Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China
Abstract
1. Introduction
2. Results
2.1. Application of Universal Nested PCR for Phytoplasma Detection
2.2. Nested PCR Primer Test and Combination
2.3. Specificity Validation of Nested PCR
2.4. Optimization of Nested PCR Annealing Temperatures
2.5. Sensitivity Determination of Nested PCR
2.6. Comparison of the Developed Nested PCR to the Universal Nested PCR
2.7. Construction and Analysis of Phylogenetic Trees
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Extraction of Total DNA from Areca Plam Leaf Samples Showing Yellow
4.3. Construction of Recombinant Plasmids for 16S rDNA of Areca Palm Yellow Leaf Phytoplasma
4.4. Design and Primary Screening of Specific Primers
4.5. Specificity Verification of Nested PCR
4.6. Optimization of Annealing Temperatures for Nested PCR with Internal Primers
4.7. Sensitivity Testing of Developed Nested PCR
4.8. Comparison of Developed Nested PCR Method with the Universal Nested PCR Method
4.9. Phylogenetic Analysis of the Fragment Amplified from Primers HNP1F/1R and HNP-2F/2R
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
YLD | yellow leaf disease of areca palm |
APYL | areca palm yellow leaf phytoplasma |
LAMP | loop-mediated isothermal amplification |
qPCR | quantitative PCR |
ddPCR | droplet digital PCR |
Appendix A
No. | Sample | Result | City/County | Year | No. | Sample | Result | City/County | Year |
---|---|---|---|---|---|---|---|---|---|
1 | J069 | Positive | Wenchang | 2022 | 169 | ZB1-2 | Negative | Wenchang | 2022 |
2 | A056 | Positive | Wenchang | 2022 | 170 | MQR4 | Negative | Ledong | 2021 |
3 | T40 | Positive | Wenchang | 2022 | 171 | HL3-4 | Negative | Ledong | 2021 |
4 | W4 | Positive | Wenchang | 2022 | 172 | HL3-1 | Negative | Ledong | 2021 |
5 | S97 | Positive | Wenchang | 2022 | 173 | HL1-2 | Negative | Ledong | 2021 |
6 | M25 | Positive | Wenchang | 2023 | 174 | HZ3-2 | Negative | Ledong | 2021 |
7 | H61 | Positive | Wenchang | 2023 | 175 | HZ5-2 | Negative | Ledong | 2021 |
8 | H64 | Positive | Wenchang | 2023 | 176 | T52 | Negative | Ledong | 2021 |
9 | R71 | Positive | Waning | 2022 | 177 | NY1-2 | Negative | Wenchang | 2022 |
10 | R72 | Positive | Waning | 2022 | 178 | ZB2-2 | Negative | Wenchang | 2022 |
11 | R61 | Negative | Waning | 2022 | 179 | M22 | Negative | Wenchang | 2022 |
12 | R62 | Negative | Waning | 2022 | 180 | M23 | Negative | Wenchang | 2022 |
13 | R63 | Negative | Waning | 2022 | 181 | M25 | Negative | Wenchang | 2022 |
14 | R64 | Negative | Waning | 2022 | 182 | M26 | Negative | Wenchang | 2022 |
15 | R65 | Negative | Waning | 2022 | 183 | M27 | Negative | Wenchang | 2022 |
16 | R66 | Negative | Waning | 2022 | 184 | M30 | Negative | Wenchang | 2022 |
17 | R67 | Negative | Waning | 2022 | 185 | M31 | Negative | Wenchang | 2022 |
18 | R68 | Negative | Waning | 2022 | 186 | L30 | Negative | Wenchang | 2021 |
19 | R69 | Negative | Waning | 2022 | 187 | L32 | Negative | Wenchang | 2021 |
20 | R70 | Negative | Waning | 2022 | 188 | L33 | Negative | Wenchang | 2021 |
21 | R73 | Negative | Waning | 2022 | 189 | S40 | Negative | Wenchang | 2023 |
22 | R74 | Negative | Waning | 2022 | 190 | S41 | Negative | Wenchang | 2023 |
23 | R75 | Negative | Waning | 2022 | 191 | S42 | Negative | Wenchang | 2023 |
24 | R76 | Negative | Waning | 2022 | 192 | S43 | Negative | Wenchang | 2023 |
25 | R77 | Negative | Waning | 2022 | 193 | S44 | Negative | Wenchang | 2023 |
26 | R78 | Negative | Waning | 2022 | 194 | S45 | Negative | Wenchang | 2023 |
27 | R79 | Negative | Waning | 2022 | 195 | S46 | Negative | Wenchang | 2023 |
