Isolation and identification of specific lytic bacteriophages as a biocontrol agent against Serratia odorifera PBA-IAUF-1 and Kluyvera intermedia PBA-IAUF-6 causing bacterial canker in the grape and Siberian pear

Abstract

Abstract Bacterial canker, a prevalent disease among fruit trees, is a significant concern. The use of phage therapy is presently seen as a dependable biological strategy to control bacterial diseases in fruits. The objective of this research was to use various biochemical and molecular techniques to determine the types of bacteria responsible for causing cankers in various fruits. Additionally, their ability to cause disease in the fruit tissues was assessed, the specific bacteriophages targeting these bacteria were isolated and identified. The bacteria were separated from different parts of the infected fruits like grapes and Siberian pears. The selection of fruit tissues showing signs of canker disease was performed, and the validation of the isolates’ pathogenicity was confirmed following Koch’s principles. Subsequently, in order to establish a conclusive identification of the bacterial species, molecular identification was conducted through the sequencing of a specific fragment within the 16S rRNA following amplification by PCR by using universal primers, RW01 and DG74. Isolation and titration of phages specific to fruit spoilage bacteria was done by spot and double-layer agar method, and the growth curve of the isolated bacteriophage was drawn. The phages were detected by transmission electron microscopy (TEM). The results of the study proved the presence of canker causing agents, Kluyvera intermedia PBA-IAUF-6 with the code Sh6 in the Siberian pears, and Serratia odorifera PBA-IAUF-1 with the code Rz3 in the grape fruits, which were deposited in GenBank with the accession numbers of KU878579 and KU168605, respectively. Isolation of the specific bacteriophages to the S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 bacterial strains were done from the effluent of South Isfahan wastewater treatment plant and Caspian Sea water, respectively. The titer of the specific phage to S. odorifera PBA-IAUF-1 and K. intermedia PBA-IAUF-6 was detected in the samples as 2.2 × 10−5 and 5 × 10−11 PFU/ml, respectively. An electron micrograph of a bacteriophage that targets two different bacterial strains revealed phages with a geometrically shaped head and a flexible tail, which resembled viruses from the Siphoviridae family.