J Phytopathol 160:755–757 (2012) © 2012 Blackwell Verlag GmbH
doi: 10.1111/jph.12007
Short Communication College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China
Detection and Identification of Group 16SrVI Phytoplasma in Willows in China Lei Zhang1,2*, Zhengnan Li1*, Chao Du3, Zhaohui Fu1 and Yunfeng Wu1 Authors’ addresses: 1State Key Laboratory of Crop Stress Biology in Arid Areas and Key Laboratory of Crop Pest Integrated Pest Management on the Loess Plateau of Ministry of Agriculture, College of Plant Protection, Northwest A&F University, Yangling, 712100, Shaanxi, China; 2College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, China; 3Garden Bureau of Dongsheng District, Ordos, 017000, Inner Mongolia, China (correspondence to Y. Wu. E-mail:
[email protected]) Received July 10, 2012; accepted August 6, 2012 Keywords: willow proliferation, group 16SrVI phytoplasma, China
Abstract In 2010 and 2011, willow proliferation disease was observed in Erdos, Inner Mongolia, China. The phytoplasma-specific 16S rRNA gene fragment of 1.2 kb was amplified by a nested PCR with universal primer pair P1/P7 followed by R16F2n/R2. Phylogenetic and virtual RFLP analyses revealed that the phytoplasma associated with willow proliferation was a member of subgroup 16SrVI-A. The field survey indicated that the incidence of willow proliferation in Erdos was approximately 36.84%. To our knowledge, this is the first record of group 16SrVI phytoplasma infecting willow in China.
Introduction Willow (Salix babylonica L.) is a broadleaf species of the Salicaceae family. A number of phytoplasma-associated willow diseases have been reported worldwide, that is, ball-like structures and small leaves associated with group 16SrXII phytoplasmas in Spain (AlfaroFerna´ndez et al. 2011), yellows associated with group 16SrI phytoplasmas in Shaanxi, China (Wei et al. 2009), and witches’ broom associated with group 16SrVI phytoplasmas in Canada (Khadhair and Hiruki 1995). In the present work, we detect a phytoplasma of group 16SrVI in association with willow proliferation disease in Erdos, Inner Mongolia, China, which is, to our knowledge, the first case reported in China.
Materials and Methods In 2010 and 2011, phytoplasma-like diseases were observed in willows in Erdos. At five locations, where willows are main roadside trees, ten branch samples of
*The first two authors contributed equally to this research.
ten symptomatic trees were collected for examining the phytoplasma-like diseases and those of ten asymptomatic trees were used as negative controls. The incidence of disease was also defined as the ratio of the number of symptomatic willows to the total number of surveyed willows. The total DNA of each sample was extracted by the cetyltrimethyl ammonium bromide (CTAB) method (Kollar et al. 1990) and dissolved in TE buffer for future use. To amplify the phytoplasma-specific 16S rRNA gene, a nested PCR was performed using primer pair P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) followed by R16F2n/R16R2 (Lee et al. 1998). Total DNA of chinaberry witches’ broom (CWB) was used as positive control, and sterilized double-distilled water was used as negative control. The nested PCR products (c. 1.2 kb) from symptomatic samples were purified using a commercial PCR Purification Kit (BioTeke Corporation, Beijing, China), cloned into pMD18-T simple vectors (TaKaRa Bio Inc., Dalian, China) and then transformed into Escherichia coli strain JM109-competent cells. At least five positive clones for each sample were sequenced by TaKaRa Bio Inc. The NCBI BLASTn program was employed to search the homologous sequences to the query sequences. A phylogenetic tree was constructed using the neighbour-joining method with a 1000-replicate bootstrap using MEGA4 (Tamura et al. 2007). Acholeplasma laidlawii JA1 was used as the outgroup. The 16S rRNA sequences obtained in this study and 24 representative strains of group 16SrI, -III, -V, -VI, -X and XII were subjected to the phylogenetic analysis. For virtual RFLP analysis, the 16S rRNA gene sequences and six representative strains of subgroup 16SrVI-A (AY390261), -B (AF036354), -C (AF409069), -D (EF186820), -E (AY270156) and -H (EF651786)
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were in silico digested with 17 restriction endonucleases, namely AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, SspI and TaqI, previously designed for phytoplasma classification (Lee et al. 1993). The RFLP pattern comparison and similarity coefficient calculation were performed using a Perl program developed by Wei et al. (2008).
