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Enterobacter cloacae, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen.

ABSTRACT: Banana (Musa spp.) is an important crop worldwide, but black Sigatoka disease caused by the fungus Pseudocercospora fijiensis threatens fruit production. In this work, we examined the potential of the endophytes of banana plants Enterobacter cloacae and Klebsiella pneumoniae, as antagonists of P. fijiensis and support plant growth in nutrient limited soils by N-transfer. The two bacterial isolates were identified by MALDI-TOF mass spectrometry and corroborated by 16S rRNA sequence analysis. Both bacteria were positive for beneficial traits such as N-fixation, indole acetic acid production, phosphate solubilization, negative for 1-aminocyclopropane 1-carboxylic acid deaminase and were antagonistic to P. fijiensis. To measure the effects on plant growth, the two plant bacteria and an E. coli strain (as non-endophyte), were inoculated weekly for 60 days as active cells (AC) and heat-killed cells (HKC) into plant microcosms without nutrients and compared to a water only treatment, and a mineral nutrients solution (MMN) treatment. Bacterial treatments increased growth parameters and prevented accelerated senescence, which was observed for water and mineral nutrients solution (MMN) treatments used as controls. Plants died after the first 20 days of being irrigated with water; irrigation with MMN enabled plants to develop some new leaves, but plants lost weight (-30%) during the same period. Plants treated with bacteria showed good growth, but E. cloacae AC treated plants had significantly greater biomass than the E. cloacae HKC. After 60 days, plants inoculated with E. cloacae AC showed intracellular bacteria within root cells, suggesting that a stable symbiosis was established. To evaluate the transference of organic N from bacteria into the plants, the 3 bacteria were grown with ¹⁵NH₄Cl or Na¹⁵NO₃ as the nitrogen source. The ¹⁵N transferred from bacteria to plant tissues was measured by pheophytin isotopomer abundance. The relative abundance of the isotopomers m/z 872.57, 873.57, 874.57, 875.57, 876.57 unequivocally demonstrated that plants acquired ¹⁵N atoms directly from bacterial cells, using them as a source of N, to support plant growth in restricted nutrient soils. E. cloacae might be a new alternative to promote growth and health of banana crops.

SUBMITTER: Macedo-Raygoza GM

PROVIDER: S-EPMC6513882 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Project description:Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Project description:New antibiotics are urgently needed due to the huge increase of multidrug-resistant bacteria. The underexplored gram-negative bacterium Enterobacter cloacae is known to cause severe urinary tract and lung infections (UTIs). The pathogenicity of E. cloacae in UTI has only been studied at the bioinformatic level, but until now not within systematic in vitro investigations. The present study assesses different human cell lines for monitoring the early steps of host-pathogen interaction regarding bacterial adhesion to and invasion into different host cells by flow cytometric adhesion assay, classical cell counting assay, gentamicin invasion assay, and confocal laser scanning microscopy. To our knowledge, this is the first report in which E. cloacae has been investigated for its interaction with human bladder, kidney, skin, and lung cell lines under in vitro conditions. Data indicate that E. cloacae exerts strong adhesion to urinary tract (bladder and kidney) and lung cells, a finding which correlates with the clinical relevance of the bacterium for induction of urinary tract and lung infections. Furthermore, E. cloacae ATCC 13047 barely adheres to skin cells (A-431) and shows no relevant interaction with intestinal cells (Caco-2, HT-29), even in the presence of mucin (HT29 MTX). In contrast, invasion assays and confocal laser scanning microscopy demonstrate that E. cloacae internalizes in all tested host cells, but to a different extent. Especially, bladder and kidney cells are being invaded to the highest extent. Defective mutants of fimH and fimA abolished the adhesion of E. cloacae to T24 cells, while csgA deletion had no influence on adhesion. These results indicate that E. cloacae has different pattern for adhesion and invasion depending on the target tissue, which again correlates with the clinical relevance of the pathogen. For detailed investigation of the early host-pathogen interaction T24 bladder cells comprise a suitable assay system for evaluation the bacterial adhesion and invasion.

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Enterobacter cloacae, an Endophyte That Establishes a Nutrient-Transfer Symbiosis With Banana Plants and Protects Against the Black Sigatoka Pathogen.

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