Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (16): 2787-2799.doi: 10.3864/j.issn.0578-1752.2019.16.005

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of the Pathogen Causing Cabbage Died in Gansu Province and Detection of Anastomosis Groups

WANG Duo,XIE XueWen,CHAI ALi,SHI YanXia(),LI BaoJu()   

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-04-04 Accepted:2019-05-17 Online:2019-08-16 Published:2019-08-21
  • Contact: YanXia SHI,BaoJu LI E-mail:shiyanxia@caas.cn;libaoju@caas.cn

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Abstract:

【Objective】 The objective of this study is to identify the pathogen causing cabbage died in Gansu Province, develop a molecular method for identification, and to provide a reference for prevention and control of the disease.【Method】Diseased samples collected from Gansu Province were purified and cultured, and isolates were characterized by colony morphology, it was initially identified the pathogen as Rhizoctonia solani. For accurate identification, the internal transcribed spacer region of the ribosomal DNA (rDNA-ITS) and translation elongation factor 1-alpha (TEF-1α) of isolates were amplified and sequenced. MEGA 7.0 was used to draw the phylogenetic tree of strains and other related sequences. Molecular identification of the pathogen was carried out by PCR amplification using a specific primer pair F-RS/R-RS. Pathogenicity on 20 cabbage cultivars was verified by spray inoculation and irrigating roots treatment. Based on the conserved region of rDNA-ITS, a pair of specific primer was designed and the SYBR Green I real-time PCR reaction system was established. The specificity, sensitivity and repeatability of conventional PCR and real-time PCR were also evaluated, and R. solani AG3-AG11, binulceate Rhizoctonia AG-A, Fusarium spp., Pythium spp. were used as control fungi. The established system was used to detect R. solani in the cabbage and rhizosphere soils. 【Result】 A total of 86 strains of R. solani were isolated. The results of conventional PCR specific test showed that most strains belonged to AG-2-1 (68/86) and others were AG-1-IB (18/86). The phylogenetic analyses showed that 50 strains were divided into AG-2-1 and AG-1-IB, and the homology of each strain in two groups was 100%. Pathogenicity test results showed that the two anastomosis groups could both cause lesions on stems, and AG-2-1 was not pathogenic to the leaves of Jinwawa and Xiaohuangbao. The pathogenicity of AG-1-IB was slightly stronger than that of AG-2-1. The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. solani strains, but not from the DNA of other fungal strains by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 8.41×10 5copies/μL plasmid, while the sensitivity of real-time PCR was 8.41×10 3copies/μL, which increased two orders of magnitude. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.9983 and with high amplification efficiency (91%). The method could successfully detect the pathogens in cabbage and rhizosphere soils collected in fields. For the indoor potted experiments, the detection results of real-time PCR of infected cabbage samples inoculated after different days showed that the stems and rhizosphere soils had the largest amount of R. solani on the 5th day after inoculation. 【Conclusion】 Through molecular biological identification and pathogenicity test of pathogen of cabbage died, it was confirmed that the causal agent of cabbage died in Gansu were R. solani AG-2-1 and AG-1-IB. The developed real-time PCR system for R. solani has strong specificity and high sensitivity, it can realize the detection and monitoring of R. solani in plant and soil, and can provide technical support for early warning and management of diseases.

Key words: cabbage, anastomosis groups of Rhizoctonia solani, real-time fluorescent quantitative PCR, detection

Table 1

Primers used in this study with sequences and sources"

基因
Gene		 片段长度
Fragment length (bp)		 引物名称
Primer name		 方向
Direction		 序列
Sequence		 引物来源
Primer source
ITS 700 ITS1 Forward TCCGTAGGTGAACCTGCGG 文献[22]
Reference [22]
ITS4 Reverse TCCTCCGCTTATTGATATGC
TEF-1α 680 RSF Forward CGTGAYTTYATCAAGAACATG 文献[23]
Reference [23]
RSR Reverse GACTTGACTTCAGTGGTCAC
28S
 F-Common Forward CTCAAACAGGCATGCTC 文献[24]
Reference [24]
300 R-AG-2-1 Reverse AGGCAATAGGTTATTGGACC
250 R-AG-1-IB Reverse AAGGTCCTTTGGGGTTGGGG

