Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Nematode Populations
2.2. DNA Extraction
2.3. RAPD-PCR-Analysis
2.4. SCAR Primer Design
2.5. Specificity Test of SCAR Primer
2.6. SCAR Primer Sensitivity Test
2.7. Duplex PCR Amplification
2.8. Direct Detection of H. schachtii in Artificially Inoculated Plant Roots and Soil
2.9. Direct Detection from Naturally Infected Roots and Soil Samples
3. Results
3.1. RAPD-PCR and Sequences Analysis
3.2. Primers Design and Species-Specific Test
3.3. Sensitivity Test
3.4. Direct Detection of H. schachtii in Inoculated Plant Roots and Soil
3.5. Direct Detection of Naturally Infected Root and Soil Samples
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | Species Code | Population Origin | Host | References |
---|---|---|---|---|
Heterodera schachtii | Hs1 | Germany | Sugar beet | [27] |
Hs2 | Xinyuan county Xinjiang, China | This study | ||
Hs3 | Xinyuan county Xinjiang, China | |||
Hs4 | Xinyuan county Xinjiang, China | |||
Hs5 | Xinyuan county Xinjiang, China | |||
Hs6 | Belgium a | [13] | ||
Hs7 | Bozova, Anliurfa Province, Turkey | [28] | ||
Hs8 | Karakprü, Anliurfa Province, Turkey | |||
Hs9 | Siverek, Anliurfa Province, Turkey | |||
H. glycines | Hg01 | Langfang, Hebei, China | Soybean | [23] |
Hg02 | Heilongjiang, China | Soybean | ||
Hg03 | Henan, China | Soybean | ||
H. sojae | Hso | Jiangxi China | Soybean | [29] |
H. cruciferae | Hc | Hubei, China | Oilseed rape | This study |
H. avenae | Ha | Baxing, Beijing, China | Wheat | [30] |
H. filipjevi | Hf | Xuchang, Henan, China | Wheat | [31] |
H. latipons | Hl | Belgium a | Wheat | [32] |
H. elachista | He | Hunan, China | Rice | [33] |
H. amaranthusiae n. sp | Ham | Xuanwei, Yunnan, China | Amaranthus retroflexus | [34] |
H. zeae | Hz | Henan, China | Maize | [35] |
Globodera rostochiensis | Gr | Belgium a | Potato | [36] |
G. pallida | Gp | Belgium a | Potato | |
Cactodera cacti | Cc | Liaoning, China | Weeds | [37] |
Meloidogyne enterolobii | Me | Wenchang, Hainan, China | Tomato | [38] |
M. graminicola | Mg | TikeGyi, Yangon, Myanmar | Rice | [39] |
M. incognita | Mi | Langfang Hebei, China | Tomato | [38] |
M. hapla | Mh | Beijing, China | Rose | [38] |
Ditylenchus Destructor | Dd | Hebei, China | Sweet potato | [40] |
Primer Name | Sequence 5′-3′ | Types | Production Sizes | References |
---|---|---|---|---|
OPA-01 | 5′-CAGGCCCTTC-3′ | RAPD primers | Random, uncertain | [9,23,41] |
OPA-02 | 5′-TGCCGAGCTG-3′ | |||
OPA-03 | 5′-AGTCAGCCAC-3′ | |||
OPA-04 | 5′-AATCGGGCTG-3′ | |||
OPA-05 | 5′-AGGGGTCTTG-3′ | |||
OPA-06 | 5′-GGTCCCTGAC-3′ | |||
OPA-09 | 5′-GGGTAACGCC-3′ | |||
OPA-13 | 5′-CAGCACCCAC-3′ | |||
OPA-18 | 5′-AGGTGACCGT-3′ | |||
OPB-15 | 5′-GGAGGGTGTT-3′ | |||
OPC-06 | 5′-AAGACCCCTC-3′ | |||
OPD-13 | 5′-GGGGTGACGA-3′ | |||
OPG-06 | 5′-GTGCCTAACC-3′ | |||
OPG-08 | 5′-TCACGTCCAC-3′ | |||
OPK-16 | 5′-GAGCGTCGAA-3′ | |||
D2A | 5′-ACAAGTACCGTGAGGGAAAGTTG-3′ | 28S-rDNA universal primers | 780 bp | [16] |
D3B | 5′-TCGGAAGGAACCAGCTACTA-3′ | |||
TW81 | 5’-GTT TCC GTA GGT GAA CCT GC-3′ | rDNA-ITS universal primers | 1027–1045 bp | [42] |
AB28 | 5’-ATA TGC TTA AGT TCA GCG GGT-3′ | |||
