Raymond K. Yokomi

Genetic diversity of Spiroplasma citri strains from different regions, hosts, and isolation dates.

Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD). Losses due to CSD in California orchards have grown over the past decade. To investigate the possibility of introduction or emergence of a new strain, a study of genetic diversity among S. citri strains from various locations was conducted using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) of 35 strains cultured from 1980 to 1993, and of 35 strains cultured from 2005 to 2006. Analysis using 20 primer pairs revealed considerable diversity among strains. However, no unique genetic signatures were associated with recently collected strains compared with those collected 15 to 28 years ago, and no geographically associated pattern was distinguishable. S. citri strains from carrot and daikon radish contain some unique DNA fragments, suggesting some host plant influence. Multiple strains from single trees also showed genetic diversity. Sequencing of five RAPD bands that differed among strains showed that diversity-related gene sequences include virus fragments, and fragments potentially encoding a membrane lipoprotein, a DNA modification enzyme, and a mobilization element. No differences in colony morphology were observed among the strains. The lack of correlation between PCR patterns and isolation date or collection site is inconsistent with the hypothesis that recent infections are due to the introduction or emergence of novel pathogen strains.

Biotype-specific expression of dsRNA in the sweetpotato whitefly

This study was conducted to evaluate the effect of two different biotypes of the sweetpotato whitefly, Bemisia tabaci (Gennadius), on the induction of squash silverleaf (SSL), and to determine if double‐stranded RNA (dsRNA) occurs in geographically remote populations of the two biotypes. Recently collected B‐biotype whiteflies from Florida, Arizona, Mississippi, and Texas (SPW‐B) all contained a 7.0 kb dsRNA molecule. Kb dsRNA molecule. Laboratory colonies of A‐biotype whiteflies that were originally collected in 1981 from cotton in Arizona and California did not contain the 7.0 Kb dsRNA. When the two biotypes were compared only the SPW‐B induced rapid onset, grade 5, SSL. DsRNA similar to that found in adult SPW‐B was concentrated in whitefly nymphs, but host plant leaf tissue did not contain any consistent dsRNA molecules. SPW‐A only induced low‐grade SSL and progeny of SPW‐A that were fed on pumpkin plants displaying SSL did not acquire the ability to express dsRNA or induce SSL. Our data suggest that dsRNA is not directly involved in the induction of SSL and that SSL is a host‐specific response to a feeding injury induced by B‐biotype whiteflies. The origin and source of the 7.0 Kb dsRNA molecule remains enigmatic but its expression is constant in the whitefly biotype that is responsible for the induction of SSL and several other plant disorders in the U.S.

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.