1、 摘要 硕士学位论文 腐烂病菌 自苹果树剪锯口侵染发病条件、动态 与防治药剂研究 Disease Conditions， Dynamics and Fungicides Control of Valsa Pathogen Infection from Apple Pruning Wounds 专 业： 植物病理学 研 究 方 向： 植物病害流行学 摘要 摘 要 苹果树腐烂病 (Valsa mali)主要危害主干、主枝，破坏皮层， 造成死枝、死树，重者毁园，在中国是苹果树上的第一大病害，严重威胁苹果产业的发展 。剪锯口是苹果树腐烂病菌的重要侵染孔口。从剪锯口侵染的腐烂病菌可以长期潜伏在木质部内，
2、木质部导管内潜伏的腐烂病病菌是导致剪锯口发病和旧病斑复发的主要菌源。为了明确腐烂病菌的侵染、扩展以及防治，本论文对腐烂病菌从剪锯口侵染木质部的所需条件、扩展动态、以及影响腐烂病病斑扩展的条件进行了研究，为苹果树腐烂病流行规律和病害防控的进一步研究提供了理理论依据和参考。 2013 年自 3 月份开始，每隔一个月一次用标记腐烂病菌的分生孢子，定期接种当年春季新修剪形成的直径 1cm 左右的剪锯口，定期检查接种剪锯口的发病情况，定期取样 分离木质部内的菌丝。 结果表明，腐烂病菌能从剪锯口侵染木质部，且能在木质部内生长扩展。 4-11 月份，腐烂病菌在木质部内都能生长扩展，但不同季节生长扩展的速度不
3、同。 6 月份，腐烂病菌在木质部内的生长扩展速度最快，一个月内的 平均扩展距离 1.46cm； 4、 5 和 7 月份次之，每月的平均扩展距离为 0.5cm、 0.67cm 和 1.1cm； 7 月份之后菌丝的扩展明显变慢，每月的平均扩展距离不超过 0.3cm。次年 6 月份，菌丝在接种枝条木质部内扩展最大距离达21cm。 2013 年 3-11 月接种的剪锯口，于 2014 年 3 月份开始发病，在 3、 4 和 8月发病数量最多，发病剪锯口分别占总接种剪锯口的 10%、 8.13%和 6.25%，其他时间发病的剪锯口占 2.51%。 3、 4、 5、 8 月份接种的剪锯口发病率较高，分别为
4、 40%、 45%、 45%和 30%，其他时间接种的剪锯口发病率较低。 在所测试 5-35C 下， 腐烂病菌的分生孢子都能从剪锯口侵染，在 5C、 10C、15C、 20C、 25C 和 30C 六 个温度下接种的技条上， 距接种点 3cm 处的枝段上，能分离出腐烂病菌，其中 20C 下病菌的分离率最高，分离率为 29.4%，与15C、 25C 和 30C 接种的枝条没 有显著差异，但与 5C 和 10C 下接种枝条上病菌的分离率存在显著差异。 35C 下接种枝条上， 3cm 处没有分离到接种的腐烂病菌，但 1cm处能分离到接种病菌，说明 35C 下腐烂病菌的分生孢子也能从剪锯口侵入，但病菌
5、的侵染率低或侵入后生长缓慢。 露时对腐烂病菌分生孢子从苹果枝条剪口侵染没有显著影响。接种后未经保湿 (0 小时 )处理枝条上，在距接种点 3cm 处木质部内病菌的分离率为 9.92%，与其他 6 个露时处理枝条上病菌的分离没有显著差异。接种后保湿 24 小时的处理摘要 枝条上，距接种点 3cm枝段上病菌的分离率最高，为 17.86%，与其他 6 个露时处理枝条上的分离率也没有显著差异。 温度对从剪锯口侵入木质部腐烂病菌的生长扩展速度影响较大。 在 25C 下培养 7 天后，距接种点 5cm 处枝段上都能分离到接种病菌； 35C 下培养的枝条上，在 3.5cm处能分离到病菌；其他温度下培养的枝条
6、上，在 4.5cm处都能分离到接种病菌 。 春季形成的伤口感病期较长。龄期为 1d、 3d 和 7d 的伤口对腐烂病菌非常敏感，接种菌饼后发病率为 100%； 15d、 30d 和 45d 的伤口接种后发病率依次降低，分别为 57%、 17%和 3%；龄期 60d 的伤口再接种腐烂病菌的菌饼后不再发 病。龄期 1d 的伤口接种病菌后，形成的病斑扩展面积最大，为 10.33cm2；随伤口龄期的增加，接种后形成伤口的面积逐渐减小。 夏季形成的伤口感病期较短。龄期为 1d 和 3d 的伤口接种腐烂病菌后的发病率为 100%， 15d 后的伤口接种菌饼后不再发病。 通过接种 2周后再施药的方法测试了
7、5种杀菌剂对侵入枝条腐烂病菌的铲除效果。腐烂病菌侵染 2 周后，喷施吡唑醚菌酯和波尔多液对侵染病菌的铲除效果较好，分别达 94.44%和 88.89%；代森锰锌、甲基硫菌灵和苯醚甲环唑 3 种铲除效果稍差，铲除效果分别为 44.44%、 44.44%、 16.67%。 通过在剪锯口上先施药后接种的方法测试了 8 种杀菌剂对剪锯口的保护效果和持效期。结果表明， 施药后第 2d， 8 种测试药剂的保护效果稍差，从剪口接种枝条内的病菌分离率较高；从施药后第 5d 开始，其中 7 种药剂的保护效果明显提高，病菌的分离率明显降低；施药 20d 后，甲基硫菌灵、吡唑醚菌酯、波尔多液、戊唑醇的保护效果仍在
8、90%以上。 通过先在剪锯口接种，然后施药的方法，测试了 3 种杀菌剂对侵染病菌的内吸治疗效果。结果表明， 3 种效果都达不到理想效果，其中甲基硫菌灵的治疗效果最好，病菌侵染 20d 后施药，治疗效果仅达 65%。 关键词 : 腐烂病；剪锯口侵染；发病动态；侵染条件；药剂防治 Abstract Abstract Valsa canker (Valsa mali) is major destructive branch diseases of apple trees in China, mainly hazards the trunks and boughs, destructing corti
9、ces, and then brings about the death of twigs, trees or the entire orchard afterwards. So seriously limits the development of fruit industry. The pruning wounds are the most susceptible sites and also the most important by Valsa mali. The mycelium of Valsa mali infected through pruning wounds can pa
