Nigris et al. BMC Microbiology (2018) 18:133 https://doi.org/10.1186/s12866-018-1306-5 RESEARCH ARTICLE Open Access Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera Sebastiano Nigris1, Enrico Baldan2, Alessandra Tondello2, Filippo Zanella2, Nicola Vitulo3, Gabriella Favaro4, Valerio Guidolin2, Nicola Bordin2, Andrea Telatin5, Elisabetta Barizza2, Stefania Marcato2, Michela Zottini2, Andrea Squartini6, Giorgio Valle2 and Barbara Baldan1* Abstract Background: Bacillus licheniformis GL174 is a culturable endophytic strain isolated from Vitis vinifera cultivar Glera, the grapevine mainly cultivated for the Prosecco wine production. This strain was previously demonstrated to possess some specific plant growth promoting traits but its endophytic attitude and its role in biocontrol was only partially explored. In this study, the potential biocontrol action of the strain was investigated in vitro and in vivo and, by genome sequence analyses, putative functions involved in biocontrol and plant-bacteria interaction were assessed. Results: Firstly, to confirm the endophytic behavior of the strain, its ability to colonize grapevine tissues was demonstrated and its biocontrol properties were analyzed. Antagonism test results showed that the strain could reduce and inhibit the mycelium growth of diverse plant pathogens in vitro and in vivo. The strain was demonstrated to produce different molecules of the lipopeptide class; moreover, its genome was sequenced, and analysis of the sequences revealed the presence of many protein-coding genes involved in the biocontrol process, such as transporters, plant-cell lytic enzymes, siderophores and other secondary metabolites. Conclusions: This step-by-step analysis shows that Bacillus licheniformis GL174 may be a good biocontrol agent candidate, and describes some distinguished traits and possible key elements involved in this process. The use of this strain could potentially help grapevine plants to cope with pathogen attacks and reduce the amount of chemicals used in the vineyard. Keywords: Grapevine, Endophytes, Biocontrol bacteria, Bacillus licheniformis, Bacterial genome sequencing Background Endophytes are bacteria that can live inside plant tissues Biological control is an increasingly successful and wide- and colonize their hosts without causing any signs of spread strategy [1] to decrease plant pathogens and the plant disease. They penetrate plants mainly from the soil negative effects of agricultural practices on the environ- and roots and spread into leaves, flowers and fruits ment. It uses beneficial microorganisms, either bacteria through the vascular plant system [3, 4]. These bacteria or fungi, that can counteract plant pathogens and limit spend part of (facultative endophytes) or all (obligate en- the use of chemicals in agriculture [2]. Among these di- dophytes) their life-cycle inside plants, exploiting this verse beneficial microorganisms, bacterial endophytes strategic interaction to their advantage [3, 5]. They may are powerful tools to protect plants from phytopatho- both promote the growth of plants and protect them gens due to their ability to enter and colonize plants. against harmful bacteria and fungi. Endophytes can en- hance plant-growth rate and biomass production largely through phytohormone synthesis, nitrogen fixation, * Correspondence: [email protected] 1Botanical Garden and Department of Biology, University of Padova, Padova, phosphate solubilization and ammonium ion production. Italy They protect hosts as biocontrol agents by interacting Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nigris et al. BMC Microbiology (2018) 18:133 Page 2 of 16 directly with pathogens and producing many antimicro- stems and leaves, showing how GFP-tagged strains may bial molecules, and/or by competing for nutrients inside be used even to check grapevine colonization [12]. the colonized tissues [3, 6]. Biocontrol bacteria also act An important characteristic of endophytes is to secrete indirectly, eliciting Induced Systemic Resistance (ISR) in into the environment lytic enzymes that degrade many their plant hosts: a plethora of metabolites produced by biological polymers. Such enzymes, in particular endophytes activates plant defense priming responses cellulose-lytic enzymes, favor the entrance of endophytes against pathogens [7]. into plant tissues and the formation of stable colonies, giv- Many endophyte taxa produce