28 | R80 | Negative | Waning | 2022 | 196 | S47 | Negative | Wenchang | 2023 |
29 | R81 | Negative | Waning | 2022 | 197 | S50 | Negative | Wenchang | 2023 |
30 | R82 | Negative | Waning | 2022 | 198 | W11 | Negative | Wenchang | 2023 |
31 | R83 | Negative | Waning | 2022 | 199 | W12 | Negative | Wenchang | 2023 |
32 | R84 | Negative | Waning | 2022 | 200 | W13 | Negative | Wenchang | 2023 |
33 | R85 | Negative | Waning | 2022 | 201 | W14 | Negative | Wenchang | 2023 |
34 | R86 | Negative | Waning | 2022 | 202 | W15 | Negative | Wenchang | 2023 |
35 | R87 | Negative | Waning | 2022 | 203 | W16 | Negative | Wenchang | 2023 |
36 | R88 | Negative | Waning | 2022 | 204 | W17 | Negative | Wenchang | 2023 |
37 | R89 | Negative | Waning | 2022 | 205 | W18 | Negative | Wenchang | 2023 |
38 | R90 | Negative | Waning | 2022 | 206 | W19 | Negative | Wenchang | 2023 |
39 | W1 | Negative | Waning | 2022 | 207 | W20 | Negative | Wenchang | 2023 |
40 | W2 | Negative | Wenchang | 2022 | 208 | W21 | Negative | Wenchang | 2023 |
41 | W3 | Negative | Wenchang | 2022 | 209 | W22 | Negative | Wenchang | 2023 |
42 | W3-4 | Negative | Wenchang | 2022 | 210 | W23 | Negative | Wenchang | 2023 |
43 | A09 | Negative | Wenchang | 2022 | 211 | W24 | Negative | Wenchang | 2023 |
44 | A12 | Negative | Wenchang | 2022 | 212 | W25 | Negative | Wenchang | 2023 |
45 | A13 | Negative | Wenchang | 2022 | 213 | W26 | Negative | Wenchang | 2023 |
46 | A14 | Negative | Wenchang | 2022 | 214 | W27 | Negative | Wenchang | 2023 |
47 | A16 | Negative | Wenchang | 2022 | 215 | W28 | Negative | Wenchang | 2023 |
48 | A19 | Negative | Wenchang | 2022 | 216 | W29 | Negative | Wenchang | 2023 |
49 | A22 | Negative | Wenchang | 2022 | 217 | W30 | Negative | Wenchang | 2023 |
50 | A24 | Negative | Wenchang | 2022 | 218 | W31 | Negative | Wenchang | 2023 |
51 | A27 | Negative | Wenchang | 2022 | 219 | W32 | Negative | Wenchang | 2023 |
52 | A30 | Negative | Wenchang | 2022 | 220 | W33 | Negative | Wenchang | 2023 |
53 | A31 | Negative | Wenchang | 2022 | 221 | W34 | Negative | Wenchang | 2023 |
54 | A32 | Negative | Wenchang | 2022 | 222 | W35 | Negative | Wenchang | 2023 |
55 | A34 | Negative | Wenchang | 2022 | 223 | W36 | Negative | Wenchang | 2023 |
56 | A36 | Negative | Wenchang | 2022 | 224 | W37 | Negative | Wenchang | 2023 |
57 | A37 | Negative | Wenchang | 2022 | 225 | W38 | Negative | Wenchang | 2023 |
58 | A39 | Negative | Wenchang | 2022 | 226 | W39 | Negative | Wenchang | 2023 |
59 | A49 | Negative | Wenchang | 2022 | 227 | B2-1 | Negative | Baoting | 2021 |
60 | A50 | Negative | Wenchang | 2022 | 228 | B2-2 | Negative | Baoting | 2021 |
61 | A51 | Negative | Wenchang | 2022 | 229 | B2-4 | Negative | Baoting | 2021 |
62 | A55 | Negative | Wenchang | 2022 | 230 | B2-5 | Negative | Baoting | 2021 |
63 | A57 | Negative | Wenchang | 2022 | 231 | B2-6 | Negative | Baoting | 2021 |
64 | A59 | Negative | Wenchang | 2022 | 232 | B2-7 | Negative | Baoting | 2021 |
65 | A60 | Negative | Wenchang | 2022 | 233 | B2-9 | Negative | Baoting | 2021 |
66 | B13 | Negative | Baoting | 2020 | 234 | B4-1 | Negative | Baoting | 2021 |
67 | B4 | Negative | Baoting | 2020 | 235 | B4-2 | Negative | Baoting | 2021 |
68 | B17 | Negative | Baoting | 2020 | 236 | B4-3 | Negative | Baoting | 2021 |
69 | B20 | Negative | Baoting | 2020 | 237 | B4-4 | Negative | Baoting | 2021 |
70 | B21 | Negative | Baoting | 2020 | 238 | B4-5 | Negative | Baoting | 2021 |
71 | B23 | Negative | Baoting | 2020 | 239 | B4-6 | Negative | Baoting | 2021 |
72 | B24 | Negative | Baoting | 2020 | 240 | B4-7 | Negative | Baoting | 2021 |
73 | B25 | Negative | Baoting | 2020 | 241 | B4-8 | Negative | Baoting | 2021 |
