳞翅目害虫的幼虫具较强的
致病性 ,对控制草地和林业生态系统的害虫种群数量, 具
有广阔的应用前景。
研究 Dh 菌株生物学特性及培养条件对菌丝生长、孢
子萌发的影响及胞外酶产生的特性等; 测定该菌株对几种
害虫的致病作用及苜蓿钩麦蛾幼虫受感染后体内保护酶
的活性变化; 利用 RAPD 多态性技术及 rDNA ITS 序列
分析, 对 Dh 菌株及其相近种进行亲缘关系和遗传性分
析。
1. Dh 菌株喜温, 生长温度范围为 15～45℃, 孢子的
萌发温度为 8～45℃, 生长发育及孢子萌发最适温度为
30℃;该菌株能利用可溶性淀粉、甘露醇、葡萄糖等多种碳
源和尿素、氯化铵等多种氮源; 光照对液培菌丝生物量和
平板培养产孢有明显的影响,长光照液培有利于菌丝生
长, 其菌丝生物量明显高于短光照和全黑暗; 光照对 Dh
菌株平板产孢具有一定的促进作用。紫外光对 Dh 菌株生
长的影响程度与照射时间有关, 在 25℃下, 在供试范围
内,照射时间越长菌落生长越慢。将含干菌丝 23. 8 mg/ ml
的母液按 1%～15% ( V / V )的比例接种后培养 60 h 以上,
其中 9%、12%和 15%接种量能产生 30 mg / ml 以上的菌
丝生物量。在一定条件培养下, Dh 菌株可产生胞外蛋白
酶、几丁质酶和淀粉酶; 该菌株在马铃薯蔗糖蛋白胨丰富
的培养液中菌丝生物量最大。研究氨基酸、维生素、糖类、
矿物质和脂肪酸对 Dh 菌株孢子萌发的影响, 结果均在不
同程度上对孢子萌发具有影响。测定 5 种杀虫剂和 5 种杀
菌剂对四脊裸孢壳 Dh 菌孢子萌发和菌丝生长的影响。结
果表明,杀虫剂对 Dh 菌的兼容性强于杀菌剂; 在杀虫剂
中, 敌杀死、双甲脒和水胺硫磷对 Dh 菌株孢子萌发和菌
丝生长的影响小,混用时孢子萌发率超过 80. 00%。
2. 用浓度为 5×107 孢子/ ml 处理苜蓿钩麦蛾幼虫,
校正死亡率达 80% ,感病虫体表皮发黑, 保湿后体壁上长
有大量 Dh 菌株的菌丝、分生孢子梗和分生孢子; 当浓度
为1. 6×108 时, 第 7 d 对小菜蛾幼虫校正死亡率为 93.
8% , 对梨尺蠖为 52. 17% ,第 5 d 对棉铃虫校正死亡率为
51. 22% ; 当浓度为 1. 6×1010时, 室内对沙枣尺蠖 (春尺
蠖)校正死亡率达 86. 42% ,田间 82. 13% ; 用 Dh 菌株处理
钩麦蛾幼虫后体内过氧化物酶 ( POD )活性和过氧化氢酶
( CAT )活性出现先降低后上升的趋势, 但酶活性明显高
于对照虫体;而 SOD 酶活性则呈先上升后下降的波动状
态,活性也高于对照虫体。
3. 运用随机扩增多态 DNA( RAPD )技术对 Dh 菌株
及其相近的 13 株曲霉菌株分析基因组 DNA 多态性, 扩
增结果表明: 14 条引物对供试菌株均能扩增出清晰的带,
而且重复性好。聚类分析结果表明:种间、变种间与形态鉴
定结果趋于一致。采用真菌核糖体基因转录间隔区( IT S)
通用引物 IT S4 和 ITS 5, PCR分别扩增了 Dh 菌株及其相
近的 5个裸孢壳菌株(无性阶段属曲霉 ) , 并对 PCR 产物
进行序列测定。根据 Mega 3. 0 软件分析排列后的绝对遗
传距离和平均遗传距离,以 Neighbour-Joining met hod 法
寻找到最简约的系统发育树, 结果 6 个裸孢壳菌可分为 3
个大组, 其中 Dh 菌株与来自定西土壤的皱折裸孢 (E .
