RESUMO
The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.
Assuntos
Antibiose , Antinematódeos/farmacologia , Enzimas/farmacologia , Nematoides/efeitos dos fármacos , Proteínas/farmacologia , Pseudomonas/fisiologia , Animais , Antinematódeos/química , Antinematódeos/isolamento & purificação , Chile , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Enzimas/química , Enzimas/isolamento & purificação , Espectrometria de Massas , Microscopia Eletrônica , Peso Molecular , Nematoides/ultraestrutura , Filogenia , Raízes de Plantas/microbiologia , Proteínas/química , Proteínas/isolamento & purificação , Pseudomonas/classificação , Pseudomonas/genética , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Microbiologia do Solo , Análise de SobrevidaRESUMO
A new plant commensal Pseudomonas veronii isolate (strain R4) was identified from a Xiphinema index biocontrol screen. Isolated from grapevine roots from vineyards in central Chile, the strain R4 exhibited a slower yet equivalently effective nematicide activity as the well-characterized P. protegens CHA0. Whole genome sequencing of strain R4 and comparative analysis among the available Pseudomonas spp. genomes allowed for the identification of gene clusters that encode putative extracellular proteases and lipase synthesis and secretion systems, which are proposed to mediate-at least in part-the observed nematicidal activity. In addition, R4 strain presented relevant gene clusters related to metal tolerance, which is typical in P. veronii. Bioinformatics analyses also showed gene clusters associated with plant growth promoting activity, such as indole-3-acetic acid synthesis. In addition, the strain R4 genome presented a metabolic gene clusters associated with phosphate and ammonia biotransformation from soil, which could improve their availability for plants.
