Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (1): 71-85.doi: 10.3864/j.issn.0578-1752.2021.01.006

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of Major Pathogenic Fungi of Soybean in Hebei Province and Screening of Control Fungicides

BI QiuYan1(),DANG ZhiHong1,ZHU WeiQi2,GAO ZhanLin1(),HAN XiuYing1(),ZHAO JianJiang1,WANG WenQiao1,LU Fen1,WU Jie1   

  1. 1Plant Protection Institute of Hebei Academy of Agricultural and Forestry Sciences/IPM Centre of Hebei Province/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs, Baoding 071000, Hebei
    2Zhaoxian Experimental Base, Shijiazhuang Academy of Agriculture and Forestry Sciences, Shijiazhuang 051530
  • Received:2020-04-14 Accepted:2020-06-25 Online:2021-01-01 Published:2021-01-13
  • Contact: ZhanLin GAO,XiuYing HAN E-mail:0304biqiuyan@163.com;zbs308@163.com;xiuyinghan@163.com

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Abstract:

【Objective】The objective of this study is to identify the fungal pathogens causing the main diseases of soybean plants in Hebei Province, screen the fungicides which can effectively inhibit the identified pathogens, and to provide a basis for chemical control of soybean disease in Hebei Province.【Method】The main pathogens of soybean collected from Hebei Province were isolated and purified according to the disease symptom. The morphology of colony, mycelium and spore was observed and identified by using 3D ultra depth of field digital microscope and Olympus Microscope. The rDNA-ITS regions of five pathogenic strains were amplified by PCR and sequenced by universal primers ITS1-ITS4. Neighbor-joining method of software MEGA 7.0 was used to construct phylogenetic trees and analyze the relationship among different pathogens. The pathogens with specific primer sequences were identified and verified. The main pathogen kinds of soybean plants in Hebei Province were identified by the combination of macromorphology, microscopic characteristics and molecular biology techniques. The method of mycelium growth rate and the control effects of different fungicides on the major diseases in vitro leaves or young stems of soybean were used to screen the fungicides.【Result】Based on the analysis of macromorphology, microstructure and sequence, the pathogens isolated from the main soybean producing areas in Hebei Province were Fusarium equiseti, Colletotrichum chlorophyti, Phoma herbarum, Alternaria alternata and Exserohilum rostratum. The corresponding diseases were soybean root rot, anthracnose, leaf spot of P. herbarum, black spot and leaf spot of E. rostratum. The results of virulence test showed that the above pathogens were all sensitive to the triazole fungicides, such as difenoconazole, triflumizole, metconazole and Qoi fungicide pyraclostrobin. F. equiseti was sensitive to Qoi fungicide trifloxystrobin, the pathogens except F. equiseti were sensitive to amide succinate dehydrogenase inhibitors fluopyram, C. chlorophyti and E. rostratum were sensitive to alkyl polyamines fungicide xinjunan, C. chlorophyti was sensitive to organic sulfur bionic fungicide ethylicin, the EC50 values of the inhibitors were less than 10 μg·mL -1. The control results of different fungicides on the major soybean diseases showed that the fungicides of difenoconazole, triflumizole, metconazole, pyraclostrobin, trifloxystrobin significantly inhibited above diseases, fluopyram significantly inhibited the diseases except soybean root rot, xinjunan and ethylicin significantly inhibited anthracnose and leaf spot of E. rostratum, the control effects were above 90%.【Conclusion】Five kinds of pathogen and disease of soybean in Hebei Province are identified. The triazole sterol biosynthesis inhibitors difenoconazole, triflumizole, metconazole and Qoi respiration inhibitor pyraclostrobin were recommended as the optimal fungicides for soybean fungal diseases in Hebei Province.

Key words: soybean, pathogenic fungus, molecular identification, fungicide screening, Hebei Province

Fig. 1

Main fungal disease symptoms and pathogen morphology from field and indoor of soybean in Hebei Province The numbers after disease A, B, C, D and E respectively represent: 1 field disease; 2 pathogenicity verification; 3 colony; 4 hypha (10 × 40 times, scale=200 μm); 5 spore (10 × 40 times, scale=20 μm)"

Fig. 2

Electrophoretic comparative identification of the pathogens isolated from the field and inoculated in the laboratory a, b and c represent rDNA of field isolated pathogens, rDNA of laboratory inoculated pathogens, and specific conservative sequence verification, respectively"

Fig. 3

The neighbor-joining (NJ) tree of pathogen A based on sequences of ITS"

Fig. 4

The neighbor-joining (NJ) tree of pathogen B based on sequences of ITS"

Fig. 5

The neighbor-joining (NJ) tree of pathogen C based on sequences of ITS"

Fig. 6

The neighbor-joining (NJ) tree of pathogen D based on sequences of ITS"

