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由杨棒盘孢菌(Coryneum populinum Bres.)侵染引起的杨树灰斑病在我国东北、华北、西北和华东地区发生普遍,尤其以幼苗和幼树受害最重,可造成杨树产量损失约16%,易感病品种损失甚至高达60%[1]。该病菌主要侵染叶片,也可侵染嫩梢和枝干。在发病初期,叶片上出现水渍状斑点,逐渐扩大成褐色不规则形,后病斑中心呈灰白色,边缘呈灰褐色,上生黑绿色霉状物。严重发病时可使叶片提前脱落,苗木顶梢和嫩枝梢死亡变黑,给林业经济带来巨大损失[2]。
目前,化学药剂仍然是防治杨树灰斑病的最主要手段,具有效果显著、成本低廉等优点,是使用最为广泛的防治措施。研究发现,赛力散、速克灵、代森锰锌、多菌灵和甲基托布津等多种化学药剂对杨树灰斑病具有一定的防治效果[3-7]。但由于化学农药过量和不合理的使用,病菌的抗药性问题日益严重,对林业健康可持续的发展以及环境安全造成了严重的影响[8-9]。随着经济发展方式的转变,社会环保意识的不断加强,低毒、高效、安全、环保的农药已成为我国农林业发展的必然趋势[10]。本文在开展杨树灰斑病菌C. populinum菌丝生长和分生孢子萌发与温度关系研究的基础上,对5种供试杀菌剂对病菌的毒力进行了试验分析,以便为筛选出有效杀菌剂及后续开展毒理机制研究提供理论依据。
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在5~35 ℃条件下病菌均可生长。在不同温度条件下,病菌菌落直径差异明显,其中最适宜菌丝生长温度为20 ℃,第25 d时菌落直径可达4.25 cm,与其它温度下菌落直径差异显著(P<0.05)。低于或高于20 ℃,菌落直径逐渐减小。在不同温度条件下,分生孢子萌发率差异显著(P<0.05)。12 h时,20 ℃下的分生孢子萌发率最大,为79.00%;在5 ℃、10 ℃及15 ℃时,病菌分生孢子的萌发率分别为30.33%、60.67%及66.00%;当温度高于30 ℃时,分生孢子萌发率明显降低,30 ℃时为23.67%,35 ℃时仅为18.33%(图1)。
图 1 不同温度条件下Coryneum populinum菌落直径和分生孢子萌发率
Figure 1. Colony diameter and conidial germination rate of Coryneum populinum under different temperature conditions
在20 ℃时,病菌在PDA培养基上菌丝生长缓慢,56 d时菌落直径5.90 cm;菌丝体发达,表面呈绒毛状,灰黑色,未见产生分生孢子盘(图2A)。在产孢培养基上,5 d左右可观察到分生孢子;分生孢子梭形,浅褐色,多数2~3个隔,少数4~6个隔,顶端尖,末端钝,中间细胞较大,孢子稍弯曲;分生孢子萌发时,多从顶端或末端(基部)生出芽管(图2B)。30 ℃时,病菌分生孢子萌发率下降,部分分生孢子的细胞异常膨大,芽管从中间细胞萌发,芽管基部或顶端膨大(图2C)。
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试验结果表明,5种供试药剂对C. populinum均有一定的抑制作用。根据不同药剂与病菌生长的毒力回归方程(相关系数r>0.95),表明试验结果可信度较高。不同药剂对病菌菌丝体生长的抑制效果存在显著差异(P<0.05),同一种药剂在不同浓度时对菌丝体生长的抑制效果也存在差异。比较发现,恶醚唑对病菌的毒力最强,EC50值为0.004 8 μg·mL−1;戊唑醇次之,EC50值为0.024 μg·mL−1;多菌灵和咪鲜胺的EC50值分别为0.047、0.25 μg·mL−1;甲基托布津的毒力相对较弱,EC50值为0.49 μg·mL−1(表1)。
表 1 5种杀菌剂对Coryneum populinum菌丝体生长的抑制效果
Table 1. Inhibitory effects of five fungicides on mycelial growth of Coryneum populinum
药剂
Fungicide浓度
Concentration/
(μg·mL−1)处理菌落直径
Diameter of colony on
fungicide medium/cm对照菌落直径
Control colony
diameter/cm抑制率
Inhibitory rate/%毒力回归方程
Toxicity regression
equation相关系数r
Correlation
coefficientEC50值
EC50 value/(
μg·mL−1)多菌灵
