Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, 'Candidatus Liberibacter asiaticus', 'Candidatus Liberibacter africanus' and 'Candidatus 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 'Ca. 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 TaqMan 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 (j = 0Á76 AE 0Á052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina 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.

TaqMan real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) using purified RNA targets or coupled to tissue-print and squash procedures was developed to detect and quantify Citrus tristeza virus (CTV) RNA-targets in plant tissues and in single aphids. With this method all CTV isolates tested from different hosts and origins were detected. The sensitivity of conventional realtime RT-PCR was 1,000 times higher than immunocapture (IC)-RT

The use of rootstocks that are less susceptible or resistant to natural Plum pox virus (PPV) infection and/or the application of mineral oil treatments are two possible strategies to reduce viral incidence in nursery plots. We evaluated the susceptibility of Prunus rootstocks used in the Spanish stone fruit industry and the effect of mineral oil treatment (Sunspray Ultrafine at 1%) on the spread of the virus at two different localities in Valencia, Spain, under different natural PPV inoculum pressures (high inoculum pressure, 2006–08; low inoculum pressure, 2006–07). Samples from both plots were analysed by double‐antibody sandwich indirect enzyme‐linked immunosorbent assay (DASI‐ELISA) and spot real‐time RT‐PCR. Under high inoculum pressure, the assayed rootstocks exhibited significant differences in their susceptibility to natural infection. The most susceptible rootstocks at the end of the experiment were Adesoto 101 and Mariana GF8‐1. Cadaman and Garnem rootstocks presented the fewest PPV‐infected plants; these infections could be detected only by spot real‐time RT‐PCR. No differences among the assayed rootstocks were found under low PPV inoculum pressure. Aphid species were monitored using Moericke yellow water traps and sticky‐plant methods at both localities in May 2006 and 2007. Aphis spiraecola was the most abundant aphid species monitored by both methods at both localities. The average percentage of A. spiraecola carrying PPV PCR‐amplifiable targets was 30.37% in the plot with high PPV inoculum pressure and only 7.98% in the plot with low inoculum pressure. We found significant differences in PPV incidence between Mariana GF8‐1 plots that were treated with mineral oil and those that were not treated after one year under natural conditions and high PPV inoculum pressure.

Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis.

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