【作者】 杨群芳；

【导师】 叶华智；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 光滑足距小蠹[Xylosandrus germanus（Blandford）],属于鞘翅目（Coleoptera）、小蠹科（Scolytidae）,是近几年来成都地区葡萄树上新发生的一种毁灭性害虫,以雌成虫在木质部中钻蛀坑道,导致树势衰弱,甚至死亡。该小蠹体小,生活隐蔽,研究和防治十分困难。为了有效地控制其危害和进一步揭示虫菌共生关系,本文对该小蠹种类进行了鉴定,并对其生物学、生态学特性及虫道真菌进行了较为系统的研究,所取得的结果如下:1光滑足距小蠹的鉴定采用体视显微和扫描电镜技术对葡萄树材小蠹外部形态特征进行了研究。该小蠹有4个关键的形态特征:1)体长:雌成虫2.0～2.3 mm,雄成虫1.0～1.3 mm:2)鞘翅前半部光亮,后半部具细长毛;3)雌成虫鞘翅后半部细长毛起自沟间部,雄成虫鞘翅不分沟中和沟间部;4)贮菌器为前-中胸背板型。这些特征表明,为害葡萄树的材小蠹为光滑足距小蠹[Xylosandrus germanus（Blandford）]。此外,扫描电镜下发现:1)成虫前胸前缘的刻点毛均从基部分成两叉,但雌成虫呈锯齿状,雄成虫呈栉齿状;2)鞘翅不具细长毛的刻点中有细短毛。首次研究了光滑足距小蠹消化道的解剖结构。该小蠹成虫前胃板由板状部和片状部组成,板状部短而简单,片状部甚长,由斜面、咀嚼刷和关闭刚毛组成。这种结构可能与其食菌习性有关。1对细管状和1对囊状的胃盲囊,着生在中肠近后端。消化道具1对细管状和1对囊状的胃盲囊是该小蠹成虫的又一识别特征。2光滑足距小蠹的生物学和生态学通过田间观察和室内饲养,明确了葡萄树光滑足距小蠹的生物学特性。该小蠹1年发生3～4代,具有局部世代现象,以雌成虫在主干基部的坑道中群集越冬。全年雌成虫有2个飞行活动高峰,其中越冬代雌成虫的飞行活动为最高峰。坑道系统包括1个侵入道、1个育幼室和3～5个分支坑道。在半人工饲料和葡萄木段中繁殖时雌雄性比分别为10.7:1和10.3:1。取食Acremonium kiliense时,幼虫有3龄。通过虫道真菌Acremonium kiliense和Ambrosiella hartigii饲养雌成虫和幼虫,首次测定了光滑足距小蠹各虫态的发育起点和有效积温,明确了温度对其生长发育的影响。16～31℃范围内,各虫态均能完成发育。卵、幼虫、蛹的发育起点温度分别为12.11、10.08和11.34℃,有效积温分别为62.65、189.02和86.20日·度,幼虫发育的最适温度为25.53℃。室内研究了光照和温度对光滑足距小蠹雌成虫起飞的影响。雌成虫具有明显的趋光性。25℃时,雌成虫在完全黑暗条件下不起飞。光照和温度两个因素中,温度是雌成虫起飞的先决条件,起飞的临界温度为21℃,只有当环境温度达到21℃,在光照条件下雌成虫才开始起飞。光滑足距小蠹雌成虫侵入孔主要分布在葡萄树0～60 cm高度的主干上,在葡萄地和葡萄树树体上均呈聚集分布,且都为负二项分布,分布的基本成分均为个体群,个体间相互吸引。结果表明,光滑足距小蠹雌成虫以聚集攻击的策略为害葡萄树。3光滑足距小蠹虫道真菌种类组成及其变化从坑道和贮菌器中共分离出4种真菌,经形态和培养特征鉴定,它们分别是:Ambrosiella hartigii、Acremonium kiliense和2种镰刀菌（Fusarium spp.）。用核糖体RNA基因序列的同源性对A.hartigii和A.kiliense进行了鉴定。形态鉴定为A.hartigii的菌株28S rRNA基因与基因库中A.hartigii和A.xylebori序列均有98%的同源性,18S rRNA基因与基因库中A.harigii和A.xylebori序列均有99%的同源性。形态鉴定为A.kiliense的菌株18S rRNA基因与基因库中A.kiliense序列有94%的同源性。结果表明,核糖体RNA基因序列的同源性不能区分A.hartigii和A.xylebori。采用分离实验和扫描电镜技术研究表明,光滑足距小蠹贮菌器和葡萄树坑道内真菌种类组成和优势种类随着蠹虫的发育阶段而有所变化。A.hartigii是光滑足距小蠹扩散、蛀孔、越冬和体壁新硬化的雌成虫贮菌器内唯一分离出的真菌,而刚羽化的雌成虫贮菌器中没有分离出任何真菌种类,体壁新硬化的雌成虫贮菌器中真菌的分离频率最低。坑道内共分离出4种真菌:A.hartigii、A.kiliense和2种镰刀菌（Fusariumspp.）,从卵期到蛹期的坑道中,A.hartigii分离频率较高,达40～60%,而在成虫期坑道中,A.kiliense和Fusarium sp.1的分离频率较高,分别都为34.6%。扫描电镜观察表明,A.hartigii为卵期到蛹期坑道中的优势种,而镰刀菌（Fusarium spp.）则是成虫期坑道中的优势种。4 Ambrosiella hartigii和Acremonium kiliense的生物学特性A.hartigii菌丝生长和产孢均以PDA为培养基最好,菌丝生长以甘露醇为碳源、KNO₃为氮源最好,产孢以蔗糖为碳源、甘氨酸为氮源最好。菌丝生长和产孢的温度范围分别为10～30℃和20～30℃,最适温均为25℃,最适pH为6。菌丝生长、产孢和分生孢子萌发需要黑暗条件。分生孢子萌发最适温度为24～28℃,致死温度为46℃10min,适宜pH为5.0～6.0。分生孢子在水中不萌发。A.kiliense菌丝生长以CMA、PDA和ME最快,产孢以YEME最好。菌丝生长以乳糖和甘露醇为碳源,蛋白胨、牛肉膏和甘氨酸为氮源最好,产孢以葡萄糖为碳源、酵母粉为氮源最好。菌丝生长和产孢最适pH为5.5,最适温为23～25℃。分生孢子萌发的适宜pH为5.0～6.0,最适温度为24～28℃,致死温度为46℃10 min。通过对峙培养研究了A.hartigii、A.kiliense和Fusarium sp.的相互关系。A.hartigii菌丝生长最快,且对A.kiliense和Fusarium sp.菌落扩展具有明显抑制作用,非挥发性代谢物对Fusarium sp.和A.kiliense气生菌丝的产生均有抑制作用。A.hartigii生长和产孢特点及其对Fusarium sp.和A.kiliense气生菌丝的抑制作用是其在坑道中占优势地位的重要原因。5 Ambrosiella hartigii、Acremonium kiliense和Fusarium sp.对光滑足距小蠹生长发育和繁殖的影响研究了A.hartigii,A.kiliense和Fusarium sp.3种虫道真菌对光滑足距小蠹生长发育和繁殖的影响。幼虫取食3种虫道真菌均能完成发育,取食A.hartigii发育最快,存活率高,而取食Fusarium sp.发育最慢,存活率最低。雌成虫取食3种虫道真菌均能繁殖后代,以取食A.hartigii最好。初步探讨了A.hartigii、Fusarium sp.和A.kiliense 3种虫道真菌的营养价值。培养3 d、5 d和10 d后,均以A.hartigii菌丝体麦角固醇含量最高,以A.kiliense可溶性蛋白质和糖含量最高。虫道真菌营养价值和蠹虫生长发育繁殖的关系有待进一步研究。更多还原

