Caracterização de isolados do Papaya ringspot virus procedentes de regiões do Brasil e de Cuba

Among the factors which reduce both fruit yield and longevity of the papaya tree, the virus-caused diseases play an important role, Papaya ringspot virus (PRSV-P), from the family Potyviridae and genus Potyvirus being the one of greatest economic importance. Among the management strategies most utilized to try to decrease both the incidence and the losses caused by PRSV-P, the construction of transgenic plants resistant to PRSV-P and the crossprotection with mild strains of the virus are thought of quite promising, but both the strategies are dependent upon the degree of genetic similarity among the isolates of each region. Thus, this work was carried out with the objective of characterizing both molecularly and biologically PRSV-P isolates collected on papaya trees form different regions of Brazil and Cuba and of a viral isolate collected on squash plant with mosaic, coming from Cuba. 21 isolates were collected in several regions of Brazil and 7 in Cuba, which were inoculated and kept dissected and multiplied on papaya plants under greenhouse conditions for total RNA extraction and doing of RT-PCR, employing primers specific to the gene region of the coat protein (CP), P1 and HC-Pro. The amplicons obtained were sequenced and analyzed with the CLUSTALW, MEGA blast, MEGA5 and RDP3 programs and compared one with another and with other PRSV sequences available in the GenBank. Those isolates were also inoculated on papaya plants cultivar Sunrise solo and squash (Cucurbita pepo L.) cultivar Caserta for evaluation of the reaction of the plants to each isolate. When the genes of the CP and of the HC-Pro were analyzed, shorter distances were found and, therefore, greater identities among the sequences of nucleotide (nts) and of aminoacids (aa) of isolates of nearby geographic regions as compared with the farthest ones. The phylogenetic trees based upon the net sequences grouped the isolates of the Americas together with the isolates of India, separately from the isolates of Asia. The Brazilian isolates were grouped per state, while the isolates of the eastern region of Cuba were separated from the ones coming from the center-western region, suggesting a possible difference in their geographic origin. In the trees based upon aa sequences, the groupings were different, forming clusters mixed with American and Asiatic isolates. The tree based on the nt sequences of genes P1 and HC-Pro confirmed the closeness of the America isolates with those of India and evidence of a possible recombinant among the CbMT-2 isolate of Cuba and BrSP-3 isolate of Brazil was found. The inoculated papaya and squash seedlings developed typical symptoms, allowing us to group the isolates together according to the differences in the intensity of the symptoms of the disease in each host. Biological analyses of the viral squash isolate collected in Cuba showed it to be capable of inducing mosaic symptoms, blistering and leaf distortion in squash cultivar Caserta, but not in the papaya tree cultivar Solo. Observations of leaf dip preparations of leaf tissues of infected squashes, under the transmission electron microscope, showed elongated and flexuous particles about 780-800 x 12nm. The genomic fragments of that virus, containing the coat protein and HC-Pro genes were amplified and sequenced, by using primers specific for those regions of the PRSV and these were analyzed and compared with other isolates available in GenBank. The identity of nucleotides and aminoacids was superior to 94% as compared with that of other PRSV-W isolates of America. In the phylogenetic tree, the isolate formed a group with other isolates of America, Australia and India and stayed farther from the Asian isolates. The data obtained revealed that to be a PRSV-W isolate, detected for the first time in Cuba. The results of this work helped, in the understanding of the genetic diversity of PRSV in Brazil and in Cuba, furnishing valued information for the management strategy of papaya ringspot virus, via cross protection and trangenesis.