Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam]

Abstract

Bacterial microorganisms which are latent in <i>in vitro</i> cultures can limit the efficiency of <i>in vitro</i> methods for the conservation of genetic resources. In this study we screened 2,373 accessions from the <i>in vitro</i> sweetpotato germplasm collection of the International Potato Center in Lima, Peru for bacteria associated with plantlets in tissue culture through a combination of morphological methods and partial 16S rDNA sequencing. Bacteria were detected in 240 accessions (10% of the accessions screened) and we were able to isolate 184 different bacterial isolates from 177 different accessions. These corresponded to at least nineteen Operational Taxonomic Units (OTUs) of bacteria, belonging to the genera <i>Sphingomonas</i>, <i>Bacillus</i>, <i>Paenibacillus</i>, <i>Methylobacterium</i>, <i>Brevibacterium</i>, <i>Acinetobacter</i>, <i>Microbacterium</i>, <i>Streptomyces</i>, <i>Staphylococcus</i>, and <i>Janibacter</i>. Specific primers were developed for PCR based diagnostic tests that were able to rapidly detect these bacteria directly from tissue culture plants, without the need of microbial sub-culturing. Based on PCR screening the largest bacterial OTUs corresponded to a <i>Paenibacillus</i> sp. closely related to <i>Paenibacillus taichungensis</i> (41.67%), and <i>Bacillus</i> sp. closely related to <i>Bacillus cereus</i> (22.22%), and <i>Bacillus pumilus</i> (16.67%). Since <i>in vitro</i> plant genetic resources must be microbe-free for international distribution and use, any microbial presence is considered a contamination and therefore it is critical to clean all cultures of these latent-appearing bacteria. To accomplish this, plantlets from <i>in vitro</i> were transferred to soil, watered with Dimanin^® (2 ml/l) weekly and then reintroduced into <i>in vitro</i>. Of the 191 accessions processed for bacterial elimination, 100% tested bacteria-free after treatment. It is suspected that these bacteria may be endosymbionts and some may be beneficial for the plants.