Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-11-13-1183-PDN • posted 01/29/2014 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
Page 1 of 3
1
Disease Note
2
First Report of a new subgroup 16Sr II- M Candidatus Phytoplasma aurantifolia
3
associated with witches’- broom disease of Tephrosia purpurea in India
4
Amit Yadav Ψ, Microbial Culture Collection, National Centre for Cell Science, Pune
5
411007, India; Udhav Bhale, Department of Botany, Arts, Science and Commerce
6
College, Naldurg, Osmanabad - 413602, India; Vipool Thorat and Yogesh Shouche,
7
Microbial Culture Collection, National Centre for Cell Science, Pune 411007, India.
8
Ψ
9
25708237; Fax: +91-20-25692259.
Corresponding author: Amit Yadav; e-mail:
[email protected]; Phone: +91-20-
10
Keywords: Candidatus Phytoplasma aurantifolia, 16Sr II group, Tephrosia purpurea,
11
Wild Indigo.
12
Wild Indigo (Tephrosia purpurea (L.) Pers.) grows as a common weed throughout the
13
Indian subcontinent. The plant has pinnate leaves, white or purplish flowers and flat
14
hairy pods and is cultivated as a green manure crop. The plant extracts contain
15
compounds, such as Tephrosin (1), an aromatic ester, prenylated flavonoid and
16
Sesquiterpene (2) that have medicinal properties. The newly recognized disease,
17
Tephrosia purpurea whites’ broom (TPWB), was characterized by chlorosis, stunting
18
and proliferative branching which were suggestive of phytoplasma infection during a
19
field survey conducted in November 2013. To determine the presence of
20
phytoplasma, 2 g of compound leaves from three symptomatic and asymptomatic
21
plants (Figure 1) were used for total DNA extraction using the CTAB method (3). The
22
phytoplasma 16S rRNA gene was detected in all three symptomatic plants using
23
nested PCR with universal phytoplasma primer pairs, P1/P7 followed by
24
R16F2n/R16R2 (4). No amplification was observed in DNA isolated from
25
asymptomatic plants. PCR fragments (1246 bp in length) generated from
26
symptomatic Tephrosia purpurea plants were sequenced directly using five different
27
primers viz. 343R, 536F, 704F, 907R and 1103F (5). TPWB phytoplasma 16S rRNA
28
gene sequence (GenBank accession number, HG792252) showed 99.12 % homology
29
with a Candidatus Phytoplasma aurantifolia strain WBDL (U15442) when compared
Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-11-13-1183-PDN • posted 01/29/2014 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
Page 2 of 3
30
using the EzTaxon 16S rRNA database (6). Virtual Restriction Fragment Length
31
Polymorphism (RFLP) analysis was carried out on the obtained sequence using
32
iPhyClassifier (7). The virtual RFLP pattern derived from the HG792252 sequence was
33
different to the reference patterns of previously established 16Sr groups and
34
subgroups. The reference pattern of the 16Sr group II, subgroup C (AJ293216) was
35
most similar with a similarity coefficient of 0.92, which placed it in a new subgroup,
36
16Sr II-M (8). Furthermore, virtual RFLP results were confirmed by digesting
37
R16F2n/R16R2 amplicon with BstUI, DraI, HinfI, HpaI and MseI restriction enzymes
38
according to manufacturer's instructions. To our knowledge, this is the first report of
39
a Candidatus Phytoplasma aurantifolia- related strain associated with witches’
40
broom disease of Tephrosia purpurea in India.
41
References: (1) Ahmad, V. U. (1999). Fitoterapia, 70: 443- 445. (2) Khalafalah, A. K. et
42
al., (2010). Pharmacognosy Res. 2(2): 72–75. (3) Doyle, J. and Doyle, J. (1990). Focus,
43
12: 13-15. (4) Smart, C. et al., (1996). Appl. and Environ. Microbiol., 62: 2988-2993.
44
(5) Baker et al., (2003). J. Microbiol. Meth., 55: 541– 555. (6) Kim et al. 2012. Int. J.
45
Syst. Evol. Microbiol. 62: 716–721. (7) Zhao, Y. et al. (2009). Int. J. Syst. Evol.
46
Microbiol., 59: 2582-2593. (8) Cai, H. et al. (2008). Int. J. Syst. Evol. Microbiol., 58 (6):
47
1448- 1457.
Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-11-13-1183-PDN • posted 01/29/2014 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
Page 3 of 3
The excised branch of Tephrosia purpurea plant showing typical witches’ broom symptoms after infection of Candidatus Phytoplasma aurantifolia at Naldurg, India (A), asymptomatic branch of Tephrosia purpurea plant (B). 400x190mm (96 x 96 DPI)
