【作者】 武德功；

【导师】 贺春贵； 刘长仲；

【作者基本信息】 甘肃农业大学 ， 草地生物多样性， 2011， 博士

【摘要】 豌豆蚜(Acyrthosiphon pisum)是苜蓿上的重要害虫之一,对苜蓿的产量和品质均有严重的影响。豌豆蚜种下分化现象已引起理论和实践层面上的高度关注。对其种群遗传结构和数量调控机制的探索有助于制定适宜的防治策略和有效的防治措施。本文利用SSR技术研究了豌豆蚜不同地理种群的遗传多样性,从两种瓢虫对其捕食功能反应和四个苜蓿品种对其抗性评价及机理两个角度,探索了豌豆蚜种群的调控机制,结果如下:1.豌豆蚜不同地理种群的遗传多样性应用SSR分子标记技术对豌豆蚜10个地理种群的基因组DNA进行了遗传多样性和遗传分化分析。选取19对引物扩增300个个体,共检测到45个多态性位点,多态性条带百分率PPB为100%。10个豌豆蚜地理种群观测等位基因数Na为1.5889,有效等位基因数Ne为1.35333,Nei′s基因多样性指数为0.20496,Shannon信息指数I为0.30538,多态位点百分率P为58.89%。呼图壁、杨陵、郑州种群的遗传多样性较高,呼和浩特、济南、民和种群相对较低。种群聚类分析结果显示,在遗传相似系数0.60附近,全部豌豆蚜种群明显聚为两大类群,济南、郑州种群为一大类群,其余为另一大类群。AMOVA分析结果表明,种群间变异占49%,种群内占51%,种群间的变异与种群内的变异相当。Mentel检测表明,遗传分化与地理距离、海拔无显著相关性。2.两种瓢虫对豌豆蚜的捕食功能反应七星瓢虫四个龄期幼虫和雌雄成虫对豌豆蚜的捕食功能反应均符合Holling-Ⅱ型;七星瓢虫雌成虫的攻击率最大,处置时间最短;同一发育时期的七星瓢虫在豌豆蚜不同密度下的捕食量差异显著;根据捕食功能反应数据,该模型预测了七星瓢虫一、二、三、四龄幼虫及雄、雌成虫对豌豆蚜的日最大捕食量分别是5.7、11.5、32.1、59.5、57.1和76.9头。龟纹瓢虫四个龄期幼虫和雌雄成虫捕食豌豆蚜的数量与豌豆蚜密度呈负加速曲线关系,符合Honing-Ⅱ型功能反应模型。龟纹瓢虫雌成虫的日捕食量最大,其次是第四龄幼虫和雄虫。模型预测了龟纹瓢虫一、二、三、四龄幼虫及雄、雌成虫对豌豆蚜的日最大捕食量分别是5.5、14.1、29.2、45.1、42.3、51.5头。在同一龄期两种瓢虫的攻击率、处置猎物所用时间、日最大捕食量均有差别,其中在同一龄期七星瓢虫的日最大捕食量高于龟纹瓢虫,而处置猎物所用时间低于龟纹瓢虫。3.四个苜蓿品种对豌豆蚜的抗性评价甘农5号(G5)在大田和室内的抗性植株百分率均显著高于其它品种,分别为50.3%、48.9%,而猎人河的抗性植株百分率在大田和室内均较低,分别为4.0%、4.3%;4个苜蓿品种在大田的抗性级别为甘农5号(G5)高抗(HR),甘农3号(G3)、金皇后(JH)、猎人河(Hu)均为感虫(S);而4个苜蓿品种在室内的抗性级别为甘农5号(G5)抗虫(R),金皇后(JH)、甘农3号(G3)低抗(LR),猎人河(Hu)感虫(S)。4.四个苜蓿品种对两种色型豌豆蚜的抗生性两种色型豌豆蚜在JH上的发育历期、净生殖率、平均世代周期差异显著(P<0.05),在G5上的内禀增长率、周限增长率、种群加倍时间差异显著(P<0.05)。以内禀增长率作为测定抗生性的指标,四个苜蓿品种对两种色型豌豆蚜的抗性表现不同,其中JH对绿色豌豆蚜的抗性较高,G5对红色豌豆蚜的抗性较高,Hu对两种色型豌豆蚜的抗性均较低,四个苜蓿品种对绿色豌豆蚜抗性的大小顺序为Hu<G3<G5<JH,对红色豌豆蚜抗性大小依次为Hu<JH<G3<G5。5.苜蓿品种对豌豆蚜的生理生化抗性机制在蚜虫刺吸诱导的过程中,感虫品种猎人河(Hu)的超氧化物歧化酶(SOD)和过氧化物酶(POD)活性低于抗虫品种甘农5号(G5)和低抗品种甘农3号(G3)、金皇后(JH);接蚜前抗虫品种(G5)的多酚氧化酶(PPO)活性高于感虫品种(Hu)和低抗品种(G3、JH),接蚜后迅速下降,其活性低于感虫品种;抗虫品种(G5)的过氧化氢酶(CAT)活性和总酚含量始终高于低抗品种和感虫品种;抗虫品种(G5)的MDA含量低于低抗品种(G3)和感虫品种(Hu);感虫品种(Hu)的叶绿素含量高于低抗品种(G3、JH),但低于抗虫品种(G5);感虫品种和抗虫品种的可溶性蛋白含量低于低抗品种(除第三天高于G3);抗虫品种(G5)和感虫品种(Hu)的单宁含量高于低抗品种(G3、JH),四个苜蓿品种品种的SOD和POD活性均表现先上升后下降的趋势,过氧化氢酶活性、叶绿素含量、可溶性蛋白含量均呈下降趋势,总酚含量呈上升趋势,而MDA和单宁含量变化无规律。因此,MDA、SOD、POD、CAT、总酚与苜蓿的抗蚜性密切相关,可作为苜蓿抗蚜性生理指标,而叶绿素、可溶性蛋白、PPO、单宁与苜蓿抗蚜性的关系有待进一步研究。更多还原

【Abstract】 Pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae), is one of most devastating insect pests of leguminous forage, which affect the quality and quantity of herbage production. Intraspecies differentiation of pea aphid has already caused the theoretical and practical attention. The exploration of this pest population genetic structure and quantity control mechanism helps formulating appropriate control strategy and effective prevention measures. Genetic diversity of A. pisum populations from different geographic regions in China was studied by SSR technology. From functional response of two coccinellid predators to A. pisum and the resistant evaluation and mechanism of four alfalfa varieties to A. pisum, the mechanism of the population regulation was explored. All results show as follows:1. Genetic diversity of A. pisum populations from different geographic regions in ChinaThe genetic diversity among ten geographic populations of A. pisum in China was investigated using Simple Sequence Repeat (SSR) markers in this study. The result showed that 45 polymorphic locus were detected using 19 pairs of SSR primers in