Detecção de espécies e patovares de pseudomonas causadoras de lesões foliares em cafeeiro empregando primers específicos

Abstract

The bacterial blight caused by Pseudomonas syringae pv. garcae (Psg), is the main bacterial disease of coffee (Coffea arabica L.) in Brazil. In addition to this bacteriosis, the bacterial leaf spot (P. syringae pv. tabaci - Psta), the bacterial leaf blight (P. cichorii - Pc) and the dark spot (Burkholderia andropogonis - Ba) occur. The diagnosis of these diseases is problematic, because it is based on symptom and biochemical tests that are not enough to identify pathogens at species and patovar levels. In addition, there are no studies described in the literature of specific primers for the detection of Psg. The objective of this work was to design specific primers for detection of Psg and primers for simultaneous detection of Psg, Psta and Pc. The primers were designed based on the sequence alignment of the rpoD gene, that encoding RNA polymerase, and hrpS. Primers specificity was tested using conventional PCR, with DNA samples extracted from Psg, Psta, Pc, as well as isolates representative of other genera and bacterial species. The sensitivity was evaluated by determination of concentration of bacterial genomic DNA in NanoDrop 2000 and its serial dilution (100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg). The primers were validated in diseased coffee leaves collected in the city of Lavras and in artificially inoculated Catuaí Vermelho 144 coffee seedling leaves. The samples were submitted to the exudation test and used in Bio-PCR. The PsgRpod F1/PsgRpod R2 primers amplified a single 287 bp band only for the Psg isolate and showed PCR reaction sensitivity of up to 100 pg. The PsgRpod F1/PgtcRpod R1 primers amplified 170 bp, 1400 bp and 250 bp fragments for Psg, Psta and Pc respectively, while the PCR reaction sensitivity for the three bacteria was up to 1 ng. Using the Bio-PCR technique, it was possible to identify Psg infected coffee leaf samples from both inoculated and field seedlings when tested with the two primer pairs designed in this study. The PsgRpod F1/PsgRpod R2 primers demonstrated to be specific for Psg identification and were able to detect all Psg isolates tested. The pair PsgRpod F1/PgtcRpod R1, made possible the simultaneous detection of bacteria of the genus Pseudomonas that cause leaf spots in coffee. The differentiation of Psg, Psta and Pc is important for the epidemiological monitoring of diseases caused by these bacteria in coffee producing regions, as they are often misdiagnosed due to similarity of symptoms.