Table 2

Strains and sequences used in this study (R. solani)"

菌株
Strain		 登录号 GenBank accession number
ITS TEF-1α
WWC18081412 MK336671 MK391084
WWC18081413 MK336672 MK391085
WWC18081623 MK336664 MK391077
WWC18081509 MK336665 MK391078
WWC18081604 MK336666 MK391079
WWC18081506 MK336696 MK391109
WWC18081510 MK336673 MK391086
WWC18081514 MK336674 MK391087
WWC18081527 MK336675 MK391088
WWC18081528 MK336684 MK391097
WWC18081606 MK336685 MK391098
WWC18081618 MK336686 MK391099
WWC18081619 MK336687 MK391100
WWC18081622 MK336668 MK391081
WWC18081625 MK336667 MK391080
WWC18081636 MK336669 MK391082
WWC18081638 MK336670 MK391083
WWC18081640 MK336676 MK391089
WWC18081641 MK336677 MK391090
WWC18081649 MK336678 MK391091
WWC18081651 MK336679 MK391092
WWC18081513 MK336688 MK391101
WWC18081515 MK336689 MK391102
WWC18081653 MK336690 MK391103
WWC18081656 MK336691 MK391104
WWC1507261101 MK336682 MK391095
WWC1507261102 MK336694 MK391107
WWC1507261103 MK336683 MK391096
WWC1507261104 MK336695 MK391108
WWC1507261105 MK336697 MK391110
WWC1608082901 MK336680 MK391093
WWC1608082902 MK336692 MK391105
WWC1608082903 MK336693 MK391106
WWC1608082904 MK336698 MK391111
WWC1608082905 MK336681 MK391094
WWC1608082501 MK336700 MK391113
WWC1608082502 MK336701 MK391114
WWC1608082503 MK336702 MK391115
WWC1608082504 MK336703 MK391116
WWC1608082505 MK336704 MK391117
WWC1608081601 MK336705 MK391118
WWC1608081602 MK336706 MK391119
WWC1608081603 MK336707 MK391120
WWC1608081604 MK336708 MK391121
WWC1608081605 MK336709 MK391122
WWC1608090401 MK336710 MK391123
WWC1608090402 MK336711 MK391124
WWC1608090403 MK336712 MK391125
WWC1608090404 MK336713 MK391126
WWC1608090405 MK336699 MK391112

Fig. 1

PCR amplification pattern of ITS and TEF-1α M：DNA分子量标准（5 000 bp）DNA Marker (5 000 bp);1—24：分离菌株经ITS和TEF-1α通用引物扩增后的产物电泳条带 The amplification of R. solani fragment by universal primers of ITS and TEF-1α;25：阴性对照 Negative control"

Fig. 2

Phylogenetic tree based on sequence of rDNA-ITS and TEF-1α regions"

Fig. 3

PCR products of R. solani strains collected from cabbage in Gansu Province using specific primer pairs of F-Common/ R-AG-2-1 and F-Common/R-AG-1-IB M：DNA分子量标准（5 000 bp） DNA marker (5 000 bp);1—8：F-Common/R-AG-2-1特异性扩增 Amplification by primer F-Common/R-AG-2-1 for AG-2-1;9—17：立枯丝核菌融合群AG-3—AG-11菌株 R. solani AG3 to AG11;18：阴性对照 Negative control;19—23：F-Common/R-AG-1-IB特异性扩增 Amplification by primer F-Common/R-AG-1-IB for AG-1-IB"

Table 3

Disease index for R. solani AG-2-1 and AG-1-IB on different cultivars of cabbage"

