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A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
Journal of Integrative Agriculture 2014, 13(8): 1707-1718
August 2014
RESEARCH ARTICLE
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group LI Zheng-nan1, 2*, ZHANG Lei1, 3*, TAO Ye1, CHI Ming1, 2, XIANG Yu2 and WU Yun-feng1 1
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, P.R.China 2 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Center, Summerland, British Columbia, V0H 1Z0, Canada 3 College of Forestry, Northwest A&F University, Yangling 712100, P.R.China
Abstract A novel phytoplasma was detected in a cherry plum (Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies (PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 µm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16S ribosomal RNA (rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows (AY) group (16SrI group). Virtual and actual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group (16SrI) by four restriction sites, Hinf I, Mse I, Sau3A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf (CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group (16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle (Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder (Cuscuta odorata) connections. Key words: phytoplasma, cherry plum yellow leaf, CPYL, phytoplasma-like bodies, 16S rRNA, restriction fragment length polymorphism, dodder-bridged transmission
INTRODUCTION Phytoplasmas are non-helical and cell-wall-less bacteria, inhabiting and propagating in both host plants and
insect vectors (Gasparich 2010). In natural conditions, phytoplasmas are mainly transmitted by phloem-feeding insects such as leafhoppers, planthoppers and psyllids (Weintraub and Beanland 2006), inducing a range of symptoms in host plants, like stunting of stems, proliferation of auxiliary shoots (witches’-broom), yellowing of
Received 4 March, 2013 Accepted 5 September, 2013 LI Zheng-nan, E-mail: [email protected]; ZHANG Lei, E-mail: [email protected]; Correspondence WU Yun-feng, Tel/Fax: +86-29-87092716, E-mail: [email protected] * These authors contributed equally to this study. © 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60600-0
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leaves, formation of sterile-deformed flowers, greening of floral tissues (virescence) and forming of leaf-like flower organs (phyllody). Since phytoplasmas have just been successfully cultured under axenic conditions (Contaldo et al. 2012), their diagnosis still mainly depends on molecular analyses of several conserved genes like 16S ribosomal RNA (rRNA), ribosomal protein (rp) and elongation factor TU (tuf) (Wei et al. 2007a). Phytoplasmas were assigned to a provisional genus, ‘Candidatus Phytoplasma’ within the class Mollicutes by the International Research Programme on Comparative Mycoplasmology (IRPCM) Phytoplasma/Spiroplasma Working Team in 2004 (IRPCM 2004), and a total of 26 phytoplasma species were proposed according to discrepancies in 16S rRNA gene sequences (IRPCM 2004). Lee et al. (1993) proposed to classify phytoplasmas based on restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S rRNA gene sequences with 17 arbitrarily designated restriction enzymes and this method has been used to differentiate a broad array of phytoplasmas (Lee et al. 1993, 1998, 2000, 2004). Recently, computer-simulated RFLP analysis of the 16S rRNA gene sequence was developed, including in silico restriction enzyme digestions, virtual RFLP pattern comparisons and calculation of similarity coefficients of the virtual RFLP patterns (Wei et al. 2007a, 2008; Zhao et al. 2009). Using the computcer-based approach, 28 phytoplasma 16S ribosomal (16Sr) groups and more than 50 subgroups have been proposed. Cherry plum (Prunus cerasifera Ehrh) is widely cultivated in China for its sweet stone fruits. For plants of genus Prunus, many phytoplasma-related diseases have been reported worldwide. In Europe, the European stone fruit yellows (ESFY) phytoplasmas caused plum leptonecrosis in Japanese plum (Prunus salicina L.) and European plum (Prunus domestica L.) (Giunchedi et al. 1978; Dosba et al. 1991; Lorenz et al. 1994). Peach yellows (Jones et al. 1974) and peach rosette (Kirkpatrick et al. 1975) phytoplasmas were detected in plum trees in different areas of the world. And phytoplasmas of peanut witches’ -broom and stolbur groups induced symptoms of little leaf, leaf rolling, rosetting and yellowing in plum trees in Iran (Zirak et al. 2009). Moreover, in a recent survey of fruit tree diseases in northwest China, we observed cherry plum diseases with symptoms resembling phytoplasma infections in several orchards located in the Yangling District, Shaanxi Province, China. One disease, plum leaf
roll (PLR), exhibited symptoms of leaf rolling and proliferation, and the associated phytoplasma was identified as a member of phytoplasma 16SrV group (Hong et al. 2011). In the present report, we identify a novel phytoplasma strain in the aster yellows (AY) group (16SrI) in a cherry plum tree mainly with yellow leaf symptom.
RESULTS Phytoplasma-like bodies observed in leaf tissues of the symptomatic cherry plum tree A cherry plum tree mainly with unusual yellow leaf symptom was observed in a cherry plum orchard in spring 2009, located in Yangling District, Shaanxi ProvS ince, China. Yellow leaf symptom presents throughout a tree branch and the number of infected branches appeared to increase over time (Fig. 1-A and B). The leaves started with chlorosis and yellowed in leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. The yellow leaves were smaller than the normal leaves. Some leaves became curled or necrotic, died on the branch or dropped prematurely over the next few weeks. The flowers in the infected branches appeared normal in the spring, but the fruits were small, immature and dropped from the trees ahead of harvest. Large numbers of typical phytoplasma-like bodies (PLBs) were found under transmission electron microscope (TEM) in the phloem cells of leaf tissues from the symptomatic tree. The PLBs were mostly spherical or elliptical vesicles in different sizes, ranging from 0.1 to 0.6 µm in diameter, with no cell wall-like structure (Fig. 1-C). The yellowing symptom in the tree and the phytoplasma-like morphology in the phloem cells resembled phytoplasma infections (Siddique et al. 1998), implying that the yellow leaf symptoms in the cherry plum tree are probably correlated to a phytoplasma-associated disease. We tentatively termed the symptoms as cherry plum yellow leaf (CPYL) in this report.
A novel phytoplasma 16S rRNA gene sequence detected in CPYL A 1 246-bp PCR fragment was amplified from all three © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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Fig. 1 Images of symptomatic cherry plum trees and the phytoplasma-like bodies. A, a cherry plum tree with yellow leaf symptoms. B, a close look of the pale yellow leaves in A. YL, yellow leaf; asm, asymptomatic leaf. C, phytoplasma-like bodies (PLBs) in two sieve element (SE) cells from the leaf tissue exhibiting yellow leaf symptom. The two SE cells were separated by a sieve plate (sp). A few PLBs were marked with arrows in the image. Scale bar=0.5 µm.
