Identification of the pathogen causing Trichosanthes kirilowii fruit rot and screening of fungicides
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摘要:目的
明确引起栝楼果实腐烂病的病原菌种类并筛选其有效防治药剂。
方法2019—2020年采集安徽省大别山区的栝楼果实腐烂病样品，采用组织分离法获得35株分离物，利用柯赫氏法则验证其致病性，依据菌株形态学特征和多基因序列分析确定病原菌种类；采用菌丝生长速率法测定7种杀菌剂的室内毒力。
结果共分离纯化得到4种不同菌落形态特征的菌株，经柯赫氏法则验证均为栝楼果实腐烂病病原菌，经形态学观察和分子系统发育分析，确定引起栝楼果实腐烂病的病原菌分别为藤仓镰孢菌Fusarium fujikuroi、层出镰孢菌F. proliferatum、果生刺盘孢Colletotrichum fructicola和辽宁刺盘孢C. liaoningense，分离频率依次为31.4%、8.6%、20.0%和40.0%。室内毒力测定结果表明，咪鲜胺、咯菌腈、苯醚甲环唑、氰烯菌酯和百菌清对F. fujikuroi和F. proliferatum的抑制效果较好，EC50为0.1046 ~ 5.1781 μg/mL；咪鲜胺、苯醚甲环唑和咯菌腈对C. fructicola和C. liaoningense的抑制效果较好，EC50为0.0097 ~ 2.1325 μg/mL。
结论栝楼果实腐烂病主要由镰孢菌属和刺盘孢属真菌侵染引起，咪鲜胺、苯醚甲环唑和咯菌腈对引起该病害的病原菌具有较好的抑制效果。
Abstract:ObjectiveTo clarify the pathogen causing Trichosanthes kirilowii fruit rot and screen effective fungicides for controlling the disease.
MethodSamples of rot fruit from T. kirilowii plants were collected from Dabie Mountain Areas, Anhui Province in 2019—2020. A total of 35 representative isolates were obtained by tissue isolation. The pathogenicity was verified according to the Koch’s postulate, and the causal agents were identified based on the morphological characteristics and multilocus sequence analysis. The indoor toxicity of seven fungicides against the pathogen was detected by the mycelium growth rate method.
ResultColonies with four different morphological characteristics were isolated and purified. The isolates were confirmed to be pathogenic to the fruit of T. kirilowii according to the Koch’s postulate. They were identified as Fusarium fujikuroi, F. proliferatum, Colletotrichum fructicola and C. liaoningense by combining morphological characteristics with phylogenetic analysis, with isolation frequencies of 31.4%, 8.6%, 20.0% and 40.0%, respectively. In addition, the indoor toxicity test results indicated that prochloraz, fludioxonil, difenoconazole, phenamacril and chlorothalonil had better inhibitory effect against F. fujikuroi andF. proliferatum, with EC50 ranging from 0.1046 to 5.1781 μg/mL. Prochloraz, fludioxonil and difenoconazole had better inhibitory effect againstC. fructiicola and C. liaoningense with EC50 ranging from 0.0097 to 2.1325 μg/mL.
ConclusionThe causal agents associated with fruit rot disease on T. kirilowii are Fusarium spp. and Colletotrichum spp.. Prochloraz, fludioxonil and difenoconazole all show good inhibitory activity against the pathogens.