Fig. 7

The neighbor-joining (NJ) tree of pathogen E based on sequences of ITS"

Fig. 8

The neighbor-joining (NJ) tree of pathogen A based on sequences of TEF1-α"

Fig. 9

The neighbor-joining (NJ) tree of pathogen B based on sequences of ACT"

Fig. 10

The neighbor-joining (NJ) tree of pathogen D based on sequences of EF-1α"

Table 1

Toxicity of different kinds of fungicide against 5 pathogens isolated from soybean"

杀菌剂种类
Fungicide species		 杀菌剂名称
Fungicide name		 病原菌Pathogen (EC50, μg·mL-1)
木贼镰孢
F. equiseti		 大豆炭疽菌
C. chlorophyti		 茎点霉菌
P. herbarum		 链格孢
A. alternata		 嘴突凸脐蠕孢
E. rostratum
三唑类
Triazole		 苯醚甲环唑Difenoconazole 1.1102 0.3129 0.9125 0.5928 0.0466
氟菌唑Triflumizole 1.5024 0.2600 0.8647 0.8581 0.0955
叶菌唑Metconazole 0.0112 0.2216 0.4206 0.3907 0.0283
甲氧基丙烯酸酯类
Strobilurin		 肟菌酯Trifloxystrobin 1.8804 26.3545 138.9770 32.0614 75.1247
吡唑醚菌酯Pyraclostrobin 2.8012 0.1775 0.5791 9.3865 2.0022
嘧菌酯Azoxystrobin 83.0320 27.0851 11.6454 10.0925 36.3156
氰烯菌酯Phenamacril 0.0109 4.4280 154.9974 164.0990 39.9152
酰胺类
Amide succinate dehydrogenase inhibitors		 氟吡菌酰胺Fluopyram 42.1129 5.8304 2.8306 7.2185 2.5741
啶酰菌胺Boscalid 40. 4705 2.5139 0.4857 155.0532 2.2585
烷基多胺类
Alkyl polyamines		 辛菌胺
Xinjunan		 12.0130 8.4517 34.3226 21.7385 7.7361
有机硫仿生类
Organic sulfur bionic fungicide		 乙蒜素
Ethylicin		 10.3906 8.1840 416.1457 180.9062 45.9120

Table 2

Control effects of different kinds of fungicide in vitro or young stems to 5 soybean fungal diseases"

杀菌剂种类
Fungicide species		 杀菌剂名称
Fungicide name		 病害Disease（病情指数Disease index /防治效果Control effect (%)）
大豆根腐病
Soybean root rot		 炭疽病
Anthracnose		 茎点霉叶斑病
Leaf spot of P. herbarum		 大豆黑斑病
Black spot		 凸脐蠕孢叶斑病
Leaf spot of E. rostratum
三唑类Triazole 苯醚甲环唑Difenoconazole 5.00/93.24a 5.00/93.15a 6.00/91.49a 5.50/92.20a 6.00/91.49ab
氟菌唑Triflumizole 6.00/91.89a 7.00/90.41a 6.50/90.78a 4.00/94.33a 4.00/94.33a
叶菌唑Metconazole 7.00/90.54a 6.00/91.78a 5.00/92.21a 4.50/93.62a 7.00/90.07ab
甲氧基丙烯酸酯类
Strobilurin		 肟菌酯Trifloxystrobin 6.00/91.89a 5.00/93.15a 5.50/92.20a 6.50/90.78a 5.50/92.20ab
吡唑醚菌酯Pyraclostrobin 4.00/94.59a 4.00/94.52a 7.00/90.07a 6.00/91.49a 5.00/92.91ab
嘧菌酯Azoxystrobin 9.00/87.84ab 12.00/83.56b 9.00/87.23ab 11.50/83.69ab 7.50/89.36b
氰烯菌酯Phenamacril 10.00/86.49ab 15.00/79.45b 12.50/82.27b 20.00/71.63c 12.50/82.27c
酰胺类
Amide succinate dehydrogenase inhibitors		 氟吡菌酰胺Fluopyram 26.00/64.86c 6.00/91.78a 6.00/91.49a 6.00/91.49a 6.50/90.78b
啶酰菌胺Boscalid 33.00/55.41d 11.00/84.93b 5.50/92.20a 13.50/80.85b 8.00/88.65b
烷基多胺类
Alkyl polyamines		 辛菌胺
Xinjunan		 20.00/72.97b 6.00/91.78a 11.00/84.40b 11.00/84.40ab 7.00/90.07b
有机硫仿生类
Organic sulfur bionic fungicide		 乙蒜素
Ethylicin		 18.00/75.68b 5.00/93.15a 12.50/82.27b 14.00/ 80.14b 4.50/93.62a
对照Control - 74.00/- 73.00/- 70.50/- 59.50/- 36.00/-
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