10、rasitize in the ducks of xylem for long time. The outbreak from pruning wounds and recurrence of old scars are caused by the mycelium of the pathogen expansion in the xylem. In order to clear infection, extension, prevention and control about the causal fungus, we study the pathogen infection, exten
11、sion dynamics, chemical control and the conditions of scar extension, which can provide theoretical basis and reference for regularity of epidemic and prevention. The pruning wounds were inoculated with conidia of Valsa mali at every month from March 2013.The diameter of pruning wounds were 1cm. The
12、 results indicated that the pathogen could infect xylem through pruning wounds and extend in xylem. The mycelium of Valsa mali could constantly extend, but the speed of expansion had difference at different months from April to November. In June the fastest growth the mycelium was 1.46cm. The myceli
13、um grew rapidly in xylem in April, May, and July; The extended distance were 0.5cm, 0.67cm and1.1cm respectively. The speed of expansion was slower after July, which was less than 0.3cm. The mycelium could extend to 21cm in xylem at next June. Pruning wounds were inoculated monthly from March to Nov
14、ember 2013 and diseased at March 2014.The number of diseased pruning wounds was maximum, inoculated at March、 April and August. The percent of diseased pruning wounds were 10%, 8.13% and 6.25% and the others was 2.51%. The percent of diseased pruning wounds inoculated at March, April, May and August
15、 was higher, 40%, 45%, 45% and 30%.The percent of diseased pruning wounds was lower. Valsa conidium could infected from pruning wounds at 5-35C. The inoculated pathogen are isolated at twig segments 3cm away from the inoculated end at 5C,10C,15C,20C,25C and 30C. The highest rate of isolated pathogen
16、 was 29.4% at 20C. Isolation rate had no difference between 20C and 15C, 25C and 30C,but had significant difference with 5C and 10C. At 35C, inoculated pathogen cannot be isolated at 3cm of twigs segments, but can be isolated at Abstract 1cm.This showed that Valsa conidium could infected form prunin
17、g wounds, but the infected rate or the speed of growth were low. Wetness have less significant effect on infection pruning wounds by Valsa conidium. The Isolation rate of pathogen at twig segments 3cm away from the inoculated end was 9.92%, without wetness. Isolation rate had no difference with othe