DNA samples from the leaves with CPYL symptoms but not from the leaves of the three asymptomatic plum samples (data not shown). DNA sequencing revealed that the 1 246-bp PCR fragments were identical (GenBank accession number HM131809). A search of NCBI nucleotide sequence databases using the BLASTn program retrieved 99 nucleotide sequences sharing high identity with HM131809 (E-value 0.0, 99% identity). All retrieved sequences were 16S rRNA gene sequences of the phytoplasmas of AY group (16SrI), with the majority from 16SrI-B subgroup (data not shown). Comparison of HM131809 to the 16S rRNA gene sequences (F2nR2 regions) of phytoplasmas (Wei et al. 2007b) of 16SrI group ranged from 98.31 to 99.4% identity. When HM131809 was compared with 28 representative sequences of other phytoplasma groups, the percent identity varied from 88.4 to 96.31% (Table 1). These data indicated that the 16S rRNA gene sequences detected in the symptomatic cherry plum tree are of phytoplasma origin and probably belong to the AY group (16SrI). A maximum-parsimony phylogenetic tree was constructed with HM131809 and the 16S rRNA gene sequences (F2nR2 region) from 28 phytoplasma groups as well as 19 subgroups of 16SrI group. The phylogenetic tree showed that HM131809 clustered with the 19 subclades of the AY group (16SrI) phytoplasmas, confirming that the CPYL associated phytoplasma (CPYLaP) is a member of the AY group (16SrI) (Fig. 2). However, the CPYLaP rRNA sequence also displayed a phylogeneticoally distinct position from all reported subgroup lineages
within the AY group in the tree topology (Fig. 2). It suggests that CPYLaP is probably a novel subgroup lineage in the AY group (16SrI). By a computer-based program (Wei et al. 2008), we carried out virtual RFLP analysis with the CPYLaP 16S rRNA gene sequence (HM131809) using 17 distinct restriction enzymes previously used to classify phytoplasmas (Lee et al. 1993). The virtual RFLP patterns of the HM131809 based on in silico restriction enzyme digestions were displayed in Fig. 3-A. The four RFLP patterns of HM131809 produced by restriction enzymes Hinf I, Mse I, Sau3A I and Taq I varied compared to the patterns of the previously identified 19 subgroups of 16SrI group (Wei et al. 2008; Gámez-Jiménez et al. 2009; Santos-Cervantes et al. 2010). The existence of the four unique RFLP patterns of the CPYLaP was confirmed by actual enzymatic digestion analysis (Fig. 3-B). The similarity coefficients of the virtual RFLP pattern of HM131809 were calculated and varied between 0.73 and 0.87 with the patterns of 19 sequences of reported 16SrI subgroups (Table 2), which is below the suggested upper threshold of 0.97 for delineating a new subgroup (Wei et al. 2008). It indicates that the CPYLaP detected here is a distinctly novel strain in the AY phytoplasma group (16SrI group). Transmission of CPYLaP via dodder bridges. An attempt was made to inoculate CPYLaP from the symptomatic cherry plum tree to healthy periwinkle plants (Catharanthus roseus) using a dodder-mediated transmission method (Marcone et al. 1997). Six healthy dodder-hosted periwinkle plants were physically
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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Table 1 16S rRNA gene sequence identities between the sweet plum yellow leaf associated phytoplasma (CPYLaP) and related phytoplasmas Group & Subgroup 16SrI-A 16SrI-B 16SrI-B 16SrI-C 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V 16SrII 16SrIII 16SrIII 16SrIV 16SrV 16SrV 16SrV-B 16SrV-B 16SrVI 16SrVII 16SrVIII 16SrIX 16SrX 16SrXI 16SrXII 16SrXII 16SrXIII 16SrXIV 16SrXV 16SrXVI 16SrXVII 16SrXVIII 16SrXIX 16SrXX 16SrXXI 16SrXXII 16SrXXIII 16SrXXIV 16SrXXV 16SrXXVI 16SrXXVII 16SrXXVIII
Acronyms
Associated disease or phytoplasma strain
AY-WB AY27 PlumLL CPhA CPhB PaWB BBS3 ACLR-AY CY STRAWB2 AV2192 AVUT LOWB 98UW166B AY-Populus CherLL StrawbPhF PeLL Toll PPT-JAL6 PPT-SON18 PnWB WX PeRP CLY EY PLR CLY5 PY-In CP AshY LfWB PPWB AP RYD STOL PJ15 I Mpv BGWL HWB ScYL PBT PPT CnWB BAWB Pin127 LDN BVGY SBS WTWB D3T1 D3T2 DerP
Aster yellows phytoplasma strain AY-WB Aster yellows-AY27 Plum little leaf Clover phyllody strain rrnA Clover phyllody strain rrnB Paulownia witches’-broom Blueberry stunt phytoplasma strain BBS3 Apricot chlorotic leaf roll Chrysanthemum yellows Phytoplasma sp. STRAWB2 Aster yellows phytoplasma strain AV2192 Aster yellows phytoplasma strain AVUT Aster yellows phytoplasma strain IOWB Aster yellows phytoplasma O isolate 98UW166B Aster yellows phytoplasma I-P Cherry little leaf Strawberry phylloid fruit Pepper little leaf Tomato little leaf Mexican potato purple top phytoplasma strain JAL6 Mexican potato purple top phytoplasma strain SON18 Peanut witches’-broom Western X phytoplasma Peach rosette Coconut lethal yellowing Elm yellow Plum leaf roll Cherry lethal yellows Peach yellows Clover proliferation Ash yellows Loofah witches’-broom Pigeon pea witches’-broom Apple proliferation Rice yellow dwarf Stolbur Iranian plum phytoplasma Mexican periwinkle virescence Bermuda grass white leaf Hibiscus witches’-broom Sugarcane yellow leaf Papaya bunchy top Nebraska potato purple top Candidatus Phytoplasma castaneae Mollicutes (from R. frangula) Candidatus Phytoplasma pini Phytoplasma sp. strain LDN Buckland valley grapevine yellows Sorghum bunchy shoot Weeping tea witches’-broom Sugar cane phytoplasma satrain D3T1 Sugar cane phytoplasma strain D3T2 Derbid phytoplasma
connected to the symptomatic cherry plum tree via the haustoria of the dodders (Cuscuta odorata). After about 2 wk’s transmission, the dodder bridges were removed. Then in the 9th wk, two of the six plants started to develop visible symptoms. Another six periwinkle plants were connected to asymptomatic cherry plum trees,
GenBank accession no. AY389828 AY180952 GU289674 AF222065 AF222066 AY265206 AY265213 AY265211 EF634457 U96616 AY180957 AY265209 AY265205 AF268405 AF503568 AY034089 AY102275 DQ092321 DQ375238 FJ914650 FJ914642 L33765 L04682 AF236121 U18747 AF189214 FJ459914 AY197659 AY197660 AY390261 X68339 AF353090 AF248957 X68375 D12581 X76427 FJ409624 AF248960 AJ550984 AF147708 AY725228 AY725234 DQ174122 AB054986 X76431 AJ632155 Y14175 AY083605 AF509322 AF521672 AJ539179 AJ539180 AY744945
16S rDNA Identity to CPYLaP (HM131809) (%) 98.88 99.40 99.28 98.80 98.80 99.00 98.88 98.48 98.96 98.31 99.20 99.28 99.20 98.48 98.80 98.96 98.88 98.48 98.80 98.64 98.48 89.04 89.74 89.98 90.08 89.99 89.99 89.99 90.15 90.01 89.77 90.41 88.4 91.89 89.50 95.67 95.51 95.83 89.82 89.11 93.21 91.6 95.51 88.62 91.27 89.68 89.29 96.31 89.98 88.68 92.96 93.19 95.00
and did not develop any visible disease symptom. The symptomatic periwinkle plants not only showed signs of leaf chlorosis and distortion, but also showed small leaves, shoot proliferation, stunted stems and phyllody (Fig. 4-A and B). Then another attempt of transmission of CPYLaP © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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Fig. 2 Phylogenetic tree constructed by parsimony analysis of the 16S rRNA gene fragments from the phytoplasma associated with CPYL (HM131809) and 53 other phytoplasmas. Numbers on tree branches indicate bootstrap values (expressed as percentages of 1 000 replications). Bar length indicates one substitution in 100 nt. Abbreviations of the phytoplasmas with GenBank accession numbers are defined in the Table 1. Group and subgroup affiliations of the phytoplasma strains are indicated in brackets.
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
LI Zheng-nan et al.
B Hinf I Sau3A I Mse I Taq I MW
C
D MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
MW
MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
MW Alu I BamH I Bfa I BstU I Dra I EcoR I Hae III Hha I Hinf I Hpa I
A
Hpa II Kpn I Sau3A I Mse I Rsa I Ssp I Taq I MW
1712
MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
E
Sau3A I
Mse I
F MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
Hinf I
Taq I
Fig. 3 Restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene product amplified from primer pairs R16F2n/R2 of the phytoplasma associated with CPYL (HM131809). A, virtual RFLP patterns derived from in silico digestions using 17 restriction enzymes: Alu I, BamH I, Bfa I, BstU I (Tha I), Dra I, EcoR I, Hae III, Hha I, Hinf I, Hpa I, Hpa II, Kpn I, Sau3A I (Mbo I), Mse I, Rsa I, Ssp I and Taq I. B, actual RFLP profiles in 2% agarose gel after digestions of the 1.2-kb nested PCR products amplified from primer pairs R16F2n/R2 with four restriction enzymes: Hinf I, Sau3A I (Mbo I), Mse I and Taq I. MW, φX174DNA-Hae III markers. Only fragments that are equal to or larger than 50 bp are shown. C to F, comparison of the virtual digestion patterns between HM131809 and 19 reported 16SrI subgroups by Hinf I, Mse I, Sau3A I (Mbo I) and Taq I.