lipopeptides (LPs) - mole- ing clear competitive advantage to bacteria with this ability cules that play a crucial role in biocontrol acting directly as [18]. Before evaluating biocontrol effects, and to develop antimicrobial/antifungal compounds and as ISR elicitors in bacteria inocula for agriculture, it is essential to show if plant hosts. These amphiphilic compounds are formed by a and how a particular strain colonizes inner plant tissues. short cyclic oligopeptide linked to a lipid tail [8]. The most In this work, the culturable strain GL174, previously iso- widely studied LPs belong to the surfactin, iturin and fengy- lated from Vitis vinifera cv. Glera [19] was investigated to cin families, according to their chemical structure. Surfac- identify both its endophytic ability and some of its biocon- tins are heptapeptides interlinked with a β-hydroxy fatty trol traits. GL174 was selected from a collection of puta- acid to form a cyclic lactone ring; due to their strong bio- tive grapevine endophytes for its plant-growth promotion surfactant activity, these molecules can readily associate abilities. Moreover, GL174 can produce ammonia and the and anchor themselves to the double layer of phospholipids plant hormone indole-3-acetic acid, and causes morpho- interfering with membrane integrity. Iturins are heptapep- logical changes to the plant roots when co-cultured with tides bound to a β-amino fatty acid chain 14–17 carbons Arabidopsis thaliana [20]. The strain was identified as Ba- long. Fengycins are lipodecapeptides with an internal lac- cillus licheniformis GL174 and its endophytic attitude was toneringinthepeptidicmoietyandaβ-hydroxy fatty acid validated by plating surface-sterilized inoculated cuttings. chain (C14-C18) that may be saturated or unsaturated [9]. Then, using confocal microscopy, we localized the These molecules, according to their chemical characteris- GFP-tagged strain within plant tissues of inoculated tics, fight bacteria, fungi, mycoplasmas and viruses. Due to grapevine Glera cuttings. Visualization of tagged bacteria their strong surfactant power, LPs enable and favor plant inside plant structures allowed us to identify the examined colonization by the producer strain hindering pathogenic strain as a true endophyte of the plants, and provided a re- tissue infection. Bacteria synthesize these families of lipo- liable protocol for cutting inoculation for further biocon- peptides in a non-ribosomal way through large enzymatic trol experiments. As the plant growth-promoting (PGP) complexes, namely, lipopeptide synthetases. These abilities of strain GL174 had already been demonstrated mega-enzymes are organized in iterative modules that [20], we focused on its potential biocontrol activity and catalyze reactions for lipopeptide production. antifungal properties. We reported both antifungal activity Visualization of bacteria inside plants is always diffi- against some grapevine fungal pathogens by an in vitro cult as plant tissues are complex and autofluorescent. bioassay and in vivo on grapevine leaves. Furthermore, we Recently, molecular techniques employing fluorescent reported an effective production of LPs, detected by mass probes that detect bacteria via hybridization have been spectrometric analyses and we also sequenced the entire used to localize and estimate microorganisms within genome. The results of this multidisciplinary approach en- plant organs [4, 10]. Inoculation with strains tagged with abled us to assess the complex pattern of biocontrol traits green fluorescent protein (GFP) and glucuronidase gene displayed by Bacillus licheniformis GL174. markers has enabled scientists to observe live bacteria inside tissues, which is particularly useful when follow- Methods ing bacterial colonization patterns and estimating endo- Bacterial strain and growth conditions phytic populations [11–13]. GFP-tagged bacteria are ThestrainGL174waspreviouslyisolatedfrom handy tools to examine endophyte–plant interactions surface-sterilized tissues of Vitis vinifera cv. Glera and iden- [14], as GFP does not require any substrate or cofactor tified as Bacillus licheniformis [19]. This strain was culti- to fluoresce. GFP cassettes for chromosomal integration vated routinely in Nutrient Broth (NB) or Nutrient Agar and expression of the reporter gene in many bacterial (NA) at 28 °C. species have been developed [15–17]. The transform- ation of bacteria with plasmids harboring