【作者】 刘珂；

【导师】 杨晓红；

【作者基本信息】 西南大学 ， 微生物学， 2021， 硕士

【摘要】 白及(Bletilla striata(Thunb.ex A.Murray)Rchb.f.),是常见的草本药用植物,一般入药部分为白及的假鳞茎。重庆缙云山适宜白及等中药材的的种植,但随着春夏雨水的增多、以及白及种植年限增长,缙云山白及的各类病害发生频率增加,抑制了当地的白及生长,严重危及到了白及的产量和品质,不利于白及产业的可持续发展。本研究的目的是确认发生在重庆缙云山的白及叶斑病的病原菌,并初步了解该病原菌的基本生物学特性、病原菌对白及叶片的侵入过程、白及叶片在病原菌侵入后的抗性响应以及进行室内毒力试验和盆栽药效试验,为重庆缙云山白及叶斑病的有效防控提供理论依据。本论文主要研究结果如下:从重庆缙云山白及种植基地采集新鲜白及病叶,从病叶取样进行病菌分离纯化,得到颜色形态有明显差异的2类菌株(F1和F2)。柯赫法则验证结果显示,F1是白及叶斑病的致病菌。观察F1分生孢子的大小和形状,初步判断该病原菌属于镰刀属。随后结合病原菌基因序列分析,鉴定F1病菌为尖孢镰刀菌(Fusarium oxysporum),该病菌是重庆缙云山的白及叶斑病的主要病原菌。对上述白及叶斑病病原菌——尖孢镰刀菌进行生物学特性研究。结果显示,尖孢镰刀菌利用效果最好和最差的碳源分别是麦芽糖和木糖;病原菌利用效果最好和最差的氮源分别是蛋白胨和赖氨酸。在实验室条件下,病原菌离体培养时最适的生长温度范围和p H范围分别25℃-30℃和在p H=7时。在供试的8种不同营养成分的培养基中,SA和Gzapek提供的营养成分最适合该病原菌吸收利用。此病原菌菌丝的致死温度是65℃。在离体的白及叶片上,接种直径为8 mm的尖孢镰刀菌的菌丝块,28℃恒温、用湿润的无菌滤纸保湿白及叶片,取样并固定不同接种时间的白及叶片病斑,观察尖孢镰刀菌侵染白及叶片的动态过程。研究结果显示:接种4h后,病原菌的分生孢子开始萌发,可观察到芽管。接种6h后,观察到特殊结构——附着胞的形成。接种10h后,萌发后的孢子已经出现了分枝菌丝。接种18h后,病原菌的菌丝开始侵入白及的表皮细胞。尖孢镰刀菌入侵白及叶片细胞的方式有两种,直接入侵和从气孔入侵。入侵白及叶片细胞时会形成二叉状分枝和附着胞等特殊结构。为了探讨病原菌侵染过程中白及植株的抗性响应,本文测定了不同的接种时间白及叶片可溶性蛋白、可溶性糖、MDA的含量以及三种抗氧化酶(POD、SOD、CAT)的活性。结果表明:刚遭受病原菌侵染时,白及叶片中的可溶性蛋白和可溶性糖的含量,以及三种抗氧化酶的活性都出现了短暂性升高。在没有防治措施和侵染时间延长的情况下,白及叶片出现发病症状,严重发病时,可溶性糖和可溶性蛋白的含量开始下降,三种抗氧化酶的活性也逐渐降低。而MDA含量则是从病原菌刚侵染白及叶片开始就是一直升高的趋势。为精准、有效地对白及叶斑病防控提供科学技术支撑,试验选择了六种化学杀菌剂对白及叶斑病病原菌开展室内毒力试验。结果表明,病菌菌落生长都受到了六种化学杀菌剂明显抑制作用。抑菌效果最好的是250g/L丙环唑,其次是抑制效果比较好的430g/L戊唑醇和45%唑醚甲硫灵。75%百菌清和30%噁霉灵的抑制效果稍差,代森锰锌的效果最差。在盆栽活体毒力试验中,于白及叶片上接种尖孢镰刀菌36h后喷药,也是丙环唑和戊唑醇对白及叶斑病的防治效果最好。更多还原

