title: Investigation of Endophytic Bacterial Community in Supposedly Axenic Cultures of Pineapple and Orchids with Evidence on Abundant Intracellular Bacteria.
abstract: Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1 year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."
authors.affiliation: Biological Science Department, "Luiz de Queiroz" Superior College of Agriculture, University of São Paulo (ESALQ/USP), Pádua Dias Avenue, 11, P.O. Box 9, Piracicaba, SP, 13418-900, Brazil. esposito.polesi@gmail.com.
authors.forename: Natalia Pimentel
authors.initials: NP
authors.lastname: Esposito-Polesi
authors.affiliation: Biological Science Department, "Luiz de Queiroz" Superior College of Agriculture, University of São Paulo (ESALQ/USP), Pádua Dias Avenue, 11, P.O. Box 9, Piracicaba, SP, 13418-900, Brazil.
authors.forename: Monita Fiori
authors.initials: MF
authors.lastname: de Abreu-Tarazi
authors.affiliation: In Vitro Palm Consultoria, Estudo e Desenvolvimento Biológico Ltda, Rua Itajobi, 421, Piracicaba, SP, 13432-000, Brazil.
authors.forename: Cristina Vieira
authors.initials: CV
authors.lastname: de Almeida
authors.affiliation: Cell Biology and Molecular Laboratory, Nuclear Energy in Agriculture Center, University of São Paulo, Centenário Avenue, 303, P.O. Box 96, Piracicaba, SP, 13416-000, Brazil.
authors.forename: Siu Mui
authors.initials: SM
authors.lastname: Tsai
authors.affiliation: Biological Science Department, "Luiz de Queiroz" Superior College of Agriculture, University of São Paulo (ESALQ/USP), Pádua Dias Avenue, 11, P.O. Box 9, Piracicaba, SP, 13418-900, Brazil.
authors.forename: Marcílio
authors.initials: M
authors.lastname: de Almeida
mesh_terms: D031230:Ananas / Q000254:growth & development / Q000382:microbiology*; D061245:Axenic Culture; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D060026:Endophytes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D029595:Orchidaceae / Q000254:growth & development / Q000382:microbiology*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S