知母（Anemarrhena asphodeloides Bunge）是我国重要的传统药用植物，在其生长过程中，由炭疽菌属真菌引起的炭疽病是知母产区的一种主要病害，影响其正常生长发育，降低药材的品质，严重时导致整株死亡，造成较大的经济损失。因此，研究知母炭疽病病原菌的种类、生物学特性、药剂敏感性以及与知母的互作过程，对知母炭疽病的防治十分重要。本研究从河北、安徽、山西省的知母产区采集知母炭疽病样品，通过形态学结合多基因分子鉴定方法，确定了知母病原菌的种类，研究其生物学特性，并通过菌丝生长速率法测定了其对7种常见杀菌剂的药剂敏感性，为知母炭疽病的防治提供了理论基础；同时，结合转录组测序技术，研究了炭疽菌与知母互作过程中的基因差异表达情况，为深入研究知母炭疽菌的致病机制和知母的防御机制奠定了基础。

（1）知母炭疽病菌的鉴定。为明确知母炭疽病菌的种类，本文从河北、安徽、山西3省知母产区的炭疽病样品中，分离到13个炭疽菌菌株，经柯赫氏法则验证，BY-1、BY-2、AG-1、AG-2、LF-1、LF-2、YC-1、JZYS-1、JZYS-2、BZ-2共10个菌株是知母炭疽病的病原菌。经形态学和多基因分子生物学鉴定，BY-1为胶孢炭疽菌（Colletotrichum gloeosporioides），BY-2为麦冬炭疽菌（C. liriopes），AG-1、AG-2、LF-1、LF-2、YC-1、JZYS-1、JZYS-2和BZ-2为白蜡树炭疽菌（C. spaethianum）。致病力最强的是YC-1，其次是JZYS-2和AG-2，致病力最弱的是BZ-2。

（2）知母炭疽病菌生物学特性。对菌株YC-1、BY-1和BY-2的生物学特性进行研究，结果表明：YC-1最适pH为4，最适生长温度为25℃ ~ 30℃，在12 h光暗交替的光照条件下，菌落出现轮纹状，最适培养基为V8培养基，最适碳源为葡萄糖，最适氮源为蛋白胨，菌丝的致死温度在50℃ ~ 55℃之间；BY-1最适pH为4，最适生长温度为25℃，在12 h光暗交替的光照条件下，产生的橘色分生孢子堆小而密集，最适培养基为Czapek培养基，可利用多种碳源，在以蔗糖、葡萄糖、果糖、麦芽糖为碳源的培养基上菌落直径无显著性差异，最适氮源为蛋白胨，菌丝的致死温度在45℃ ~ 50℃之间；BY-2最适pH为6，最适生长温度为20℃ ~ 30℃，光照条件的改变对菌丝的生长速率和菌落形态没有显著影响，最适培养基为OA培养基，最适碳源为葡萄糖，最适氮源为蛋白胨，菌丝的致死温度在50℃ ~ 55℃之间。

（3）知母炭疽病菌药剂敏感性。通过菌丝生长速率法，分析多菌灵、戊唑醇、苯醚甲环唑、咪鲜胺、咯菌腈、吡唑醚菌酯、嘧菌酯，对YC-1、BY-1和BY-2的抑制效果。7种杀菌剂对YC-1的EC₅₀范围为0.018~1.805 mg/L，对BY-1的EC₅₀范围为0.016 ~ 55.684 mg/L，对BY-2的EC₅₀范围为0.019 ~ 3.746 mg/L，仅BY-1对嘧菌酯的EC₅₀较高，为55.684 mg/L。

（4）知母炭疽病菌与寄主的互作关系初探。对炭疽菌和知母叶片的互作过程进行转录组分析发现，精氨酸和脯氨酸代谢在3株炭疽菌的样品中均显著富集，说明精氨酸和脯氨酸代谢可能在知母炭疽菌的侵染过程中发挥重要作用。黑色素生成与甘油的产生在炭疽菌附着胞成熟过程中十分重要，在BY-1和BY-2的样品中分别富集到黑色素生成通路和甘油酯类代谢通路，表明在48 h时炭疽菌仍处于侵染前期。在3株炭疽菌侵染的知母叶片中黄酮、类黄酮代谢和次级代谢的生物合成通路均被富集，BY-1和YC-1侵染的叶片中还富集到类胡萝卜素代谢通路，表明受到炭疽菌侵染时，可能通过产生黄酮、类黄酮、类胡萝卜素等次级代谢产物来清除活性氧，以抵御炭疽菌带来的氧化伤害。YC-1和BY-1侵染的知母叶片中富集到植物激素信号通路，BY-2中富集到MAPK信号通路，说明知母在应答麦冬炭疽菌的侵染时，调控方式与应对其他白蜡树炭疽菌、胶孢炭疽菌可能有所不同。