Carbendazim0.08 0.68 ± 0.03 m 1.73 ± 0.06 ab 92.7 ± 2.44 a y=6.085 9x + 13.100 0 0.981 8 0.047 0.07 0.80 ± 0.00 kl 1.70 ± 0.00 abc 81.8 ± 0.00 bc 0.06 0.80 ± 0.00 kl 1.65 ± 0.00 abc 81.0 ± 0.00 cd 0.05 1.12 ± 0.08 d 1.73 ± 0.06 ab 54.5 ± 4.55 hi 0.04 1.35 ± 0.05 a 1.73 ± 0.06 ab 33.8 ± 2.31 l 戊唑醇
Tebuconazole0.2 0.68 ± 0.03 m 1.68 ± 0.03 abc 92.3 ± 2.76 a y=1.282 8x + 7.086 1 0.953 6 0.024 0.1 0.93 ± 0.06 ghi 1.72 ± 0.08 ab 70.3 ± 3.19 ef 0.05 0.98 ± 0.10 fgh 1.68 ± 0.03 abc 64.6 ± 9.26 fg 0.025 1.15 ± 0.05 cd 1.68 ± 0.03 abc 49.3 ± 3.52 ij 0.012 5 1.25 ± 0.05 b 1.70 ± 0.00 abc 40.9 ± 4.55 k 甲基托布津
Thiophanate-methyl0.9 0.72 ± 0.08 lm 1.60 ± 0.17 c 88.8 ± 6.23 ab y=4.556 2x + 6.421 6 0.981 3 0.49 0.8 0.82 ± 0.08 jk 1.75 ± 0.05 a 81.3 ± 6.00 bcd 0.7 0.80 ± 0.05 kl 1.63 ± 0.08 bc 80.6 ± 4.47 cd 0.6 1.00 ± 0.10 efg 1.72 ± 0.03 ab 64.2 ± 9.14 fg 0.5 1.15 ± 0.05 cd 1.73 ± 0.06 ab 51.5 ± 2.62 hi 咪鲜胺
Prochloraz2 0.80 ± 0.00 kl 1.68 ± 0.03 abc 81.5 ± 0.50 bcd y=0.827 4x + 5.495 7 0.956 0 0.25 1 1.00 ± 0.00 efg 1.68 ± 0.03 abc 63.1 ± 1.00 fg 0.5 1.07 ± 0.06 def 1.68 ± 0.03 abc 56.9 ± 5.97 ghi 0.25 1.13 ± 0.06 d 1.68 ± 0.03 abc 50.8 ± 4.75 hi 0.125 1.23 ± 0.03 bc 1.70 ± 0.00 abc 42.4 ± 2.62 jk 恶醚唑
Difenoconazole0.1 0.88 ± 0.03 ijk 1.68 ± 0.03 abc 73.9 ± 2.00 de y=0.564 5x + 6.310 7 0.988 0 0.004 8 0.05 0.90 ± 0.00 hij 1.70 ± 0.00 abc 72.7 ± 0.00 e 0.025 0.93 ± 0.06 ghi 1.70 ± 0.00 abc 69.7 ± 5.25 ef 0.01 1.08 ± 0.03 de 1.73 ± 0.06 ab 57.3 ± 2.44 gh 0.000 5 1.42 ± 0.03 a 1.73 ± 0.06 ab 27.9 ± 1.09 l 注:表中数据为平均值 ± 标准误,同一列中字母相同表示不同处理差异不显著( P> 0.05);字母不同表示差异显著(P< 0.05).下同
Notes:Notes: The data in the table are mean ± SE. The same letters in the same column indicate that there is no significant difference between different treatments ( P > 0.05). Different letters indicated significant difference ( P < 0.05).The same below. -
试验结果表明,5种供试药剂对病菌分生孢子萌发具有不同程度的抑制作用,同一种药剂对分生孢子萌发的抑制率随药剂浓度的增加而增大。根据不同药剂对分生孢子萌发抑制率的毒力回归方程(相关系数r>0.96),表明试验结果可信度较高。恶醚唑对病菌分生孢子萌发的抑制作用最强,EC50值最小,为85.07 μg·mL−1;咪鲜胺、甲基托布津及多菌灵的EC50值分别为101.23、123.28和254.80 μg·mL−1;戊唑醇对分生孢子萌发的抑制作用最差,EC50值为327.78 μg·mL−1(表2)。
表 2 5种杀菌剂对Coryneum populinum分生孢子萌发的抑制效果
Table 2. Inhibitory effect of five fungicides on conidial germination of Coryneum populinum
