Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (3): 579-588.DOI: 10.3969/j.issn.1004-1524.20230859
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Identification and biological characteristics of pathogen causing branch dieback on Styphnolobium japonicum (L.) Schott
LUO Zhihan1(
), LIU Pengfei1, YU Jun2, QI He3, CHEN Xiaoguang4, LOU Binggan1,*()
- 1. Ministry of Agriculture and Rural Affairs Key Lab of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
2. College of Plant Science, Tarim University, Alar 843399,Xinjiang, China
3. Forestry and Grassland Administration of Bayingol Mongolian Autonomous Prefecture, Korla 841000, Xinjiang, China
4. Landscaping Affair Center of Korla,Korla 841000, Xinjiang, China
-
Received:2023-07-13Online:2024-03-25Published:2024-04-09
CLC Number:
Cite this article
LUO Zhihan, LIU Pengfei, YU Jun, QI He, CHEN Xiaoguang, LOU Binggan. Identification and biological characteristics of pathogen causing branch dieback on Styphnolobium japonicum (L.) Schott[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 579-588.
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Table 1 Primers for amplification of gene fragments
| 基因 Gene | 引物名称 Primer | 核苷酸序列 Nucleotide sequence |
|---|---|---|
| rDNA-ITS | ITS1 | 5'-TCCGTAGGTGAACCTGCGG-3' |
| ITS4 | 5'-TCCTCCGCTTATTGATATGC-3' | |
| EF1-α | EF1-728F | 5'-CATCGAGAAGTTCGAGAAGG-3' |
| EF1-986R | 5'-TACTTGAAGGAACCCTTACC-3' | |
| LSU | LROR | 5'-ACCCGCTGAACTTAAGC-3' |
| LR5 | 5'-TCCTGAGGGAAACTTCG-3' |
Table 1 Primers for amplification of gene fragments
| 基因 Gene | 引物名称 Primer | 核苷酸序列 Nucleotide sequence |
|---|---|---|
| rDNA-ITS | ITS1 | 5'-TCCGTAGGTGAACCTGCGG-3' |
| ITS4 | 5'-TCCTCCGCTTATTGATATGC-3' | |
| EF1-α | EF1-728F | 5'-CATCGAGAAGTTCGAGAAGG-3' |
| EF1-986R | 5'-TACTTGAAGGAACCCTTACC-3' | |
| LSU | LROR | 5'-ACCCGCTGAACTTAAGC-3' |
| LR5 | 5'-TCCTGAGGGAAACTTCG-3' |
Table 2 Botryosphaeriaceae isolates used in the phylogenetic analyses with its GenBank accession number
| 菌株名称 Strain | 序列登录号GenBank accession numbers | ||
|---|---|---|---|
| rDNA-ITS | EF1-α | LSU | |
| GH01 | OP379832 | OQ136617 | OP389048 |
| GH02 | OQ096643 | OQ586044 | OQ127403 |
| GH03 | OQ096644 | OQ586045 | OQ127404 |
| GH04 | OQ096645 | OQ586046 | OQ127405 |
| GH05 | OQ096646 | OQ586047 | OQ127406 |
| GH06 | OQ096647 | OQ586048 | OQ127407 |
| GH07 | OQ096648 | OQ586049 | OQ127408 |
| GH08 | OQ096649 | OQ586050 | OQ127409 |
| GH09 | OQ096650 | OQ586051 | OQ127410 |
| GH10 | OQ096651 | OQ586052 | OQ127411 |
| GH11 | OQ096652 | OQ586053 | OQ127412 |
| GH12 | OQ096653 | OQ586054 | OQ127413 |
| GH13 | OQ096654 | OQ586055 | OQ127414 |
| GH14 | OQ096655 | OQ586056 | OQ127415 |
| GH15 | OQ096656 | OQ586057 | OQ127416 |
| GH16 | OQ096657 | OQ586058 | OQ127417 |
| 7b | OL376284 | OL455961 | OL376301 |
| BMP13 | MN067769 | MN078938 | MK968808 |
| CBS 115206 | KF766226 | GU349030 | DQ377882 |
| MFLUCC 17-0359 | MK480514 | MK495819 | MK478925 |
| Mlty_Ma02 | MT195553 | MT362603 | MT195555 |