【Abstract】 Bletilla striata(Thunb.ex A.Murray)Rchb.f.is a perennial herbaceous medicinal plant.The medicinal part is the pseudobulb of Bletilla striata in soil.The Jinyun Mountain Area of Chongqing is suitable for the cultivation of Bletilla striata and other traditional Chinese medicine.However,with the extendation of planting years,the expansion of planting scale,and the increase of spring-summer rainfall,the occurrence frequency of various diseases increases.The trend is increasing year by year.These diseases inhibit the growth of B.striata,lead to the decline of yield and quality,and are not conducive to the sustainable development of Bletilla striata industry.The purpose of this study is to identify the pathogen on leaf spots of B.striata in Jinyun Mountain Area of Chongqing,to understand the biological characteristics of the pathogen and the invasion process of the pathogen to B.striata leaves,the resistance response of B.striata leaves triggered by the invasion of the pathogen.Finnally,indoor toxicity test and pot efficacy test were carried out so as to further provide theoretical basis for the accurate control the disease of.striata leaf spot.The main results are as follows.Fresh disease leaves of B.striata were collected from the planting base of Chongqing Jinyun Mountain.Two strains(F1 and F2)were isolated and purified by plate marking method.The results of Koch’s rule test clarified that F1 was the pathogen on leaf spot of B.striata.Through morphological observation of the shape and size of large conidia and small conidia,it was preliminarily concluded that the pathogen belonged to the Fusarium.Combined with gene sequence analysis,the pathogen on leaf spot of Bletilla striata in Jinyun Mountain of Chongqing was identified as Fusarium oxysporum.The biological characteristics of the pathogen on leaf spot of B.striata were determined.Among the carbon sources in tests,the pathogen made use of the best utilization of maltose and the worst utilization of xylose.Among the nitrogen sources in tests,the pathogen made use of the best utilization of peptone from organic nitrogen sources,and the worst utilization of lysine and ammonium sulfate from inorganic nitrogen sources.The pathogen can grow in the temperature range of 10 ℃～40 ℃,and the optimal growth temperature is between 25 ℃ and 30 ℃.The pathogen can grow in the range of p H4～9,and the colony growth diameter is the largest at p H7.SA and Gzapek medium were the most suitable for mycelial growth.The lethal temperature of the mycelium was65℃.The leaves of B.striata were inoculated in vitro with mycelia.The samples of different inoculation time were collected for tissue transparency,staining and observation to clarify the infection process of pathogenic fungi on plants.The results showed that the conidia began to germinate and form germ tubes for 4 hours later after inoculation.Appressorium was formed for 6 hours after inoculation.After 10 hours of inoculation,the spores produced branching hyphae.After 18 hours of inoculation,the hyphae of the pathogen began to invade the epidermal cells of B.striata leaves.There were two ways for F.oxysporum to invade Bletilla striata leaves.They were direct invasion and stomatal invasion.The bifurcate branches and appressorium were special structures when F.oxysporum invaded leaves B.striata.In order to study the resistance response of B.striata to pathogen infection,the contents of soluble sugar,soluble protein,MDA and the activities of three antioxidant enzymes were detected at different inoculation time.The results showed that the physiological indexes,such as the contents of soluble sugar and soluble protein,and the activities of POD,CAT and SOD,were increased transiently at the beginning of infection.As the infection continued,the leaf pot symptoms of B.striata appeared.The physiological indexes,such as the contents of soluble sugar and soluble protein,the activities of POD,CAT and SOD,began to decrease.But the content of MDA has been increased.In order to provided scientific and technical support to control the leaf spot of Bletilla striata accurately and effectively,six chemical fungicides were selected for the indoor virulence test on the pathogen in the leaf spots of Bletilla striata.The results of PDA plate bacteriostasis test showed that the six chemicals had certain inhibitory effect on the mycelial growth of the pathogen.The inhibition effectsof Propiconazole and Tebuconazole were the best.The results of vivo toxicity test of potted plants showed that the control effects of Propiconazole and Tebuconazole were also the best.更多还原