J.K. Uyemoto

A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay

The complexity of most nucleic acid extraction procedures limits the number of samples that can be easily processed for analysis by polymerase chain reaction (PCR). A simple, small-scale procedure was developed which can be carried out entirely in 1.5-ml microfuge tubes whereby the container and contents are frozen with liquid nitrogen, tissue is pulverized, and targeted nucleic acids are extracted. DNA of bacterial and phytoplasmal plant pathogens was extracted in hot CTAB buffer followed by chloroform clarification. Following centrifugation, the DNA in the aqueous fraction was precipitated with isopropanol and resuspended in water. RNA originating from viruses and viroids was extracted from triturated tissue using STE buffer and phenol. The nucleic acid fraction was purified using CF-11 cellulose. All purified preparations were used as PCR or RT-PCR templates to detect DNA or RNA, respectively. These procedures were used to detect Xylella fastidiosa, peach yellow leaf roll phytoplasma, sour cherry green ring mottle virus, and peach latent mosaic viroid by agarose gel electrophoresis.

Complete Genome Sequence of a Novel Vitivirus Isolated from Grapevine

ABSTRACT A novel virus-like sequence from grapevine was identified by Illumina sequencing. The complete genome is 7,551 nucleotides in length, with polyadenylation at the 3′ end. Translation of the sequence revealed five open reading frames (ORFs). The genomic organization was most similar to those of vitiviruses. The polymerase (ORF1) and coat protein (ORF4) genes shared 31 to 49% nucleotide and 40 to 70% amino acid sequence identities, respectively, with other grapevine vitiviruses. The virus was tentatively named grapevine virus F (GVF).

Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

Phytoplasmal diseases of peach and associated phytoplasma taxa

Phytoplasmal diseases occur wherever peach (Prunus persica) trees are grown. However, the causal agents differ considerably in taxonomy, insect vector relationships and geographic locations. X-disease of peach is widespread in North America, but does not occur elsewhere in the world. X-disease is induced by ‘Candidatus Phytoplasma pruni’, a member of the X-disease phytoplasma group (16SrIII group, subgroup 16SrIII-A). Peach rosette, peach red suture, and peach yellows, which occur in eastern United States and Canada are all caused by the X-disease phytoplasma. Another North America disease, peach yellow leaf roll (PYLR) is present in a limited area of northern California. Its causal agent is classified in the apple proliferation (AP) group, 16SrX group, as a subtype of the pear decline phytoplasma. In Europe, phytoplasmal diseases of peach are reported under the name European stone fruit yellows and incited by ‘Ca. P. prunorum’, a member of the AP group, subgroup 16SrX-B. ‘Ca. P. prunorum’ is closely related to the PYLR agent. In Lebanon and Iran, peach trees are affected by almond witches’ broom, a lethal disease incited by ‘Ca. P. phoenicium’, a member of the 16SrIX group, subgroup 16SrIX-B. Phytoplasmas of other phylogenetic groups, known to infect a wide range of plant hosts, have been identified in declining peach trees in several fruit-growing areas of the world. The pathological relevance of several ‘nonpeach’ phytoplasmas requires further investigations as their presence was ascertained by nested PCR assays only.