Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity.
ABSTRACT: Many actinobacteria live in close association with eukaryotes such as fungi, insects, animals and plants. Plant-associated actinobacteria display (endo)symbiotic, saprophytic or pathogenic life styles, and can make up a substantial part of the endophytic community. Here, we characterised endophytic actinobacteria isolated from root tissue of Arabidopsis thaliana (Arabidopsis) plants grown in soil from a natural ecosystem. Many of these actinobacteria belong to the family of Streptomycetaceae with Streptomyces olivochromogenes and Streptomyces clavifer as well represented species. When seeds of Arabidopsis were inoculated with spores of Streptomyces strain coa1, which shows high similarity to S. olivochromogenes, roots were colonised intercellularly and, unexpectedly, also intracellularly. Subsequent exposure of endophytic isolates to plant hormones typically found in root and shoot tissues of Arabidopsis led to altered antibiotic production against Escherichia coli and Bacillus subtilis. Taken together, our work reveals remarkable colonization patterns of endophytic streptomycetes with specific traits that may allow a competitive advantage inside root tissue.
Project description:We report for the first time the isolation of endophytic actinobacteria associated with wild populations of the Chinese medicinal herb Thymus roseus Schipcz obtained from the arid land in Ili and Tacheng of the Xinjiang Province, China. Strains were isolated by special pretreatment of plant tissues and identified based on their 16S rRNA gene sequences, and their antimicrobial activities in vitro were evaluated. A total of 126 endophytic actinobacteria belonging to two classes, eight orders, 14 families, and 24 genera were isolated from different organs at the Ili and Tacheng sites. In addition, the diversity of culturable endophytic actinobacteria genera was higher at Tacheng site (n = 71, 56.35%) than the Ili site (n = 55, 43.65%). A neighbor-joining tree of 126 isolated actinobacteria showing the phylogenetic relationships based on 16S rRNA gene sequences and the genus Streptomyces was the most dominant isolate. The number of endophytic actinobacteria genera obtained from root tissues (n = 54, 42.86%) was higher compared to stem (n = 35, 27.78%) and leaf tissue (n = 37, 29.36%). Among 126 endophytic actinobacteria, 54 strains were antagonistic against at least one or more indicator organisms in vitro. Notably, most strains of Streptomyces proved antagonistic activities. For example, strain T4SB028, namely Streptomyces polyantibioticus, showed the highest inhibition ratio reached 67.06, 64.20, and 70.55% against Alternaria solani, Valsa malicola, and Valsa mali, respectively. The results demonstrate that about 30.95%, 23.01% of the tested endophytic actinobacteria were capable of producing siderophores and chitinase, respectively. Additionally, the results of the amplification of biosynthetic genes polyketide synthetase (PKS-I) and non-ribosomal peptide synthetase (NRPS) indicated that at least one antibiotic biosynthetic gene was detected in 27 (50%) of the tested strains. Our result emphasizes that the endophytic actinobacteria communities are different based on the plant tissues and the geographical environment of the sampled area. Thus, we conclude that T. roseus Schipcz. provided a rich source of endophytic actinobacteria that exhibited a broad-spectrum antimicrobial agent.
Project description:The diversity and functional roles of the plant associated endophytic actinobacteria in unique habitats remain poorly understood. In this paper, we examined the phylogenetic diversity and community composition of endophytic actinobacteria associated with native coastal salt marsh plants in Jiangsu, China using a combination of cultivation and 16S rRNA gene-based high-throughput sequencing (HTS) methods. Further, we evaluated the antifungal, fibrinolytic activities and the secondary metabolite biosynthesis potential of isolates via gene screening. A total of 278 actinobacterial isolates were isolated from 19 plant samples. 16S rRNA gene sequencing revealed that the isolates were highly diverse and belonged to 23 genera within the Actinomycetales order, with Streptomyces, Saccharopolyspora, and Pseudonocardia comprising the most abundant genera. In addition, more than 10 of the isolates were novel actinobacterial taxa distributed across eight genera. HTS analyses of seven representative plant root samples revealed that Actinobacteria phylum constituted 0.04-28.66% of root endophytic bacterial communities. A total of four actinobacterial classes, 14 orders, 35 families, and 63 known genera were detected via HTS, and these communities were found to be dominated by the members of the order Actinomycetales including the genera Streptomyces, Mycobacterium, Arthrobacter, Nocardioides, and Micromonospora. In addition, 30.4% of the representative isolates exhibited antifungal activities, 40.5% of them showed fibrinolytic activities, while 43.0% of the strains harbored secondary metabolite biosynthesis genes. These results demonstrated that coastal salt marsh plants in the Jiangsu Province represented an underexplored new reservoir of diverse and novel endophytic actinobacteria that may be of potential interest in the discovery of bioactive compounds with potential as biocontrol agents and for fibrinolytic enzyme production.
