Table 1 Specificity of find more CBC-LAMP assay Species Strain Detection Method     Gel LFD SYBRGreen Xanthomonas citri subsp. citri 306 + + + Xylella fastidiosa 9a5c – - – Candidatus Liberibacter asiaticus * – - – Xanthomonas campestris pv. campestris 8004 – - – Xanthomonas campestris Staurosporine mouse pv. vesicatoria 85-10 – - – Pseudomonas syringae DC3000 – - – Botrytis cinerea B-191 – - – Phytophthora citricola * – - – Guignardia citricarpa * – - – Elsinoe fawcettii * – - – For each dilution CBC-LAMP reaction was performed in triplicate. Gel: gel electrophoresis. LFD: lateral flow dipstick.+:

Positive reaction.-: Negative reaction. * Performed with DNA from an infected plant without symptoms of CBC. Figure 1 CBC-LAMP reaction optimization. Temperature, time and primer combinations applied to CBC-LAMP to determine the optimal reaction conditions. An aliquot of 15 μl of CBC-LAMP reaction aliquot was applied to 1.5% agarose gel electrophoresis and stained with ethidium bromide. C – : negative control without DNA. M: 100-bp DNA ladder. Figure 2 Direct analysis of CBC-LAMP products. Direct visual evaluation methods were used as follows. A-CBC-LAMP positive and negative reaction tubes were stained

with SYBRGreen I and inspected under daylight. B-CBC-LAMP positive and negative reactions were subjected to lateral flow dipstick visual detection. The CBC-LAMP detection limit was determined using Xanthomonas citri subsp. citri strain 306. The detection limit for Xcc pure DNA was 10 fg (Table 2), 5 CFU of Xcc cultured PIK-5 cells and 18 CFU from infected leave selleck chemicals tissues according to the detection method used (Table 3). Positive amplification was obtained for every CBC-causing Xanthomonas strains from different regions in Argentina and around the world, including CBC types A, B and C strains. Xanthomonas axonopodis pv. citrumelo, the causative agent of Citrus Bacterial Spot, a non canker producing citrus associated bacteria, did not produced any amplification (Table 4). Table 2 CBC-LAMP assay sensitivity from pure DNA Detection method Purified Xanthomonas

citri subsp. citri DNA   100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg 10 fg 1 fg Gel + + + + + + + + – LFD + + + + + + + + – SYBRGreen + + + + + + Nc Nc – For each dilution the CBC-LAMP reaction was performed in triplicate. Gel: gel electrophoresis. LFD: lateral flow dipstick.+: Positive reaction.-: Negative reaction. Nc: The colour developed in the test tube was not clearly distinguishable between a positive or negative reaction. Table 3 CBC-LAMP assay sensitivity from cultured cells and infected tissue Strain Specimen source Detection method CFU per reaction (10-fold dilutions) X. citri pv. citri Pure culture   395.3 37.6 5.2 0.7     Gel + + + –     LFD + + + –     SYBRGreen + + + – X. citri pv. citri Infected tissue   248.4 18.7 3.3 0.