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Keywords:
- Trichosanthes kirilowii /
- Fruit rot disease /
- Fusarium /
- Colletotrichum /
- Pathogenicity /
- Fungicide screening
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图 1 栝楼果实腐烂病田间发病症状
Figure 1. Sympthoms of fruit rot on Trichosanthes kirilowii in the field
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图 3 4种不同类型菌株对栝楼果实的致病性
A~D分别接种菌株HP017、QSC2、HP002、SSA9；E：对照
Figure 3. Pathogenicity of four different isolates on the fruit of Trichosanthes kirilowii
A−D: Inoculated with HP017, QSC2, HP002, SSA9, respectively; E: Control
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图 4 基于TEF-1α基因序列对镰孢菌属菌株构建的系统发育树
分支位置中的数字表示自举值；标尺表示每个核苷酸位点上的0.02替换值
Figure 4. Phylogenetic tree of the strains of Fusarium spp. based on TEF-1α gene sequences
The numbers in each branch points denote the percentages supported by bootstrap; The scale bar represents 0.02 substitutions per nucleotide position
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图 5 基于4个位点序列对辽宁刺盘孢及其近缘种构建的系统发育树
“*”为模式菌株；节点上的数字为50%以上的自举值
Figure 5. Phylogenetic tree of the strains of Colletotrichum liaoningense and related species based on the concatenated sequences of four loci
“*” represent type strain; Bootstrap support values above 50% are shown at the nodes
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图 6 基于6个位点序列对果生刺盘孢及其近缘种构建的系统发育树
“*”为模式菌株；节点上的数字为50%以上的自举值
Figure 6. Phylogenetic tree of the strains of Colletotrichum fructicola and related species based on the concatenated sequences of six loci
“*” represents type strain; Bootstrap support values above 50% are shown at the nodes
表 1 菌株信息及目的基因序列
Table 1 Strain information and target gene sequences
物种 Species 菌株 Strain GenBank登录号 GenBank accession number ITS ACT CAL CHS-1 GAPDH TUB2 TEF-1α Fusarium fujikuroi HP016 MW357640 HP017 MW357641 Fusarium proliferatum QSC2 MW357642 Colletotrichum fructicola DMC2 MW349792 MW357613 MW357616 MW357619 MW357622 MW357625 HP009 MW349793 MW357614 MW357617 MW357620 MW357623 MW357626 HP002 MW349794 MW357615 MW357618 MW357621 MW357624 MW357627 Colletotrichum liaoningense DMC1 MW349983 MW357628 MW357632 MW357636 QSA6 MW349984 MW357629 MW357633 MW357637 SSA9 MW349985 MW357630 MW357634 MW357638 HKC10 MW349986 MW357631 MW357635 MW357639 
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表 2 7种杀菌剂对镰孢病菌和炭疽病菌的室内毒力测定
Table 2 Determination of the virulence of seven fungicides to Fusarium spp. and Collrtotrichum spp.
菌株 Strain 药剂 Fungicide w(有效成分)/% Active ingredient content 毒力回归方程 Virulence regression equation 相关系数 Correlation coefficient EC50/ (μg·mL−1) F. fujikuroi HP017 春雷霉素 Kasugamycin 70 y=3.5923+0.8234x 0.9518 51.2516 咪鲜胺 Prochloraz 98 y=6.0341+1.2900x 0.9985 0.1579 咯菌腈 Fludioxonil 95 y=5.3824+0.5183x 0.8821 0.1829 嘧菌酯 Azoxystrobin 95 y=4.1359+0.4202x 0.9695 113.8843 苯醚甲环唑 difenoconazole 95 y=5.2198+0.6339x 0.9788 0.4500 氰烯菌酯 Phenamacril 95 y=5.4272+0.8213x 0.9223 0.3019 百菌清 Chlorothalonil 99 y=4.4958+0.9701x 0.9701 5.1781 F. proliferatum QSC2 春雷霉素 Kasugamycin 70 y=3.2031+0.9861x 0.8280 66.3944 咪鲜胺 Prochloraz 98 y=5.7667+0.8081x 0.9814 0.1125 咯菌腈 Fludioxonil 95 y=5.1877+0.4394x 0.9718 0.3740 嘧菌酯 Azoxystrobin 95 y=4.0472+0.4645x 0.9547 112.4802 苯醚甲环唑 Difenoconazole 95 y=5.4574+0.4666x 0.9914 0.1046 氰烯菌酯 Phenamacril 95 y=5.2917+0.8950x 0.8174 0.4721 百菌清 Chlorothalonil 99 y=4.7491+0.4570x 0.9933 3.5409 C. fructicola HP002 春雷霉素 Kasugamycin 70 y=3.6179+0.9751x 0.9507 26.1436 咪鲜胺 Prochloraz 98 y=6.1584+1.2283x 0.9815 0.1140 咯菌腈 Fludioxonil 95 y=4.8670+0.4045x 0.9151 2.1325 嘧菌酯 Azoxystrobin 95 苯醚甲环唑 Difenoconazole 95 y=5.4561+0.9250x 0.9937 0.3213 氰烯菌酯 Phenamacril 95 y=1.3563+2.9354x 0.9472 17.4313 百菌清 Chlorothalonil 99 y=3.5858+0.7712x 0.9917 68.2058 C. liaoningense SSA9 春雷霉素 Kasugamycin 70 y=3.1409+1.4118x 0.9916 20.7440 咪鲜胺 Prochloraz 98 y=6.2287+1.1271x 0.9966 0.0813 咯菌腈 Fludioxonil 95 y=5.4883+0.2428x 0.8546 0.0097 嘧菌酯 Azoxystrobin 95 y=5.1603+0.2492x 0.9395 0.2274 苯醚甲环唑 Difenoconazole 95 y=5.1997+0.9780x 0.9563 0.6249 氰烯菌酯 Phenamacril 95 y=0.6025+2.8622x 0.9879 34.3876 百菌清 Chlorothalonil 99 y=3.4277+0.7099x 0.9859 164.0227
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