Results and Discussion The whole or part of branches of the phytoplasmainfected willows had numerous buds in irregular structures (Fig. 1). These buds did not grow into leaves or branches and did not abscise as leaves usually do in winter (Fig. 1b). The same symptom was observed also in the following June or July. We named the disease willow proliferation-Erdos (WiP-E). The incidence of WiP-E was approximately 36.84% (Table 1). A PCR product of 1.2 kb was amplified only from the all WiP-E samples and CWB positive control, but not from the healthy samples or negative control. The sequences derived from the same location were identical, but those from different ones showed a few single-
(a)
(d)
(b)
base mutations. The five sequences shared an identity of 99.73%. These query sequences were determined using NCBI BLASTn program, and the partial 16S rRNA gene sequences from the existing phytoplasmas of group 16SrVI were returned for each query sequence. All had the highest identity with 16S rRNA gene of ‘Candidatus (Ca.) Phytoplasma trifolii’ strain LB002 (accession no. HM745928). The results demonstrated those as belonging to 16S rRNA gene of phytoplasmas. The five sequences of WiP-E were deposited in GenBank (accession numbers JX123319– JX123323). In the phylogenetic tree, which contained the WiPE-associated phytoplasmas and 24 representative strains and A. laidlawii as outgroup, the five WiP-Eassociated phytoplasmas clustered with members of group 16SrVI and were closely related to subgroup 16SrVI-A (Fig. 2). The classification was further confirmed by the virtual RFLP analysis; the RFLP pattern similarity coefficient of the five phytoplasmas was 1.0 with clover proliferation phytoplasma strain CPR (AY390261), a representative strain of subgroup 16SrVI-A (data not shown).
(c)
(e)
(f)
Fig. 1 Phytoplasma-infected willows with proliferative buds. (a) in summer, (b) in winter, (c) a higher magnification of proliferative buds, which did not grow into leaves or branches. (d), (e) and (f) show three typical irregular structures
Occurrence of 16SrVI Phytoplasma in China
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Table 1 Incidence survey of willow proliferation disease Tree age
The total number of willows surveyed
The number of symptomatic willows
Incidence (%)
40 30 20 15 10 –
130 35 269 594 503 1531
129 27 90 196 122 564
99.23 77.14 33.45 32.99 24.25 36.84
Phytoplasmas of groups 16SrI, -II, -V, -XII, -XIV and -XXX have been reported to be plant pathogens in China. Groups 16SrI and -V are important in northern areas of China, and group 16SrII is important in southern areas of China (Li et al. 2012). This, however, is the first record of group 16SrVI phytoplasma in China, which, to our knowledge, has great significance for plant pathology research in China. Acknowledgements This research was supported by the 111 project and the Ph.D. Program Foundation from the Ministry of Education of China (Grant no. B07049 and 20100204110004), National Natural Science Foundation of China (Grant no. 30970133) and PhD Student Academic Newcomer Award of China Ministry of Education (Grant no. Z107021202).
References
Fig. 2 A phylogenetic tree inferred from neighbour-joining analysis of the F2nR2 region of 16S rDNA sequences. Numbers on the tree branches indicate bootstrap values. Acholeplasma laidlawii JA1 was used as the outgroup reference. WiP-E-a, -b, -c, -d and -e (accession numbers JX123319-JX123323) were the sequences of phytoplasmas associated with willow proliferation
Our work revealed that WiP-E is a very serious disease in Erdos and it is associated with phytoplasmas of subgroup 16SrVI-A. Interestingly, the older willows had a higher WiP-E incidence than the younger ones: incidence of 40-year-old willows was 99.23%; for >30year-old ones, 77.14%; for >20-year-old ones, 33.45%; >for 15-year-old ones, 32.99%; and for >10-year-old ones, 24.25% (Table 1). This might be explained by the older trees having more chances to be infected by transmitting insects or their defence may be weak, or that the number of phytoplasmas in the plant host might increase over time. The resistance or tolerance showed by this willow variety is lost with age.
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