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张梨梨 罗庭 李彦忠

引用本文: 张梨梨,罗庭,李彦忠. 黑龙江苜蓿炭疽病病原鉴定及其生物学特性. 草业科学, 2020, 37(0): 1-13 doi: shu
Citation:  ZHANG L L, LUO T, LI Y Z. Identification and biological characteristics of causing alfalfa anthracnose in Heilongjiang. Pratacultural Science, 2020, 37(0): 1-13 doi: shu

黑龙江苜蓿炭疽病病原鉴定及其生物学特性

    作者简介: 张梨梨(1991-),女,甘肃古浪人,在读硕士。E-mail: .cn;
    通讯作者: 李彦忠, .cn
  • 基金项目:博亚平台 长江学者和创新团队发展计划资助(IRT_17R50);甘肃省科技重大专项计划草类植物种质创新与品种选育(19ZD2NA002);公益性行业(农业)科研专项经费项目(201303057);国家现代农业产业技术体系(CARS-34);南志标院士工作站(2018IC074)

摘要: 2019年对黑龙江省2龄栽培紫花苜蓿(Medicago sativa)进行病害调查,发现部分苜蓿茎秆表面出现大量卵圆形或不规则形的黑色小病斑,病斑发展后期相互融合,严重破坏韧皮组织。本研究鉴定了其病原,并对病原菌生物学特性进行了研究。经形态学鉴定、多基因联合测序分析和致病性测定,将该病病原鉴定为北美炭疽菌(Colletotrichum americae-borealis)。生物学特性研究结果表明,病原菌生长和产孢最适培养基为PDA,生长速率和产孢量分别为1.10 cm·d–1和1.3 × 106个·mL–1,显著高于其它培养基(P < 0.05);最适氮源蛋白胨,分别为0.91 cm·d–1和1.8 × 105个·mL–1,显著高于其它氮源(P < 0.05)。以葡萄糖为碳源时,产孢量显著高于其他碳源(P < 0.05),为3.6 × 106个·mL–1;以可溶性淀粉和蔗糖为碳源,菌丝生长速率显著高于其他碳源(P < 0.05),为0.82 cm·d–1。

English

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  • 博亚平台

    图 1  博亚平台 苜蓿炭疽病的田间症状和病原形态特征

    Figure 1.  The symptoms of alfalfa anthracnose and morphology of Colletotrichum americae-borealis

    图 2  多基因(ITS、CHS-1和GADPH)联合系统发育树

    Figure 2.  Polygene (ITS, CHS-1 and GADPH) combined phylogenetic tree

    图 3  北美炭疽菌(C. americae-borealis博亚平台)在9种培养基上的动态生长曲线

    Figure 3.  The dynamic growth curve of C. americae-borealis博亚平台 on nine kinds medium

    图 4  北美炭疽菌(C. americae-borealis博亚平台)在5种碳源上的动态生长曲线

    Figure 4.  The dynamic growth curve of C. americae-borealis博亚平台 on five carbon sources

    图 5  北美炭疽菌(C. americae-borealis)在5种氮源上的动态生长曲线

    Figure 5.  The dynamic growth curve of C. americae-borealis on five nitrogen sources

    表 1  本研究所用引物序列[7]

    Table 1.  Primer and used in this study

    基因
    Target gene
    引物
    Primer
    引物序列 (5’–3’)Primer
    DNA sequence(5’–3’)
    ITSITS1TCCGTAGGTGAACCTGCGG
    ITS4TCCTCCGCTTATTGATATGC
    CHS1CHS-79FTGGGGCAAGGATGCTTGGAAGAAG
    CHS-354RTGGAAGAACCATCTGTGAGAGTTG
    GAPDHGDFGCCGTCAACGACCCCTTCATTGA
    GDRGGGTGGAGTCGTACTTGAGCATGT
    下载: 导出CSV

    表 2  构建炭疽病(C. americae-borealis博亚平台)系统发育树菌种信息及登记号

    Table 2.  Information on the phylogenetic tree species and registration numbers of C. americae-borealis

