AREA TECNICO SCIENTIFICA

29° cicloCorso di dottorato in Scienze e Biotecnologieagrarie

The genome of Lysobacter capsici AZ78:

a biocontrol agent of Plasmopara viticolaIntroductionThe genus Lysobacter (Family Xanthomonadaceae) encompasses bacterial strains that share physiological features, such as the release of lytic enzymes and antibiotics, that make them capable to control plant diseases (Hayward et al., 2010). Recently, we showed that the application of L. capsici AZ78 cells on grapevine plants drastically reduces the infections of Plasmopara viticola (Puopolo et al., 2014). Additionally, we provided the evidence that L. capsici AZ78 can be combined with copper fungicides, thus increasing the efficacy against this pathogen (Puopolo et al., 2014).

Fig.1 Phylogenetic analysis of the gene coding for B-lactamase in Lysobacter capsici AZ78. The sequenceswere aligned by using Clustal X and the bestphylogenetic tree was constructed by applyingKimura’s two-parameter model and the neighbor-joining method implemented in the MEGA5 program.

Production of antibioticsL. capsici AZ78 genome has genes involved in the production of secondary metabolites toxic to Gram-positive bacteria, phytopathogenic fungi and oomycetes. Specifically, the AZ78_1098 gene has homology with the gene of L. enzymogenes C3 involved in the synthesis of a macrocyclic lactam antibiotic (Fig. 1) (Puopolo et al., 2016).

Objective and future prospectiveThe aim of my PhD project was to analyze the genome of L. capsici AZ78 in order to have information useful for its possible employment as a new active ingredient in biofungicides.

Resistance to copper and toxic compoundsThe genome of L. capsici AZ78 contains genes responsible for the transport, homeostasis, uptake and resistance to copper ions (Tab.2). All these data are in agreement with the in vitro experiments, which showed that the combination of this bacterial strain with copper based fungicides is possible (Fig.3) (Puopolo et al., 2014; 2016). Moreover, the genome analysis revealed the presence of genes coding for efflux pumps involved in the resistance to antibiotics, heavy metals and toxic compounds (Tab.2).

Cell motility and host colonizationThe mining of L. capsici AZ78 genome allowed the identification of 43 genes related to the biogenesis of Type IV pili (Fig.4) and 21 putative genes encoding components of flagellar apparatus. The presence of motility mechanisms may play a key role in the bacterial colonization of leaves and P. viticola hyphae.

Fig.3 Resistance of Lysobacter capsici AZ78 to plant protection products. The survival of L. capsici AZ78 cellsin the presence of fungicides was assessed by growing the bacterium on LBA amended with plant protectionproducts at concentrations commonly applied in the field. The reduction in cell viability was calculated as theratio between the CFU difference for L capsici AZ78 grown on LBA and L capsici AZ78 grown on LBA amendedwith plant protection products, over L capsici AZ78 CFU grown on LBA.

ConclusionAll the information provided by the genome of L. capsici AZ78 will be helpful to determine the mechanisms involved in the biological control of phytopathogenic fungi and oomycetes. This knowledge is important for the future development of L. capsici AZ78 as an active ingredient in new biofungicides.

Fig.2 Characterization of Lysobacter capsici

AZ78. L. capsici AZ78 produces A) cellulases; B)

glucanases; C) chitinases and D) siderophores.

The genomeThe L. capsici AZ78 genome (JAJA00000000) consists of 6,315,650 bases assembled into three contigs, and the G+C content is 66.4%. The genome contains 5,292 coding sequences, 93 predicted RNAs of which 1 is a tmRNA, 7 rRNAs and 85 tRNAs.

Biological activity Gene ID

Proteolytic activity AZ78_4508,4509,4511,4512

AZ78_4514,4516

AZ78_269,271,272

Degradation of glucans AZ78_1531 gluC

AZ78_4722 gluB

AZ78_4006 cel8A

AZ78_4352 celA

Degradation of cellulose AZ78_3685 cel5G

Degradation of chitin AZ78_0055 chiB

Synthesis of catechol siderophores

AZ78_407,408,409,410,411,412

Tab.1 List of genes of Lysobacter capsici AZ78 associated

with the production of lytic enzymes and siderophores.

Biological activity Gene ID

Resistance to copper ions

AZ78_560,561,562

Resistance to toxic compounds

AZ78_906,1192 AZ78_266,1103

AZ78_3698,3949

AZ78_4767

Resistance to antibiotics

AZ78_3393

AZ78_238,2665

AZ78_3627,4028

Tab.2 List of genes of Lysobacter capsiciAZ78 associated with resistance toenvironmental stressors.

Fig.4 Type 4 pili geneorganization in Lysobactercapsici AZ78 genome. Genesencoding structuralcomponents (grey) regulatorycomponents (white) of Type 4pili. The gene names arereported above the arrows,and the correspondingaccession number is reportedunder each gene.

Lytic enzyme and siderophore productionThe direct biocontrol activity of L. capsici AZ78 is based on the presence of several lytic enzymes. Specifically, the genome has genes coding for chitinases, glucanases, lipases, proteases, and xylanases (Tab.1). The in vitro experiments confirmed the outcome of the genome analysis (Fig.2). Moreover, L. capsici AZ78 compete with other microorganisms for the iron resources in the environment by the production of siderophores.

Dott. Selena Tomada

Prof. Nazia Loi

Dott. Ilaria Pertot

Dott. Gerardo PuopoloInfo:

Tel. +39 0461615502

tomada.selena@spes.uniud.it

ReferencesPuopolo, G., Tomada, S., Sonego, P., Moretto, M., Engelen, K., Perazzolli, M., & Pertot, I. (2016). The Lysobacter capsici AZ78 genome has a gene pool enabling it to interact successfully with phytopathogenic microorganisms and environmental factors. Front. Microbiol., 7. Puopolo, G., Giovannini, O., and Pertot, I. (2014). Lysobacter capsici AZ78 can be combined with copper to effectively control Plasmopara viticola on grapevine. Microbiol. Res. 169, 633–642.Hayward, A.C., Fegan, N., Fegan, M., and Stirling, G.R. (2010). Stenotrophomonas and Lysobacter: ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology. J. App. Microbiol. 108, 756–770.

AcknowledgmentsThis project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 289497 (project CO-FREE, theme KBBE.2011.1.2-06).

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