Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants
Abstract
:1. Introduction
2. Materials and Methods
2.1. Test Strains
2.2. Reagents and Seeds
2.3. DNA Extraction
2.4. Primer and Probe Design
2.5. Optimization of LAMP-LFD Reaction Conditions
2.6. Specificity Assay
2.7. Sensitivity Assay
2.8. Evaluation of LAMP-LFD Assay for Detection of A. citrulli in Seeds
2.9. Detection of A. citrulli in Leaf Tissues
3. Results
3.1. Optimization of A. citrulli LAMP-LFD Conditions and Establishment of Detection Method
3.2. LAMP-LFD Specificity
3.3. LAMP-LFD Sensitivity
3.4. Artificially and Commercially Available Seeds Assay
3.5. LAMP-LFD Validation with Leaf Samples
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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No. | Species/Strains | Source/Origin | Host | LAMP-LFD |
---|---|---|---|---|
1 | Acidovorax citrulli Ac-1 | IPPFAAS | Citrullus lanatus | + |
2 | A. citrulli Ac-2 | IPPFAAS | Cucumis melo | + |
3 | A. citrulli Ac-3 | FAFU | Citrullus lanatus | + |
4 | A. citrulli Ac-4 | FAFU | Cucumis melo | + |
5 | A. citrulli Ac-5 | PPSX | Cucumis melo | + |
6 | A. citrulli Ac-6 | PPSX | Cucumis melo | + |
7 | A. oryzae OS-1 | CGMCC | Oryza sativa | − |
8 | A. delafieldii UK-1 | CGMCC | Unknown | − |
9 | A. avenae subsp. avenae AS-1 | CGMCC | Avena sativa | − |
10 | A. caeni UK-1 | CGMCC | Unknown | − |
11 | A. avenae AS-1 | CGMCC | Avena sativa | − |
12 | Bacillus amyloliquefaciens UK-1 | IPPFAAS | Unknown | − |
13 | B. atrophaeus UK-1 | IPPFAAS | Unknown | − |
14 | B. megatherium CS-1 | IPPFAAS | Cucumis sativus | − |
15 | B. mojavensis UK-1 | IPPFAAS | Unknown | − |
16 | B. licheniformis CA-1 | IPPFAAS | Capsicum annuum | − |
17 | B. pumilus CA-1 | IPPFAAS | Capsicum annuum | − |
18 | B. velezensis CS-1 | IPPFAAS | Cucumis sativus | − |
19 | Burkholderia vietnamiensis UK-1 | IPPFAAS | Unknown | − |
20 | Escherichia coli CS-1 | IPPFAAS | Cucumis sativus | − |
21 | Lysinibacillus fusiformis CR-1 | IPPFAAS | Citrus reticulata | − |
22 | Paenibacillus polymyxa UK-1 | IPPFAAS | Unknown | − |
23 | Pseudomonas syringae pv. Actinidiae AC-1 | SICAU | Actinidia chinensis | − |
24 | P. viridiflava UK-1 | IPPFAAS | Unknown | − |
25 | P. cichorii UK-1 | IPPFAAS | Unknown | − |
26 | P. xiamenensis UK-1 | IPPFAAS | Unknown | − |
27 | P. syringae pv. tomato SL-1 | IPPFAAS | Solanum lycopersicon | − |
28 | Ralstonia solanacearum NT-1 | FAFU | Nicotiana tabacum | − |
29 | R. solanacearum DE-1 | IPPFAAS | Dioscorea esculenta | − |
30 | Xanthomonas oryzae pv. oryzae OS-1 | SICAU | Oryza sativa | − |
31 | X. oryzae pv. oryzicola OS-1 | IPPFAAS | Oryza sativa | − |
32 | X. axonopodis pv. citri CR-1 | IPPFAAS | Citrus reticulata | − |
No. | Variety | Manufacturing Enterprise/Origin | LAMP-LFD Results of Five Replicates | ||||
---|---|---|---|---|---|---|---|
1 | Watermelon cv. ‘Xiaodelei’ | Changchun Jixiangdi Seed Industry Co., Ltd., Changchun, China | + | + | + | + | + |
2 | Watermelon cv. ‘Lanhanteida’ | Hebei Qingfeng Agricultural Technology Co., Ltd., Qingxian, China | + | + | − | + | + |
3 | Watermelon cv. ‘Zhaojia 84-24’ | Qingxian Chunfeng Seed Industry Co., Ltd., Qingxian, China | − | − | − | − | − |
4 | Watermelon cv. ‘Hongxiaoyu’ | Shenyang Bosite Agricultural Technology Co., Ltd., Shenyang, China | + | − | + | − | + |
5 | Watermelon cv. ‘Lvlongbawang’ | Qingxian Aisen Vegetable Variety Promotion Center, Qiangxian, China | + | + | + | − | − |
6 | Watermelon cv. ‘Jingmihuanshuai’ | Qingxian Chunfeng Seed Industry Co., Ltd., Qingxian, China | − | − | − | − | − |
7 | Watermelon cv. ‘Zhimacui’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
8 | Watermelon cv. ‘Lanhanxigua’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
9 | Watermelon cv. ‘Gailianjingxin’ | Qingxian Chunfeng Seed Industry Co., Ltd., Qingxian, China | − | − | − | − | − |
10 | Watermelon cv. ‘Lanhangxiwang’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
11 | Watermelon cv. ‘Lanhanxinong 8’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
