Molecular Diagnosis Of Phytoplasma Diseases On Common Plants In Northwest China And Diversity Study Of The Associated Phytoplasmas

Phytoplasmas are cell-wall-less prokaryotes, and strictly inhabit in the phloem tissue of host plants, causing thousands of plant diseases and economic loss. In nature phytoplasmas are mainly transmitted by piercing-sucking mouthpart insects. In the present study, we survey phytoplasmas associated plant diseases in Shaanxi, China and detected and identified the taxonomic positions of these phytoplasmas using molecular technique. The 16 S r RNA genes of all the strains were cloned as well as rp and tuf genes of some strains. Based on these genes, the phytoplasmas were correctly classified into different 16 Sr groups and subgroups. Moreover, several strains were tested and examined under transmission electron microscope(TEM) for the morphological properties. Four phytoplasma strains were transmitted from their natural host plants to periwinkle plants by dodder bridge or insect feeding.1. Two hundreds and eighteen samples of plant tissues, according to the phytoplasma-like symptoms, were collected in Shaanxi, Gansu, Ningxia, Qinghai, Xinjiang and Erdos of Inner Mongolia. PCR tests were conducted using the phytoplasma universal primer pairs P1/P7 and R16F2n/R2 for detecting phytoplasmas in these samples. The positive PCR results(201 of 218 samples positive) indicated that there are 39 phytoplasma-associated diseases, including Paulownia witches’ broom(Pau WB), Wild jujube witches’ broom(Wj WB), Jujube witches’ broom(JWB), Cynodon dactylon white leaves(Cd WL), Chinaberry witches’ broom(Cb WB), Chinaberry yellows(CY), Apple decline(AD), Cherry plum yellows(Cp Y), China ixeris flat stem(Ci FS), Rose Balsam phyllody(RBP), Pepper witches’ broom(Pep WB), Cactus witches’ broom(Ca WB), Puna chicory witches’ broom(Pc WB), Japanese maple witches’ broom(Jm WB), Alfalfa witches’ broom(AWB), Peach yellows(PY), Chinese trumpet creeper witches’ broom(Ctc WB), Sweet cherry witches’ broom(Sc WB), Sophora japonica witches’ broom(Sj WB), Robinia pseudoacacia witches’ broom(Rp WB), Squash witches’ broom(SWB), Phyllostachys propinqua witches’ broom(Pp WB), Sasa fortune witches’ broom(Sf WB), Brassica rapa virescence(Br V), Green bristlegrass virescence(Gb V), Rosa chinensis virescence(Rc V), Chrysanthemum virescence(CV), Sakura virescence(SV), Sakura yellows(SY), Digitaria yellows(DY), Vitis vinifera yellows(Vv Y), Millet yellows(MY), Platycodon grandiflorum flat stem(Pg FS), Sophora japonica flat stem(Sj FS), Lagerstroemia indica flat stem(Li FS), sesame flat stem(SFS), Millet reddening(MR), Green bristlegrass reddening(Gb R) and Potato purple top(Pp T). The results suggested that Paulownia witches’ broom, jujube witches’ broom and wild jujube witches’ broom are the most common phytoplasma associated diseases in Northwestern China. Nine out of the 39 were the first reports in world that Ctc WB, Sf WB, Pp WB, Ci FS, Pc WB, Pg FS, MY, MD and AD. Another eight ones were the first reports in China, including SWB, Gb R, Gb V, Br V, Jm WB, RBP, CV, DY and Vv Y.2. Phylogeny analysis and restriction fragment length polymorphism(RFLP) analysis of 16 S r RNA gene sequences indicated that phytoplasma associated with AD belongs to subgroup 16 Sr V-B as well as AWB to 16 Sr V-B, Br V to 16 Sr VI-A, Ca WB to 16 Sr II-A, Cb WB to 16 Sr I-B, Cd WL to 16 Sr XIV-A, Ci FS to 16 Sr I-C, Cp Y to a novel subgroup in group 16 Sr I, Ctc WB to 16 Sr I-C, CV to 16 Sr I-B, CY to 16 Sr I-B, DY to 16 Sr V-B, Gb R to 16 Sr I-B, Gb V to 16 Sr I-B, Jm WB to 16 Sr I-D, JWB to 16 Sr V-B, Li FS to a novel subgroup in group 16 Sr I, MR to 16 S r I-B, MY to 16 Sr I-C, Pau WB to 16 Sr I-D, Pc WB to 16 Sr V-B, Pep WB to 16 Sr I-B, Pg FS to a novel subgroup in group 16 Sr I, Pp T to 16 Sr VI-A, Pp WB to a novel subgroup in group 16 Sr I, PY to 16 Sr V-B, RBP to 16 Sr I-B, Rc V to 16 Sr I-B, Rp WB to 16 Sr V-B, Sc WB to 16 Sr V-B, SFS to 16 Sr I-B, Sf WB to 16 Sr I-C, Sj FS to a novel subgroup in group 16 Sr I, Si WB to 16 Sr V-B, SV to a novel subgroup in group 16 Sr I, SWB1 to 16 SXr II-A, SWB2 to a novel subgroup in group 16 Sr XII, SY to 16 Sr V-B, Vv Y to 16 Sr I-B and Wj WB to 16 Sr V-B.3. Ten samples were selected and dested by transmission electron microscope. The samples are witches’-broom-symptomatic paulownia, alfalfa, Japanese maple and pepper; Cynodon dactylon showing white leaves; yellows-symptomatic chinaberry, peach and cherry plum; flat-stem-symptomatic Puna chicory and apple decline. Under TEM, the phytoplasma particles were observed in the phloem tissue of host plants, inside both sieve tube elements and companion cells. The diameter of the particles ranged from 200-850 nm, and disassociated with the species of host plants as well as symptoms.4. The phytoplasmas associated with Jujube witches’ broom and Sesame flat stem were transmitted to healthy periwinkles, respectively, by dodder bridges. The phytoplasma associated with wheat blue dwarf was transmitted to periwinkle by Psammotettix striatus as phytoplasma associated with paulownia witches’ broom by Empoasca flavescens. The infected periwinkle plants were then maintained and proliferated by graft on new healthy periwinkle plants. After insect-feeding transmission, the periwinkle plants developed symptoms in one week to two, while the times were 2 to 3 months and 2-3 weeks for dodder transmission and graft, respectively.