Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum

Background The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. Result The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. Conclusions We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

Aspects of pharmacognostic evaluation of the herbs for a traditional antimalarial (maloff-hb) powdered formulation in ogbomoso, Nigeria

Maloff-HB is a documented traditional oral powdered herbal drug in Ogbomoso, Nigeria whose botanical constituents, ascorbic acid and mineral elements composition have been quantified but there is inadequate information on the pharmacognostic properties of the nine herbal materials for its formulation. This study therefore sought to elucidate the bark and wood anatomy of eight of the nine herbs used in the formulation, and identify the diagnostic markers for their authentication. The conventional anatomical techniques of transverse sectioning (TS) and tissue maceration (TIM) were used to draw out 21 characteristics from the root barks of the eight woody species studied. In addition, 41 features of the wood in the roots of three of the species were drawn using TS, transverse longitudinal sectioning (TLS), radial longitudinal sectioning (RLS) and TIM. Following staining, mounting and microscopic examinations, the observed qualitative and quantitative features were taxonomically described in accordance with the provisions of International Association of Wood Anatomists, and their diagnostic values among the medicinal herbs were explored. Bark anatomical markers that are clearly diagnostic of the species studied included features of the secondary cortex, phloem rays, axial parenchyma, sclereids and resin ducts. In the wood, these included features of the vessels in the TS and variable ray characteristics in the TLS. The two artificial keys obtained from discontinuities in qualitative and quantitative features observed in the barks and the woods are useful tools for reliable identification of the herbal materials studied.

Evaluation of some pharmacognostic parameters of the herbs for a traditional hematinic (Haematol -B) powdered formulation in Ogbomoso, Nigeria

The traditional hematinic oral powdered herbal drug (Haematol-B) from Ogbomoso, Nigeria had been characterized in terms of its botanical constituents, ascorbic acid and mineral elements composition, but there is inadequate information on the pharmacognostic properties of the ten herbal materials for its formulation. Therefore the objective of this study was to evaluatethe bark and wood anatomy (as appropriate) of seven of these herbs with a view to highlighting the diagnostic markers for their authentication. Following the conventional anatomical procedures of transverse sectioning (TS) and tissue maceration (TIM), a total of 21 characters were drawn from the barks (root or stem as appropriate) of the seven woody species studied, while 41 characteristics of the wood in the roots of three of the species were compiled using TS, transverse longitudinal sectioning (TLS), radial longitudinal sectioning (RLS) and TIM techniques. Staining, mounting and microscopic examinations followed, and the observed features were taxonomically described in accordance with the provisions of International Association of Wood Anatomists. Their diagnostic values among the medicinal herbs studied were also explored. The bark anatomical features that can be used to diagnose the species studied were those of the secondary cortex, phloem rays, axial parenchyma, sclereids and resin ducts. The wood anatomical markers included features of the vessels in the TS and variable ray characteristics in the TLS. The two artificial keys constructed from discontinuities in qualitative and quantitative anatomical features of the barks and the wood are useful tools for avoiding misidentification of the herbal materials studied.

Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum

The non-vascular plant Marchantia polymorpha has emerged as a valuable model for studying evolutionarily conserved microbial infection strategies and plant immune responses. However, only a handful of fungal pathogens of Marchantia have been described so far. Here we establish a new pathosystem using the root-infecting vascular wilt fungus Fusarium oxysporum. On angiosperms, this fungus exhibits exquisite adaptation to the plant vascular niche and host-specific pathogenicity, both of which are conferred by lineage-specific effectors secreted during growth in the xylem. We show that F. oxysporum isolates with different lifestyles - pathogenic or endophytic - are able to infect this non-vascular liverwort causing tissue maceration and plant cell killing. Similar to bacterial pathogens, F. oxysporum induces a PAMP-triggered immune response in M. polymorpha. Analysis of isogenic fungal mutants established that infection of Marchantia requires conserved fungal pathogenicity mechanisms such as mitogen activated protein kinases, transcriptional regulators and cell wall remodeling enzymes. Remarkably, lineage-specific virulence effectors are dispensable for infection, most likely due to the absence of xylem tissue in this non-vascular plant. The F. oxysporum - M. polymorpha system provides new insights into the mechanism and evolution of pathogenic and endophytic fungus-plant interactions.

An Exo-Polygalacturonase Pgc4 Regulates Aerial Hyphal Growth and Virulence in Fusarium oxysporum f. sp. cubense race 4

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein−1 ·min−1, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4.