Zhi mu (Anemarrhena asphodeloides Bunge) is an important traditional medicinal plant in China. Its growth is greatly threatened by the fungal disease anthracnose, which can be caused by a number of species of Colletotrichum in zhi mu producing areas. Zhi mu anthracnose affects the normal growth of Zhi mu development, reduce the quality of crops, and even lead to the death of the whole plant, causing serious economic losses. It is very important to study the species, biological characteristics, chemical susceptibility and the interaction process with zhi mu. In this study, samples of Zhi mu anthracnose leaves and stems were collected from Hebei, Anhui and Shanxi provinces, and the species of Zhi mu anthracnose were identified by multi-gene molecular identification combined with morphological methods, and their biological characteristics were studied. Its fungicides susceptibility to seven common fungicides was studied, which provided a theoretical basis for the control and further research of zhi mu anthracnose. At the same time, through transcriptome sequencing technology, the gene differences in the interaction between anthracnose and Zhi mu were studied. The expression situation has laid a foundation for the depth study of the pathogenic mechanism and the defense mechanism of zhi mu.

（1）Classifying the status of the pathogens of a total of Anemarrhena asphodeloides anthracnose. Anthracnose samples were obtained from Hebei, Anhui, Shanxi provinces and anthracnose strains were analyzed based on multi-locus phylogenetic analyses, micromorphological characteristics and the characteristics of colonies. BY-1, BY-2, AG-1, AG-2, LF-1, LF-2, YC-1, JZYS-1, JZYS-2 and BZ-2 were the pathogenic of zhi mu anthracnose. BY-1 is C. gloeosporioides, BY-2 is C. liriopes and AG-1, AG-2, LF-1, LF-2, YC-1, JZYS-1, JZYS-2 and BZ-2 is C. spaethianum. YC-1 was the most virulent, followed by JZYS-2 and AG-2. The weakest is the BZ-2.

（2）The biological characteristics of pathogens of Zhi mu. The biological characteristics of three pathogens of zhi mu anthracnose were studied, and the results showed that the optimum pH of YC-1 and BY-1 was 4, and the optimum pH of BY-2 was 6. The optimum growth temperature of YC-1 was 25℃ ~ 30℃, the optimum growth temperature of BY-1 was 25℃, and the colony diameter of BY-2 had no significant difference between 20℃ and 30℃. The change of light conditions had no significant effect on the growth rate of hyphae. Under the light condition of 12 h light-dark alternation, the colonies of YC-1 appeared rotundiform; the aerial hyphae of BY-1 became less than under 24 h dark condition, and resulted small and dense orange conidia; the colony morphology of BY-2 was not greatly affected by light conditions. The optimum medium for YC-1 is V8 medium, the optimum medium for BY-1 is Czapek medium, and the optimum medium for BY-2 is OA medium. In terms of carbon source utilization, YC-1 and BY-2 have the largest colony diameter on the medium with glucose as carbon source; BY-1 can utilize a variety of carbon sources, and there was no significant difference on the medium with sucrose, glucose, fructose and maltose. In terms of the utilization rate of nitrogen source, YC-1, BY-1 and BY-2 all had the largest colony diameter on the medium with peptone as nitrogen source. The lethal temperature of YC-1 and BY-2 mycelium was between 50℃ and 55℃, and the lethal temperature of BY-1 mycelium was between 45℃ and 50℃.

（3）The fungicides sensitivity of pathogens of Zhi mu. Analysis the effect of carbendazim, tebuconazole, difenoconazole, prochloraz, fludioxonil, pyraclostrobin, azoxystrobin to YC-1, BY-1 and BY-2 by mycelial growth rate method. The EC₅₀s of the 7 fungicides ranged from 0.018 to 1.805 mg/L for YC-1, 0.016 to 55.684 mg/L for BY-1, and 0.019 to 3.746 mg/L for BY-2. Only BY-1 had a higher EC₅₀ to azoxystrobin, which was 55.684 mg/L.

（4）The pathogenic mechanism of pathogens of Zhi mu. Transcriptome analysis of the interaction process between pathogens and Zhi mu leaves showed that arginine and proline metabolism were significantly enriched in the samples of the three strains, indicating that arginine and proline metabolism in Zhi mu anthracnose pathogens might play an important role in the infection process. Melanin production and glycerol production are very important in the maturation of appressors. The melanin production pathway and glyceride metabolism pathway were enriched in BY-1 and BY-2 samples, respectively, indicating that the pathogen was still in the pre-infection process of 48 h. The biosynthetic pathways of flavonoids, flavonoid metabolism and secondary metabolism were all enriched in the leaves infected by three pathogen strains, and carotenoid metabolism was also enriched in the leaves infected by BY-1 and YC-1, indicating that when the leaves were infected by pathogen, secondary metabolites were produced to scavenge reactive oxygen to resist the oxidative damage caused by pathogen. The phytohormone signaling pathway was enriched in the leaves infected by YC-1 and BY-1, and the MAPK signaling pathway was enriched in BY-2, indicating that zhi mu in response to the infection of C. liriopes, might be different from that of C. gloeosporioides and C. spaethianum.