18、r six wet treatments. The highest isolation rate of pathogen was 17.86% at twig segments 3cm away from the inoculated end after inoculated kept wetness for 24h and had no difference with other six wet treatments. Temperature had significant effect on the speed of pathogenic infected xylem form pruni
19、ng wounds by Valsa conidium. The inoculated pathogenic could be isolated at twig segments 5cm away from the inoculated end after cultivated for seven days at 25C; At 35C, inoculated pathogen could be isolated at 3.5cm of twigs segments; At others temperature treatments inoculated pathogen could be i
20、solated at 4.5cm. The period susceptible wounds were longer formed at spring. Wounds were infected more easily by the pathogen and the rate of diseased was 100%, when the age of wounds were 1d, 3d and 7d; The rate of diseased wounds was low growly, 57%, 17% and 3%; The wounds could not disease at 60
21、d. The average expanding area of disease spots was largest at 1d after the wounds formed and the largest expanding area was 10.33cm2.With the age of wounds increasing, expanding area of disease spots decreased growly. The period susceptible wounds were shorter formed at summer .The rate of diseased
22、wounds was 100% when the age of wounds only were 1d and 3d, Inoculated wounds could not disease until 15days after the wounds formed. Five kinds of fungicides were tested for eradication of pathogen in branches by sprayed fungicides after two weeks. Eradication of Pyraclostrobin and Bordeaux mixture
23、 were better when pathogen infected after two weeks and eradication were 94.44% and 88.89% respectively. Eradication ofMancozeb, Thiophanate-methyl and Difenoconazole were not good, and were 44.44%, 44.44% and 16.67% respectively. Protective effects and persistent period of 8 kinds of fungicides wer
24、e tested by inoculated on the pruning wounds after sprayed fungicides. The test showed that protective effects of 8 kinds of fungicides were not good on the second days after sprayed fungicides and pathogen isolation rate were higher; Protective effects of 7 kinds of fungicides were improved and pat
25、hogen isolation rate Abstract were lower from the fifth days after sprayed fungicides; Protective effects of Thiophanate-methyl, Pyraclostrobin ,Bordeaux mixture and Tebuconazole were remain above 90%. Protective effect of 3kinds of fungicides were tested by sprayed fungicides after inoculated on th
26、e pruning wounds。 The test showed that 3 kinds of fungicides also had not ideal effect, but protective effect of Thiophanate-methyl was the best.Protective effect of Thiophanate-methyl was only 65% when was sprayed on the twentieth day after infected. Key words: Valsa mali; pruning infection; diseas
