Detection and transmission of Candidatus Liberibacter sp. causing leaf mottling of citrus species grown in three provinces in Southern Luzon, Philippines

The different citrus species grown in Laguna, Batangas and Quezon provinces in Southern Luzon Philippines showed similar mottling, yellowing and zinc deficiency-like symptoms. Three methods, namely: gentisic acid detection, iodostarch reaction (IR) test and polymerase chain reaction (PCR) were used in testing leaf mottling infection and in detecting the leaf mottling pathogen. Gentisic acid was detected by thin layer chromatography and spectrophotometry in bark tissues of field-grown Citrofortunella microcarpa (Bunge) (Calamansi) and Citrus reticulata Blanco (Mandarin orange) with leaf mottling or yellowing symptoms. By spectrophotometry, highest gentisic acid concentration (3.68 ppm) was observed in C. microcarpa with leaf mottling symptom. Gentisic acid was also detected on the apparently healthy C. microcarpa but the concentration was lower. The iodostarch reaction test detected leaf mottling infection in field-collected C. microcarpa and C. reticulata. Eighty percent of C. microcarpa samples from San Pablo, Laguna and 95% of C. reticulata samples reacted positively in IR test. Similar symptoms were observed in other samples but these were negative to leaf mottling infection in the IR test. Among the methods used for testing leaf mottling infection, PCR was found to be the most sensitive. The Candidatus Liberibacter sp. was detected by PCR in field-collected samples of four citrus species. Samples with positive reaction in PCR showed the expected amplified DNA fragment of about 1160 bp which corresponds to the targeted 16S rDNA sequence of C. Liberibacter sp. The C. Liberibacter sp. was detected by PCR in all the C. microcarpa samples from San Pablo, Laguna and Padre Garcia, Batangas and in 60% samples from Los Banos, Laguna. The bacterium was also detected by PCR in all samples of Fortunella sp. and in 75% of C. reticulata and 40% of C. grandis (L.) Osbeck samples. Grafted C. microcarpa and C. reticulata developed mottling symptoms. The C. Liberibacter sp. was detected by PCR in five of six C. microcarpa and four of six of C. reticulata grafted plants. The expression of mottling symptoms and the detection of C. Liberibacter sp. in grafted plants showed that the causal agent of leaf mottling was infectious and transmissible and it determined that leaf mottling in the Philippines is caused by bacterium, C. Liberibacter sp.