74 | B27 | Negative | Baoting | 2020 | 242 | B4-9 | Negative | Baoting | 2021 |
75 | B30 | Negative | Baoting | 2020 | 243 | B4-10 | Negative | Baoting | 2021 |
76 | B31 | Negative | Baoting | 2020 | 244 | B4-11 | Negative | Baoting | 2021 |
77 | B32 | Negative | Baoting | 2020 | 245 | B4-12 | Negative | Baoting | 2021 |
78 | B33 | Negative | Baoting | 2020 | 246 | B4-13 | Negative | Baoting | 2021 |
79 | B37 | Negative | Baoting | 2020 | 247 | B4-14 | Negative | Baoting | 2021 |
80 | B41 | Negative | Baoting | 2020 | 248 | B4-15 | Negative | Baoting | 2021 |
81 | B44 | Negative | Baoting | 2020 | 249 | B4-16 | Negative | Baoting | 2021 |
82 | B46 | Negative | Baoting | 2020 | 250 | B4-17 | Negative | Baoting | 2021 |
83 | B47 | Negative | Baoting | 2020 | 251 | B4-18 | Negative | Baoting | 2021 |
84 | B49 | Negative | Baoting | 2020 | 252 | B4-19 | Negative | Baoting | 2021 |
85 | B53 | Negative | Baoting | 2020 | 253 | B5-4 | Negative | Baoting | 2021 |
86 | B54 | Negative | Baoting | 2020 | 254 | B5-5 | Negative | Baoting | 2021 |
87 | B56 | Negative | Baoting | 2020 | 255 | B5-6 | Negative | Baoting | 2021 |
88 | B57 | Negative | Baoting | 2020 | 256 | B5-10 | Negative | Baoting | 2021 |
89 | B61 | Negative | Baoting | 2020 | 257 | B6-1 | Negative | Baoting | 2021 |
90 | B63 | Negative | Baoting | 2020 | 258 | B6-2 | Negative | Baoting | 2021 |
91 | B76 | Negative | Baoting | 2020 | 259 | B6-4 | Negative | Baoting | 2021 |
92 | C21 | Negative | Qionghai | 2020 | 260 | B6-5 | Negative | Baoting | 2021 |
93 | C26 | Negative | Qionghai | 2020 | 261 | B6-6 | Negative | Baoting | 2021 |
94 | C27 | Negative | Qionghai | 2020 | 262 | B6-8 | Negative | Baoting | 2021 |
95 | C41 | Negative | Qionghai | 2020 | 263 | B6-10 | Negative | Baoting | 2021 |
96 | C47 | Negative | Qionghai | 2020 | 264 | B6-11 | Negative | Baoting | 2021 |
97 | C61 | Negative | Qionghai | 2020 | 265 | F04-2 | Negative | Tunchang | 2020 |
98 | C68 | Negative | Qionghai | 2020 | 266 | F05-2 | Negative | Tunchang | 2020 |
99 | C69 | Negative | Qionghai | 2020 | 267 | F05-1 | Negative | Tunchang | 2020 |
100 | C70 | Negative | Qionghai | 2020 | 268 | F06-1 | Negative | Tunchang | 2020 |
101 | C73 | Negative | Qionghai | 2020 | 269 | F07-1 | Negative | Tunchang | 2020 |
102 | C85 | Negative | Qionghai | 2020 | 270 | F022-1 | Negative | Tunchang | 2020 |
103 | C87 | Negative | Qionghai | 2020 | 271 | F028-2 | Negative | Tunchang | 2020 |
104 | C89 | Negative | Qionghai | 2020 | 272 | F023-2 | Negative | Tunchang | 2020 |
105 | C90 | Negative | Qionghai | 2020 | 273 | F035-1 | Negative | Tunchang | 2020 |
106 | C91 | Negative | Qionghai | 2020 | 274 | F036-1 | Negative | Tunchang | 2020 |
107 | C95 | Negative | Qionghai | 2020 | 275 | F036-2 | Negative | Tunchang | 2020 |
108 | C99 | Negative | Qionghai | 2020 | 276 | F037-1 | Negative | Tunchang | 2020 |
109 | C100 | Negative | Qionghai | 2020 | 277 | F037-2 | Negative | Tunchang | 2020 |
110 | BSL-1 | Negative | Ding’an | 2023 | 278 | F038-1 | Negative | Tunchang | 2020 |
111 | BSL-2 | Negative | Ding’an | 2023 | 279 | F038-2 | Negative | Tunchang | 2020 |
112 | BSL-3 | Negative | Ding’an | 2023 | 280 | F039-1 | Negative | Tunchang | 2020 |
113 | BSL-4 | Negative | Ding’an | 2023 | 281 | F040-1 | Negative | Tunchang | 2020 |
114 | BSL-5 | Negative | Ding’an | 2023 | 282 | F040-2 | Negative | Tunchang | 2020 |
115 | BSL-6 | Negative | Ding’an | 2023 | 283 | F041-2 | Negative | Tunchang | 2020 |
116 | BSL-7 | Negative | Ding’an | 2023 | 284 | F044-1 | Negative | Tunchang | 2020 |
117 | BSL-8 | Negative | Ding’an | 2023 | 285 | F044-2 | Negative | Tunchang | 2020 |