from symptomatic periwinkle plants to a healthy 1-yrold cherry plum was also made by dodder bridges. The symptomatic periwinkle plant, which has been infected by CPYLaP via the dodder bridges, was set near the healthy cherry plum tree in insect-proof greenhouse,
and some vines were artificially carefully twisted on the young branches of cherry plum tree to help connect. After approximately 3 wk, the periwinkle-dodder-cherry plum bridge was removed. And after another 10 wk, the young cherry plum tree developed symptoms of
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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Table 2 Similarity coefficients derived from analysis of virtual RFLP patterns of 16S rRNA genes from the representative phytoplasma strains in the aster yellow group (16SrI) Strain/GenBank accession
1
1
Serial
AY389828(16SrI-A)
1.00
2
3
4
5
2
AY180952(16SrI-B)
0.92 1.00
3
AF222066(16SrI-C)
0.90 0.92 1.00
4
AY265206(16SrI-D)
0.91 0.97 0.89 1.00
5
AY265213(16SrI-E)
0.91 0.93 0.91 0.90 1.00
6
7
6
AY265211 (16SrI-F)
0.86 0.88 0.88 0.85 0.87 1.00
7
EF634457(16SrI-H)
0.89 0.97 0.89 0.94 0.90 0.85 1.00
8
9
8
U96616 (16SrI-K)
0.86 0.86 0.86 0.85 0.90 0.90 0.83 1.00
9
AY180957(16SrI-L)
0.89 0.97 0.89 0.94 0.90 0.85 0.94 0.83 1.00
10
11
10
AY265209(16SrI-M)
0.92 1.00 0.92 0.97 0.93 0.88 0.97 0.86 0.97 1.00
11
AY265205(16SrI-N)
0.92 1.00 0.92 0.97 0.93 0.88 0.97 0.86 0.97 1.00 1.00
12
13
12
AF268405(16SrI-O)
0.86 0.87 0.80 0.85 0.80 0.74 0.84 0.80 0.83 0.87 0.87 1.00
13
AF503568(16SrI-P)
0.92 0.94 0.94 0.91 0.93 0.94 0.91 0.92 0.91 0.94 0.94 0.80 1.00
14
15
14
AY034089(16SrI-Q)
0.84 0.92 0.84 0.89 0.85 0.86 0.89 0.78 0.89 0.92 0.92 0.80 0.86 1.00
15
AY102275(16SrI-R)
0.91 0.93 0.93 0.90 0.92 0.87 0.90 0.85 0.90 0.93 0.93 0.80 0.93 0.89 1.00
16
17
16
DQ092321 (16SrI-S)
0.88 0.96 0.88 0.93 0.90 0.84 0.93 0.83 0.93 0.96 0.96 0.85 0.90 0.90 0.90 1.00
17
DQ375238(16SrI-T)
0.85 0.83 0.85 0.90 0.87 0.81 0.91 0.80 0.90 0.93 0.93 0.82 0.87 0.87 0.87 0.97 1.00
18
19
20
18
FJ914650 (16SrI-U)
0.87 0.93 0.87 0.90 0.87 0.81 0.91 0.80 0.90 0.93 0.93 0.84 0.87 0.87 0.88 0.97 0.94 1.00
19
FJ914642(16SrI-V)
0.85 0.93 0.85 0.90 0.87 0.81 0.92 0.80 0.92 0.93 0.93 0.82 0.87 0.87 0.87 0.97 0.94 0.94 1.00
20
HM131809
0.79 0.87 0.81 0.84 0.80 0.75 0.86 0.73 0.86 0.87 0.87 0.75 0.81 0.85 0.82 0.85 0.82 0.84 0.84 1.00
chlorosis and yellowing alongside leaf veins and edges (Fig. 4-D and E). A ~1.2-kb nested PCR product was respectively amplified using the primer pairs R16mF2/R16mR1 and R16F2n/R16R2 from the DNA samples extracted from the symptomatic periwinkle plants and the young cherry plum tree. Asymptomatic plants, including the controls did not produce a similar PCR product using the nested primer pairs (data not shown). When digested with restriction enzymes Hinf I, Mse I, Sau3A I and Taq I, respectively, the ~1.2-kb DNA fragment showed identical RFLP patterns with the CPYLaP 16S rRNA gene (Fig. 3-B, data not shown), indicating that the CPYLaP was transmitted to the periwinkle plants from the naturally infected cherry plum tree, and also from the symptomatic periwinkle plant back to the healthy young cherry plum tree.
DISCUSSION The AY group (16SrI) is the largest and the most diverse phytoplasma group. It includes over 100 identified isolates that are widespread in many places of the world and infect a broad range of plant species (Marcone et al. 2000; Santos-Cervantes et al. 2010). In total 19 subgroups including 16SrI-A to -F, -H, and -K to -V have
been identified in the AY group based on results of RFLP analysis of the 16S rRNA gene sequence (Marcone et al. 2000; IRPCM 2004; Wei et al. 2007a; Gámez-Jiménez et al. 2009; Santos-Cervantes et al. 2010). It has been suggested that a new subgroup would be recognized if only one restriction site differed within the 16S rRNA gene F2nR2 region, or if the new phytoplasma strain’s RFLP pattern of the 16S rRNA gene F2nR2 region had a similarity coefficient of 0.97 or lower with each known subgroups (Lee et al. 1998; Wei et al. 2008). The CPYLaP could be easily distinguished from all existing 19 subgroups in the AY group (16SrI) by four restriction sites (Hinf I, Mse I, Sau3A I and Taq I) within its 16S rRNA gene F2nR2 region (Fig. 3); its similarity coefficient of the RFLP pattern ranges varied from 0.73 to 0.87 with those of the 19 described subgroups (Table 2). The results suggested that CPYLaP is probably a previously un-described subgroup lineage in the AY group (16SrI). Phylogenetic analysis of the 16S rRNA genes indicated that the CPYLaP has a cladistically similar ancestor to the 19 subgroups within the AY group (16SrI) phytoplasmas, but it does not form a common subclade with any of the 19 subgroup lineages (Fig. 2). In addition, query of the CPYLaP 16S rRNA gene sequence using an interactive online phytoplasma classification tool, iPhyClassifier (http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier. html, Zhao et al. 2009), also concludes that CPYLaP © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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Fig. 4 Periwinkle (Catharanthus roseus) plants infected with CPYLaP at 11 wk after dodder connections with the cherry plum tree with the yellow leaf symptoms and young cherry plum tree infected by CPYLaP from the symptomatic periwinkle at 13 wk after dodder connections. A, symptoms of leaf chlorosis, distortion, small leaves and stunting in periwinkle. B, symptoms of phyllody, shoot proliferation and small leaves produced on new growth of shoots and leaves in periwinkle. C, a healthy periwinkle plant. D, construction of the periwinkle-dodder-young cherry plum tree bridge. E, symptoms of chlorosis and yellowing alongside leaf veins and edges in the young cherry plum tree.
