Identification Of Phytoplasma And Molecular Characterization Of Immunodominant Membrane Protein Gene From Wheat Blue Dwarf Disease

Wheat blue dwarf (WBD) is an important disease of winter wheat disseminated by Psammotettix striatus L. in Shaanxi and Northwest area. Using primers (Rm16F2/Rm16R1) for phytoplasma 16S rDNA, a 1.4 kb DNA fragment was amplified by polymerase chain reaction (PCR) in DNA samples extracted from infected wheats but not in those extracted from healthy plants. Homology analysis of nucleotide sequences showed that the amplified 16S rDNA fragment from WBD was closely related to those of clover phyllody (CPh), aster yellows(AY), hydrangea phyllody (HP), blueberry stunt (BBS) and tomato big bud (TBB), with homology rate 99.2%～99.9%. Our results suggest that WBD is caused by a phytoplasma in 16SrⅠg roup.Using primers (fTufu/rTufu) for phytoplasma tuf gene (EF-Tu) to amplify DNA samples extracted from infected wheats, a 850bp DNA fragment was amplified in DNA samples from infected wheats but not in those extracted from healthy plants. The sequence result revealed that the tuf gene of phytoplasma from wheat blue dwarf consists of 842bp, which encoding 280 amino acids. Homology analysis for sequences of tuf gene from WBD and other 14 kinds of phytoplasmas showed that the tuf gene of WBD was closely related to those of clover phyllody (KVF), homology rate of nucleotide sequences and amino acids is 99.9% and 100%, respectively. Homology analysis for sequences of tuf gene can differentiate and classify phytoplasma of wheat blue dwarf from subgroup.Inoculating test plants by vector, observing symptom and using universal primers (R16mF2/R16mR1) for phytoplasma 16S rDNA, 1.4 kb DNA fragments were amplified by polymerase chain reaction (PCR) in DNA samples extracted from inoculated wheat and viruliferous P. striatus L. respectively. Seven new host plants of WBD were discovered. Nine kinds of weed and wheat(Triticum aestivum L.) were determined to be naturally infected WBD plants by nested-PCR for 20 naturally infected kinds of weed in the field. They are Descurainia sophia L., Veronica didyma Tenore, Erysimum cheiranthoides L., Avena fatua L., Lithospermum arvense, Aegilops squarrosa, Amaranthus retroflexus L., Tritolium repens L., Eragrostis cilianensis (AII) Link., and Triticum aestivum L., which become important mid-host and infected resources. Restriction fragment length polymorphism (RFLP) analysis of nested-PCR products of 16S rDNA with six special restriction enzymes, which are AluⅠ,EcoRⅠ,HpaⅡ,Sau3AⅠ,RsaⅠand TaqⅠ, revealed that most of the digestion patterns by restriction endonucleases coincided with those of Aster yellows (AY) phytoplasma ,which belong to the same 16Sr I group phytoplasma. It was showed that WBD phytoplasma exits in 9 naturally infected kinds of weed and wheat, which had not differentiation of host biology.Phytoplasma was observed in the phloem sieve elements of wheat and periwinkle leaf tissues and hind intestine of P. striatus L. under the electron microscope but no phytoplasma was found in healthy plants, viruliferous P. striatus L. and eggs of viruliferous P .striatus L. Using transmission experiments and PCR assys, the desirable acquisition access period (AAP) of the vector is 7 days. The latent period (LP) in the vector ranged from 15 to 17 days after the start of the acquisition and inoculation access periods (IAPs) is 2 to 3 days. In general, infectivity of the vector lasted for life. The different life stage of the vector is not very critical for WBD acquisition. Host-plant species influenced WBD acquisition.The aim of the experiment is to get the basic molecular information for the mechanism of vector-pathogen-host plant relationships. DNA was extracted from infected wheat and periwinkle samples and the target fragments were amplified using primers Imp1051/Imp2265 designated based on conserved regions of immundominant membrane protein gene from phytoplasmas. A 1.0kb DNA fragment was amplified by PCR in DNA samples extracted from infected wheats and periwinkles but not in those extranted from healthy plants. The amplified fragment is analysed for open reading frame, homology matrix and phylogentic tree; Encoding protein is anlysised for transmembrane region, hydrophilic and hydrophobic region and leader signal sequence. It was showed that immundominant membrane protein gene from wheat blue dwarf (WBD) phytoplasma was 495 bp in length, encoding a predicted protein of 164 amino acids. Homology analysis of sequence from WBD and 10 phytoplasmas showed that immundominant membrane protein gene of WBD phytoplasma was closely related to those of clover phyllody (CPh) phytoplasma, with homology rate of nucleotide and amino acids sequences, 98.4% and 95.1%. Prediction of protein structure showed the immunodominant membrane proteins from WBD phytoplasma possess N-terminal hydrophobic transmembrane regions with export leader signal sequence, a C-terminal hydrophobic transmembrane anchor, and a central hydrophilic region, possibly external to the phytoplasma cell. Immundominant membrane protein of WBD phytoplasma belong to the same type with those of CPh, aster yellow (AY), onion yellow (OY) and paulownia witches'-broom (PaWB) phytoplasmas.