【Abstract】 Xylosandrus germanus（Blandford）（Coleoptera:Scolytidae） is a new destructive pest attacking grapevine in Chendu.The adult females excavate galleries in wood.As a result, the vigor of grapevine descends,even the trees die off.It is very difficult to study and control the beetle because of its small body and secluded life.Aiming to provide a basis for the pest management and further reveal symbiosis between the beetle and fungi,the biology,ecology and ambrosia fungi of the beetle were studied,and the results are as follows.1 Identification of X.germanusMorphological characteristics of X.germanus were studied by stereo and scanning electron microscopy.The key characteristicses to identify the beetle are:1) the adult female is 2.02 to 2.26 mm long,and the adult male is 1.00 to 1.30 mm long;2) the anterior half of elytron is shining,while there is long hair on the hind half;3) there is long hair only in the interstriae of the posterior half of elytron,whereas the adult males’ elytrons can not be divided into striae and interstriae;4) the mycangium is pro-mesonotal.These characteristicses showed that the beetle attacking grapevine is Xylosandrus germanus （Blandford）.Furthermore,two morphological characteristics were found by scanning electron microscopy,they are:1) hair in the sculpture of the costal margin of the adult’s prothorax is divided into two branches,the female’s is serrate while the male’s is pectinate; 2) there is very short hair in the sculptures which have not long hair in the elytrons.The anatomy of the alimentary cannal of X.germanus was described for the first time home and abroad.The adult’s proventriculus plate is composed of anterior plate and posterior plate,the former is shorter and simpler,and the later is longer,composed of declivity,masticatory brush and stopping bristles.The plate structure is likely related to the beetle’s mycetophagous habit.The adults have a pair of tuble-shaped and a pair of sac-shaped gastric caeca,being located in the posterior portion of midgut.That there are a pair of tuble-shaped and a pair of sac-shaped gastric caeca in the alimentary cannal is one of the identification characteristics of the adult.2 The biology and ecology of X.germanusThe biology of X.germanus were clarified by field observation and indoor breeding. It had three to four generations per year with partial generation,and overwintered as the adult females clustering in the galleries of bole base of grapvines.There were two peaks of flight activity in a year,the bigger one was the overwintered females’ flight activity.The gallery system was composed of one entrance tunnel,one brood chamber and three to five branch tunnels.When the adult females were fed in semi-artificial diets and grapevine logs, the sex ratio was 10.7 female:lmale,and 10.3 female:lmale,respectively.When they fed on Acremonium kiliense at 25℃,the larvae had three instars.Adult females and larvae were fed with Acremonium kiliense and Ambrosiella hartigii, temperature thresholds and thermal