Crops ›› 2019, Vol. 35 ›› Issue (6): 168-176.doi: 10.16035/j.issn.1001-7283.2019.06.027

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A Rapid Detection Method for Potato Late Blight Caused by Phytophthora infestans Based on qPCR and LAMP Assays

Zhu Juli1,2,3,Liang Jingsi1,2,3,Zhang Pei1,Wang Weiwei1,2,3,Lin Tongsiqi1,Xie Xinyu1,Su Rui1,Tang Wei1,2,3   

  1. 1School of Life Science, Yunnan Normal University, Kunming 650500, Yunnan, China
    2Joint Academy of Potato Science, Yunnan Normal University, Kunming 650500, Yunnan, China
    3Key Laboratory of Potato Biology of Yunnan Province, Kunming 650500, Yunnan, China
  • Received:2019-05-24 Revised:2019-08-26 Online:2019-12-15 Published:2019-12-11
  • Contact: Wei Tang

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Abstract:

Phytophthora infestans can infect many kinds of Solanaceae plants. Potato late blight (PLB) caused by P. infestans is a most severe disease in the potato production. To better and fast diagnose PLB in the fields, quantitative PCR (qPCR) and Loop-mediated isothermal amplification (LAMP) primers were designed based on the P. infestans T30-4 whole genome shotgun contig 1.18131. After amplification conditions optimized, both specificity and sensitivity were detected. Finally, developed qPCR and LAMP methods were used to detect PLB incidence and compared with traditional isolation method on harvested potato tubers in the fields. Specific detection showed that qPCR and LAMP were positive amplification only in the presence of P. infestans DNA template, and were no amplification in the presence of host and other microorganism DNA. Moreover, as low as 1×10 -6ng/μL gDNA was detected using both qPCR and LAMP methods, while the sensitivity of primers showed no significant difference in the presence of P. infestans DNA template, host and other microorganism DNA. Detecting harvested tubers in the field using two methods in Dali, Lijiang and Kunming, qPCR and LAMP showed no significant difference but extremely significant difference between two fast detection methods and morphology based identification. Additionally, in Dali, Lijiang and Kunming, PLB incidence of the two molecular detection methods were both higher than morphology based identification method, in which qPCR detection method was 12.00%, 2.00% and 8.70% higher than morphology based identification method, and LAMP detection method was 11.30%, 2.00% and 8.70% higher than morphology based identification method, respectively.

Key words: Quantitative PCR (qPCR), Loop-mediated isothermal amplification (LAMP), Phytophthora infestans, Latent infection, Detection

Table 1

Strain information of oomycetes and other potato related pathogens for qPCR and LAMP detection"

分类
Clossify		 编号
No.		 菌株
Strain		 种
Species		 寄主(分离部位)
Hosts (Isolation source)		 来源
Source		 ITS序列
ITS sequence
卵菌 1 110P Phytophthora infestans Solanum tuberosum (leaf) 中国云南 LS479123
Oomycetes 2 XD1213 P. infestans S. tuberosum(leaf) 中国云南 LS479124
3 XD15 P. infestans S. tuberosum(leaf) 中国云南 LS479193
4 JC1207 P. infestans S. tuberosum(leaf) 中国云南 LS479126
5 XD1314 P. infestans S. tuberosum(leaf) 中国云南 LS479127
6 XA-4 P. infestans S. tuberosum(tuber) 中国云南 LS479171
7 DN111 P. infestans S. tuberosum(unknown) 日本北海道 LS479169
8 80029 P. infestans S. tuberosum(unknown) 荷兰瓦格宁根 LS479173
9 88069
 P. infestans
 Lycopersicon esculentum(leaf) 荷兰瓦格宁根 LS479172
10 Pds13 P. sojae Glycine max(leaf) 中国云南 LS479895
11 Ddr1231 P. parasitica Nicotiana tabacum (leaf) 中国云南 LS479896
12 LL2480 P. capsica Capsicum annuum(fruit) 中国云南 LS479897
13 Fuz37 Pythium acanthicum Saccharum officiarum L. (root) 中国云南 LS479899
真菌 14 AT1289 Alternaria alternate S. tuberosum(leaf) 中国云南 LS479189
Fungus 15 Hz1177 Synchytrium endobioticum S. tuberosum(root) 中国云南 LS479191
16 Hz1608 Fusarium solani S. tuberosum(leaf) 中国云南 LS479188
细菌 17 Hh1503
 Clavibacter michiganensissubsp. Sepedonicus S. tuberosum(tuber) 中国云南 LS479190
Bacteria 18 Hhrf1
 Pectobacterium carotovorum subsp.Carotovorum S. tuberosum(tuber)
 中国云南 LS479898
19 Hhqk1 Ralstonia solanacearum S. tuberosum(stem) 中国云南 LS479192