118 | BSL-9 | Negative | Ding’an | 2023 | 286 | F045-1 | Negative | Tunchang | 2020 |
119 | BSL-10 | Negative | Ding’an | 2023 | 287 | F045-2 | Negative | Tunchang | 2020 |
120 | BSL-11 | Negative | Ding’an | 2023 | 288 | F049-2 | Negative | Tunchang | 2020 |
121 | BSL-12 | Negative | Ding’an | 2023 | 289 | F055-2 | Negative | Tunchang | 2020 |
122 | BSL-13 | Negative | Ding’an | 2023 | 290 | F099-1 | Negative | Tunchang | 2020 |
123 | BSL-14 | Negative | Ding’an | 2023 | 291 | C002-1 | Negative | Qionghai | 2020 |
124 | BSL-15 | Negative | Ding’an | 2023 | 292 | C007-2 | Negative | Qionghai | 2020 |
125 | BSL-16 | Negative | Ding’an | 2023 | 293 | C013-2 | Negative | Qionghai | 2020 |
126 | BSL-17 | Negative | Ding’an | 2023 | 294 | C018Y-1 | Negative | Qionghai | 2020 |
127 | BSL-18 | Negative | Ding’an | 2023 | 295 | C018Y-2 | Negative | Qionghai | 2020 |
128 | BSL-19 | Negative | Ding’an | 2023 | 296 | C021-1 | Negative | Qionghai | 2020 |
129 | BSL-20 | Negative | Ding’an | 2023 | 297 | C023-1 | Negative | Qionghai | 2020 |
130 | XS-1 | Negative | Ding’an | 2023 | 298 | C026-1 | Negative | Qionghai | 2020 |
131 | XS-2 | Negative | Ding’an | 2023 | 299 | C026-2 | Negative | Qionghai | 2020 |
132 | XS-3 | Negative | Ding’an | 2023 | 300 | C031-2 | Negative | Qionghai | 2020 |
133 | XS-4 | Negative | Ding’an | 2023 | 301 | C048-1 | Negative | Qionghai | 2020 |
134 | XS-5 | Negative | Ding’an | 2023 | 302 | C064-1 | Negative | Qionghai | 2020 |
135 | XS-6 | Negative | Ding’an | 2023 | 303 | C065-2 | Negative | Qionghai | 2020 |
136 | XS-7 | Negative | Ding’an | 2023 | 304 | C071-2 | Negative | Qionghai | 2020 |
137 | XS-8 | Negative | Ding’an | 2023 | 305 | C072-2 | Negative | Qionghai | 2020 |
138 | XS-9 | Negative | Ding’an | 2023 | 306 | C076-2 | Negative | Qionghai | 2020 |
139 | XS-10 | Negative | Ding’an | 2023 | 307 | C077-2 | Negative | Qionghai | 2020 |
140 | XS-11 | Negative | Ding’an | 2023 | 308 | C078-1 | Negative | Qionghai | 2020 |
141 | LK-1 | Negative | Ding’an | 2023 | 309 | C080-2 | Negative | Qionghai | 2020 |
142 | LK-2 | Negative | Ding’an | 2023 | 310 | C056-2 | Negative | Qionghai | 2020 |
143 | LK-3 | Negative | Ding’an | 2023 | 311 | C065-2 | Negative | Qionghai | 2020 |
144 | LK-4 | Negative | Ding’an | 2023 | 312 | D008-2 | Negative | Ding’an | 2020 |
145 | LK-5 | Negative | Ding’an | 2023 | 313 | D039-1 | Negative | Ding’an | 2020 |
146 | LK-6 | Negative | Ding’an | 2023 | 314 | D062-2 | Negative | Ding’an | 2020 |
147 | LK-7 | Negative | Ding’an | 2023 | 315 | D093-2 | Negative | Ding’an | 2020 |
148 | LK-8 | Negative | Ding’an | 2023 | 316 | ZY2-1 | Negative | Wenchang | 2020 |
149 | LK-9 | Negative | Ding’an | 2023 | 317 | 7-4-2 | Negative | Wenchang | 2020 |
150 | LK-10 | Negative | Ding’an | 2023 | 318 | 003-2 | Negative | Wenchang | 2020 |
151 | LK-11 | Negative | Ding’an | 2023 | 319 | B021-2 | Negative | Baoting | 2020 |
152 | LK-12 | Negative | Ding’an | 2023 | 320 | B027-2 | Negative | Baoting | 2020 |
153 | LK-13 | Negative | Ding’an | 2023 | 321 | B029-2 | Negative | Baoting | 2020 |
154 | LK-14 | Negative | Ding’an | 2023 | 322 | B030-1 | Negative | Baoting | 2020 |
155 | LK-15 | Negative | Ding’an | 2023 | 323 | B032-1 | Negative | Baoting | 2020 |
156 | LK-16 | Negative | Ding’an | 2023 | 324 | B037-2 | Negative | Baoting | 2020 |
157 | LK-17 | Negative | Ding’an | 2023 | 325 | B038-2 | Negative | Baoting | 2020 |
158 | LK-18 | Negative | Ding’an | 2023 | 326 | B042-1 | Negative | Baoting | 2020 |
159 | LK-19 | Negative | Ding’an | 2023 | 327 | B042-2 | Negative | Baoting | 2020 |
