When genome-based approach meets the “Old but Good”: revealing genes involved in the antibacterial activity of Pseudomonas sp. P482 against soft rot pathogens

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYS(T). Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis

The antimicrobial volatile power of the rhizospheric isolate Pseudomonas donghuensis P482.

Soil and rhizosphere bacteria produce an array of secondary metabolites including a wide range of volatile organic compounds (VOCs). These compounds play an important role in the long-distance interactions and communication between (micro)organisms. Furthermore, bacterial VOCs are involved in plant pathogens inhibition and induction of soil fungistasis and suppressivenes. In the present study, we analysed the volatile blend emitted by the rhizospheric isolate Pseudomonas donghuensis P482 and evaluated the volatile effect on the plant pathogenic fungi and bacteria as well as one oomycete. Moreover, we investigated the role of the GacS/GacA system on VOCs production in P. donghuensis P482. The results obtained demonstrated that VOCs emitted by P. donghuensis P482 have strong antifungal and antioomycete, but not antibacterial activity. The production of certain volatiles such as dimethyl sulfide, S-methyl thioacetate, methyl thiocyanate, dimethyl trisulfide, 1-undecan and HCN is depended on the GacS/GacA two-component regulatory system. Apparently, these compounds play an important role in the pathogens suppression as the gacA mutant entirely lost the ability to inhibit via volatiles the growth of tested plant pathogens

Data obtained from MetaboAnalyst 3.0 and GC-MS results.

<p>(A) PLS-DA plot of VOC’s produced by <i>P</i>. <i>donghuensis</i> P482 and KN3318 mutant with media as a control (triplicates); (B) TIC chromatographs from GC-MS analysis showing different peaks between <i>P</i>. <i>donghuensis</i> P482 and KN3318 mutant. Significant peaks that differ between wild type and mutant are indicated by asterisk. Dimethyl disulfide (DMDS) is indicated by “#”.</p

Number of colony forming units (cfu) of <i>Pseudomonas fluorescens</i> AD21 and <i>Agrobacterium</i> sp. AD140 after overnight exposure to volatiles emitted by <i>P</i>. <i>donghuensis</i> P482 and control without bacterial volatiles.

<p>Error bars represents standard deviation of the mean.</p

The list of the bacterial, fungal and oomycetes strains used in this study.

<p>The list of the bacterial, fungal and oomycetes strains used in this study.</p

Volatile compounds identified in the headspace <i>P</i>. <i>donghuensis</i> P482 wt not present in the KN3318 gacA mutant culture.

<p>Volatile compounds identified in the headspace <i>P</i>. <i>donghuensis</i> P482 wt not present in the KN3318 gacA mutant culture.</p

Average hyphae radius after 4 days (<i>Rhizoctonia solani</i>), 7 days (<i>Fusarium cilmorum</i>) and 5 days (<i>Pythium ultimum</i>) of incubation under influence of volatiles emitted by <i>P</i>. <i>donghuensis</i> P482 wt, KN3318 mutant and non-treated control (NC).

<p>Significant difference between sample and control are indicated by asterisk (one-way ANOVA), error bars represents standard deviation of the mean.</p

The antimicrobial volatile power of the rhizospheric isolate <i>Pseudomonas donghuensis</i> P482 - Fig 4

<p>(A) Average hyphae radius of <i>P</i>. <i>ultimum</i> and <i>R</i>. <i>solani</i> after 4 days of incubation exposed and not exposed (NC) to pure volatile organic compounds: S-methyl thioacetate (MTA), dimethyl disulfide (DMDS) and combination of MTA and DMDS. Significant difference between samples are indicated by different letters (one-way ANOVA, post hoc t-test p < 0.05), error bars represents standard deviation of the mean. (B) Photos of <i>R</i>. <i>solani</i> cultures exposed (+) and not exposed (-) to MTA.</p

Plastic mulch film residues in agriculture : impact on soil suppressiveness, plant growth, and microbial communities

Plastic mulch film residues have been accumulating in agricultural soils for decades, but so far, little is known about its consequences on soil microbial communities and functions. Here, we tested the effects of plastic residues of low-density polyethylene and biodegradable mulch films on soil suppressiveness and microbial community composition. We investigated how plastic residues in a Fusarium culmorum suppressive soil affect the level of disease suppressiveness, plant biomass, nutrient status, and microbial communities in rhizosphere using a controlled pot experiment. The addition of 1% plastic residues to the suppressive soil did not affect the level of suppression and the disease symptoms index. However, we did find that plant biomasses decreased, and that plant nutrient status changed in the presence of plastic residues. No significant changes in bacterial and fungal rhizosphere communities were observed. Nonetheless, bacterial and fungal communities closely attached to the plastisphere were very different from the rhizosphere communities with overrepresentation of potential plant pathogens. The plastisphere revealed a high abundance of specific bacterial phyla (Actinobacteria, Bacteroidetes, and Proteobacteria) and fungal genera (Rhizoctonia and Arthrobotrys). Our work revealed new insights and raises emerging questions for further studies on the impact of microplastics on the agroecosystems

Effects of plastic mulch film residues on wheat rhizosphere and soil properties

Plastic residues could accumulate in soils as a consequence of using plastic mulching, which results in a serious environmental concern for agroecosystems. As an alternative, biodegradable plastic films stand as promising products to minimize plastic debris accumulation and reduce soil pollution. However, the effects of residues from traditional and biodegradable plastic films on the soil-plant system are not well studied. In this study, we used a controlled pot experiment to investigate the effects of macro- and micro- sized residues of low-density polyethylene and biodegradable plastic mulch films on the rhizosphere bacterial communities, rhizosphere volatile profiles and soil chemical properties. Interestingly, we identified significant effects of biodegradable plastic residues on the rhizosphere bacterial communities and on the blend of volatiles emitted in the rhizosphere. For example, in treatments with biodegradable plastics, bacteria genera like Bacillus and Variovorax were present in higher relative abundances and volatile compounds like dodecanal were exclusively produced in treatment with biodegradable microplastics. Furthermore, significant differences in soil pH, electrical conductivity and C:N ratio were observed across treatments. Our study provides evidence for both biotic and abiotic impacts of plastic residues on the soil-plant system, suggesting the urgent need for more research examining their environmental impacts on agroecosystems