| NeNo1 | KX370828 | MF662595 | MH899579 |
| PTD-MA | MW605153 | MW605154 | MW605155 |
| URF_Pt01 | MT010216 | MT362600 | MT010293 |
| CBS456.78 | KF766192 | EF622063 | KF766362 |
| CMW 26167 | EU144055 | EU144070 | KF766333 |
Table 2 Botryosphaeriaceae isolates used in the phylogenetic analyses with its GenBank accession number
| 菌株名称 Strain | 序列登录号GenBank accession numbers | ||
|---|---|---|---|
| rDNA-ITS | EF1-α | LSU | |
| GH01 | OP379832 | OQ136617 | OP389048 |
| GH02 | OQ096643 | OQ586044 | OQ127403 |
| GH03 | OQ096644 | OQ586045 | OQ127404 |
| GH04 | OQ096645 | OQ586046 | OQ127405 |
| GH05 | OQ096646 | OQ586047 | OQ127406 |
| GH06 | OQ096647 | OQ586048 | OQ127407 |
| GH07 | OQ096648 | OQ586049 | OQ127408 |
| GH08 | OQ096649 | OQ586050 | OQ127409 |
| GH09 | OQ096650 | OQ586051 | OQ127410 |
| GH10 | OQ096651 | OQ586052 | OQ127411 |
| GH11 | OQ096652 | OQ586053 | OQ127412 |
| GH12 | OQ096653 | OQ586054 | OQ127413 |
| GH13 | OQ096654 | OQ586055 | OQ127414 |
| GH14 | OQ096655 | OQ586056 | OQ127415 |
| GH15 | OQ096656 | OQ586057 | OQ127416 |
| GH16 | OQ096657 | OQ586058 | OQ127417 |
| 7b | OL376284 | OL455961 | OL376301 |
| BMP13 | MN067769 | MN078938 | MK968808 |
| CBS 115206 | KF766226 | GU349030 | DQ377882 |
| MFLUCC 17-0359 | MK480514 | MK495819 | MK478925 |
| Mlty_Ma02 | MT195553 | MT362603 | MT195555 |
| NeNo1 | KX370828 | MF662595 | MH899579 |
| PTD-MA | MW605153 | MW605154 | MW605155 |
| URF_Pt01 | MT010216 | MT362600 | MT010293 |
| CBS456.78 | KF766192 | EF622063 | KF766362 |
| CMW 26167 | EU144055 | EU144070 | KF766333 |
Fig.1 Infection symptoms of branch dieback on S. japonicum A-C,Symptoms on current-year twigs ; D, Branches dieback, leaves falling off; E, Visible black protrusions on epidermis surface ; F, Black conidia formed under the epidermis; G, Most of the shoots are diseased and dry; H, Trees about to die completely.
Fig.1 Infection symptoms of branch dieback on S. japonicum A-C,Symptoms on current-year twigs ; D, Branches dieback, leaves falling off; E, Visible black protrusions on epidermis surface ; F, Black conidia formed under the epidermis; G, Most of the shoots are diseased and dry; H, Trees about to die completely.
Fig.2 Results of pathogenicity test of conidia and mycelial plugs on S. japonicum branch A, Sprayed conidial; B, Sprayed sterile water; C, Mycelial inoculation; D, PDA medium control.
Fig.2 Results of pathogenicity test of conidia and mycelial plugs on S. japonicum branch A, Sprayed conidial; B, Sprayed sterile water; C, Mycelial inoculation; D, PDA medium control.
Fig.3 Morphology characters of isolates pathogen A, Cultured with PDA medium for 2 d; B, Cultured with PDA medium for 7 d; C, Mycelium; D, Arthrospore; E-F, Septospore and single septospore ;G, Double septospore; H, Lattice spore.
Fig.3 Morphology characters of isolates pathogen A, Cultured with PDA medium for 2 d; B, Cultured with PDA medium for 7 d; C, Mycelium; D, Arthrospore; E-F, Septospore and single septospore ;G, Double septospore; H, Lattice spore.