160 | LK-20 | Negative | Ding’an | 2023 | 328 | B055-2 | Negative | Baoting | 2020 |
161 | I027 | Negative | Qionghai | 2022 | 329 | B059-1 | Negative | Baoting | 2020 |
162 | N01 | Negative | Lingshui | 2022 | 330 | B059-2 | Negative | Baoting | 2020 |
163 | N06 | Negative | Lingshui | 2022 | 331 | B056-1 | Negative | Baoting | 2020 |
164 | N07 | Negative | Lingshui | 2022 | 332 | B056-2 | Negative | Baoting | 2020 |
165 | N012 | Negative | Lingshui | 2022 | 333 | B061-1 | Negative | Baoting | 2020 |
166 | N013 | Negative | Lingshui | 2022 | 334 | B069-1 | Negative | Baoting | 2020 |
167 | ZH-40 | Negative | Lingshui | 2022 | 335 | B069-2 | Negative | Baoting | 2020 |
168 | NLT | Negative | Lingshui | 2022 |
References
- Kumar, S.N.; Bai, K.V.K.; Rajagopal, V.; Aggarwal, P.K. Simulating coconut growth, development and yield with the infocrop-coconut model. Tree Physiol. 2008, 28, 1049–1058. [Google Scholar] [CrossRef] [PubMed]
- Sun, H.J.; Gong, M. Current development status and countermeasures of Arecanut planting and processing industry in Hainan. Chin. J. Trop. Agric. 2019, 39, 91–94. [Google Scholar]
- Chen, F.; Liu, T.; Li, J.J.; Chen, J.X. The medical value of areca nut. China Trop. Med. 2014, 14, 243–245. [Google Scholar]
- Tang, Q.H.; Meng, X.L.; Yu, S.S.; Lin, Z.W.; Niu, X.Q.; Song, W.W.; Qin, W.Q. Forty years of research on “yellow leaf disease of areca palm” in China: New progress of the causal agent and the management. Chin. J. Trop. Crops 2022, 43, 1010–1022. [Google Scholar]
- Tang, Q.H.; Song, W.W.; Yu, S.S.; Niu, X.Q.; Qin, W.Q. Questions and foresight on the causal agent of Arecanut yellow leaf disease researches and it’s management. Plant Prot. 2021, 47, 6–11. [Google Scholar]
- Abeysinghe, S.; Abeysinghe, P.D.; Kanatiwela-de Silva, C.; Udagama, P.; Warawichanee, K.; Aljafar, N.; Kawicha, P.; Dickinson, M. Refinement of the taxonomic structure of 16SrXI and 16SrXIV phytoplasmas of gramineous plants using multilocus sequence typing. Plant Dis. 2016, 100, 2001–2010. [Google Scholar] [CrossRef] [PubMed]
- Jin, K.X.; Sun, F.S.; Chen, M.R.; Luo, D.Q.; Cai, X.Z. Yellows disease of betel nut palm in Hainan, China. Sci. Silvae Sin. 1995, 31, 556–558+560. [Google Scholar]
- Kanatiwela-de Silva, C.; Damayanthi, M.; de Silva, R.; Dickinson, M.; de Silva, N.; Udagama, P. Molecular and scanning electron microscopic proof of phytoplasma associated with areca palm yellow leaf disease in Sri Lanka. Plant Dis. 2015, 99, 1641. [Google Scholar] [CrossRef]
- Lin, Z.W.; Tang, Q.H.; Meng, X.L.; Song, W.W.; Yu, F.Y.; Huang, S.C.; Niu, X.Q.; Qin, W.Q. Occurrence and distribution of areca pathological yellowing in Hainan, China and Its phytoplasma detection analysis. Chin. J. Trop. Crops 2022, 43, 2106–2113. [Google Scholar]
- Nair, S.; Roshna, O.M.; Soumya, V.P.; Hegde, V.; Suresh Kumar, M.; Manimekalai, R.; Thomas, G.V. Real-Time PCR technique for detection of Arecanut yellow leaf disease phytoplasma. Australas. Plant Pathol. 2014, 43, 527–529. [Google Scholar] [CrossRef]
- Chaithra, M.; Priya, M.; Kumar, S.; Manimekalai, R.; Rao, G. Detection and characterization of 16SrI-B phytoplasmas associated with yellow leaf disease of Arecanut palm in India. Phytopathog. Mollicutes 2015, 4, 77–82. [Google Scholar]
- Sumi, K.; Priya, M.; Kumar, S.; Rao, G.; Rao, K. Molecular confirmation and interrelationship of phytoplasmas associated with diseases of palms in South India. Phytopathog. Mollicutes 2015, 4, 41–52. [Google Scholar] [CrossRef]
- Zhou, Y.K.; Gan, B.C.; Zhang, Z.; Sui, C.; Wei, J.H.; Yang, Y.; Yang, X.Q. Detection of the phytoplasmas associated with yellow leaf disease of Areca catechu L. in Hainan province of China by nested PCR. Chin. Agric. Sci. Bull. 2010, 26, 381–384. [Google Scholar]