the 300 individuals of 10 populations,percentage of polymorphic bands were 100%. The number of observed alleles was 1.5889, and that of effective alleles was1.35333, the Nei’s genetic diversity and Shannon-Wiener diversity indexes were respectively 0.20496 and 0.30538, percentage of polymorphic locus were 58.89%. The population genetic diversity of Hutubi, Yangling and Zhengzhou were higher, Huhehaote, Jinan and Minhe was lower. A cluster analysis showed that genetic similarity coefficient around 0.60, the populations could be divided into two groups, one containing Jinan and Zhengzhou populations, and one containing the rest of the populations. Analysis of AMOVA showed that there were about 49% of the total variations among populations, 51% presented within populations. Mantel-test showed that genetic distances had no significant correlation with geographic distance and also with the gap of elevation among populations.2. Functional response of two coccinellid predators, Coccinella septempunctata and Propylaea japonica, to A. pisumPlotting prey density against prey killed by four larval instars, and adult males and females of C. septempunctata fit well with the type-II. The adult females had the highest attack rate and shortest handling time. The prey consumption differences of C. septempunctata in the same stage under various prey densities were significant. Based on the functional response data, the model predicted a maximum of 5.7, 11.5, 32.1, 59.5, 57.1, and 76.9 nymphs to be consumed per day by individual first, second, third, fourth instars, adult male, and adult female, respectively.Plotting prey density against prey killed by four larval instars, and adult males and females of P. japonica fit well with the typeⅡmodel of Holling’s disc equation. Adult females consumed the highest number of prey, followed by fourth instars and Adult males. Based on the functional response data, the model predicted a maximum of 5.5, 14.1, 29.2, 45.1, 42.3 and 51.5 nymphs to be consumed per day by and individual first, second, third, fourth instars, adult male and adult female, respectively.Rate of attack, handling time and the daily maximum amount were differences between C. septempunctata and P. japonica in the same instar. The daily maximum amount of C. septempunctata were higher than P. japonica, while the handling time of C. septempunctata were lower than P. japonica, when two ladybirds were in the same stages.3. Evaluation on resistance of 4 Medicago cultivars to A. pisumResistance of 4 alfalfa cultivars to A. pisum was evaluated by mass-infesting young seedlings in field and lab, in order to select high resistant material to pea aphid. The result showed that % of resistant plant of Gannong No.5(HA-3)was significantly higher than the others in field and lab, 50.3%,48.9%, respectively. However, % of resistant plant of Hunter River was low in field and lab, 4.0%, 4.3%, respectively. Resistant class of 4 alfalfa cultivars in field were Gannong No.5 (High Resistance, HR), Gannong No.3(Susceptible, S), Golden Empress (Susceptible, S), Hunter River (Susceptible, S), while resistant class of 4 alfalfa cultivars in lab were Gannong No.5 (Resistance, R), Gannong