Quorum Quenching in a Novel Acinetobacter sp. XN-10 Bacterial Strain against Pectobacterium carotovorum subsp. carotovorum

Quorum sensing (QS) is a cell density-dependent mechanism that regulates the expression of specific genes in microbial cells. Quorum quenching (QQ) is a promising strategy for attenuating pathogenicity by interfering with the QS system of pathogens. N-Acyl-homoserine lactones (AHLs) act as signaling molecules in many Gram-negative bacterial pathogens and have received wide attention. In this study, a novel, efficient AHL-degrading bacterium, Acinetobacter sp. strain XN-10, was isolated from agricultural contaminated soil and evaluated for its degradation efficiency and potential use against QS-mediated pathogens. Strain XN-10 could effectively degrade N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-hexanoyl-L-homoserine lactone (C6HSL), N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL), and N-(3-oxooctanoyl)-L-homoserine lactone (3OC8HSL), which all belong to the AHL family. Analysis of AHL metabolic products by gas chromatography–mass spectrometry (GC-MS) led to the identification of N-cyclohexyl-propanamide, and pentanoic acid, 4-methyl, methyl ester as the main intermediate metabolites, revealing that AHL could be degraded by hydrolysis and dehydroxylation. All intermediates were transitory and faded away without any non-cleavable metabolites at the end of the experiment. Furthermore, strain XN-10 significantly attenuated the pathogenicity of Pectobacterium carotovorum subsp. carotovorum (Pcc) to suppress tissue maceration in carrots, potatoes, and Chinese cabbage. Taken together, our results shed light on the QQ mechanism of a novel AHL-degrading bacterial isolate, and they provide useful information which show potential for biocontrol of infectious diseases caused by AHL-dependent bacterial pathogens.

The metabolic shift in highly and weakly virulent Dickeya solani strains is more affected by temperature than by mutations in genes encoding global virulence regulators

ABSTRACT Global warming may shortly increase the risk of disease development on plants. Significant differences in the metabolic activity screened with Phenotype Microarray at 22°C and 28°C were observed between D. solani strains with high and low virulence level. Highly virulent D. solani was characterized by a higher number of metabolized compounds and a faster metabolism and was more tolerant to non-favorable pH and osmolarity. Metabolic phenotyping showed for the first time that the mutation in pecT gene, which encodes a global repressor of virulence, affects several pathways of the basic cell metabolism. PecT mutants had a higher maceration capacity of potato tissue and showed a higher pectinolytic activity than the wild-type strains. On the contrary, mutation in expI gene, which encoded the signaling molecules synthase crucial for quorum sensing, had an insignificant effect on the cell metabolism, although it slightly reduced the potato tissue maceration. The ability to utilize most of the tested compounds was higher at 28°C, while the survival at non-favorable pH and osmolarity was higher at 22°C. These results proved that the temperature of incubation had the most significant impact on the D. solani metabolic profiles.

Characterization of Endophytic Microbial Communities in Store-Bought Kale Evaluated by Different Plant Tissue Homogenization Methods

Endophytic microorganisms live in intercellular and vascular spaces of plants and span the continuum from beneficial to pathogenic in both plants and animals. Increasing human consumption of fruits and vegetables has placed an emphasis on identifying and studying those microbes that colonize the internal tissues of plants, with a goal of limiting populations of enteric pathogens in store-bought foods meant for raw consumption, such as leafy greens. Culture-independent (i.e., metagenomic) methods are increasingly used to obtain an accurate snapshot of plant microbial communities, yet technical hurdles limit the accuracy and throughput of these methods. This includes the low-throughput nature of plant tissue maceration, and the prevalence of plant plastid DNA in metagenomic DNA extracts, which is typically coamplified via PCR strategies that target the bacterial 16S rRNA gene. In this study, we use kale (Brassica oleracea) as a model to explore the leafy green endophytic microbiome and to compare how two tissue maceration techniques used in traditional endophyte research compare in culture-independent microbiome studies using the Illumina Miseq platform. Three different brands of store-bought kale were surface sterilized and subjected to two tissue maceration strategies: enzyme digestion and blender processing. Analysis of 16S rRNA gene amplicon libraries revealed two highly abundant operational taxonomic units present in all libraries, one classified to the genus Pseudomonas and one to the family Enterobacteriaceae. Community structure and membership were highly similar among brands and between tissue maceration strategies, suggesting that both enzyme digestion and blender processing are suitable methods. Nonetheless, enzyme digestion may increase sample throughput and minimize steps involved in sample processing.

Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µL (7 days in transport buffer) to 702.00 ng/µL (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µL (30 days in silica gel) to 2,850.00 ng/µL (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

Compatible Mixture of Bacterial Antagonists Developed to Protect Potato Tubers from Soft Rot Caused by Pectobacterium spp. and Dickeya spp.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.