constants for development were calculated for the first time,and the effect of temperature on development of the beetle was clarified.The results showed that egg,larva and pupa could complete the development from 16 to 31℃.For egg, larva and pupa stage development,the temperature thresholds were 12.11,10.08 and 11.34℃,the thermal constants were 62.65,189.02 and 86.20 degree-days,respectively.The optimum temperature for larval development was 25.53℃.The effect of light and temperature on X.germanus adult females’ take-off was studied in the laboratory.A relative remarkable phototaxis of the adult females was proved.At 25℃,no beetle took off in the dark.Temperature was prerequisite for adult females’ take-off,and the threshold temperature was 21℃.Only when temperature reached 21℃, began female adults to take off in the light.The entrance holes excavated by X.germanus adult females mainly distributed in the 0-60 cm high stem,the spatial distribution patterns fitted the negative binomial distribution and belonged to the aggregated pattern both in orchards and within trees,the basic element for the distribution was clusters of individuals,and the individuals attracted each other. These results indicated that aggregatively attack strategy was used to attack grapevine by X. germanus adult females.3 Composition and variety of the ambrosia fungi associated with X.germanus4 species of fungi were isolated from galleries and mycagium.According to the morphological and cultural characteristics,they were identified as Ambrosiella hartigii Batra,A cremonium kiliense Gr（u|¨）tz and two species of Fusarium.The sequence homologies of ribosome RNA gene was also used to identify A.hartigii and A.kiliense.The sequence homologies of 28S ribosome RNA were all 98%when A. hartigii identified by morphology was compared with the sequences of A.hartigii and that of Ambrosiella xylebori retrieved from genebank,whereas that of 18S ribosome RNA were all 99%.The sequence homologies of 18S ribosome RNA between A.kiliense identified by morphology and the sequence retrieved from genebank was 94%.These results indicated that the sequence homologies of ribosome RNA gene could not be used to distinguish A. hartigii from A.xylebori.The composition and the relative dominance of the fungi in the gallery system and in the mycangium of X.germanus varied with developmental stages of the beetle by isolation experiments and scanning electron microscopy.A.hartigii was the only fungus isolated from the mycangia of the dispersal,boring,overwintering and newly sclerotized adults but not the callow adults.The lowest frequency of the fungus isolated from the mycangium occurred in the newly sclerotized adults.The fungi isolated from galleries consisted of A. hartigii,A.kiliense and two species of Fusarium.A.hartigii occurred in a higher percentage of about 40-60%during the period from egg to pupal stage,while A.kiliense and Fusarium sp.1 accounted for 34.6%and 34.6%from the adult galleries,respectively. A.hartigii was always isolated from galleries from