- Che, H.Y.; Wu, C.T.; Fu, R.Y.; Wen, Y.S.; Ye, S.B.; Luo, D.Q. Molecular identification of pathogens from Areca nut yellow leaf disease in Hainan. Chin. J. Trop. Crops 2010, 31, 83–87. [Google Scholar]
- Lin, Z.W.; Song, W.W.; Tang, Q.H.; Meng, X.L. First Report of 16SrII group-related phytoplasma associated with areca palm yellow leaf disease on Areca catechu in China. Plant Dis. 2023, 107, 3275. [Google Scholar] [CrossRef]
- Yu, S.S.; Zhu, A.N.; Che, H.Y.; Song, W.W. Molecular identification of ‘Candidatus Phytoplasma Malaysianum’-related strains associated with Areca Catechu palm yellow leaf disease and phylogenetic diversity of the phytoplasmas within the 16SrXXXII group. Plant Dis. 2024, 108, 1331–1343. [Google Scholar] [CrossRef]
- Nayar, R.; Seliskar, C.E. Mycoplasma like organisms associated with yellow leaf disease of Areca Catechu L. For. Pathol. 1978, 8, 125–128. [Google Scholar] [CrossRef]
- Rajeev, G.; Prakash, V.R.; Vaganan, M.M.; Sasikala, M.; Solomon, J.J.; Nair, G.M. Microscopic and polyclonal antibody-based detection of yellow leaf disease of Arecanut (Areca Catechu L.). Arch. Phytopathol. Plant Prot. 2011, 44, 1093–1104. [Google Scholar] [CrossRef]
- Meng, X.L.; Tang, Q.H.; Lin, Z.W.; Niu, X.Q.; Liu, B.; Song, W.W. Developing efficient primers to detect phytoplasmas in areca palms infected with yellow leaf disease. Mol. Plant Breed. 2022, 20, 4624–4633. [Google Scholar]
- Yu, S.S.; Che, H.Y.; Wang, S.J.; Lin, C.L.; Lin, M.X.; Song, W.W.; Tang, Q.H.; Yan, W.; Qin, W.-Q. Rapid and efficient detection of 16SrI group areca palm yellow leaf phytoplasma in China by Loop-Mediated Isothermal Amplification. J. Plant Pathol. 2020, 36, 459–467. [Google Scholar] [CrossRef]
- Yu, S.S.; Zhang, X.; Song, W.W.; Qin, W.Q. Accurate and sensitive detection of areca palm yellow leaf phytoplasma in China by Droplet Digital PCR targeting tuf gene sequence. Ann. Appl. Biol. 2022, 181, 152–159. [Google Scholar] [CrossRef]
- Lin, Z.W.; Tang, Q.H.; Long, S.D.; Niu, X.Q.; Liu, F.; Song, W.W. Establishment of real-time fluorescent quantitative PCR method for detection of areca yellows phytoplasma 16SrI group. Mol. Plant Breed. 2024, 1–15. Available online: https://link.cnki.net/urlid/46.1068.S.20240528.1048.002. (accessed on 28 May 2024).
- Lin, Z.W.; Meng, X.L.; Tang, Q.H.; Niu, X.Q.; Song, W.W. Establishment of TaqMan probe real-time fluorescent quantitative PCR detection method for areca palm yellow leaf phytoplasma. Chin. J. Trop. Crops 2024, 45, 1120–1126. [Google Scholar]
- Fan, X.X.; Zhao, Y.G.; Li, L.; Wu, X.D.; Wang, Z.L. Research progress of recombinant enzyme polymerase amplification (RPA) in rapid detection of diseases. China Anim. Health Insp. 2016, 33, 72–77. [Google Scholar]
- Lu, P.P.; Wu, W.H.; Zheng, J.L.; Wang, G.H.; He, C.P.; Lin, P.Q.; Huang, X.; Liang, Y.Q.; Yi, K.X. Establishment and optimization of single-tube nested PCR detection technique for phytoplasma related to sisal purple leafroll disease. J. Agric. Biotechnol. 2021, 29, 1426–1434. [Google Scholar]
- Lin, Z.W.; Long, S.D.; Meng, X.L.; Tang, Q.H.; Niu, X.Q.; Wang, Y.N.; Song, W.W. Cloning and sequence analysis of tuf, secA and rp genes of areca yellows phytoplasma. Mol. Plant Breed. 2024, 1–15. Available online: https://kns.cnki.net/kcms2/article/abstract?v=mV2q5OJ_OLyzrfmpxyfzS87acE0GRBiYf9aOKKEc7RUYhQrZR-UfrKTU9Fb9xpuHkmi9wzDk6U-e5Tqj6jz8qvRd9XD8WnirZ-3fZmPYnR8eLErnquV-7GC39rVITyHQwIDG7d9kaOZRfbK-grks73pxH__JL2F25h71JYQXpNJCSsM32_EalA==&uniplatform=NZKPT&language=CHS (accessed on 30 April 2024).