SHF6 | 5′-GTTCTTACGTTACTTCCA-3′ | Specific primer | 255 bp | [12] |
rDNA2 | 5′-TTTCACTCGCCGTTACTAAGG-3′ | rDNA-ITS universal primer | ||
OPA06-HsF | 5′-GGACCCTGACGACCAGAATA-3′ | SCAR primers | 922 bp | This study |
OPA06-HsR | 5′-GACAACACGAAGGAGCGAGC-3′ |
Treatments | Mixed Populations | Detection Results |
---|---|---|
Group 1 | H. schachtii, H. glycines, H. sojae, H. avenae and H. flipjevi | + |
Group 2 | H. schachtii, G. rostochiensis and G. pallida | + |
Group 3 | H. schachtii, M. enterolobii, M. incognita, M. hapla and M. graminicola | + |
Group 4 | H. schachtii, D. ditylenchus | + |
Group 5 | H. glycines, H. avenae, H. flipjevi, G. pallida and G. rostochiensis | − |
Group 7 | M. enterolobii, M. incognita, M. hapla and M. graminicola | − |
Group 8 | D. ditylenchus | − |
Positive Control | H. schachtii | + |
Samples | Host | Location | GPS | Nematode Density in Root a | Cyst Density b | Detection Results | ||
---|---|---|---|---|---|---|---|---|
SCAR-PCR | PCR | ITS-Sequencing | ||||||
1 | Sugar beet | Huocheng County-1, Xinjiang | N44°4′7″, E80°44′36″ | 0 | 0 | - | - | N/A |
2 | Huocheng County-2, Xinjiang | N44°2′43″, E80°50′7″ | 0 | 0 | - | - | N/A | |
3 | Qapqal County-1, Xinjiang | N43°45′50″, E80°59′34″ | 0 | 4 | - | - | H. avenae | |
4 | Qapqal County-2, Xinjiang | N43°46′26″, E81°2′6″ | 0 | 0 | - | - | N/A | |
5 | Qapqal County-3, Xinjiang | N43°45′40″, E81°9′18″ | 0 | 0 | - | - | N/A | |
6 | Xinyuan County-1, Xinjian | N43°27′09″, E 82°59′56″ | 22 ± 7 | 132 | + | + | H. schachtii | |
7 | Xinyuan County-2, Xinjian | N43°29′55”, E83°59′25” | 44 ± 6 | 144 | + | + | H. schachtii | |
8 | Xinyuan County-3, Xinjian | N43°44′67”, E83°06′39” | 12 ± 3 | 287 | + | + | H. schachtii | |
9 | Xinyuan County-4, Xinjian | N43°31′04”, E83°11′13” | 23 ± 7 | 92 | + | + | H. schachtii | |
10 | Xinyuan County-5, Xinjian | N42°88′3252”, E83°39′710” | 33 ± 5 | 54 | + | + | H. schachtii | |
11 | Emin County-1, Xinjiang | N46°46′460”, E83°53′902” | 8 ± 3 | 11 | + | + | H. schachtii | |
12 | Emin County-2, Xinjiang | N46°46′468”, E83°53′949” | 45 ± 11 | 64 | + | + | H. schachtii | |
13 | Keyouqian Banner, Inner Mongolia | N/A | 0 | 3 | - | - | H. avenae | |
14 | Urad Front Banner, Inner Mongolia | N/A | 0 | 12 | - | - | H. glycines | |
15 | Linxi County, Inner Mongolia | N/A | 0 | 9 | - | - | H. avenae H. glycines |
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Jiang, C.; Zhang, Y.; Yao, K.; Abdulsalam, S.; Li, G.; Gao, H.; Li, K.; Huang, W.; Kong, L.; Peng, D.; et al. Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples. Life 2021, 11, 1358. https://doi.org/10.3390/life11121358
Jiang C, Zhang Y, Yao K, Abdulsalam S, Li G, Gao H, Li K, Huang W, Kong L, Peng D, et al. Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples. Life. 2021; 11(12):1358. https://doi.org/10.3390/life11121358
Chicago/Turabian StyleJiang, Chen, Yingdong Zhang, Ke Yao, Sulaiman Abdulsalam, Guangkuo Li, Haifeng Gao, Kemei Li, Wenkun Huang, Lingan Kong, Deliang Peng, and et al. 2021. "Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples" Life 11, no. 12: 1358. https://doi.org/10.3390/life11121358