品种
Cultivar		 AG-2-1 AG-1-IB
叶片 Leaf 茎基部 Stem 叶片 Leaf 茎基部 Stem
贝贝皇 Beibeihuang 30.4 45.0 39.2 61.7
炎童 Yantong 11.9 36.7 54.2 63.3
黄孩 Huanghaier 3.0 11.7 42.1 58.3
全新玉皇大帝 Quanxinyuhuangdadi 2.2 6.7 33.1 65.0
瑞丽 Ruili 0.7 3.3 52.1 60.0
纯皇 Chunhuang 5.2 38.3 49.3 66.7
兴苑 Xingyuan 12.6 36.7 37.1 46.7
黄金 Huangjin 30.4 50.0 59.1 60.0
卓生黄金 Zhuoshenghuangjin 23.7 41.7 48.0 35.0
金宝 Jinbao 38.5 20.0 62.1 46.7
京春娃2号 Jingchunwa 2 0.7 6.7 55.1 53.3
沃德春娃 Wodechunwa 3.0 50.0 63.1 66.7
金娃娃 Jinwawa 0 8.3 15.0 21.7
金贝贝 Jinbeibei 0.7 26.7 7.0 28.3
小皇宝 Xiaohuangbao 0 5.0 6.0 25.0
绿亨 Lvheng 17.8 51.7 2.0 5.0
金旺 Jinwang 21.5 41.7 2.0 3.3
格莱美 Gelaimei 18.5 36.7 4.0 13.3
芭比 Babi 4.4 26.7 2.0 5.0
金童秀 Jintongxiu 12.6 18.3 26.0 26.6

Fig. 4

The amplification of R. solani fragment by conventional PCR M：DNA分子量标准（5 000 bp） DNA marker (5 000 bp);1：阳性对照Positive control;2：感染立枯丝核菌的白菜样品R. solani-infected cabbage sample;3—21：立枯丝核菌融合群AG-3—AG-11菌株、水稻纹枯病菌、双核丝核菌AG-A、小麦纹枯病菌、茄镰孢、尖镰孢、胶孢炭疽菌、瓜果腐霉、终极腐霉、芸薹生链格孢菌和致病疫霉 R. solani AG-3 to AG-11, R. solani AG-1-IA, binulceate Rhizoctonia AG-A, R. cerealis, F. solani, F. oxysporum, C. gloeosporioides, P. aphanidermatum, P. ultimum, A. brassicicola, P. infestans;22：健康白菜样品 Healthy cabbage sample;23：空白对照 Blank control"

Fig. 5

The standard curve of real-time PCR for R. solani"

Fig. 6

The detection sensitivity of R. solani by real-time PCR 1—7：8.41×109—8.41×103 copies/μL质粒标准品8.41×109-8.41×103 copies/μL standard plasmids;8：空白对照 Blank control"

Fig. 7

The detection sensitivity of R. solani by conventional PCR M：DNA分子量标准（5 000 bp） DNA marker (5 000 bp);1—7：8.41×109—8.41×103 copies/μL质粒标准品8.41×109-8.41×103 copies/μL standard plasmids;8：空白对照 Blank control"

Table 4

The detection results of real-time PCR of infected cabbage samples and rhizosphere soils"

接种后天数
Days after inoculation (d)		 平均Ct 值
The average cycle threshold value		 DNA平均拷贝数的对数值
Logarithm of average copy number of DNA (lg copies/μL)
茎基部 Stem 根际土壤 Soil 茎基部 Stem 根际土壤 Soil
1 23.46 31.28 5.85 3.66
5 11.92 18.84 9.11 7.16
10 20.86 23.57 6.59 5.76
15 28.92 26.88 4.31 4.90
20 30.40 32.85 3.90 3.21

Fig. 8

The R. solani detection of field samples by real-time PCR A：阳性对照 Positive control;B—Q：田间样品 Samples in field;S：阴性对照 Negative control;T：空白对照 Blank control"

Fig. 9

The R. solani detection of field samples by conventional PCR M：DNA分子量标准（5 000 bp） DNA marker（5 000 bp）;1：阳性对照 Positive control;2—8：田间死棵病株 Died plants in fields;9—18：田间病株根际土壤 Soils on died plants;19：阴性对照Negative control;20：空白对照 Blank control"

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