probably represents a new subgroup in the AY group (16SrI) (data not shown). Since most previous efforts to isolate infectious phytoplasmas on artificial media have not been successful, it is difficult to confirm the etiology of phytoplasma-caused diseases by fulfilling Koch’s postulates. Nonetheless the detection of the CPYLaP by TEM, PCR and the positive transmission of phytoplasma to periwinkle plants then back to the young cherry plum tree suggested that CPYLaP was a probable causal agent of the cherry plum yellow leaf symptoms. Phytoplasma-like bodies (PLBs) were the only type of microbe detected in the yellow leaf tissues and the PLBs were primarily distributed in the phloem cells (Fig. 1-C). No other pathogenic agents such as viruses, fungi or other bacteria were observed in the tissues. The transmission of CPYLaP to perih winkle plants from the naturally infected plum tree was confirmed by PCR and RFLP analysis. The symptoms in the periwinkle plants included not only leaf chlorosis and distortion, but also shoot proliferation, small leaf, stunting stem and phyllody in the newly growing organs which were not observed in the symptomatic cherry plum tree (Fig. 4-A and B). Additionally, the young cherry plum tree was successfully infected by the CPYLaP in
periwinkle plants via dodder bridges, and developed the same symptoms with that naturally infected cherry plum tree (Fig. 4-D and E). RFLP analysis of the PCR fragments amplified from the dodder-mediated infected periwinkle plants and the young cherry plum tree indicated that only CPYLaP was identified in the plants. Many phytoplasmas have been experimentally transmitted to periwinkle plants from their naturally infected herbaceous or woody plant hosts by dodder-bridged transmissions and often caused visible symptoms in the periwinkles. However, the periwinkles, when infected with phytoplasmas from wild hosts, did not always exhibit the exact same symptoms as showed in their wild hosts (Marcone et al. 1997, 1999; Kamińska and Korbin 1999; Kamińska et al. 2001) . Recently, we reported that plum leaf roll (PLR) disease was associated with a phytoplasma belonging to elm yellows (16SrV) group (Hong et al. 2011). The PLR disease was found in cherry plum trees in an ornamental garden in Yangling, Shaanxi, China. The garden is approximately 3 km away from the other plum orchards where the CPYLaP was discovered. SympL toms and symptom development in PLR are different from CPYL. PLR showed leaf rolling/curling and leaf
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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proliferation symptoms and it took longer for leaves to become chlorotic compared to CPYL. The 16S rRNA gene sequence of the PLR-associated phytoplasma has a high homology and shares an identical RFLP pattern to the jujube witches’-broom phytoplasma (JWB). JWB is a widespread phytoplasma strain infecting jujube trees in Northwest China (Hong et al. 2011). Our experimental result confirmed that the PLR-associated phytoplasma was not detectable in the CPYL tissues (Data not shown). The CPYLaP is the first report of an AY group (16SrI) phytoplasma infecting cherry plum (P. cerasifera Ehrh) trees in China. In addition, to our knowledge, a phytoplasma of 16SrI-B subgroup, plum little leaf phytoplasma strain PlumLL (GenBank accession number GU289674) was recently identified in Lithuania in European plum (P. domestica L.) trees with symptoms of proliferation of shoots and abnormally small leaves (Valiunas et al. 2009). Plum leptonecrosis (PLN) in Japanese plum (P. salicina L.) and European plum in Europe was caused by EFSY phytoplasma which contained mainly 16SrX-B and also a number of other phytoplasmas belonging to 16SrX-A, 16SrI-B, 16SrIII and 16SrV groups (Lee et al. 1995). EFSY phytoplasmas produce a complex disease syndrome, such as early bud burst, leaf rolling, leaf chlorosis, deformed fruits, phloem necrosis and plant decline in plum, peach, apricot, almond and sweet cheery (Carraro et al. 1998). Phytoplasmas belonging to 16SrII and 16SrXII were recently detected in Japanese plum trees with symptoms of little leaf, leaf rolling, rosetting and yellowing in Iran (Zirak et al. 2009). Collectively, these data show that Prunus spp. are apparently susceptible to a number of phytoplasma lineages of broad genetic diversities and geographical distributions (Gámez-Jiménez et al. 2009).
transmission back to healthy young cherry plum tree according to Koch’s postulates. Subsequent molecular analyses of the 16S rRNA gene sequence suggested that the CPYL-associated phytoplasma (CPYLaP) is probably a distinct phytoplasma strain belonging to AY group (16SrI).
CONCLUSION
Total DNA was extracted from plant leaf samples using a cetyltrimethyl ammonium bromide (CTAB) based method (Angelini et al. 2001; Deng et al. 2012). Briefly, 0.15 g of leaf tissue was ground in 500 μL of preheated CTAB extraction buffer with 0.2% β-mercaptoethanol in a 1.5-mL micro-centrifuge tube. The CTAB extraction buffer consisted of 2% (w/v) CTAB, 1.4 mol L-1 NaCl, 20 mmol L-1 EDTA (pH 8.0) and 100 mmol L-1 Tris-HCl (pH 8.0). The treated tissue was incubated at 60°C for 30 min and extracted with chloroform-isoamyl alcohol (24:1). The aqueous DNA layer was precipitated with isopropanol, washed with 70% ethanol
In this report, we described a cherry plum tree with yellow leaf symptom growing in a plum orchard located in the Yangling District, Shaanxi Province, China. We provided evidence that the symptomatic tree was associated with a phytoplasma based on TEM observations, positive PCR results, RFLP analysis, the dodder-mediated transmission to periwinkle plants and followed
MATERIALS AND METHODS Plant materials Cherry plum (P. cerasifera Ehrh) leaf samples mainly with or without yellow leaf symptom was collected from a plum orchard located in Yangling District, Shaanxi Province, China in June 2009. Dodder (Cuscuta odorata) and periwinkle (Catharanthus roseus) plants were grown from seeds in an insect-proof greenhouse. The healthy 1-yr-old cherry plum tree (P. cerasifera Ehrh) was kindly provided by the College of Horticulture, Northwest A&F University, China.
Transmission electron microscope (TEM) Fresh cherry plum leaf tissues with or without yellow leaf symptoms were immediately fixed in 0.1% cacodylate buffer (pH 7.2) containing 3% (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde at 4°C for 4 h. After that, the samples were post-fixed in 1% (w/v) osmium tetroxide for 2 h at room temperature. Then the fixed samples were dehydrated in concentration gradients of ethanol (10-70%) or acetone (0-100%) and embedded in Epson 812 resin. Ultra-thin sections (70 nm) were cut with a diamond knife and mounted onto nickel grids. The grids were doubly stained with uranyl acetate and lead citrate, and then examined under TEM (JEM1200EXII, JEOL) (Siddique et al. 1998).
Nucleic acid extraction and PCR detection of phytoplasmas
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and resuspended in 30 μL of sterile deionized water. A nested PCR method was used to amplify the 16S rRNA gene fragment of phytoplasmas as previously described (Wu et al. 2010) using two phytoplamsa universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2 (Lee et al. 1993; Gundersen and Lee 1996). PCR products were separated by electrophoresis in a 1% agarose gel.
DNA cloning, sequencing, sequence analysis and phylogenetic analysis Nested PCR products were purified using the 1% agarose gel, cloned into pMD18-T simple vector (TaKaRa Bio, Japan) and sequenced from both 5´- and 3´-end by automatic DNA sequencing (TaKaRa Bio., Japan). Nucleotide sequences were analyzed using LaserGene (DNAStar, Madison, USA). The National Center for Biotechnology (NCBI) BLASTn program (http://blast.ncbi.nlm.nih.gov/) was used to search against the NCBI nucleotide collection (nr/nt) database and align the query sequences with 16S rRNA gene sequences from other phytoplasmas in the database (Ye et al. 2006). Phylogenetic trees were constructed by neighbor-joining method with a 1 000-replicate bootstrap search using MEGA4 (Tamura et al. 2007) .
Virtual RFLP pattern analysis and actual enzymatic digestion analysis In silico digestion analysis of the 16S rRNA gene sequences with the 17 restriction endonucleases previously designed for phytoplasma classification (Lee et al. 1993), virtual RFLP pattern identification and the similarity coefficient calculation were performed with a Perl program developed by Wei et al. (2008). Actual enzymatic digestion analyses were performed by digesting gel-purified PCR products of the nested PCR amplifications using four restriction endonucleases, Hinf I, Mse I, Sau3A I and Taq I in separate reactions. Digestion products were separated by electrophoresis in a 2% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator.
Dodder transmission Dodder transmission of the phytoplasmas from naturally infected cherry plum tree to healthy periwinkle plants or from infected periwinkle plants to healthy young cherry plum tree was adapted from a previously reported method (Marcone et al. 1999). Dodder was first established on healthy periwinkle by planting dodder seeds with young periwinkle plants in pots
(25 cm in diameter) in an insect-free greenhouse for about 4 wk. The pots containing the dodder-parasitic periwinkle plants were moved adjacent to the donor plum trees in the plum orchard where the yellow leaf symptoms were found. The dodders from the periwinkles were wrapped around the branch-tips of both symptomatic and asymptomatic cherry plum trees, creating a plum tree-dodder-periwinkle bridge. The bridge was kept for approximately 2 wk under insect-proof netting before the dodder-parasitic periwinkle plants were removed from the plum trees and returned to the greenhouse to allow for symptom development. When the periwinkle plants showed symptoms, a symptomatic one was placed next to a healthy young cherry plum tree for creating a periwinkle-dodder-plum tree bridge. The bridge was kept for approximately three weeks before the tree was separated from the dodder-parasitic periwinkle plant and still grew in the greenhouse to allow for symptom development. Total DNA was extracted from infected and uninfected periwinkle plants and the young cherry plum tree using the CTAB-based method (Angelini et al. 2001). Nested PCR amplification using primer pairs R16mF2/R16mR1 and R16F2n/R16R2 was performed as described above. Actual enzymatic RFLP analysis of PCR products from dodder transmitted DNA samples were also performed as described previously in this report.
Acknowledgements
This work was supported by the 111 Project from the Ministry of Education of China (B07049), the PhD Program Foundation from the Ministry of Education of China (20100204110004) and the National Natural Science Foundation of China (31371913).