药剂
Fungicide浓度
Concentration/
(μg·mL−1)孢子萌发率
Spore germination
rate/%抑制率
Inhibitory
rate/%毒力回归方程
Toxicity regression
equation相关系数r
Correlation
coefficientEC50值
EC50 value/
(μg·mL−1)对照(无菌水)
Control (sterile water)— 79.00 ± 2.00 a — — — — 多菌灵
Carbendazim350 28.00 ± 1.00 p 64.6 ± 1.14 a y=2.741 8x−1.597 3 0.996 1 254.80 300 34.00 ± 3.00 n 57.0 ± 3.36 cd 250 40.33 ± 2.31 lm 49.0 ± 1.89 e 200 46.67 ± 1.53 jk 40.9 ± 0.55 gh 150 59.00 ± 1.00 cd 25.3 ± 0.63 mn 戊唑醇
Tebuconazole350 35.33 ± 1.15 n 55.3 ± 1.88 d y=2.002 6x-0.037 7 0.975 9 327.78 300 42.67 ± 0.58 l 46.0 ± 0.81 ef 250 49.67 ± 0.58 ghi 37.1 ± 1.02 hij 200 52.67 ± 1.15 f 33.3 ± 0.85 jk 150 58.33 ± 1.53 de 26.2 ± 0.37 mn 甲基托布津
Thiophanate-methyl120 39.67 ± 0.58 m 49.8 ± 1.93 e y=2.487 6x-0.201 3 0.990 0 123.28 100 48.00 ± 1.00 ij 39.2 ± 0.27 gh 80 52.33 ± 1.53 f 33.8 ± 0.42 ijk 70 58.67 ± 1.15 d 25.7 ± 3.25 mn 60 61.33 ± 1.53 bc 22.3 ± 3.89 no 咪鲜胺
Prochloraz120 30.33 ± 1.53 op 61.6 ± 0.99 ab y=3.721 4x-2.462 6 0.994 2 101.23 100 40.00 ± 1.00 m 49.4 ± 1.29 e 80 51.33 ± 1.15 fgh 32.2 ± 6.28 kl 70 56.00 ± 1.00 e 29.1 ± 2.69 lm 60 63.00 ± 1.00 b 20.2 ± 1.79 o 恶醚唑
Difenoconazole100 31.33 ± 1.15 o 60.3 ± 1.80 bc y=3.169 3x-1.116 0 0.967 2 85.07 90 35.67 ± 0.58 n 54.8 ± 1.80 d 80 45.33 ± 2.52 k 42.6 ± 1.74 fg 70 49.33 ± 1.53 hi 37.5 ± 1.50 hi 60 52.00 ± 1.73 fg 34.1 ± 2.79 ijk
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与对照相比,添加恶醚唑使C. populinum菌落生长缓慢,菌丝体生长明显受到抑制。随着恶醚唑浓度升高,C. populinum菌落生长直径逐渐减小,药剂对菌丝体生长的抑制率逐渐增大。如恶醚唑0.000 5 μg·mL−1,菌落直径为1.42 cm,抑制率27.9%;0.01 μg·mL−1时,菌落直径为1.08 cm,抑制率57.3%;0.1 μg·mL−1时,菌落直径为0.88 cm,抑制率73.9%(图3)。
图 3 不同浓度恶醚唑对Coryneum populinum菌落生长的影响(10 d)
Figure 3. Effects of different concentrations of Difenoconazole on colony growth of Coryneum populinum (10 d)
对照中C. populinum菌丝生长粗细均匀,表面光滑,分支正常,产孢正常,芽管从分生孢子顶端或者末端(基部)正常萌发。经恶醚唑作用后,C. populinum菌丝表面变得粗糙、分支处膨大,大多数分生孢子萌发异常,产生的芽管较短,尖端膨大(图4)。
杨树灰斑病菌(Coryneum populinum Bres.)对5种杀菌剂的敏感性
Sensitivity of Coryneum populinum Bres. to Five Fungicides
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摘要:
