Publications

4374

ABSTRACT: Apple plants from France introduced in Brazil for research purposes were subjected to a phytosanitary analysis at the Plant Quarantine Laboratory of Embrapa Genetic Resources and Biotechnology (Cenargen). After grafting onto healthy apple rootstock, some plants showed phytoplasma-infection symptoms. Polymerase Chain Reaction (PCR) tests yielded DNA fragments of the expected size for phytoplasmas. DNA sequencing revealed an identity of the 16S rDNA nucleotide sequence of 98-99% with ‘Candidatus Phytoplasma solani’. This phytoplasma species is responsible for losses in European apple orchards and has not been reported in Brazil. According to the Federal Legislation on Plant Protection, the plants were incinerated to avoid the introduction of this exotic pest in Brazil.

AbstractThe incidence of a bacterium “Candidatus Liberibacter asiaticus” was assessed in a Florida population of Asian citrus psyllid, Diaphorina citri. The bacterium is the presumed causal agent of Asiatic huanglongbing, a serious citrus disease. Adult D. citri were periodically collected between May 2010 and September 2012 in a block of diseased trees located in east‐central Florida. The psyllids were individually subjected to molecular analyses (quantitative polymerase chain reaction assays using HLBaspr primers) to determine whether the bacterium was present and, if so, the population level of the pathogen based on qPCR cycle threshold (CT) values. Significantly greater percentages of females tested positive for the pathogen than males, but there were no significant differences between females and males with respect to population levels of the pathogen within the psyllids. No significant differences were found among the three D. citri colour morphs with respect to percentages of adults testing positive for the pathogen. Among 47 sample dates, a mean of 17.5% of adults per sample date tested positive (CT < 36) for the pathogen with a mean CT value of 31.1. The incidence of the pathogen was generally higher during late fall or early winter and often lower during mid‐ to late summer. There was a significant negative correlation between percentages of D. citri testing positive and air temperature. Increases in the incidence of the pathogen may not necessarily correspond to increases in transmission, as a number of factors both internal and external to D. citri can influence transmission. Transmission rates may be highest during periods when D. citri infestation levels are large, a high percentage of adults carry a high population of the pathogen in their salivary glands, and citrus flush is abundant.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of ‘Candidatus Liberibacter asiaticus’, a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of ‘Ca. L. asiaticus’ with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of ‘Ca. L. asiaticus’.