Eduardo Vidal

Direct systems to process samples allow high-throughput testing or identification of Citrus tristeza virus (CTV) by the sensitive real-time reverse transcription coupled to polymerase chain reaction (RT-PCR) neither with extract preparation nor nucleic acid purification. Immobilized CTV targets are amplified from fresh sections of plant tissues or squashes of fresh or already caught vectors onto paper, nitrocellulose, or positively charged nylon membranes. The printed or squashed support can be stored or mailed at room temperature. These validated user-friendly methods are recommended by IPPC-FAO standard for CTV diagnosis, detection, and identification. The methods are safe, not under current quarantine regulations because they do not involve any risk of introduction of exotic CTV isolates or vectors and are discrete and useful for epidemiological studies or screening for large-scale analyses. In this chapter, tissue-printing and squashing capture methods for direct sample preparation without extract preparation or nucleic acid extraction and purification were coupled with validated real-time RT-PCR detection protocols based on TaqMan chemistry for CTV detection.

ISHS III International Symposium on Plum Pox Virus Sharka, a vector-borne disease caused by Plum pox virus: vector species, transmission mechanism, epidemiology and mitigation strategies to reduce its natural spread

Grapevine leafroll-associated virus 3 (GLRaV-3) has a worldwide distribution and is the most economically important virus that causes grapevine leafroll disease. Reliable, sensitive, and specific methods are required for the detection of the pathogen in order to assure the production of healthy plant material and control of the disease. Although different serological and nucleic acid-based methods have been developed for the detection of GLRaV-3, diagnostic parameters have not been established, and there is no gold standard method. Therefore, the main aim of this work was to determine the sensitivity, specificity, and likelihood ratios of three commonly used methods, including one serological test (double-antibody sandwich enzyme-linked immunosorbent assay [DAS-ELISA]) and two nucleic acid-based techniques (spot and conventional real-time reverse transcription-polymerase chain reaction [RT-PCR]). Latent class models using a Bayesian approach have been applied to determine diagnostic test parameters and to facilitate decision-making regarding diagnostic test selection. Statistical analysis has been based on the results of a total of 281 samples, which were collected during the dormant period from three different populations. The best-fit model out of the 49 implemented models revealed that DAS-ELISA was the most specific method (value = 0.99) and provided the highest degree of confidence in positive results. Conversely, conventional real-time RT-PCR was the most sensitive method (value = 0.98) and produced the highest degree of confidence in negative results. Furthermore, the estimation of likelihood ratios showed that in populations with low GLRaV-3 prevalence the most appropriate method could be DAS-ELISA, while conventional real-time RT-PCR could be the most appropriate method in medium or high prevalence populations. Combining both techniques significantly increases detection accuracy. The flexibility and power of Bayesian latent class models open new possibilities for the evaluation of diagnostic tests for plant viruses.

ISHS II International Symposium on Plum Pox Virus DIAGNOSIS AND DETECTION OF PLUM POX VIRUS: STATE-OF-THE-ART AND FUTURE OPTIONS

Huanglongbing ( HLB ) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘ C andidatus Liberibacter asiaticus’, ‘ C andidatus Liberibacter africanus’ and ‘ C andidatus  Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR . Universal detection of ‘ C a . Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with T aq M an chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR . This simplified method was validated and could detect HLB ‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB ‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA ( κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature D iaphorina citri that fed on leaves of HLB ‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.

Epidemiology of Citrus tristeza virus in nursery blocks of Citrus macrophylla and evaluation of control measures