12 | Watermelon cv. ‘Bintanxiaoxigua’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
13 | Watermelon cv. ‘Bintanxiaoxigua 6’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
14 | Watermelon cv. ‘Fangzheng 209’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
15 | Watermelon cv. ‘Fangzhengheilong’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
16 | Watermelon cv. ‘Bintan 8424’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
17 | Watermelon cv. ‘Fangzheng 208’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
18 | Watermelon cv. ‘Fangzhengmuzhi’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
19 | Muskmelon cv. ‘Balixian’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
20 | Muskmelon cv. ‘Ribentiaobao’ | Shouguang Meinong Information Technology Co., Ltd., Shouguang, China | − | − | − | − | − |
21 | Muskmelon cv. ‘Fangzhengyanjiaomi’ | Henan Fangzhen Seed Co., Ltd., Xingxian, China | − | − | − | − | − |
22 | Muskmelon cv. ‘Huancuili F1’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
23 | Muskmelon cv. ‘Xiantianxuemi 208’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
24 | Muskmelon cv. ‘Jinhongrui’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
25 | Muskmelon cv. ‘Tianla 008’ | Shouguang Meinong Information Technology Co., Ltd., Shouguang, China | − | + | + | + | + |
26 | Muskmelon cv. ‘Jinhuabao F1’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
27 | Muskmelon cv. ‘Jinmi No.1 F1’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
28 | Muskmelon cv. ‘Xianmangyuan F1’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
29 | Muskmelon cv. ‘Teixuanyanjiaocui’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
30 | Muskmelon cv. ‘Cuixiansu’ | Hebei Maohua Seed Industry Co., Ltd., Cangzhou, China | − | − | − | − | − |
Primer/ Probe | Length (bp) | Sequence (5′–3′) | Purpose |
---|---|---|---|
F3 | 17 | CGGAAGGCGAACAGCAG | LAMP |
B3 | 19 | GGTATCACTGCAGGGTCGA | LAMP |
FIP | 36 | Biotin-GATGCGCTCGTAGCGCAGCCCGCGTCGTAGGCATCA | LAMP |
BIP | 36 | 6-FAM-CCGGGACAGCACAGACAAGTTGCAGTCCACGCCCAC | LAMP |
HP | 18 | GGATGCCGGGACAGCACA | LAMP-LFD |
Seed Carrying Different Bacterial Cells | Seed with Different Infestation Rates | |||
---|---|---|---|---|
Bacterial Cells (CFU·g−1) | Results | Infestation Rate (%) | Frequency (%) (Positive/All Replicates) | Statistical Significance # |
104 | + | 0.1 | 100(10/10) | a |
103 | + | 0.09 | 100(10/10) | a |
102 | + | 0.08 | 90(9/10) | b |
101 | + | 0.07 | 80(8/10) | c |
5 | + | 0.06 | 50(5/10) | d |
2 | − | 0.05 | 20(2/10) | e |
1 | − | 0.04 | 0(0/10) | f |
0.5 | − | 0.03 | 0(0/10) | f |
0.2 | − | 0.02 | 0(0/10) | f |
0.1 | − | 0.01 | 0(0/10) | f |
0 | − | 0 | 0 | / |
Sample Code | Location | Collection Time | Varieties | Number of Samples | Frequency of Positives (%) |
---|---|---|---|---|---|
XP1~XP10 | Ningde Xiapu, China | 2023-7-14 | Watermelon cv. ‘Xinhuanghou’ | 10 | 20.00 |
SM1~SM12 | Sanming Sanyuan, China | 2023-8-9 | Watermelon cv. ‘Heimeireng’ | 12 | 0 |
NP1~NP8 | Nanping Jianyang, China | 2023-8-22 | Muskmelon cv. ‘Balixian’ | 8 | 12.50 |
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Lan, C.; Luo, M.; Gan, L.; Hu, M.; Ruan, H.; Dai, Y.; Liu, X.; Yang, X. Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants. Agronomy 2024, 14, 1804. https://doi.org/10.3390/agronomy14081804
Lan C, Luo M, Gan L, Hu M, Ruan H, Dai Y, Liu X, Yang X. Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants. Agronomy. 2024; 14(8):1804. https://doi.org/10.3390/agronomy14081804
Chicago/Turabian StyleLan, Chengzhong, Minsang Luo, Lin Gan, Meiling Hu, Hongchun Ruan, Yuli Dai, Xiaofei Liu, and Xiujuan Yang. 2024. "Application of Loop-Mediated Isothermal Amplification Assay Combined with Lateral Flow Dipstick (LAMP-LFD) for Specific and Sensitive Detection of Acidovorax citrulli (Schaad et al.) Causing Bacterial Fruit Blotch in Cucurbit Plants" Agronomy 14, no. 8: 1804. https://doi.org/10.3390/agronomy14081804