Project description:This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.
Project description:Endophytic and rhizosphere actinobacteria isolated from the root system of 1-year-old grafted Vitis vinifera plants were evaluated for their activities against fungi that cause grapevine trunk diseases. A total of 58 endophytic and 94 rhizosphere isolates were tested. Based on an in vitro bioassay, 15.5% of the endophytic isolates and 30.8% of the rhizosphere isolates exhibited antifungal activity against the fungal pathogen Diplodia seriata, whereas 13.8% of the endophytic isolates and 16.0% of the rhizosphere isolates showed antifungal activity against Dactylonectria macrodidyma (formerly Ilyonectria macrodidyma). The strains which showed the greatest in vitro efficacy against both pathogens were further analyzed for their ability to inhibit the growth of Phaeomoniella chlamydospora and Phaeoacremonium minimum (formerly Phaeoacremonium aleophilum). Based on their antifungal activity, three rhizosphere isolates and three endophytic isolates were applied on grafts in an open-root field nursery in a 3-year trial. The field trial led to the identification of one endophytic strain, Streptomyces sp. VV/E1, and two rhizosphere isolates, Streptomyces sp. VV/R1 and Streptomyces sp. VV/R4, which significantly reduced the infection rates produced by the fungal pathogens Dactylonectria sp., Ilyonectria sp., P. chlamydospora, and P. minimum, all of which cause young grapevine decline. The VV/R1 and VV/R4 isolates also significantly reduced the mortality level of grafted plants in the nursery. This study shows that certain actinobacteria could represent a promising new tool for controlling fungal trunk pathogens that infect grapevine plants through the root system in nurseries.IMPORTANCE Grapevine trunk diseases are a major threat to the wine and grape industry worldwide. They cause a significant reduction in yields as well as in grape quality, and they can even cause plant death. Trunk diseases are caused by fungal pathogens that enter through pruning wounds and/or the root system. Although different strategies have recently been developed to protect pruning wounds using antifungal compounds (natural or synthetic) or biocontrol agents, no tools are yet available for controlling soil pathogens that infect plants through their root system. This study shows that different actinobacterial isolates, when applied to grafts in a nursery, can significantly reduce the infection rate caused by fungal pathogens that enter through the root system. This is a new, promising, and green alternative for preventing the decline of young grapevines in nurseries and vineyards.
Project description:Actinobacteria associated with insects represent one potentially rich source of novel natural products with antifungal activity. Here, we investigated the phylogenetic diversity and community composition of actinobacteria associated with ants using a combination of culture-dependent and -independent methods. Further, we assessed the antagonistic activity against phytopathogenic fungi and identified the secondary metabolites from isolates with bioactivity. A total of 416 actinobacterial isolates were obtained from three ant species (Camponotus japonicus, Lasius fuliginosus, and Lasius flavus) located in five nests. The largest amount of isolates were observed in the head samples. 16S rRNA gene sequencing showed that the isolates were diverse and belonged to ten genera within the phylum Actinobacteria, with Streptomyces and Micromonospora comprising the most abundant genera. High-throughput sequencing analyses revealed that the actinobacterial communities were more diverse and dominated by the families Nocardioidaceae, Nocardiaceae, Dermacoccaceae, Intrasporangiaceae, and Streptomycetaceae. In addition, 52.3% of the representative isolates had inhibitory properties against phytopathogenic fungi. Chemical analysis of one Streptomyces strain led to the discovery of two known compounds and one new compound. These results demonstrated that ant-derived actinobacteria represented an underexplored bioresource library of diverse and novel taxa that may be of potential interest in the discovery of new agroactive compounds.