目的 在测定杨树灰斑病菌最适生长温度的基础上,开展病菌对5种杀菌剂的敏感性试验,为进一步筛选出可用于杨树灰斑病防治的有效药剂和明确杀菌剂对病菌的作用机制提供理论参考。 方法 采用菌丝生长速率法和分生孢子萌发法测定5种供试杀菌剂对C. populinum菌丝生长和分生孢子萌发的抑菌效果,构建毒力回归方程并计算EC50值;同时,利用光学显微镜观察杀菌剂对菌丝生长和分生孢子萌发的影响。 结果 C. populinum的最适生长温度为20 ℃,20 ℃条件下,第25 d菌落直径为4.25 cm,12 h分生孢子萌发率为79.00%;30 ℃条件下,病菌菌丝生长和分生孢子萌发均受到抑制。室内试验发现5种供试杀菌剂对C. populinum菌丝生长的抑制作用效果表现为恶醚唑>戊唑醇>多菌灵>咪鲜胺>甲基托布津,平均EC50值分别为0.0048、0.024、0.047、0.25和0.49 μg·mL−1;对分生孢子萌发率的抑制作用效果表现为恶醚唑>咪鲜胺>甲基托布津>多菌灵>戊唑醇,平均EC50值分别为85.07、101.23、123.28、254.80和327.78 μg·mL−1。恶醚唑作用后病菌菌丝表面粗糙、分支处膨大,不产孢;分生孢子形态异常,出现芽管分支、膨大或不伸长等畸形特征。 结论 恶醚唑对杨树灰斑病菌具有较高的抑菌活性,是田间防治杨树灰斑病的有效候选药剂。 Abstract:Objective On the basis of the optimum mycelial growth temperature investigation of Coryneum populinum Bres, the pathogen was measured for susceptibility to five fungicides. The approach provides a theoretical reference for disease control of poplar gray spot and fungicide acting mechanism against the pathogen. Method The mycelium growth rate method and conidia germination method were used to determine the antibacterial effects of five tested fungicides on C. populinum mycelium growth and conidia germination, and the toxicity regression equation was constructed and the EC50 value was calculated. The morphological changes of mycelia and conidial germination were observed after fungicide treatment. Result The optimum mycelial growing temperature of C. populinum was 20 ℃ with the colony diameter of 4.25cm after 25 days, and the conidial germination rate was 79.00% after 12h on PDA. The mycelial growth and conidial germination were both inhibited at above 30 ℃. The inhibitory effects of the five fungicides on mycelial growth of the pathogen showed the trend of Difenoconazole > Tebuconazole > Carbendazim > Prochloraz > Thiophanate-methyl, with the average EC50 values of 0.0048, 0.024, 0.047, 0.25 and 0.49μg·mL −1, respectively. The virulence of the five fungicides to the pathogen’s conidial germination showed the pattern of Difenoconazole > Prochloraz > Thiophanate-methyl > Carbendazim > Tebuconazole, with the average EC50 values of 85.07, 101.23, 123.28, 254.80 and 327.78μg·mL −1, respectively. The hyphal surface of the pathogen became rough with enlarged