Molecular detection of citrus bacterial pathogen (Xanthomonas axonopodis) and incorporation of AMP gene in citrus cultivar for disease resistance against citrus canker.

Abstract

Citrus is one of the most popular and major commercial fruit crops of Pakistan. It belongs to family Rutaceae that comprises of 158 genera and 1900 species. Pakistan is the 12th largest producer of citrus, which is cultivated on an area of 206,569 hectares with 2.36 million tons annual production in 2016. The yield and poor quality of citrus fruits is threatened by many fungal, viral and bacterial diseases. Among these diseases, Citrus bacterial canker (CBC) caused by Gram negative, rod shape, phytopathogen Xanthomonas axonopodis. subsp citri (Xac) is most important which reduce the cosmetic and market values of the fruit. It is a leaf-spotting and fruit rind blemishing disease which resulted into defoliation, shoot dieback, and fruit drop in severe infection. In current study, almost 500 samples belonging to different citrus groups such as Mandarin, Grape fruit, Lemon and limes, Sweet oranges and Rootstocks were collected from “Citrus Research Institute, Sargodha and Bursha Citrus Research & Development Center, Sillanwali. In the Symptomatic leaves and fruit samples the early symptoms appeared as slightly raised, tiny, blister-like lesions. In the fruit the lesions appeared as dark brown to black with brown to black sunken, corky centers on maturity. The present study was conducted to identify the citrus bacterial canker pathogen (Xac) on the basis of microscopy, morphological, biochemical and molecular assays. The pathogen was identified as Gram negative and colonies were appeared as creamish light yellow with mucoid surface and sticky texture. The 20 different biochemical tests were performed to characterize the Xac. In decarboxylase of Lysine, Ornithine and Arginine dihydrolase tests, all Xac isolates showed negative results while positive results were observed in case of Citrate utilization, Urea hydrolysis, Acetoin production, Gelatin liquefaction tests, Rhaminose and Sucrose fermentation. The identity of the isolates was further confirmed by PCR amplification using primer specific to 16S rDNA and ITS region of the pathogen. Out of 55 samples 27 samples were found to be positive for the pathogen by using 16S rDNA primers. Further 500 samples were screened by ITS based primer out of which 196 were positive for the disease. The disease severity was different in all seven groups of citrus. Lemon and Lime group of citrus collected from CRI recorded the highest percentage of disease xii susceptibility (81%) followed by Grapefruit (71%), whereas rootstocks exhibited the least disease susceptibility (12%). While in Silanwali, the highest percentage (frequency) of disease susceptibility was in grapefruit (39%) following by Mandarin (26%) and the lowest disease severity 15% in Sweet oranges. The Silanwali and CRI localities recorded a disease incidence of 62% and 38% respectively. The phylogenetic tree constructed by neighbor joining method by Mega 7.0 method clustered all Xanthomonas axonopodis taxa into a monophyletic group. The bacterium isolate from shamber (3C_XCF) showed 100% homology to reference sequence of Xac isolate from China (Accesion No. CP011827.2). Considering the results of the performed tests, sequencing and phylogenetic tree confirmed the bacterial isolates to be X. axonopodis subsp. citri which is an infectious pathogen of A type citrus canker. In order to obtain resistance against CBC disease, insect derived antimicrobial peptide gene (AMP) attacin A (attA) included the native signal peptide responsible for directing the insect protein to the extracellular space was successfully cloned into pFF19 cloning vector to from pFFattA vector. Further expression cassette 35S-35S-attA-35ST was restricted from pFFattA vector and was successfully transformed into binary pCambia 2300 vector to form pCattA recombinant vector. The transformation of the expression cassette was confirmed by restriction digestion and sequencing. The pCattA was successfully transformed into citrus cultivar (Rough lemon) by using Agrobacterium mediated transformation using epicotyl region as an explant collected from seedling germinated in vitro under 16hrs light period and 8hrs darkness. The best co-cultivation condition was the incubation of the explant for three days in co-culture medium supplemented with 200μM Acetosyringone. Genetically transformed plants were identified by conventional polymerase chain reaction (PCR) by using attA gene specific primers. Quantitative real-time PCR assay was also used to detect the presence of mRNA in all transgenic lines and the threshold values of positive samples was in the range of 14-19.