    种类 Species 序列编号
    Accession No.
    寄主
    Host
    国家
    County
    基因序列及类型 Gen bank accessions
    ITS GAPDH CHS-1
    C. fuscum CBS 133701* Digitalis lutea Germany KM105174 KM105524 KM105244
    CBS 133703 Digitalis obscura Netherlands KM105175 KM105525 KM105245
    C. vignae CBS 501.97, LARS 56* Vign aunguiculata Nigeria KM105183 KM105534 KM105253
    C. bryoniicola CBS 109849* Bryonia dioica Netherlands KM105181 KM105532 KM105251
    C. utrechtense CBS 130243* Trifolium pratense Netherlands KM105201 KM105554 KM105271
    C. higginsianum Abp 2-2, CPC 19365 Brassica rapa Japan KM105185 KM105537 KM105255
    Abc 6-2, CPC 19368 Brassica chinensis Japan KM105187 KM105539 KM105257
    C. americae-borealis CBS 136232* Medicago sativa USA KM105224 KM105579 KM105294
    C. ocimi CBS 298.94* Ocimum basilicum Italy KM105222 KM105577 KM105292
    C. destructivum CBS 511.97, LARS 202 Medicago sativa Morocco KM105216 KM105571 KM105286
    CBS 114801, AR 4031 Crupina vulgaris Greece KM105219 KM105574 KM105289
    CBS 157.83 Medicago sativa Serbia KM105215 KM105570 KM105285
    CBS 128509, LARS 320 Medicago sativa Canada KM105214 KM105569 KM105284
    C. antirrhinicola CBS 102189* Antirrhinum majus New Zealand KM105180 KM105531 KM105250
    C. tabacum CBS 124249, MUCL44942 Centellaasiatica Madagascar KM105206 KM105560 KM105276
    C. lentis CBS 127605, DAOM Lens culinaris Canada KM105241 KM105598 KM105311
    235317, CT26
    CBS 127604, DAOM Lens culinaris Canada JQ005766 KM105597 JQ005787
    235316, CT21*
    C. tanaceti CBS 132693, BRIP Tanacetum Australia JX218228 JX218243 JX259268
    57314, UM01* cinerariifolium
    C. pisicola CBS 724.97, LARS 60* Pisum sativum USA KM105172 KM105522 KM105242
     注: “*”代表模式菌株
     Note: "*" stands for model strain.
    下载: 导出CSV

    表 3  32个苜蓿品种田间发病率和病情指数

    Table 3.  Disease incidence and index of 32 alfalfa varieties in filed

    品种名 Varities原产地 Source发病率 Disease incidence (%)病情指数 Index
    WL343HQ美国 USA83.02 ± 7.0428.18 ± 0.01
    WL354HQ美国 USA83.02 ± 5.8247.20 ± 0.01
    北极熊 Polar bear美国 USA78.36 ± 6.8030.11 ± 0.01
    甘农5号 Gannong No. 5中国 China37.01 ± 9.7227.95 ± 0.01
    甘农9号 Gannong No. 9中国 China63.82 ± 11.3233.20 ± 0.01
    皇冠 Imperial crown加拿大 Canada84.48 ± 5.4659.86 ± 0.01
    旱地 Dryland美国 USA73.13 ± 7.5844.66 ± 0.01
    前景 Foreground加拿大 Canada61.18 ± 8.8028.89 ± 0.01
    雷霆 Thunder美国 USA91.70 ± 3.4055.52 ± 0.01
    巨能2 Magnum 2美国 USA66.50 ± 7.2739.82 ± 0.04
    巨能7 Magnum 7美国 USA75.33 ± 7.4136.40 ± 0.21
    康赛 Kangsai美国 USA92.71 ± 5.0447.22 ± 0.01
    耐盐之星 Salt-tolerant star美国 USA69.19 ± 8.0234.64 ± 0.01
    骑士 Tknight T美国 USA67.34 ± 8.1731.03 ± 0.00
    WL319美国 USA85.05 ± 5.9941.48 ± 0.01
    甘农6号 Gannong No. 6中国 China77.71 ± 6.7172.28 ± 0.01
    阿尔冈金 Algonquin加拿大 Canada92.81 ± 3.5042.20 ± 0.01
    敖汉苜蓿 Aohan中国 China78.91 ± 10.1043.06 ± 0.01
    中苜1号 Zhongmu No. 1中国 China77.57 ± 11.0947.44 ± 0.01
    中苜3号 Zhongmu No. 3中国 China88.53 ± 6.4484.00 ± 0.01
    龙威3010Longwei 3010美国 USA85.46 ± 6.0879.08 ± 0.01
    公农5号 Gongnong No. 5中国 China97.22 ± 1.8751.92 ± 0.01
    陇东苜蓿 Longdong alfalfa中国 China73.27 ± 10.9343.44 ± 0.01
    WL363HQ美国 USA78.81 ± 6.5543.51 ± 0.01
    WL168HQ美国 USA93.19 ± 4.1890.98 ± 0.01
    阿迪娜 Adina美国 USA90.00 ± 10.0093.10 ± 0.01
    甘农4号 Gannong No. 4中国 China64.81 ± 14.8157.58 ± 0.01
    甘农3号 Gannong No. 3中国 China71.45 ± 11.1675.00 ± 0.01
    SR4030加拿大 Canada82.22 ± 7.2940.83 ± 0.01
    新牧4号 Xinmu No. 4中国 China74.38 ± 8.6148.36 ± 0.01
    MF4020加拿大 Canada85.15 ± 5.6866.19 ± 0.01
    冲击波 Shockwave美国 USA66.62 ± 12.1868.06 ± 0.01
    P < 0.05P < 0.05
    下载: 导出CSV