the egg to pupal stage.Scanning electron microscopy showed that the conidiophores and conidia of A.hartigii were predominantly occurred at the bottom of galleries from the egg to pupal stage,whereas Fusarium spp.were predominantly occurred in the mature adult galleries.4 Biological characteristics of Ambrosiella hartigii and Acremonium kilienseA.hartigii could produce many spores and grow very well on the PDA and PSA medium.The mannitol carbon source and KNO₃ nitrogen source were the best for mycelial growth,while sucrose and glycin was favorable for spore formation.The mycelium could grow between10-30℃,while the spores could form between 20-30℃. For mycelial growth and sporulation,the optimum temperatures was 25℃,the optimum pH was 6.0.The mycelial growth,sporulation and spore germination all need darkness. For spore germination,the optimum temperatures were 24-28℃,pH was 5.0-6.0,the lethal temperature was 46℃（10min）.The conidia could not germinate in water.A.kiliense could grow very well on the CMA,PDA and ME medium,and produce many spores on the YEME medium.The lactose and mannitol carbon source,and the peptone,beef extract and glycin nitrogen source were the best for mycelial growth,while glucose and yeast extract were favorable for spore formation.For the mycelial growth and sporulation,the optimum pH was 5.0,the optimum temperatures was 23-25℃.For spore germination,the optimum pH,the optimum temperatures and lethal temperature were 5.0-6.0,24-28℃and 46℃（10min）,respectively.Interrelation of A.hartigii,A.kiliense and Fusarium sp.were studied in dual culture. A.hartigii grew faster than the other two species of fungi,and it significantly inhibited the growth of the other fungi.The nonvolatile significantly prevent A.kiliense and Fusarium sp.from growth of aerial hypha.These results indicated that the growth and sporulation characteristics of A.hartigii as well as its prevention from aerial hypha growth of the other fungi made it predominant in the galleries.5 Effect of AmbrosieUa hartigii,Acremonium kiliense and Fusarium sp.on development and reproduction of X.germanusThe effect of A.hartigii,A.kiliense and Fusarium sp.on the X.germanus development and reproduction were studied.The larvae could complete development living on three species of fungi,the larvae grew fastest and had the highstest survival living on A.hartigii,while the larvae grew slowest and had the lowest survival feeding on Fusarium sp..The adult females could reproduce offsprings feeding on three species of fungi,however,its reproductivity feeding on A.hartigii was highest of three species of ambrosia fungi.The nutritional value of A.hartigii,A.kiliense and Fusarium sp.was primarily studied.After ambrosia fungi being cultured 3,5 and 10 d,Ergosterol content in A.hartigii were higher than that in the other fungi.Soluble protein and sugar content in A.kiliense were higher than that in the other fungi.The correlation between nutritional value of ambrosia fungi and development and reproduction of the beetle is to be studied further.更多还原