cor rugata)之间的靴带支持强度为 88% , 其间的碱基差异
为 11 bp,两者的亲缘关系相对较近; 与来自张掖土壤的褶
皱裸孢(E . rugulosa)的靴带支持强度为 73% , 其间的碱基
差异为 16～17 bp,两者间的亲缘关系相对较远;与其它供
试菌株间亲缘关系相对更远。上述结果表明, 对裸孢壳菌
rDNA IT S 进行测序分析,可清晰地区分裸孢壳的形态学
种。
关键词: 虫生菌; Dh 菌株; 生理学; 致病性; 分子生物学
中图分类号: S 812. 6; Q943　　　文献标识码: A
完成时间: 2005 年 11 月
收稿日期: 2006-02-14; 修回日期: 2006-03-14
基金项目: 农业部草地生态系统学重点开放实验室开放基金;甘肃省农牧厅生物技术专项( GNSW-2004-08)
作者简介: 朱建兰( 1956-) ,女,博士,教授,研究方向为植物保护, E -m ail : zh ujl@ gsau . edu. cn
导师简介: 张自和( 1944-) ,男,教授,研究方向为草业科学, E-mail: z hzihe@ lzu . edu. cn
第 14卷　第 2期
　Vo l. 14　 No . 2
草　地　学　报
ACT A AGRESTIA SIN ICA
　　　2006年　 6 月
　June　　 2006
Physiological Characteristics, Pathogenicity and Molecular Biology of the
Dh Strain of Entomopathogenic Fungi( Emericella Quadriclineata)
Candidate: ZHU Jian-lan
1　Advisor s: ZHANG Zi-he1, 2
( 1. Gan su Agricu lture Un iversi ty, Lanzhou, Gansu Pr ovin ce 730070, China;
2. Key Lab or atory of Gras sland Agro-Ecosystem s, Lanzhou University and Minis t ry of Agricul ture, Lanzhou, Gansu Pr ovin ce 730020, China)
The Dh strain of Marcella quadriclineata, w hich exerts st rong pathogenicity on pests of cankerw orm of
Lepidopter a and lar va of Ap r oaerema anthy lidel la, is believed to be a desirable cho ice for cont roll ing insect
pests and of gr eat use value in the g rassland and for estr y ecolog ical system .
The experiment w as conducted to make a sy stematic and profound research on Dh str ains biolog ical
characterist ics, on the effect of culture condit ions on its hyphae grow ing and spore germinat ing, and the
exocellular enzyme act ivity, etc. The experiment w as also designed to evaluate the pathogenic ef fectiveness of
Dh st rain on several different pests, and to determine the variat ion of some pro tection enzymes act ivity level in
larva of Ap r oaerema anthy lidel la on alfalfa infected by Dh. In addit ion, thr ough RAPD and rDNA ITS
analy sis, the genet ic relationship betw een Dh st rain and some other kindr ed species w as explo red.
Results of the r esearch indicate that Dh st rain pr efer s moderate temperatur e, gr ow ing normally in a
temperature ranging fr om 15 to 45℃. T he Dh st rain spores g erminate in the condit ion of 8～45℃, and 30℃
was the opt imum condit ion fo r its gr ow th and spo re germinat ion. In addit ion, the results also show that Dh
st rain could ut ilize several different types of carbon and nit ro gen, more par ticularly urea and NH4Cl, and could
gr ow vig orously in the media of soluble starch, mannite and maltose as carbon sources. T he study r eveals that
light plays a signif icant role in Dh st rain g row ing and spo re gener at ing. Liquid-cul ture under lengthened
illuminat ion could ef fectively promote the gr ow th of the hyphae, the biomass yield of w hich w as remarkably
higher than that of lim ited illum inat ion t ime or that o f no-l ight cultur e at al l. T he ef fect of ult raviolet light on
Dh grow th w as closely related to the length of t reatment t ime; in the exper iment site of a temperatur e o f below
25℃, the longer the time, the more slow ly the co lony g rew . T he ef fect of culture t ime length and inoculat ion
volume w as tested w ith six differ ent levels f rom 1% to 15%( V/ V ) mother so lut ion of dry Dh hyphae of an
orig inal concentr at ion of 23. 8 mg / ml, on the biomass o f Dh hyphae shake-cul tured on PD medium under g iv en
condit ions of 12 L∶12 D light at 22℃±2℃. The results showed that the biomass yield could r each 30mg/ m l
w ithin 60 hours after inoculat ion w hen the inoculat ion volume w as over 9% o f mother so lut ion. T he Dh str ain
could generate exocellular enzyme, such as proteinase, chit inase, and amylase, in a special culture medium and
given condit ions. The biomass yield o f Dh hyphae incubated in nutritious medium made of potato+ sacchar ose
+ peptone w as much higher than that incubated in any other sampled liquid media . The study indicates that
various o rganic components, such as amino acids, v itamins, sugars, minerals, and fat ty acid, each played a
dif ferent part in Dh spore germination. An assay concerning the effect o f fung icide and insect icide on Dh spore
germ inat ion and g row th show ed that insect icides sampled w ere of mo re compatibility w ith Dh than germicides
in the harm ful org anism management . Among the f iv e sampled insecticides: decis, amit raz, and iso carbophos
optunal had litt le effect on Dh spore germinat ion and grow th, and when Dh w as ut ilized tog ether w ith those
chemicals, the eff iciency of spore germinat ion r ate could exceed 80. 00% .