No.3(Low Resistance, LR), Golden Empress(Low Resistance, LR), Hunter River(Susceptible, S).4. Antibiosis of alfalfa cultivars to two color morphs of A. pisumBiology of two color morphs of A. pisum was studied on four cultivars of alfalfa (Medicago sativa L.) in order to identified antibiosis of alfalfa cultivars to two color morphs of pea aphids. The results showed that two color morphs of pea aphids were different in mean development time, net reproductive rate, generation time on JH, in intrinsic rate of increase, population doubling time, infinite rate of increase on G5. Using the innate capacity of increase (r_m) as the index of alfalfa antibiosis to the aphid, antibiosis of four alfalfa cultivars were different to two color morphs of pea aphid, resistance of JH to green aphid was higher and resistance of G5 to pink aphid was higher, while resistance of Hu was lower to two morphs of pea aphids. The level of alfalfa resistance to green aphid and to pink aphid were in order of Hu<G3<G5<JH, and Hu<JH<G3<G5, respectively.5. Physiological and biochemical resistance mechanism of M. sativa to A. pisumThe SOD and POD activities in the susceptible variety (Hu) appeared lower than that of the resistant variety (G5) and low resistant varieties (G3, JH) when their leaves were piercing-sucked by the aphid. The activities of PPO of resistant variety (G5) were higher than that of the susceptible variety (Hu) and low resistant varieties (G3, JH) before invaded by pea aphids, whereas the activities of PPO of resistant variety (G5) decreased rapidly, were lower than that of the susceptible variety (Hu) after damaged by pea aphids. In the period of the aphid attacks, the MDA contents of resistant variety (G5) were lower than that of the susceptible variety (Hu) and low resistant varieties (G3), the total phenolic content and CAT activity of resistant variety (G5) were higher than that of the susceptible variety (Hu) and low resistant varieties (G3, JH), the chlorophyll content of the susceptible variety (Hu)was higher than that of low resistant varieties (G3, JH), lower than that of resistant variety (G5), and that the soluble protein content of resistant variety (G5) and the susceptible variety (Hu) were lower than that of low resistant cultivars (G3, JH), except on the third day were higher than soluble protein content of G3, the Tannin contents of resistant variety (G5) and the susceptible variety (Hu) displayed lower than that of the low resistant ones (G3 and JH). Throughout the period of the aphid attacks, the activities of SOD and POD of all varieties rose firstly and then declined, while PPO of all varieties rose firstly and then declined. The chlorophyll and soluble protein contents and the activities of CAT displayed a decreasing trend in all of the varieties, while total phenolic content appeared increasing trend. The dynamics of MDA and Tannin contents of four alfalfa cultivars showed irregular. It indicated that the SOD, POD, CAT, MDA and total phenolic content were positive correlation with the resistance of M. sativa to pea aphid, while the chlorophyll, soluble protein contents, Tannin contents, PPO were not explicit correlation with that.更多还原