branches, and no spore was produced after application of Difenoconazole. The conidial germination became morphologically abnormal with branching and enlargement or no enlagement of germ tubes. Conclusion The fungicide difenoconazole has high virulence to C. populinum, and it shows a potentially effective candidate agent for management of popular grey spot. -
Key words:
- Coryneum populinum
- / temperature
- / fungicide
- / EC50
- / micro-morphology
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表 1 5种杀菌剂对Coryneum populinum菌丝体生长的抑制效果
Table 1. Inhibitory effects of five fungicides on mycelial growth of Coryneum populinum
药剂
Fungicide浓度
Concentration/
(μg·mL−1)处理菌落直径
Diameter of colony on
fungicide medium/cm对照菌落直径
Control colony
diameter/cm抑制率
Inhibitory rate/%毒力回归方程
Toxicity regression
equation相关系数r
Correlation
coefficientEC50值
EC50 value/(
μg·mL−1)多菌灵
Carbendazim0.08 0.68 ± 0.03 m 1.73 ± 0.06 ab 92.7 ± 2.44 a y=6.085 9x + 13.100 0 0.981 8 0.047 0.07 0.80 ± 0.00 kl 1.70 ± 0.00 abc 81.8 ± 0.00 bc 0.06 0.80 ± 0.00 kl 1.65 ± 0.00 abc 81.0 ± 0.00 cd 0.05 1.12 ± 0.08 d 1.73 ± 0.06 ab 54.5 ± 4.55 hi 0.04 1.35 ± 0.05 a 1.73 ± 0.06 ab 33.8 ± 2.31 l 戊唑醇
Tebuconazole0.2 0.68 ± 0.03 m 1.68 ± 0.03 abc 92.3 ± 2.76 a y=1.282 8x + 7.086 1 0.953 6 0.024 0.1 0.93 ± 0.06 ghi 1.72 ± 0.08 ab 70.3 ± 3.19 ef 0.05 0.98 ± 0.10 fgh 1.68 ± 0.03 abc 64.6 ± 9.26 fg 0.025 1.15 ± 0.05 cd 1.68 ± 0.03 abc 49.3 ± 3.52 ij 0.012 5 1.25 ± 0.05 b 1.70 ± 0.00 abc 40.9 ± 4.55 k 甲基托布津
Thiophanate-methyl0.9 0.72 ± 0.08 lm 1.60 ± 0.17 c 88.8 ± 6.23 ab y=4.556 2x + 6.421 6 0.981 3 0.49 0.8 0.82 ± 0.08 jk 1.75 ± 0.05 a 81.3 ± 6.00 bcd 0.7 0.80 ± 0.05 kl 1.63 ± 0.08 bc 80.6 ± 4.47 cd 0.6 1.00 ± 0.10 efg 1.72 ± 0.03 ab 64.2 ± 9.14 fg 0.5 1.15 ± 0.05 cd 1.73 ± 0.06 ab 51.5 ± 2.62 hi 咪鲜胺
Prochloraz2 0.80 ± 0.00 kl 1.68 ± 0.03 abc 81.5 ± 0.50 bcd y=0.827 4x + 5.495 7 0.956 0 0.25 1 1.00 ± 0.00 efg 1.68 ± 0.03 abc 63.1 ± 1.00 fg 0.5 1.07 ± 0.06 def 1.68 ± 0.03 abc 56.9 ± 5.97 ghi 0.25 1.13 ± 0.06 d 1.68 ± 0.03 abc 50.8 ± 4.75 hi 0.125 1.23 ± 0.03 bc 1.70 ± 0.00 abc 42.4 ± 2.62 jk 恶醚唑