On pathogenicity and the control ling ef fectiv eness of Dh on some field pests, the study indicates that larva
of Ap r oaerema anthyl idella on alfalfa treated w ith Dh of 5×107 conidia m l- 1 displayed a lethal rate of 80%. T he
body of infected Ap roaerema anthy lidel la lar va turned black and when they w ere kept under a humid condit ion,
large amount of Dh hyphae, conydiophor e, and conidia w ould appear on them . Dh of 5×107 conidia per
milliliter could gener ate a lethality of 65% of larva of diamondback moth w ithin eight days after t reatment ,
65%of pear geometrid, and 54. 76% of co t ton bo llw orm w ithin f ive days. Results also show that indoors,
1. 6×1010 of Dh conidia could lead to a lethality up to 86. 42% of cankerw orm( Apocheima cinerarius) larv a, and
199第 2期 朱建兰等:虫生菌四脊裸胞壳Dh菌株生理特性、致病性和分子生物学研究
《草地学报》编委会
主　任: 洪绂曾
副主任: 任继周　石元春　陈文新　张子仪　刘更另　张新时
顾　问: 王　培　许　鹏　张自和　李建东　苏大学　苏加楷　陈佐忠　周寿荣
孟昭仪　姜　恕　祝廷成　胡兴宗　胡自治　黄文惠
主　编: 洪绂曾
副主编: 周　禾　云锦凤　韩建国　南志标　龙瑞军　李向林　徐　柱　阿不来提
宗锦耀　才　杰
编　委: 于　卓* 　才　杰*　云锦凤* 　王　涛　王　 *　王宗礼　王明玖* 　王德利*
卢欣石* 龙瑞军* 刘国道 孙启忠 朱进忠 毕玉芬 张新全 李向林
汪诗平* 　沈益新 阿不来提 周　禾* 周道玮 呼天明 孟庆祥 宗锦耀
南志标* 　姚爱兴 施大钊* 洪绂曾* 徐　柱 郭振飞 高洪文* 龚元石
程　序　 程积民　 董宽虎 谢应忠 韩建国* 韩烈保 赖志强* 樊江文
盐见正卫(日本 Japan)　Jerrold L . Dodd(美国 USA )
* 常务编委
顾问、编委均以姓氏笔划为序
82. 13% o f the w orm in the f ield. Furthermore, results indicate that in the t reated larv a body of alfalfa leaf-
fo lder ( Ap roaer ema anthill idella) both POD and CAT init ially show ed a decline in act ivity level, and then a
rise, but both w er e much higher than that of the contr ol as a whole. While compared w ith the control , the
act ivity level of SOD though fluctuated, w as st ill higher than the control .
Po lymo rphisms of Dh genom ic DNA and thirteen analog ous st rains of asp er gillus were analy zed by RAPD
( Random Amplif ied Polymorphism DNA ) techno logy . The r esults show that the selected fourteen primers
could all serve as specific ones to amplify dist inct bands w ith high duplicat ion, and the total 211 RAPD sites
w er e amplif ied, 77. 73% of w hich, o r 166 sites, could be specific genet ic ones. T hr ough clustering analysis, it
w as ascertained that the result coincided basically w ith that o f morpholog ic determinat ion.
Through PCR techno logy , genomic DNA of Dh and f ive analog ous genus of Emericella were ampl if ied w ith
tw o common primers that w as designed from IT S, and also the sequence of DN A amplif ied w as determ ined. In
addit ion, through the analy sis of M ega 3. 0 so ftw are, their absolute and average heredity distances were
measured and the simple phylog enet ic development t ree w as establ ished by N eighbor-Joining method. It
show ed that six Emericel la strains could be classified into three pools ( g roups ) . T here are 11 bases of
dif ferences and a boo tst rap support intensity o f 88% betw een Dh and E . corrugata sampled f rom Ding-xi, and
16～17 bp differ ences and a bootstr ap support intensity of 73% betw een Dh and E . corr ugata fr om Zhang-ye,
based on which it could be inferred that there ex isted, in comparison w ith E . corr ugata f rom Zhang-ye, a clo ser
genet ic relat ionship betw een Dh and E . corrugata fr om Ding-x i. As such, it also showed that morpholog y
species o f Emer icella could be dist inct ly dif ferent iated by analysis of rDN A ITS determ inat ion.
Key words: Entomopathogenic fungi; Dh st rain; Physiolog ical characteristics; Pathogenicity ; M olecular
bio logy
(责任编辑　孟昭仪)
200 草　地　学　报 第 14卷