- Deng, S.; Hiruki, C. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. J. Microbiol. Methods 1991, 14, 53–61. [Google Scholar] [CrossRef]
- Schneider, B.; Seemüller, E.; Smart, C.D.; Kirkpatrick, B.C. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In Molecular and Diagnostic Procedures in Mycoplasmology, 1st ed.; Razin, S., Tully, J.G., Eds.; Academic Press: San Diego, CA, USA, 1995; Volume 1, pp. 369–380. [Google Scholar]
- Gundersen, D.E.; Lee, I.-M. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol. Mediterr. 1996, 35, 144–151. [Google Scholar]
- Mou, H.Q.; Zhu, S.F.; Xu, X.; Zhao, W.J. An overview of research on phytoplasma-induced diseases. Plant Prot. 2011, 37, 17–22. [Google Scholar]
- Bertaccini, A. Plants and phytoplasmas: When bacteria modify plants. Plants 2022, 11, 1425. [Google Scholar] [CrossRef]
- Wei, W.; Zhao, Y. Phytoplasma taxonomy: Nomenclature, classification, and identification. Biology 2022, 11, 1119. [Google Scholar] [CrossRef] [PubMed]
- Song, X.B.; Huang, F.; Tang, Y.F.; Cui, Y.P.; Ling, J.F.; Chen, X. First report of; little leaf disease caused by phytoplasma on Breynia disticha in China. Plant Prot. 2024, 50, 272–277+292. [Google Scholar]
- Wang, G.; Wu, W.; Tan, S.; Liang, Y.; He, C.; Chen, H.; Huang, X.; Yi, K. Development of a specific nested PCR assay for the detection of 16SrI group phytoplasmas associated with sisal purple leafroll disease in sisal plants and mealybugs. Plants 2022, 11, 2817. [Google Scholar] [CrossRef] [PubMed]
Primer | Primer Sequence (5′-3′) | Annealing Temperature (°C) | Target Fragment Length (bp) | References |
---|---|---|---|---|
P1 | AAGAATTTGATCCTGGCTCAGGATT | 55 | 1800 | [27] |
P7 | CGTCCTTCATCGGCTCTT | [28] | ||
R16mF2 | CATGCAAGTCGAACGGA | 50 | 1500 | [29] |
R16mR1 | CTTAACCCCAATCATCGA | |||
HNP-1F | TTCTTGTTTTTAAAAGACCT | 44 | 1072 | This study |
HNP-1R | AAACTTGCGCTTCAGCT | |||
HNP-2F | TGTGGTCTAAGTGCAAT | 48 | 429 | This study |
HNP-2R | CTGATAACCTCCACTGTGTT | |||
HNP-3F | TTCTTGTTTTTAAAAGACCT | 50 | 837 | This study |
HNP-3R | ATAACCTCCACTGTGTTTCT | |||
HNP-4F | AATGCTCAACATTGTGATGCT | 48 | 652 | This study |
HNP-4R | AAACTTGCGCTTCAGCT |
No | Sample | City/County | Year | No | Sample | City/County | Year |
---|---|---|---|---|---|---|---|
1 | C002-1 | Tunchang | 2020 | 16 | D093-2 | Tunchang | 2020 |
2 | C007-2 | Tunchang | 2020 | 17 | ZY2-1 | Tunchang | 2020 |
3 | C013-2 | Tunchang | 2020 | 18 | 7-4-2 | Tunchang | 2020 |
4 | C018Y-1 | Tunchang | 2020 | 19 | 003-2 | Tunchang | 2020 |
5 | C018Y-2 | Tunchang | 2020 | 20 | B021-2 | Tunchang | 2020 |
6 | C021-1 | Tunchang | 2020 | 21 | B027-2 | Tunchang | 2020 |
7 | C023-1 | Tunchang | 2020 | 22 | B029-2 | Tunchang | 2020 |
8 | C026-1 | Tunchang | 2020 | 23 | B030-1 | Tunchang | 2020 |
9 | C026-2 | Tunchang | 2020 | 24 | B032-1 | Tunchang | 2020 |
10 | C031-2 | Tunchang | 2020 | 25 | B037-2 | Tunchang | 2020 |
11 | C042-1 | Tunchang | 2020 | 26 | B038-2 | Tunchang | 2020 |
12 | C064-1 | Tunchang | 2020 | 27 | B042-1 | Tunchang | 2020 |
13 | 12-3-2 | Tunchang | 2020 | 28 | B042-2 | Tunchang | 2020 |
14 | D039-1 | Tunchang | 2020 | 29 | B055-2 | Tunchang | 2020 |
15 | D062-2 | Tunchang | 2020 | 30 | B059-1 | Tunchang | 2020 |
Strain | Phytoplasma Identified | GenBank Accession No | Host Plant | Location |
---|---|---|---|---|
J069 | ‘Ca. P. asteris’ 16SrI | PV760294 | Areca palm Areca catechu | Wenchang, Hainan |
S97 | ‘Ca. P. asteris’ 16SrI | PV760296 | Areca palm A. catechu | Wenchang, Hainan |
T40 | ‘Ca. P. asteris’ 16SrI | PV760293 | Areca palm A. catechu | Wenchang, Hainan |
H-9 | ‘Ca. P. asteris’ 16SrI | PV760299 | Periwinkle Catharanthus roseus | Wenchang, Hainan |
A056 | ‘Ca. P. australasiae’ 16SrII | PV760298 | Areca palm A. catechu | Wenchang, Hainan |
W4 | ‘Ca. P. australasiaticum’ 16SrII | PV760297 | Areca palm A. catechu | Wenchang, Hainan |
SHM-1 | ‘Ca. P. malaysianum’ 16SrXXXII | PV759645 | Trema tomentosa | Qionghai, Hainan |
MW1-1 | Burkholderia andropogonis | PV759631 | Areca palm A. catechu | Wenchang, Hainan |