Table 2

LAMP and real-time fluorescent qPCR primers"

扩增类型PCR type 引物名称Primer name 序列Sequence (5'-3') 目标长度Target length (bp)
qPCR infnew1-F ACCACACCTAAAAACTTTCCACGT 241
infnew1-R CATCCACTGCTGAAAGTTGC
LAMP F3 ACCCAATAGTTGGGGGTCTT
B3 AGCGTTCTTCATCGATGTGC
BIP CTGTGGGGACGAAAGTCTCTGCGC
CTAGACATCCACTGCTGA
FIP CGAAGTCCAAACGCTCGCCTTGCT
TTATTGCTGGCGGCTA

Fig.1

Neighbor-joining analysis of Blast results Middle brackets represent the position of contig 1.18131"

Fig.2

P. infestans species-specific test results by optimized qPCR"

Fig.3

Specific test and sensitive test by qPCR for species A: P. infestans species specific test by qPCR, 1: P. infestans gDNA; 2: Mixed solution with P. infestans gDNA; 3: Mixed solution but without P. infestans gDNA; 4: ddH2O. B: Sensitive test, sensitive test profiles of 10-fold serial dilutions of P. infestans (1×10-6~1×10-1ng/μL) to create standard curve; E: Reaction efficiency (103%). C: Sensitive test with the presentation of host DNA, quantitative real-time PCR (qPCR) amplification profiles of 10-fold serial dilutions of P. infestans (1×10-6~1×10-1ng/μL) using infnew1 primers with host gDNA; E: Reaction efficiency (108%)"

Fig.4

Specific test and sensitive test by LAMP for species A: LAMP using 19 isolates; B: Observation of intensity change of positive amplification by SYBR Green staining under ultraviolet light; C: LAMP products analyzed by 2.0% agarose gel"

Table 3

PLB incidence of potato tubers caused by P. infestans determined by LAMP, qPCR and morphology observation"

地区
Region		 PLB发病率a (%)
PLB incidencea		 样品数
Number of samples		 症状b
Disease symptomsb		 LAMP qPCR 形态学c
Morphologyc
大理Dali 32.50±10.80 150 77 (51.30%) 78 (52.00%) 79 (52.70%) 61 (40.70%)
丽江Lijiang 12.66±4.25 150 21 (14.00%) 21 (14.00%) 21 (14.00%) 18 (12.00%)
昆明Kunming 22.71±11.62 150 48 (32.00%) 46 (30.70%) 46 (30.70%) 33 (22.00%)

Table 4

Number of various microbe isolated from tuber samples"

地点Site 致病疫霉
P. infestans		 腐皮镰孢霉菌
Fusarium solani		 枯草芽孢杆菌
Bacillus subtilis		 赖氏菌属
Leifsoniasp.		 类芽孢杆菌属
Paenibacillussp.		 副球菌属
Paracoccussp.
大理Dali 61 7 13 3 11 13
丽江Lijiang 18 0 10 0 1 5
昆明Kunming 33 5 18 7 5 10
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