ExpansinB3 as a marker for detecting pine wood nematode-infected pine trees

To develop a method for recognizing pine trees infected with pine wood nematodes (PWN), Bursaphelenchus xylophilus, we have constructed a monoclonal antibody (Mab) library for Expansin B3 (EXPB3), which is one of the known proteins that is secreted by PWN. We conducted a primary screening by using the EXPB3 peptides and selected 96 fusion cell lines with a high immunoreactivity. A secondary screening was performed using the PBS extracts of the PWN-infected pine trees (PWNIPTs). Thirteen clones, including 9 clones with high immunoreactivities, were selected, and limited dilutions were performed to establish Mab-secreting hybridoma lines. A total of 74 PWN infected pine trees collected from 9 PWN-affected areas in the Republic of Korea were analyzed by using EXPB3-Mab-3-1-H3-C11-E4 (EXPB3-C11-E4). EXPB3-C11-E4 recognized all of the infected pine trees with some variations. This Mab demonstrated a significantly higher reactivity to the black pines when compared to the red pines. This Mab recognized both PWN-infected red and black pine trees collected from different regions, but were able to displayed regional differences with the black pine trees. In addition, the EXPB3-C11-E4 immunoreactivity in black pine trees had a significant correlation regarding the degree of PWN infection and the perimeters of the trees. The results of this study reveal that EXPB3, an antigen secreted by PWN, and its specific Mabs may be used for the diagnosis of PWNIPTs.