I027 | Areca palm velarivirus 1 | OQ423126 | Areca palm A. catechu | Wenchang, Hainan |
TC-1 | Pantoea ananatis | PV759632 | Areca palm A. catechu | Tunchang, Hainan |
Reference Strain | GenBank Accession Number | Application |
---|---|---|
‘Ca. P. asteris’ 16SrI | FJ998269 | Primer Design/Phylogenetic analysis |
‘Ca. P. asteris’ 16SrI | FJ694685 | Primer Design/Phylogenetic analysis |
‘Ca. P. australasaticum’ 16SrII | OQ586085 | Primer Design |
Areca catechu chloroplast | NC_050163 | Primer Design |
Burkholderia andropogonis | NR_104960 | Primer Design |
Pantoea ananatis | MW174802 | Primer Design |
Chrysophyllum albidum | LC110196 | Primer Design |
Curtobacterium citreum | MF319766 | Primer Design |
Curtobacterium luteum | JX437941 | Primer Design |
Sphingomonas yantingensis | MF101149 | Primer Design |
Bacillus cereus (Robbsia andropogonis) | HQ833025 | Primer Design |
Staphylococcus epidermidis | CP040883 | Primer Design |
Xanthomonas sacchari | MN889285 | Primer Design |
Xanthomonas campestris | JX415480 | Primer Design |
‘Ca. P. aurantifolia’ WBDL 16SrII | U15442 | Primer Design |
‘Ca. P. australasiae’ PpYC 16SrII | Y10097 | Primer Design |
‘Ca. P. pruni’ PX11CT1 rrnA 16SrIII | JQ044393 | Primer Design |
‘Ca. P. ziziphi’ JWB-G1 16SrV | AB052876 | Primer Design |
‘Ca. P. ulmi’ EY1 16SrV | AY197655 | Primer Design |
‘Ca. P. rubi’ RuS 16SrV | AY197648 | Primer Design |
‘Ca. P. trifolii’ CP 16SrVI | AY390261 | Primer Design |
‘Ca. P. sudamericanum’ PassWB-Br3 16SrVI | GU292081 | Primer Design |
‘Ca. P. fraxini’ AshY1 16SrVII | AF092209 | Primer Design |
‘Ca. P. luffae’ LfWB rrnA 16SrVIII | AF248956 | Primer Design |
‘Ca. P. luffae’ LfWB rrnB 16SrVIII | AF353090 | Primer Design |
‘Ca. P. phoenicium’ AlmWB 16SrIX | AF515636 | Primer Design |
‘Ca. P. pyri’ PD1 16SrX | AJ542543 | Primer Design |
‘Ca. P. spartii’ SpaWB 16SrX | X92869 | Primer Design |
‘Ca. P. mali’ AP15 16 SrX | AJ542541 | Primer Design |
‘Ca. P. oryzae’ RYD-Th 16SrXI | AB052873 | Primer Design |
‘Ca. P. cirsii’ CirYS 16SrXI | KR869146 | Primer Design |
‘Ca. P. solani’ STOL11 16SrXII | AF248959 | Primer Design |
‘Ca. P. japonicum’ JPH 16SrXII | AB010425 | Primer Design |
‘Ca. P. convolvuli’ BY-S5711 16SrXII | JN833705 | Primer Design |
‘Ca. P. hispanicum’ MPV 16SrXIII | AF248960 | Primer Design |
‘Ca. P. meliae’ ChTY-Mo3 16SrXIII | KU850940 | Primer Design |
‘Ca. P. cynodontis’ BGWL-C1 16SrXIV | AJ550984 | Primer Design |
‘Ca. P. brasiliense’ HibWB26 16SrXV | AF147708 | Primer Design |
‘Ca. P. graminis’ SCYLP 16SrXVI | AY725228 | Primer Design |
‘Ca. P. caricae’ PAY 16SrXVII | AY725234 | Primer Design |
‘Ca. P. castaneae’ CnWB 16SrXIX | AB054986 | Primer Design |
‘Ca. P. omanense’ IM-1 16SrXXIX | EF666051 | Primer Design |
‘Ca. P. costaricanum’ SoyST1c1 16SrXXXI | HQ225630 | Primer Design |
‘Ca. P. malaysianum’ MaPV 16SrXXXII | OP237525 | Primer Design |
‘Ca. P. malaysianum’ MaPV 16SrXXXII | EU371934 | Primer Design |
‘Ca. P. wodyetiae’ FPYD Bangi-2 16SrXXXVI | KC844879 | Primer Design |
Acholeplasma laidlawii | CP000896 | Phylogenetic analysis |
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Ge, H.; Meng, X.; Lin, Z.; Jan, S.; Song, W.; Qin, W.; Tang, Q.; Zhu, X. Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China. Plants 2025, 14, 2144. https://doi.org/10.3390/plants14142144
Ge H, Meng X, Lin Z, Jan S, Song W, Qin W, Tang Q, Zhu X. Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China. Plants. 2025; 14(14):2144. https://doi.org/10.3390/plants14142144
Chicago/Turabian StyleGe, Huiyuan, Xiuli Meng, Zhaowei Lin, Saad Jan, Weiwei Song, Weiquan Qin, Qinghua Tang, and Xiaoqiong Zhu. 2025. "Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China" Plants 14, no. 14: 2144. https://doi.org/10.3390/plants14142144
APA StyleGe, H., Meng, X., Lin, Z., Jan, S., Song, W., Qin, W., Tang, Q., & Zhu, X. (2025). Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China. Plants, 14(14), 2144. https://doi.org/10.3390/plants14142144