Phenotypic and Molecular Characterization of the Beetle Pathogens Paenibacillus popilliae and Paenibacillus lentimorbus

Abstract

DNA similarity studies were used to determine the species of thirty-one strains of bacteria isolated from the hemolymph of infected larvae from Mexico and throughout Central and South America. Twenty-one of the strains were determined to be Paenibacillus popilliae and ten were found to be more closely related to Paenibacillus lentimorbus. Only one of the P. popilliae strains, an isolate from Mexico, was resistant to the antibiotic vancomycin, a trait characteristic of P. popilliae strains from other geographic areas. As expected, all P. lentimorbus strains were sensitive to vancomycin. The polymerase chain reaction (PCR) was used to amplify a portion of a ligase gene necessary for vancomycin resistance in the Mexican strain. Sequencing of the amplicon revealed a sequence identical to that obtained from a North American strain of P. popilliae previously described. The ability of P. popilliae and the inability of P. lentimorbus to grow on medium supplemented with 2% sodium chloride has been used as a phenotypic trait for differentiating between the two species. Approximately 86% of the P. popilliae strains were capable of growth on medium supplemented with 2% sodium chloride and 60% of the P. lentimorbus strains were not capable of growth on this medium. Microscopic examination revealed that all of the Mexican and Central and South American strains of P. popilliae and P. lentimorbus produced a parasporal body. PCR was used to amplify two different regions of the cry18Aa1 gene encoding the paraspore in all of the isolates. One primer pair, CryBP2, detected the cry18Aa1 gene in 17 of the 21 P. popilliae strains and in all ten of the P. lentimorbus strains. The second primer pair, CryBP4, detected the parasporal gene in 20 of the 21 P. popilliae strains and in all ten of the P. lentimorbus strains. Thirty of the thirty-one P. popilliae and P. lentimorbus strains produced amplicons of approximately 616 bp with the CryBP4 primers. The CryBP4 primers did not detect the paraspore gene in one of the P. popilliae strains. The CryBP2 primer pair produced amplicons of three different sizes, indicating possible variability in the parasporal proteins of P. popilliae and P. lentimorbus. Eleven of the P. popilliae strains produced CryBP2 amplicons approximately 660 bp in size and six of the P. popilliae strains produced CryBP2 amplicons approximately 1100 bp in size. The cry gene was not detected in four of the P. popilliae strains with the CryBP2 primers. The P. lentimorbus strains produced CryBP2 amplicons approximately 750 bp in size. Three PCR products representing the variable CryBP2 amplicon sizes were sequenced and compared to the published cry18Aa1 gene sequence. Sequencing data revealed that the Central and South American CryBP2 amplicons are similar to the published cry18Aa1 sequence, however, the 1100 bp amplicon has a 453 bp insert that is not found in the published cry18Aa1 gene sequence. Paraspores are produced by P. popilliae and P. lentimorbus and is not a reliable phenotypic trait for differentiation between the two species. The ability of Mexican and Central and South American strains of P. lentimorbus to produce paraspores supports the previous findings of a North American group of P. lentimorbus strains that were also capable of producing paraspores. Except for one Mexican strain of P. popilliae, the Central and South American strains of P. popilliae are sensitive to vancomycin. This was unexpected since all North American strains of P. popilliae are vancomycin resistant. Vancomycin resistance could be useful in identifying strains of P. popilliae from North America but not for identifying strains of P. popilliae from Central and South America. So far, no vancomycin resistant strains of P. lentimorbus have been identified. There was variability in the ability of these organisms to grow on medium supplemented with 2% sodium chloride so the usefulness of this trait is debatable. However, the majority of P. popilliae strains from Mexico and Central and South America will grow on medium supplemented with 2% sodium chloride and the majority of the P. lentimorbus strains from these same areas will not grow on this medium. North American strains of P. popilliae and P. lentimorbus also showed variability of growth on medium supplemented with 2% sodium chloride.