References
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A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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August 2014
RESEARCH ARTICLE
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group LI Zheng-nan1, 2*, ZHANG Lei1, 3*, TAO Ye1, CHI Ming1, 2, XIANG Yu2 and WU Yun-feng1 1
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, P.R.China 2 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Center, Summerland, British Columbia, V0H 1Z0, Canada 3 College of Forestry, Northwest A&F University, Yangling 712100, P.R.China
Abstract A novel phytoplasma was detected in a cherry plum (Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies (PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 µm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16S ribosomal RNA (rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows (AY) group (16SrI group). Virtual and actual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group (16SrI) by four restriction sites, Hinf I, Mse I, Sau3A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf (CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group (16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle (Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder (Cuscuta odorata) connections. Key words: phytoplasma, cherry plum yellow leaf, CPYL, phytoplasma-like bodies, 16S rRNA, restriction fragment length polymorphism, dodder-bridged transmission
INTRODUCTION Phytoplasmas are non-helical and cell-wall-less bacteria, inhabiting and propagating in both host plants and
insect vectors (Gasparich 2010). In natural conditions, phytoplasmas are mainly transmitted by phloem-feeding insects such as leafhoppers, planthoppers and psyllids (Weintraub and Beanland 2006), inducing a range of symptoms in host plants, like stunting of stems, proliferation of auxiliary shoots (witches’-broom), yellowing of
Received 4 March, 2013 Accepted 5 September, 2013 LI Zheng-nan, E-mail: [email protected]; ZHANG Lei, E-mail: [email protected]; Correspondence WU Yun-feng, Tel/Fax: +86-29-87092716, E-mail: [email protected] * These authors contributed equally to this study. © 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60600-0
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leaves, formation of sterile-deformed flowers, greening of floral tissues (virescence) and forming of leaf-like flower organs (phyllody). Since phytoplasmas have just been successfully cultured under axenic conditions (Contaldo et al. 2012), their diagnosis still mainly depends on molecular analyses of several conserved genes like 16S ribosomal RNA (rRNA), ribosomal protein (rp) and elongation factor TU (tuf) (Wei et al. 2007a). Phytoplasmas were assigned to a provisional genus, ‘Candidatus Phytoplasma’ within the class Mollicutes by the International Research Programme on Comparative Mycoplasmology (IRPCM) Phytoplasma/Spiroplasma Working Team in 2004 (IRPCM 2004), and a total of 26 phytoplasma species were proposed according to discrepancies in 16S rRNA gene sequences (IRPCM 2004). Lee et al. (1993) proposed to classify phytoplasmas based on restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S rRNA gene sequences with 17 arbitrarily designated restriction enzymes and this method has been used to differentiate a broad array of phytoplasmas (Lee et al. 1993, 1998, 2000, 2004). Recently, computer-simulated RFLP analysis of the 16S rRNA gene sequence was developed, including in silico restriction enzyme digestions, virtual RFLP pattern comparisons and calculation of similarity coefficients of the virtual RFLP patterns (Wei et al. 2007a, 2008; Zhao et al. 2009). Using the computcer-based approach, 28 phytoplasma 16S ribosomal (16Sr) groups and more than 50 subgroups have been proposed. Cherry plum (Prunus cerasifera Ehrh) is widely cultivated in China for its sweet stone fruits. For plants of genus Prunus, many phytoplasma-related diseases have been reported worldwide. In Europe, the European stone fruit yellows (ESFY) phytoplasmas caused plum leptonecrosis in Japanese plum (Prunus salicina L.) and European plum (Prunus domestica L.) (Giunchedi et al. 1978; Dosba et al. 1991; Lorenz et al. 1994). Peach yellows (Jones et al. 1974) and peach rosette (Kirkpatrick et al. 1975) phytoplasmas were detected in plum trees in different areas of the world. And phytoplasmas of peanut witches’ -broom and stolbur groups induced symptoms of little leaf, leaf rolling, rosetting and yellowing in plum trees in Iran (Zirak et al. 2009). Moreover, in a recent survey of fruit tree diseases in northwest China, we observed cherry plum diseases with symptoms resembling phytoplasma infections in several orchards located in the Yangling District, Shaanxi Province, China. One disease, plum leaf
roll (PLR), exhibited symptoms of leaf rolling and proliferation, and the associated phytoplasma was identified as a member of phytoplasma 16SrV group (Hong et al. 2011). In the present report, we identify a novel phytoplasma strain in the aster yellows (AY) group (16SrI) in a cherry plum tree mainly with yellow leaf symptom.
RESULTS Phytoplasma-like bodies observed in leaf tissues of the symptomatic cherry plum tree A cherry plum tree mainly with unusual yellow leaf symptom was observed in a cherry plum orchard in spring 2009, located in Yangling District, Shaanxi ProvS ince, China. Yellow leaf symptom presents throughout a tree branch and the number of infected branches appeared to increase over time (Fig. 1-A and B). The leaves started with chlorosis and yellowed in leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. The yellow leaves were smaller than the normal leaves. Some leaves became curled or necrotic, died on the branch or dropped prematurely over the next few weeks. The flowers in the infected branches appeared normal in the spring, but the fruits were small, immature and dropped from the trees ahead of harvest. Large numbers of typical phytoplasma-like bodies (PLBs) were found under transmission electron microscope (TEM) in the phloem cells of leaf tissues from the symptomatic tree. The PLBs were mostly spherical or elliptical vesicles in different sizes, ranging from 0.1 to 0.6 µm in diameter, with no cell wall-like structure (Fig. 1-C). The yellowing symptom in the tree and the phytoplasma-like morphology in the phloem cells resembled phytoplasma infections (Siddique et al. 1998), implying that the yellow leaf symptoms in the cherry plum tree are probably correlated to a phytoplasma-associated disease. We tentatively termed the symptoms as cherry plum yellow leaf (CPYL) in this report.
A novel phytoplasma 16S rRNA gene sequence detected in CPYL A 1 246-bp PCR fragment was amplified from all three © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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Fig. 1 Images of symptomatic cherry plum trees and the phytoplasma-like bodies. A, a cherry plum tree with yellow leaf symptoms. B, a close look of the pale yellow leaves in A. YL, yellow leaf; asm, asymptomatic leaf. C, phytoplasma-like bodies (PLBs) in two sieve element (SE) cells from the leaf tissue exhibiting yellow leaf symptom. The two SE cells were separated by a sieve plate (sp). A few PLBs were marked with arrows in the image. Scale bar=0.5 µm.