    表 4  Colletotrichum americae-borealis 致病性测定

    Table 4.  Pathogenicity tests of Colletotrichum americae-borealis

    处理 Different treatment株高 Plant height(cm)根长 Root length(cm)鲜重 Fresh weight干重 Dry weight
    地上 On the ground(g) 地下 Underground(g) 地上 On the ground(g) 地下 Underground(g)
    接病 Inoculation19.58b 8.03 b 4.98 b 4.16 b 1.62 b 1.41 b
    对照 CK22.07a9.20 a6.78 a5.38 a2.51 a2.28 a
     注: 同列数据不同字母者表示在P < 0.05水平上存在显著差异。
     Note: Comparing the data in the same column, the different letter indicate significant difference (P < 0.05).
    下载: 导出CSV

    表 5  北美炭疽菌(C. americae-borealis博亚平台)在9种培养基的产孢量和生长速率

    Table 5.  Spore production and growth rate of C. americae-borealis in 9 kinds of media

    培养基种类 Kinds of medium菌落净增 Net colony growth (cm)生长速率 Growth rate (cm·d–1)产孢数量 Number of spores (个·mL–1)
    OMA6.00 ± 0.06cd0.86 ± 0.01cd0
    PSA7.13 ± 0.04b1.02 ± 0.01b1.6 × 105
    WHDA4.80 ± 0.06e0.69 ± 0.01e0
    CA5.70 ± 0.04d0.81 ± 0.01d5 × 102
    WA2.83 ± 0.18f0.41 ± 0.03f0
    V86.13 ± 0.02c0.88 ± 0.00c1.1 × 106
    PCA5.90 ± 0.26cd0.84 ± 0.04cd0
    PDA7.70 ± 0.00a1.10 ± 0.00a1.3 × 106
    CMA7.00 ± 0.13b1.00 ± 0.02b1.1 × 103
     注: 同列数据不同字母者表示在P < 0.05水平上存在显著差异。
     Note: Comparing the data in the same column, the different letter indicate significant difference (P < 0.05).
    下载: 导出CSV

    表 6  北美炭疽菌(C. americae-borealis博亚平台)在5种碳源的产孢量和生长速率

    Table 6.  Spore production and growth of C. americae-borealis in 5 kinds of carbon

    氮源种类 Kinds of carbon菌落净增 Net colony growth (cm)生长速率 Growth rate (cm·d–2)产孢数量 Number of spores (个·mL–1)
    可溶性淀粉 (soluble starch)5.77 ± 0.04a0.82 ± 0.01a3.3 × 105
    葡萄糖 (glucose)5.13 ± 0.02b0.73 ± 0.00b3.6 × 106
    乳糖 (lactose)4.03 ± 0.04c0.58 ± 0.01c0
    麦芽糖 (maltose)5.07 ± 0.14b0.72 ± 0.02b1.8 × 102
    蔗糖 (sucrose)5.73 ± 0.21a0.82 ± 0.03a1.1 × 102
     注: 同列数据不同字母者表示在P < 0.05水平上存在显著差异。
     Note: Comparing the data in the same column, the different letter indicate significant difference (P < 0.05).
    下载: 导出CSV

    表 7  北美炭疽菌(C. americae-borealis)在5种氮源的产孢量和生长速率

    Table 7.  Spore production and growth of C. americae-borealis in 5 kinds of nitrogen sources

    氮源种类 Kinds of nitrogen菌落净增 Net colony growth (cm)生长速率 Growth rate (cm·d–2)产孢数量 Number of spores (个·mL–1)
    硫酸铵 ammonia vitriol3.60 ± 0.29c0.51 ± 0.04c0
    蛋白胨 peptone6.37 ± 0.09a0.91 ± 0.01a1.8 × 105
    氯化铵 ammonium chloride3.23 ± 0.06c0.46 ± 0.01cd0
    硝酸钠 sodium nirirate5.07 ± 0.09b0.73 ± 0.01b5.1 × 103
    尿素 urea2.97 ± 0.09d0.43 ± 0.01d0
     注: 同列数据不同字母者表示在P < 0.05水平上存在显著差异。
     Note: Comparing the data in the same column, the different letter indicate no significant difference (P < 0.05).
    下载: 导出CSV
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                • 通讯作者:  李彦忠, .cn
                • 收稿日期:  2021-06-28
                • 网络出版日期:  2021-09-11
                通讯作者: 陈斌,
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