Send Orders for Reprints to reprints@benthamscience.net 60 Current Biotechnology, 2014, 3, 60-75 Biotechnological Applications of Bacterial Endophytes Jesús Mercado-Blanco *,1 and Ben J.J. Lugtenberg 2 1 Department of Crop Protection, Institute for Sustainable Agriculture (CSIC), Campus ‘Alameda del Obispo’, Avda. Menéndez Pidal, S/N, 14004 Córdoba, Spain 2 Leiden University, Institute of Biology, Sylvius Laboratory, Sylviusweg 72, 2333 BD Leiden, The Netherlands Abstract: This review starts with a brief introduction on bacterial endophytes. Only small fractions of rhizosphere and phyllosphere bacteria are able to live inside the plant. Endophytes are bacteria and fungi that can be detected at a particular moment within the tissues of apparently healthy plant hosts without producing symptoms in or on the plant. Possible traits required by these bacteria to enter the plant and live inside will be discussed. Furthermore, we will focus on possible biotechnological applications of bacterial endophytes and present case studies as examples. Endophytes can promote plant growth, for example by the production of hormones or by making nutrients (such as nitrogen, phosphate and ferric ions) available to the plant. Endophytes can also promote plant growth indirectly, for example by suppression of plant diseases, by inactivating environmental pollutants, and by alleviating stresses of the plant caused by excess of the hormone ethylene, by heavy metals, by draught and by salinated soil. Some endophytic bacteria can produce nanoparticles which have numerous applications. At the end of the review we will discuss aspects involved in the commercialization of microbes. Keywords: Biological control, commercial microbial products, disease suppression, endophyte, microbiome, nanoparticles, plant growth-promotion, plant-microbe interaction, rhizoremediation, root hair, stress tolerance. 1. INTRODUCTION We define the word endophyte as “those bacteria and fungi that can be detected at a particular moment within the tissues of apparently healthy plant hosts without producing symptoms” [1]. This definition excludes pathogens and nodule-producing microbes. Although fungal endophytes also exist, we will focus in this review on bacterial endophytes. The vast majority of bacterial endophytes is (presently) non-culturable or viable but non-culturable (VBNC). Therefore metagenomic approaches are used to uncover the broad diversity of endophytic communities [2- 5]. Since endophytes which presently cannot be produced at a large scale are not suitable for biotechnological applications we will focus on culturable endophytes. We will start this review with a brief introduction on bacterial endophytes. Only small fractions of rhizosphere and phyllosphere bacteria are able to live inside the plant. Possible traits required to enter the plant and live endophytically will be discussed. In the rest of this review we will focus on possible biotechnological applications of bacterial endophytes and present case studies as examples. 2. BACTERIAL ENDOPHYTES Presently, the general belief is that bacterial and fungal endophytes are present in all plants [6]. In order to isolate endophytes, microbes present on the plant surface have to be *Address correspondence to this author at the Department of Crop Protection, Institute for Sustainable Agriculture (CSIC), Campus ‘Alameda del Obispo’, Avda. Menéndez Pidal, S/N, 14004 Córdoba, Spain; Tel: + 34 957 499261; E-mail: jesus.mercado@ias.csic.es fully eliminated first. So, requirements for sterilisation protocols are (i) that all microbes present on the plant’s surface are killed but also (ii) that these procedures have an as small as possible negative effect on the endophyte population. It is clear that the more stringent the used sterilisation protocol is, the lesser endophytes will be found. So, values reported for cfu’s (colony forming units) are minimal values. Criteria for the correct identification of endophytic bacteria have been established elsewhere [6, 7]. These go beyond isolation from surface-disinfected plant tissues and require further support, for instance, by microscopic proof and by the ability of the putative endophyte to re-infect disinfected seedlings [8]. On the one hand, the plant’s interior can be considered as a protective environment compared to the highly competitive/predatory environment found outside plant tissues [9]. On the other hand, the interior can be viewed as an hostile environment considering the multiple defence responses that plants deploy against the “invasion” of bacteria that are able to become endophytic [10]. Perhaps, the prevalence of nonculturable and/or VBNC states for most endophytes points towards a survival strategy to overcome stresses operating in plant tissues such as defensive responses [11]. From this point of view, endophytes might have found an evolutionary solution to cope with an extreme environment. Bacterial endophytes can be found at many sites in the plant, such as root, stem, leave, berry, seed, and xylem sap [6, 8, 12-14] (Fig. 1). The population density of endophytes is higher in roots than in any other plant organ. In the root the average density is 10 5 cfu per g fresh weight whereas average values of 10 4 and 10 3 are reported for stem and for 2211-551X/14 $58.00+.00 © 2014 Bentham Science Publishers