DNA samples from the leaves with CPYL symptoms but not from the leaves of the three asymptomatic plum samples (data not shown). DNA sequencing revealed that the 1 246-bp PCR fragments were identical (GenBank accession number HM131809). A search of NCBI nucleotide sequence databases using the BLASTn program retrieved 99 nucleotide sequences sharing high identity with HM131809 (E-value 0.0, 99% identity). All retrieved sequences were 16S rRNA gene sequences of the phytoplasmas of AY group (16SrI), with the majority from 16SrI-B subgroup (data not shown). Comparison of HM131809 to the 16S rRNA gene sequences (F2nR2 regions) of phytoplasmas (Wei et al. 2007b) of 16SrI group ranged from 98.31 to 99.4% identity. When HM131809 was compared with 28 representative sequences of other phytoplasma groups, the percent identity varied from 88.4 to 96.31% (Table 1). These data indicated that the 16S rRNA gene sequences detected in the symptomatic cherry plum tree are of phytoplasma origin and probably belong to the AY group (16SrI). A maximum-parsimony phylogenetic tree was constructed with HM131809 and the 16S rRNA gene sequences (F2nR2 region) from 28 phytoplasma groups as well as 19 subgroups of 16SrI group. The phylogenetic tree showed that HM131809 clustered with the 19 subclades of the AY group (16SrI) phytoplasmas, confirming that the CPYL associated phytoplasma (CPYLaP) is a member of the AY group (16SrI) (Fig. 2). However, the CPYLaP rRNA sequence also displayed a phylogeneticoally distinct position from all reported subgroup lineages
within the AY group in the tree topology (Fig. 2). It suggests that CPYLaP is probably a novel subgroup lineage in the AY group (16SrI). By a computer-based program (Wei et al. 2008), we carried out virtual RFLP analysis with the CPYLaP 16S rRNA gene sequence (HM131809) using 17 distinct restriction enzymes previously used to classify phytoplasmas (Lee et al. 1993). The virtual RFLP patterns of the HM131809 based on in silico restriction enzyme digestions were displayed in Fig. 3-A. The four RFLP patterns of HM131809 produced by restriction enzymes Hinf I, Mse I, Sau3A I and Taq I varied compared to the patterns of the previously identified 19 subgroups of 16SrI group (Wei et al. 2008; Gámez-Jiménez et al. 2009; Santos-Cervantes et al. 2010). The existence of the four unique RFLP patterns of the CPYLaP was confirmed by actual enzymatic digestion analysis (Fig. 3-B). The similarity coefficients of the virtual RFLP pattern of HM131809 were calculated and varied between 0.73 and 0.87 with the patterns of 19 sequences of reported 16SrI subgroups (Table 2), which is below the suggested upper threshold of 0.97 for delineating a new subgroup (Wei et al. 2008). It indicates that the CPYLaP detected here is a distinctly novel strain in the AY phytoplasma group (16SrI group). Transmission of CPYLaP via dodder bridges. An attempt was made to inoculate CPYLaP from the symptomatic cherry plum tree to healthy periwinkle plants (Catharanthus roseus) using a dodder-mediated transmission method (Marcone et al. 1997). Six healthy dodder-hosted periwinkle plants were physically
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Table 1 16S rRNA gene sequence identities between the sweet plum yellow leaf associated phytoplasma (CPYLaP) and related phytoplasmas Group & Subgroup 16SrI-A 16SrI-B 16SrI-B 16SrI-C 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V 16SrII 16SrIII 16SrIII 16SrIV 16SrV 16SrV 16SrV-B 16SrV-B 16SrVI 16SrVII 16SrVIII 16SrIX 16SrX 16SrXI 16SrXII 16SrXII 16SrXIII 16SrXIV 16SrXV 16SrXVI 16SrXVII 16SrXVIII 16SrXIX 16SrXX 16SrXXI 16SrXXII 16SrXXIII 16SrXXIV 16SrXXV 16SrXXVI 16SrXXVII 16SrXXVIII
Acronyms
Associated disease or phytoplasma strain
AY-WB AY27 PlumLL CPhA CPhB PaWB BBS3 ACLR-AY CY STRAWB2 AV2192 AVUT LOWB 98UW166B AY-Populus CherLL StrawbPhF PeLL Toll PPT-JAL6 PPT-SON18 PnWB WX PeRP CLY EY PLR CLY5 PY-In CP AshY LfWB PPWB AP RYD STOL PJ15 I Mpv BGWL HWB ScYL PBT PPT CnWB BAWB Pin127 LDN BVGY SBS WTWB D3T1 D3T2 DerP
Aster yellows phytoplasma strain AY-WB Aster yellows-AY27 Plum little leaf Clover phyllody strain rrnA Clover phyllody strain rrnB Paulownia witches’-broom Blueberry stunt phytoplasma strain BBS3 Apricot chlorotic leaf roll Chrysanthemum yellows Phytoplasma sp. STRAWB2 Aster yellows phytoplasma strain AV2192 Aster yellows phytoplasma strain AVUT Aster yellows phytoplasma strain IOWB Aster yellows phytoplasma O isolate 98UW166B Aster yellows phytoplasma I-P Cherry little leaf Strawberry phylloid fruit Pepper little leaf Tomato little leaf Mexican potato purple top phytoplasma strain JAL6 Mexican potato purple top phytoplasma strain SON18 Peanut witches’-broom Western X phytoplasma Peach rosette Coconut lethal yellowing Elm yellow Plum leaf roll Cherry lethal yellows Peach yellows Clover proliferation Ash yellows Loofah witches’-broom Pigeon pea witches’-broom Apple proliferation Rice yellow dwarf Stolbur Iranian plum phytoplasma Mexican periwinkle virescence Bermuda grass white leaf Hibiscus witches’-broom Sugarcane yellow leaf Papaya bunchy top Nebraska potato purple top Candidatus Phytoplasma castaneae Mollicutes (from R. frangula) Candidatus Phytoplasma pini Phytoplasma sp. strain LDN Buckland valley grapevine yellows Sorghum bunchy shoot Weeping tea witches’-broom Sugar cane phytoplasma satrain D3T1 Sugar cane phytoplasma strain D3T2 Derbid phytoplasma
connected to the symptomatic cherry plum tree via the haustoria of the dodders (Cuscuta odorata). After about 2 wk’s transmission, the dodder bridges were removed. Then in the 9th wk, two of the six plants started to develop visible symptoms. Another six periwinkle plants were connected to asymptomatic cherry plum trees,
GenBank accession no. AY389828 AY180952 GU289674 AF222065 AF222066 AY265206 AY265213 AY265211 EF634457 U96616 AY180957 AY265209 AY265205 AF268405 AF503568 AY034089 AY102275 DQ092321 DQ375238 FJ914650 FJ914642 L33765 L04682 AF236121 U18747 AF189214 FJ459914 AY197659 AY197660 AY390261 X68339 AF353090 AF248957 X68375 D12581 X76427 FJ409624 AF248960 AJ550984 AF147708 AY725228 AY725234 DQ174122 AB054986 X76431 AJ632155 Y14175 AY083605 AF509322 AF521672 AJ539179 AJ539180 AY744945
16S rDNA Identity to CPYLaP (HM131809) (%) 98.88 99.40 99.28 98.80 98.80 99.00 98.88 98.48 98.96 98.31 99.20 99.28 99.20 98.48 98.80 98.96 98.88 98.48 98.80 98.64 98.48 89.04 89.74 89.98 90.08 89.99 89.99 89.99 90.15 90.01 89.77 90.41 88.4 91.89 89.50 95.67 95.51 95.83 89.82 89.11 93.21 91.6 95.51 88.62 91.27 89.68 89.29 96.31 89.98 88.68 92.96 93.19 95.00
and did not develop any visible disease symptom. The symptomatic periwinkle plants not only showed signs of leaf chlorosis and distortion, but also showed small leaves, shoot proliferation, stunted stems and phyllody (Fig. 4-A and B). Then another attempt of transmission of CPYLaP © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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Fig. 2 Phylogenetic tree constructed by parsimony analysis of the 16S rRNA gene fragments from the phytoplasma associated with CPYL (HM131809) and 53 other phytoplasmas. Numbers on tree branches indicate bootstrap values (expressed as percentages of 1 000 replications). Bar length indicates one substitution in 100 nt. Abbreviations of the phytoplasmas with GenBank accession numbers are defined in the Table 1. Group and subgroup affiliations of the phytoplasma strains are indicated in brackets.
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LI Zheng-nan et al.
B Hinf I Sau3A I Mse I Taq I MW
C
D MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
MW
MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
MW Alu I BamH I Bfa I BstU I Dra I EcoR I Hae III Hha I Hinf I Hpa I
A
Hpa II Kpn I Sau3A I Mse I Rsa I Ssp I Taq I MW
1712
MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
E
Sau3A I
Mse I
F MW HM131809 16SrI-A 16SrI-B 16SrI-C 16SrI-D 16SrI-E 16SrI-F 16SrI-H 16SrI-K 16SrI-L 16SrI-M 16SrI-N 16SrI-O 16SrI-P 16SrI-Q 16SrI-R 16SrI-S 16SrI-T 16SrI-U 16SrI-V MW
Hinf I
Taq I
Fig. 3 Restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene product amplified from primer pairs R16F2n/R2 of the phytoplasma associated with CPYL (HM131809). A, virtual RFLP patterns derived from in silico digestions using 17 restriction enzymes: Alu I, BamH I, Bfa I, BstU I (Tha I), Dra I, EcoR I, Hae III, Hha I, Hinf I, Hpa I, Hpa II, Kpn I, Sau3A I (Mbo I), Mse I, Rsa I, Ssp I and Taq I. B, actual RFLP profiles in 2% agarose gel after digestions of the 1.2-kb nested PCR products amplified from primer pairs R16F2n/R2 with four restriction enzymes: Hinf I, Sau3A I (Mbo I), Mse I and Taq I. MW, φX174DNA-Hae III markers. Only fragments that are equal to or larger than 50 bp are shown. C to F, comparison of the virtual digestion patterns between HM131809 and 19 reported 16SrI subgroups by Hinf I, Mse I, Sau3A I (Mbo I) and Taq I.