Table 3 Effects of different culture media on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01
| 培养基 Medium | 菌落直径 Colony diameter/cm | 产孢数量 Spore yield/105 |
|---|---|---|
| 马铃薯葡萄糖琼脂培养基 | 3.69±0.09 a | 166.40±34.26 a |
| Potato dextrose agar, PDA | ||
| 固体查氏培养基Czapek | 3.74±0.11 a | 21.20±3.92 c |
| 甘薯葡萄糖琼脂培养基 | 3.70±0.16 a | 17.80±3.31 c |
| Sweet potato dextrose agar, SPDA | ||
| 玉米粒琼脂培养基 | 3.32±0.18 b | 68.60±16.70 b |
| Corn meal agar, CMA | ||
| 基本培养基 | 2.98±0.19 c | 27.00±7.82 c |
| Complete medium, CM | ||
| 燕麦片琼脂培养基 | 2.62±0.24 d | 85.60±18.78 b |
| Oat meal agar, OA | ||
| 改良营养琼脂培养基 | 2.29±0.14 e | 16.20±3.92 c |
| Nutrient agar, NA | ||
| 水琼脂培养基 | 1.94±0.13 f | 2.00±0.67 d |
| Water agar, WA |
Table 3 Effects of different culture media on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01
| 培养基 Medium | 菌落直径 Colony diameter/cm | 产孢数量 Spore yield/105 |
|---|---|---|
| 马铃薯葡萄糖琼脂培养基 | 3.69±0.09 a | 166.40±34.26 a |
| Potato dextrose agar, PDA | ||
| 固体查氏培养基Czapek | 3.74±0.11 a | 21.20±3.92 c |
| 甘薯葡萄糖琼脂培养基 | 3.70±0.16 a | 17.80±3.31 c |
| Sweet potato dextrose agar, SPDA | ||
| 玉米粒琼脂培养基 | 3.32±0.18 b | 68.60±16.70 b |
| Corn meal agar, CMA | ||
| 基本培养基 | 2.98±0.19 c | 27.00±7.82 c |
| Complete medium, CM | ||
| 燕麦片琼脂培养基 | 2.62±0.24 d | 85.60±18.78 b |
| Oat meal agar, OA | ||
| 改良营养琼脂培养基 | 2.29±0.14 e | 16.20±3.92 c |
| Nutrient agar, NA | ||
| 水琼脂培养基 | 1.94±0.13 f | 2.00±0.67 d |
| Water agar, WA |
Fig.4 Phylogenetic tree of S. japonica pathogens and other Botryosphaeriales strains based on combined ITS-EF1α-LSU sequences
Fig.4 Phylogenetic tree of S. japonica pathogens and other Botryosphaeriales strains based on combined ITS-EF1α-LSU sequences
Fig.5 Effects of different temperature on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01 Same color blocks marked without the same lowercase letters indicate signficant (P<0.05) difference.
Fig.5 Effects of different temperature on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01 Same color blocks marked without the same lowercase letters indicate signficant (P<0.05) difference.
Fig.6 Effects of different relative humidity on spores’ germination of Neoscytalidium dimidiatum isolate GH01 Dots marked without the same lowercase letters indicate signficant (P<0.05) difference.
Fig.6 Effects of different relative humidity on spores’ germination of Neoscytalidium dimidiatum isolate GH01 Dots marked without the same lowercase letters indicate signficant (P<0.05) difference.
Fig.7 Effects of different pH value on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01 Data in the same line without the same lowercase letters indicate signficant (P<0.05) difference.
Fig.7 Effects of different pH value on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01 Data in the same line without the same lowercase letters indicate signficant (P<0.05) difference.
Table 4 Effects of different C sources on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01
| 碳源 C sources | 菌落直径 Colony diameter/cm | 干重 Dry weight/mg | 产孢数量 Spore yield/105 |
|---|---|---|---|
| 甘露糖Mannose | 5.95±0.16 a | 218.50±5.20 a | 24.01±1.62 d |
| 果糖Fructose | 4.82±0.26 b | 219.70±9.00 a | 37.26±0.89 b |
| 半乳糖Galactose | 4.47±0.41 c | 204.30±1.50 c | 11.21±0.30 gh |
| 蔗糖Sucrose | 4.38±0.16 c | 205.00±0.50 c | 13.37±1.24 fg |
| 葡萄糖Glucose | 4.35±0.26 c | 207.20±3.00 ab | 14.57±1.02 f |
| 乳糖Lactose | 3.97±0.17 d | 198.40±1.90 cd | 30.88±0.83 c |
