Investigation of Endophytic Bacterial Community in Supposedly Axenic Cultures of Pineapple and Orchids with Evidence on Abundant Intracellular Bacteria Natalia Pimentel Esposito-Polesi 1 • Monita Fiori de Abreu-Tarazi 1 • Cristina Vieira de Almeida 2 • Siu Mui Tsai 3 • Marcı ´lio de Almeida 1 Received: 4 November 2015 / Accepted: 19 July 2016 Ó Springer Science+Business Media New York 2016 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 microor- ganism growth in the culture medium. These microorgan- isms 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 asymp- tomatic 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-depen- dent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bac- teria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scan- ning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the pres- ence 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.’’ Introduction Micropropagation is a technique associated with large- scale production of healthy seedlings in a limited physical space, in which asepsis and the absence of microbial contamination are essential conditions [1]. This microbial contamination ordinarily occurs by ineffective surface disinfestation of explants, as well as by handling failures during sub-culturing procedures, resulting in excessive loss of propagules, thus making this a very expensive technique [2, 3]. However, in the absence of contamination, microprop- agation is a very successful procedure, resulting in what generally known as axenic microplants. However, this same microplant after long-term in vitro culture, or after a mechanical, physical, or nutritional stress, may present the growth of small microorganism colonies on the culture medium or on the microplant surface that was previously theretofore asymptomatic [4–6]. These microorganisms are actually latent endophytes inside the plant tissues that do not show any symptoms of colonization and are not eliminated by the usual disinfes- tation processes [3–5]. Therefore, their growth on the culture medium is only in response to some in vitro con- dition imbalance [7, 8], and not specifically due to failures in micropropagation as previously mentioned [9]. & Natalia Pimentel Esposito-Polesi esposito.polesi@gmail.com 1 Biological Science Department, ‘‘Luiz de Queiroz’’ Superior College of Agriculture, University of Sa ˜o Paulo (ESALQ/ USP), Pa ´dua Dias Avenue, 11, P.O. Box 9, Piracicaba, SP 13418-900, Brazil 2 In Vitro Palm Consultoria, Estudo e Desenvolvimento Biolo ´gico Ltda, Rua Itajobi, 421, Piracicaba, SP 13432-000, Brazil 3 Cell Biology and Molecular Laboratory, Nuclear Energy in Agriculture Center, University of Sa ˜o Paulo, Centena ´rio Avenue, 303, P.O. Box 96, Piracicaba, SP 13416-000, Brazil 123 Curr Microbiol DOI 10.1007/s00284-016-1163-0