from symptomatic periwinkle plants to a healthy 1-yrold cherry plum was also made by dodder bridges. The symptomatic periwinkle plant, which has been infected by CPYLaP via the dodder bridges, was set near the healthy cherry plum tree in insect-proof greenhouse,
and some vines were artificially carefully twisted on the young branches of cherry plum tree to help connect. After approximately 3 wk, the periwinkle-dodder-cherry plum bridge was removed. And after another 10 wk, the young cherry plum tree developed symptoms of
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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Table 2 Similarity coefficients derived from analysis of virtual RFLP patterns of 16S rRNA genes from the representative phytoplasma strains in the aster yellow group (16SrI) Strain/GenBank accession
1
1
Serial
AY389828(16SrI-A)
1.00
2
3
4
5
2
AY180952(16SrI-B)
0.92 1.00
3
AF222066(16SrI-C)
0.90 0.92 1.00
4
AY265206(16SrI-D)
0.91 0.97 0.89 1.00
5
AY265213(16SrI-E)
0.91 0.93 0.91 0.90 1.00
6
7
6
AY265211 (16SrI-F)
0.86 0.88 0.88 0.85 0.87 1.00
7
EF634457(16SrI-H)
0.89 0.97 0.89 0.94 0.90 0.85 1.00
8
9
8
U96616 (16SrI-K)
0.86 0.86 0.86 0.85 0.90 0.90 0.83 1.00
9
AY180957(16SrI-L)
0.89 0.97 0.89 0.94 0.90 0.85 0.94 0.83 1.00
10
11
10
AY265209(16SrI-M)
0.92 1.00 0.92 0.97 0.93 0.88 0.97 0.86 0.97 1.00
11
AY265205(16SrI-N)
0.92 1.00 0.92 0.97 0.93 0.88 0.97 0.86 0.97 1.00 1.00
12
13
12
AF268405(16SrI-O)
0.86 0.87 0.80 0.85 0.80 0.74 0.84 0.80 0.83 0.87 0.87 1.00
13
AF503568(16SrI-P)
0.92 0.94 0.94 0.91 0.93 0.94 0.91 0.92 0.91 0.94 0.94 0.80 1.00
14
15
14
AY034089(16SrI-Q)
0.84 0.92 0.84 0.89 0.85 0.86 0.89 0.78 0.89 0.92 0.92 0.80 0.86 1.00
15
AY102275(16SrI-R)
0.91 0.93 0.93 0.90 0.92 0.87 0.90 0.85 0.90 0.93 0.93 0.80 0.93 0.89 1.00
16
17
16
DQ092321 (16SrI-S)
0.88 0.96 0.88 0.93 0.90 0.84 0.93 0.83 0.93 0.96 0.96 0.85 0.90 0.90 0.90 1.00
17
DQ375238(16SrI-T)
0.85 0.83 0.85 0.90 0.87 0.81 0.91 0.80 0.90 0.93 0.93 0.82 0.87 0.87 0.87 0.97 1.00
18
19
20
18
FJ914650 (16SrI-U)
0.87 0.93 0.87 0.90 0.87 0.81 0.91 0.80 0.90 0.93 0.93 0.84 0.87 0.87 0.88 0.97 0.94 1.00
19
FJ914642(16SrI-V)
0.85 0.93 0.85 0.90 0.87 0.81 0.92 0.80 0.92 0.93 0.93 0.82 0.87 0.87 0.87 0.97 0.94 0.94 1.00
20
HM131809
0.79 0.87 0.81 0.84 0.80 0.75 0.86 0.73 0.86 0.87 0.87 0.75 0.81 0.85 0.82 0.85 0.82 0.84 0.84 1.00
chlorosis and yellowing alongside leaf veins and edges (Fig. 4-D and E). A ~1.2-kb nested PCR product was respectively amplified using the primer pairs R16mF2/R16mR1 and R16F2n/R16R2 from the DNA samples extracted from the symptomatic periwinkle plants and the young cherry plum tree. Asymptomatic plants, including the controls did not produce a similar PCR product using the nested primer pairs (data not shown). When digested with restriction enzymes Hinf I, Mse I, Sau3A I and Taq I, respectively, the ~1.2-kb DNA fragment showed identical RFLP patterns with the CPYLaP 16S rRNA gene (Fig. 3-B, data not shown), indicating that the CPYLaP was transmitted to the periwinkle plants from the naturally infected cherry plum tree, and also from the symptomatic periwinkle plant back to the healthy young cherry plum tree.
DISCUSSION The AY group (16SrI) is the largest and the most diverse phytoplasma group. It includes over 100 identified isolates that are widespread in many places of the world and infect a broad range of plant species (Marcone et al. 2000; Santos-Cervantes et al. 2010). In total 19 subgroups including 16SrI-A to -F, -H, and -K to -V have
been identified in the AY group based on results of RFLP analysis of the 16S rRNA gene sequence (Marcone et al. 2000; IRPCM 2004; Wei et al. 2007a; Gámez-Jiménez et al. 2009; Santos-Cervantes et al. 2010). It has been suggested that a new subgroup would be recognized if only one restriction site differed within the 16S rRNA gene F2nR2 region, or if the new phytoplasma strain’s RFLP pattern of the 16S rRNA gene F2nR2 region had a similarity coefficient of 0.97 or lower with each known subgroups (Lee et al. 1998; Wei et al. 2008). The CPYLaP could be easily distinguished from all existing 19 subgroups in the AY group (16SrI) by four restriction sites (Hinf I, Mse I, Sau3A I and Taq I) within its 16S rRNA gene F2nR2 region (Fig. 3); its similarity coefficient of the RFLP pattern ranges varied from 0.73 to 0.87 with those of the 19 described subgroups (Table 2). The results suggested that CPYLaP is probably a previously un-described subgroup lineage in the AY group (16SrI). Phylogenetic analysis of the 16S rRNA genes indicated that the CPYLaP has a cladistically similar ancestor to the 19 subgroups within the AY group (16SrI) phytoplasmas, but it does not form a common subclade with any of the 19 subgroup lineages (Fig. 2). In addition, query of the CPYLaP 16S rRNA gene sequence using an interactive online phytoplasma classification tool, iPhyClassifier (http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier. html, Zhao et al. 2009), also concludes that CPYLaP © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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Fig. 4 Periwinkle (Catharanthus roseus) plants infected with CPYLaP at 11 wk after dodder connections with the cherry plum tree with the yellow leaf symptoms and young cherry plum tree infected by CPYLaP from the symptomatic periwinkle at 13 wk after dodder connections. A, symptoms of leaf chlorosis, distortion, small leaves and stunting in periwinkle. B, symptoms of phyllody, shoot proliferation and small leaves produced on new growth of shoots and leaves in periwinkle. C, a healthy periwinkle plant. D, construction of the periwinkle-dodder-young cherry plum tree bridge. E, symptoms of chlorosis and yellowing alongside leaf veins and edges in the young cherry plum tree.