Difenoconazole0.1 0.88 ± 0.03 ijk 1.68 ± 0.03 abc 73.9 ± 2.00 de y=0.564 5x + 6.310 7 0.988 0 0.004 8 0.05 0.90 ± 0.00 hij 1.70 ± 0.00 abc 72.7 ± 0.00 e 0.025 0.93 ± 0.06 ghi 1.70 ± 0.00 abc 69.7 ± 5.25 ef 0.01 1.08 ± 0.03 de 1.73 ± 0.06 ab 57.3 ± 2.44 gh 0.000 5 1.42 ± 0.03 a 1.73 ± 0.06 ab 27.9 ± 1.09 l 注:表中数据为平均值 ± 标准误,同一列中字母相同表示不同处理差异不显著( P> 0.05);字母不同表示差异显著(P< 0.05).下同
Notes:Notes: The data in the table are mean ± SE. The same letters in the same column indicate that there is no significant difference between different treatments ( P > 0.05). Different letters indicated significant difference ( P < 0.05).The same below.表 2 5种杀菌剂对Coryneum populinum分生孢子萌发的抑制效果
Table 2. Inhibitory effect of five fungicides on conidial germination of Coryneum populinum
药剂
Fungicide浓度
Concentration/
(μg·mL−1)孢子萌发率
Spore germination
rate/%抑制率
Inhibitory
rate/%毒力回归方程
Toxicity regression
equation相关系数r
Correlation
coefficientEC50值
EC50 value/
(μg·mL−1)对照(无菌水)
Control (sterile water)— 79.00 ± 2.00 a — — — — 多菌灵
Carbendazim350 28.00 ± 1.00 p 64.6 ± 1.14 a y=2.741 8x−1.597 3 0.996 1 254.80 300 34.00 ± 3.00 n 57.0 ± 3.36 cd 250 40.33 ± 2.31 lm 49.0 ± 1.89 e 200 46.67 ± 1.53 jk 40.9 ± 0.55 gh 150 59.00 ± 1.00 cd 25.3 ± 0.63 mn 戊唑醇
Tebuconazole350 35.33 ± 1.15 n 55.3 ± 1.88 d y=2.002 6x-0.037 7 0.975 9 327.78 300 42.67 ± 0.58 l 46.0 ± 0.81 ef 250 49.67 ± 0.58 ghi 37.1 ± 1.02 hij 200 52.67 ± 1.15 f 33.3 ± 0.85 jk 150 58.33 ± 1.53 de 26.2 ± 0.37 mn 甲基托布津
Thiophanate-methyl120 39.67 ± 0.58 m 49.8 ± 1.93 e y=2.487 6x-0.201 3 0.990 0 123.28 100 48.00 ± 1.00 ij 39.2 ± 0.27 gh 80 52.33 ± 1.53 f 33.8 ± 0.42 ijk 70 58.67 ± 1.15 d 25.7 ± 3.25 mn 60 61.33 ± 1.53 bc 22.3 ± 3.89 no 咪鲜胺
Prochloraz120 30.33 ± 1.53 op 61.6 ± 0.99 ab y=3.721 4x-2.462 6 0.994 2 101.23 100 40.00 ± 1.00 m 49.4 ± 1.29 e 80 51.33 ± 1.15 fgh 32.2 ± 6.28 kl 70 56.00 ± 1.00 e 29.1 ± 2.69 lm 60 63.00 ± 1.00 b 20.2 ± 1.79 o 恶醚唑
Difenoconazole100 31.33 ± 1.15 o 60.3 ± 1.80 bc y=3.169 3x-1.116 0 0.967 2 85.07 90 35.67 ± 0.58 n 54.8 ± 1.80 d 80 45.33 ± 2.52 k 42.6 ± 1.74 fg 70 49.33 ± 1.53 hi 37.5 ± 1.50 hi 60 52.00 ± 1.73 fg 34.1 ± 2.79 ijk
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