| 麦芽糖Maltose | 3.95±0.46 d | 216.80±7.20 ab | 9.52±0.41 h |
| 可溶性淀粉Soluble starch | 3.77±0.09 d | 198.00±1.50 cd | 14.10±0.83 f |
| 阿拉伯糖Arabinose | 3.44±0.24 e | 190.50±2.70 de | 16.83±1.55 e |
| CK | 3.11±0.12 f | 178.00±3.50 de | 125.97±1.39 a |
| 甘油Glycerol | 3.11±0.28 f | 207.20±3.00 de | 10.48±0.41 h |
| 木糖Xylose | 3.07±0.30 f | 191.10±6.20 de | 10.48±0.41 h |
| 山梨糖Sorbose | 1.82±0.29 g | 185.30±2.00 e | 24.77±1.28 d |
Table 4 Effects of different C sources on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01
| 碳源 C sources | 菌落直径 Colony diameter/cm | 干重 Dry weight/mg | 产孢数量 Spore yield/105 |
|---|---|---|---|
| 甘露糖Mannose | 5.95±0.16 a | 218.50±5.20 a | 24.01±1.62 d |
| 果糖Fructose | 4.82±0.26 b | 219.70±9.00 a | 37.26±0.89 b |
| 半乳糖Galactose | 4.47±0.41 c | 204.30±1.50 c | 11.21±0.30 gh |
| 蔗糖Sucrose | 4.38±0.16 c | 205.00±0.50 c | 13.37±1.24 fg |
| 葡萄糖Glucose | 4.35±0.26 c | 207.20±3.00 ab | 14.57±1.02 f |
| 乳糖Lactose | 3.97±0.17 d | 198.40±1.90 cd | 30.88±0.83 c |
| 麦芽糖Maltose | 3.95±0.46 d | 216.80±7.20 ab | 9.52±0.41 h |
| 可溶性淀粉Soluble starch | 3.77±0.09 d | 198.00±1.50 cd | 14.10±0.83 f |
| 阿拉伯糖Arabinose | 3.44±0.24 e | 190.50±2.70 de | 16.83±1.55 e |
| CK | 3.11±0.12 f | 178.00±3.50 de | 125.97±1.39 a |
| 甘油Glycerol | 3.11±0.28 f | 207.20±3.00 de | 10.48±0.41 h |
| 木糖Xylose | 3.07±0.30 f | 191.10±6.20 de | 10.48±0.41 h |
| 山梨糖Sorbose | 1.82±0.29 g | 185.30±2.00 e | 24.77±1.28 d |
Table 5 Effects of different N sources on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01
| 氮源 N sources | 菌落直径 Colony diameter/cm | 干重 Dry weight/mg | 产孢数量 Spore yield/105 |
|---|---|---|---|
| 牛肉膏Beef Extract | 4.63±0.12 a | 252.60±6.00 a | 53.61±0.44 c |
| CK | 4.22±0.20 b | 180.60±9.00 e | 20.05±0.87 e |
| 蛋白胨Peptone | 4.11±0.14 bc | 243.10±4.90 ab | 138.10±0.70 a |
| 氯化铵NH4Cl | 4.05±0.10 cd | 212.10±2.00 c | 8.85±0.65 i |
| 硫酸铵(NH4)2SO4 | 3.69±0.09 cd | 230.00±7.40 b | 15.30±0.50 f |
| 苯丙氨酸Phenylalanine | 3.87±0.34 de | 202.00±4.90 cd | 137.43±1.23 a |
| 硝酸铵NH4NO3 | 3.78±0.13 e | 193.00±6.00 de | 13.24±0.88 g |
| 硝酸钠NaNO3 | 3.76±0.17 ef | 191.70±6.30 de | 13.24±0.88 g |
| 草酸铵(NH4)2C2O4 | 3.61±0.18 g | 191.80±6.60 de | 84.87±0.66 b |
| 甘氨酸Glycine | 3.60±0.20 g | 178.50±6.00 e | 10.25±0.89 hi |
| 精氨酸Arginine | 3.39±0.13 h | 236.10±4.00 b | 42.08±0.82 d |
| 硝酸钾KNO3 | 3.29±0.22 h | 185.90±4.10 e | 11.64±0.46 h |
| 尿素Urea | 1.90±0.12 i | 149.40±7.30 f | 8.65±0.46 i |
Table 5 Effects of different N sources on mycelial growth and sporulation of Neoscytalidium dimidiatum isolate GH01
| 氮源 N sources | 菌落直径 Colony diameter/cm | 干重 Dry weight/mg | 产孢数量 Spore yield/105 |
|---|---|---|---|
| 牛肉膏Beef Extract | 4.63±0.12 a | 252.60±6.00 a | 53.61±0.44 c |
| CK | 4.22±0.20 b | 180.60±9.00 e | 20.05±0.87 e |
| 蛋白胨Peptone | 4.11±0.14 bc | 243.10±4.90 ab | 138.10±0.70 a |
| 氯化铵NH4Cl | 4.05±0.10 cd | 212.10±2.00 c | 8.85±0.65 i |
| 硫酸铵(NH4)2SO4 | 3.69±0.09 cd | 230.00±7.40 b | 15.30±0.50 f |
| 苯丙氨酸Phenylalanine | 3.87±0.34 de | 202.00±4.90 cd | 137.43±1.23 a |
| 硝酸铵NH4NO3 | 3.78±0.13 e | 193.00±6.00 de | 13.24±0.88 g |
| 硝酸钠NaNO3 | 3.76±0.17 ef | 191.70±6.30 de | 13.24±0.88 g |
| 草酸铵(NH4)2C2O4 | 3.61±0.18 g | 191.80±6.60 de | 84.87±0.66 b |
| 甘氨酸Glycine | 3.60±0.20 g | 178.50±6.00 e | 10.25±0.89 hi |
| 精氨酸Arginine | 3.39±0.13 h | 236.10±4.00 b | 42.08±0.82 d |
| 硝酸钾KNO3 | 3.29±0.22 h | 185.90±4.10 e | 11.64±0.46 h |
| 尿素Urea | 1.90±0.12 i | 149.40±7.30 f | 8.65±0.46 i |
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