probably represents a new subgroup in the AY group (16SrI) (data not shown). Since most previous efforts to isolate infectious phytoplasmas on artificial media have not been successful, it is difficult to confirm the etiology of phytoplasma-caused diseases by fulfilling Koch’s postulates. Nonetheless the detection of the CPYLaP by TEM, PCR and the positive transmission of phytoplasma to periwinkle plants then back to the young cherry plum tree suggested that CPYLaP was a probable causal agent of the cherry plum yellow leaf symptoms. Phytoplasma-like bodies (PLBs) were the only type of microbe detected in the yellow leaf tissues and the PLBs were primarily distributed in the phloem cells (Fig. 1-C). No other pathogenic agents such as viruses, fungi or other bacteria were observed in the tissues. The transmission of CPYLaP to perih winkle plants from the naturally infected plum tree was confirmed by PCR and RFLP analysis. The symptoms in the periwinkle plants included not only leaf chlorosis and distortion, but also shoot proliferation, small leaf, stunting stem and phyllody in the newly growing organs which were not observed in the symptomatic cherry plum tree (Fig. 4-A and B). Additionally, the young cherry plum tree was successfully infected by the CPYLaP in
periwinkle plants via dodder bridges, and developed the same symptoms with that naturally infected cherry plum tree (Fig. 4-D and E). RFLP analysis of the PCR fragments amplified from the dodder-mediated infected periwinkle plants and the young cherry plum tree indicated that only CPYLaP was identified in the plants. Many phytoplasmas have been experimentally transmitted to periwinkle plants from their naturally infected herbaceous or woody plant hosts by dodder-bridged transmissions and often caused visible symptoms in the periwinkles. However, the periwinkles, when infected with phytoplasmas from wild hosts, did not always exhibit the exact same symptoms as showed in their wild hosts (Marcone et al. 1997, 1999; Kamińska and Korbin 1999; Kamińska et al. 2001) . Recently, we reported that plum leaf roll (PLR) disease was associated with a phytoplasma belonging to elm yellows (16SrV) group (Hong et al. 2011). The PLR disease was found in cherry plum trees in an ornamental garden in Yangling, Shaanxi, China. The garden is approximately 3 km away from the other plum orchards where the CPYLaP was discovered. SympL toms and symptom development in PLR are different from CPYL. PLR showed leaf rolling/curling and leaf
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A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group
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proliferation symptoms and it took longer for leaves to become chlorotic compared to CPYL. The 16S rRNA gene sequence of the PLR-associated phytoplasma has a high homology and shares an identical RFLP pattern to the jujube witches’-broom phytoplasma (JWB). JWB is a widespread phytoplasma strain infecting jujube trees in Northwest China (Hong et al. 2011). Our experimental result confirmed that the PLR-associated phytoplasma was not detectable in the CPYL tissues (Data not shown). The CPYLaP is the first report of an AY group (16SrI) phytoplasma infecting cherry plum (P. cerasifera Ehrh) trees in China. In addition, to our knowledge, a phytoplasma of 16SrI-B subgroup, plum little leaf phytoplasma strain PlumLL (GenBank accession number GU289674) was recently identified in Lithuania in European plum (P. domestica L.) trees with symptoms of proliferation of shoots and abnormally small leaves (Valiunas et al. 2009). Plum leptonecrosis (PLN) in Japanese plum (P. salicina L.) and European plum in Europe was caused by EFSY phytoplasma which contained mainly 16SrX-B and also a number of other phytoplasmas belonging to 16SrX-A, 16SrI-B, 16SrIII and 16SrV groups (Lee et al. 1995). EFSY phytoplasmas produce a complex disease syndrome, such as early bud burst, leaf rolling, leaf chlorosis, deformed fruits, phloem necrosis and plant decline in plum, peach, apricot, almond and sweet cheery (Carraro et al. 1998). Phytoplasmas belonging to 16SrII and 16SrXII were recently detected in Japanese plum trees with symptoms of little leaf, leaf rolling, rosetting and yellowing in Iran (Zirak et al. 2009). Collectively, these data show that Prunus spp. are apparently susceptible to a number of phytoplasma lineages of broad genetic diversities and geographical distributions (Gámez-Jiménez et al. 2009).
transmission back to healthy young cherry plum tree according to Koch’s postulates. Subsequent molecular analyses of the 16S rRNA gene sequence suggested that the CPYL-associated phytoplasma (CPYLaP) is probably a distinct phytoplasma strain belonging to AY group (16SrI).
CONCLUSION
Total DNA was extracted from plant leaf samples using a cetyltrimethyl ammonium bromide (CTAB) based method (Angelini et al. 2001; Deng et al. 2012). Briefly, 0.15 g of leaf tissue was ground in 500 μL of preheated CTAB extraction buffer with 0.2% β-mercaptoethanol in a 1.5-mL micro-centrifuge tube. The CTAB extraction buffer consisted of 2% (w/v) CTAB, 1.4 mol L-1 NaCl, 20 mmol L-1 EDTA (pH 8.0) and 100 mmol L-1 Tris-HCl (pH 8.0). The treated tissue was incubated at 60°C for 30 min and extracted with chloroform-isoamyl alcohol (24:1). The aqueous DNA layer was precipitated with isopropanol, washed with 70% ethanol
In this report, we described a cherry plum tree with yellow leaf symptom growing in a plum orchard located in the Yangling District, Shaanxi Province, China. We provided evidence that the symptomatic tree was associated with a phytoplasma based on TEM observations, positive PCR results, RFLP analysis, the dodder-mediated transmission to periwinkle plants and followed
MATERIALS AND METHODS Plant materials Cherry plum (P. cerasifera Ehrh) leaf samples mainly with or without yellow leaf symptom was collected from a plum orchard located in Yangling District, Shaanxi Province, China in June 2009. Dodder (Cuscuta odorata) and periwinkle (Catharanthus roseus) plants were grown from seeds in an insect-proof greenhouse. The healthy 1-yr-old cherry plum tree (P. cerasifera Ehrh) was kindly provided by the College of Horticulture, Northwest A&F University, China.
Transmission electron microscope (TEM) Fresh cherry plum leaf tissues with or without yellow leaf symptoms were immediately fixed in 0.1% cacodylate buffer (pH 7.2) containing 3% (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde at 4°C for 4 h. After that, the samples were post-fixed in 1% (w/v) osmium tetroxide for 2 h at room temperature. Then the fixed samples were dehydrated in concentration gradients of ethanol (10-70%) or acetone (0-100%) and embedded in Epson 812 resin. Ultra-thin sections (70 nm) were cut with a diamond knife and mounted onto nickel grids. The grids were doubly stained with uranyl acetate and lead citrate, and then examined under TEM (JEM1200EXII, JEOL) (Siddique et al. 1998).
Nucleic acid extraction and PCR detection of phytoplasmas
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and resuspended in 30 μL of sterile deionized water. A nested PCR method was used to amplify the 16S rRNA gene fragment of phytoplasmas as previously described (Wu et al. 2010) using two phytoplamsa universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2 (Lee et al. 1993; Gundersen and Lee 1996). PCR products were separated by electrophoresis in a 1% agarose gel.
DNA cloning, sequencing, sequence analysis and phylogenetic analysis Nested PCR products were purified using the 1% agarose gel, cloned into pMD18-T simple vector (TaKaRa Bio, Japan) and sequenced from both 5´- and 3´-end by automatic DNA sequencing (TaKaRa Bio., Japan). Nucleotide sequences were analyzed using LaserGene (DNAStar, Madison, USA). The National Center for Biotechnology (NCBI) BLASTn program (http://blast.ncbi.nlm.nih.gov/) was used to search against the NCBI nucleotide collection (nr/nt) database and align the query sequences with 16S rRNA gene sequences from other phytoplasmas in the database (Ye et al. 2006). Phylogenetic trees were constructed by neighbor-joining method with a 1 000-replicate bootstrap search using MEGA4 (Tamura et al. 2007) .
Virtual RFLP pattern analysis and actual enzymatic digestion analysis In silico digestion analysis of the 16S rRNA gene sequences with the 17 restriction endonucleases previously designed for phytoplasma classification (Lee et al. 1993), virtual RFLP pattern identification and the similarity coefficient calculation were performed with a Perl program developed by Wei et al. (2008). Actual enzymatic digestion analyses were performed by digesting gel-purified PCR products of the nested PCR amplifications using four restriction endonucleases, Hinf I, Mse I, Sau3A I and Taq I in separate reactions. Digestion products were separated by electrophoresis in a 2% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator.
Dodder transmission Dodder transmission of the phytoplasmas from naturally infected cherry plum tree to healthy periwinkle plants or from infected periwinkle plants to healthy young cherry plum tree was adapted from a previously reported method (Marcone et al. 1999). Dodder was first established on healthy periwinkle by planting dodder seeds with young periwinkle plants in pots
(25 cm in diameter) in an insect-free greenhouse for about 4 wk. The pots containing the dodder-parasitic periwinkle plants were moved adjacent to the donor plum trees in the plum orchard where the yellow leaf symptoms were found. The dodders from the periwinkles were wrapped around the branch-tips of both symptomatic and asymptomatic cherry plum trees, creating a plum tree-dodder-periwinkle bridge. The bridge was kept for approximately 2 wk under insect-proof netting before the dodder-parasitic periwinkle plants were removed from the plum trees and returned to the greenhouse to allow for symptom development. When the periwinkle plants showed symptoms, a symptomatic one was placed next to a healthy young cherry plum tree for creating a periwinkle-dodder-plum tree bridge. The bridge was kept for approximately three weeks before the tree was separated from the dodder-parasitic periwinkle plant and still grew in the greenhouse to allow for symptom development. Total DNA was extracted from infected and uninfected periwinkle plants and the young cherry plum tree using the CTAB-based method (Angelini et al. 2001). Nested PCR amplification using primer pairs R16mF2/R16mR1 and R16F2n/R16R2 was performed as described above. Actual enzymatic RFLP analysis of PCR products from dodder transmitted DNA samples were also performed as described previously in this report.
Acknowledgements
This work was supported by the 111 Project from the Ministry of Education of China (B07049), the PhD Program Foundation from the Ministry of Education of China (20100204110004) and the National Natural Science Foundation of China (31371913).
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