Isolation,Identification And Molecular Detection Of Citrus Black Spot And Citrus Brown Spot

Citrus black spot and Citrus brown spot caused by Guignardia citricarpa Kiely and Alternaria alternata (Fr.) Keissle, respectively。They are economically important citrus diseases in the world and could cause black or brown spots on fruits, even fruit drop.In this study, the samples collected from Sichuan, Guangxi, Fujian, Chongqing, Brazil were indentified. The results showed as follows:1、14 strains of G.citricarpa were obtained from 48 suspect samples with Citrus black spot.(1) According to the morphological characteristics on the PDA medium, the colonies were divided into four types. Using the conidial suspension to inoculate, only the pummel showed symptoms two months later, and was tested into a positive result with Citrus black spot by PCR.(2) Specific primers were designed on the basis of internal transcribed spacer of ribosomal DNA(rDNA-ITS) and used to amplify by PCR. The homology of the positive samples with G.citricarpa was up to 99%. Therefore, Gcitricarpa is the pathogen of Citrus black spot. According to the phylogenetic tree on rDNA-ITS of the 14 G.citricarpa strains, the results showed that the pathogen of Citrus black spot has great diversity.(3) A pair of specific primer set RT-1/RT-2 was designed according to the differences on ITS alignments among Stenella citri-grisea、Colletotrichum gloeosporioides and G.citricarpa. To optimize the polymerase chain reaction (PCR) detection system, L9(34) orthogonal design was used. Eight temperature gradients were set to optimize the annealing temperature, among which 54.7℃was confirmed to be the most suitable one. The specific band 210 bp amplified by primer set RT-1/RT-2 could detect the existence of Citrus black spot. The sensitivity of specific primer set RT-1/RT-2 and general primer set ITS1/ITS4 were respectively 0.1 pg and 0.1 ng, respectively. This study provided a quick, convenient and high sensitive molecular detection method for Citrus black spot, and was importance to its control.2、Four strains of A.alternata were obtained from eight suspect samples with Citrus brown spot.The culture characteristics of the pathogen were as follows:gray colonies, developed, septated and multi-branched aerial mycelium. The conidiophore was single or gathered, upright or slightly bended top, septated but no branch, and the colour varied from dark brown to hazel from the root to the top. The endopolygalacturonase gene (endoPG) was amplified with the primer set PG2/PG3, the amplified fragment length was 481 bp. After comparing, the homology was 99%with the A.alternata (Fr.) Keissle, which caused the Citrus brown spot. Combined with the culture characteristics, we can make clear that A.alternata was the pathogen which cause Citrus brown spot.