27、e dynamics; infection conditions; fungicides control. 目录 目录 第一章 文献综述 . 1 1.1 苹果树腐烂病的发生和危害症状 . 1 1.2 苹果树腐烂病的病原学及致病力研究 . 2 1.2.1 病原菌的来源和多样性研究 . 2 1.2.2 病原菌的致病力机制的研究 . 3 1.3 病原菌的侵染 . 4 1.3.1 侵染循环 . 4 1.3.2 潜伏侵染 . 4 1.4 影响苹果腐烂病大流行的因素 . 5 1.5 病害的防治 . 6 1.6 本研究的目的和意义 . 8 第二章 腐烂病菌从剪口侵染的时期、所需条件及在木质部内生长扩展研究
28、. 9 2.1 材料与方法 . 9 2.1.1 病原菌及孢子悬浮液的制备 . 9 2.1.2 腐烂病菌从剪口木质部侵染后的扩展动态 . 9 2.1.3 剪口龄期对腐烂病菌从剪锯口侵染的影响 . 10 2.1.4 露温、露时对腐烂病菌分生孢子自剪锯口侵染的影响 . 10 2.1.5 温度对腐烂病菌在木质部内生长扩展的影响 . 11 2.2 结果与分析 .11 2.2.1 剪口木质部内腐烂病菌的扩展动态 . 11 2.2.2 剪口龄期对木质部接种腐烂病菌分生孢子后生长扩展的影响 . 12 2.2.3 露温、露时对腐烂病菌分生孢子自剪锯口侵染的影响 . 14 2.2.4 温度对腐烂病菌在木质部内扩展
29、的影响 . 16 2.3 结论与讨论 . 17 第三章 影响腐烂病病斑扩展和发病的研究 . 20 3.1 材料和方法 . 20 3.1.1 供试菌株及孢子悬浮液的制备 . 20 3.1.2 皮层伤口龄期对腐烂病病斑扩展的影响 . 20 目录 3.1.3 枝条龄期对腐烂病病斑扩展的影响 . 20 3.1.4 低温冻害、干旱失水影响腐烂病菌发病的研究 . 20 3.2 结果与分析 . 21 3.2.1 春夏季节伤口龄期对腐烂病病斑扩展的影响 . 21 3.2.2 枝条龄期对腐烂病病斑扩展的影响 . 23 3.2.3 腐烂病 菌潜伏部位以及低温冻害、干旱失水影响腐烂病菌发病的研究 . 24 3.3
30、结论与讨论 . 25 第四章 不同杀菌剂对苹果树腐烂病的防治效果 . 28 4.1 材料与方法 . 28 4.1.1 供试病原菌 . 28 4.1.2 供试杀菌剂 . 28 4.1.3 对枝条上潜伏病菌的铲除效果 . 28 4.1.4 杀菌剂对从剪锯口侵染病菌的铲除效果 . 29 4.1.5 保护性杀菌剂持效期 . 30 4.2 结果与分析 . 30 4.2.1 对枝条上潜伏病菌的铲除效果 . 30 4.2.2 杀菌剂对从剪锯口侵染病菌的铲除效果 . 31 4.2.3 保 护性杀菌剂持效期 . 32 4.3 结论与讨论 . 35 全文小结 . 37 参考文献 . 40 Contents Con
31、tents Chapter 1 Literature Review 1 1.1The damage symptom of apple Valsa canker . 1 1.2 Studies on etiology and pathogenicity of Valsa mali. . 2 1.2.1 Studies on pathogen srouce and diversity .2 1.2.2 Studies on pathogenicity of pathogen. 3 1.3 Infection of pathogen. .4 1.3.1Infection cycle . . 4 1.
32、3.2Latent infection. . .4 1.4 Influence factors on epidemic of Valsa mali . .5 1.5 Control of apple Valsa canker. .6 1.6The purpose and significance of this study. 8 Chapter 2 Studies on infection time and conditions of Valsa mali from pruning wounds and expansion in xylem . .9 2.1Materials and meth
33、ods. 9 2.1.1Pathogen and spores. .9 2.1.2 Extension dynamics of Valsa mali in xylem after infected from pruning wounds .9 2.1.3 Effect of pruning wounds age on infection of Valsa mali from pruning wounds .10 2.1.4 Effect of temperature and RH on infection of Valsa conidial from pruning wounds. .10 2
34、.1.5 Effect of temperature on extension of pathogen in xylem .11 2.2 Results and analysis .11 2.2.1 Effect of pruning wounds age on infection of Valsa mali from pruning wounds .11 2.2.2 The effect of pruning wounds age on xylem after inoculated with Valsa conidium . .12 2.2.3 Effect of temperature and RH on infection of Va