Project description:The phylum Actinobacteria constitutes one of the largest and anciently divergent phyla within the Bacteria domain. Actinobacterial diversity has been thoroughly researched in various environments due to its unique biotechnological potential. Such studies have focused mostly on soil communities, but more recently marine and extreme environments have also been explored, finding rare taxa and demonstrating dispersal limitation and biogeographic patterns for Streptomyces. To test the distribution of Actinobacteria populations on a small scale, we chose the extremely oligotrophic and biodiverse Cuatro Cienegas Basin (CCB), an endangered oasis in the Chihuahuan desert to assess the diversity and uniqueness of Actinobacteria in the Churince System with a culture-dependent approach over a period of three years, using nine selective media. The 16S rDNA of putative Actinobacteria were sequenced using both bacteria universal and phylum-specific primer pairs. Phylogenetic reconstructions were performed to analyze OTUs clustering and taxonomic identification of the isolates in an evolutionary context, using validated type species of Streptomyces from previously phylogenies as a reference. Rarefaction analysis for total Actinobacteria and for Streptomyces isolates were performed to estimate species' richness in the intermediate lagoon (IL) in the oligotrophic Churince system. A total of 350 morphologically and nutritionally diverse isolates were successfully cultured and characterized as members of the Phylum Actinobacteria. A total of 105 from the total isolates were successfully subcultured, processed for DNA extraction and 16S-rDNA sequenced. All strains belong to the order Actinomycetales, encompassing 11 genera of Actinobacteria; the genus Streptomyces was found to be the most abundant taxa in all the media tested throughout the 3-year sampling period. Phylogenetic analysis of our isolates and another 667 reference strains of the family Streptomycetaceae shows that our isolation effort produced 38 unique OTUs in six new monophyletic clades. This high biodiversity and uniqueness of Actinobacteria in an extreme oligotrophic environment, which has previously been reported for its diversity and endemicity, is a suggestive sign of microbial biogeography of Actinobacteria and it also represents an invaluable source of biological material for future ecological and bioprospecting studies.
Project description:We examined abundance, bioactivity, and endophytism of cultivable actinobacteria isolated from plant interiors of two Thai pigmented rice cultivars: Hom Nin (HN) rice and Luem Pua (LP) glutinous rice. Both rice cultivars housed the same amount of endophytic actinobacteria (33 isolates each). Microbispora (76%) and Streptomyces (73%) were the predominant endophytic actinobacteria of LP glutinous rice and HN rice, respectively. Sphaerisporangium (9%) was found only in LP glutinous rice. Twelve percent of endophytic actinobacteria was the possibility of discovering novel species from both rice cultivars. Most endophytic actinobacteria exhibited plant growth-promoting potentials, including antimicrobial activity against test bacteria and phytopathogenic fungi, solubilization of phosphate, and production of biostimulants (i.e., ammonia, indole-3-acetic acid, and siderophore) and biocatalysts (i.e., amylase, cellulase, chitinase, lipase, and protease). Our findings revealed that seed phytochemicals of pigmented rice (e.g., anthocyanin, ?-oryzanol, phytate, antioxidants, and content of amylose) were effectors, shaping the community structures and biofunctions of endophytic actinobacteria. We conclude that pigmented rice is yet a challenging source for discovery of bioactive and novel actinobacteria. This study also provides new insights into the plant-endophyte interactions by which seed phytochemicals act as a primary checkpoint in the natural selection for establishing unique plant endophytomes.
Project description:The present study was conducted to examine the influence of a pathogen Sclerotinia sclerotiorum (Lib.) de Bary on the actinobacterial community associated with the soybean roots. A total of 70 endophytic actinobacteria were isolated from the surface-sterilized roots of either healthy or diseased soybeans, and they were distributed under 14 genera. Some rare genera, including Rhodococcus, Kribbella, Glycomyces, Saccharothrix, Streptosporangium and Cellulosimicrobium, were endemic to the diseased samples, and the actinobacterial community was more diverse in the diseased samples compared with that in the heathy samples. Culture-independent analysis of root-associated actinobacterial community using the high-throughput sequencing approach also showed similar results. Four Streptomyces strains that were significantly abundant in the diseased samples exhibited strong antagonistic activity with the inhibition percentage of 54.1-87.6%. A bioactivity-guided approach was then employed to isolate and determine the chemical identity of antifungal constituents derived from the four strains. One new maremycin analogue, together with eight known compounds, were detected. All compounds showed significantly antifungal activity against S. sclerotiorum with the 50% inhibition (EC50) values of 49.14-0.21 mg/L. The higher actinobacterial diversity and more antifungal strains associated with roots of diseased plants indicate a possible role of the root-associated actinobacteria in natural defense against phytopathogens. Furthermore, these results also suggest that the root of diseased plant may be a potential reservoir of actinobacteria producing new agroactive compounds.
Project description:Cave moonmilk deposits host an abundant and diverse actinobacterial population that has a great potential for producing novel natural bioactive compounds. In our previous attempt to isolate culturable moonmilk-dwelling Actinobacteria, only Streptomyces species were recovered, whereas a metagenetic study of the same deposits revealed a complex actinobacterial community including 46 actinobacterial genera in addition to streptomycetes. In this work, we applied the rehydration-centrifugation method to lessen the occurrence of filamentous species and tested a series of strategies to achieve the isolation of hard-to-culture and rare Actinobacteria from the moonmilk deposits of the cave "Grotte des Collemboles". From the "tips and tricks" that were tested, separate autoclaving of the components of the International Streptomyces Project (ISP) medium number 5 (ISP5) medium, prolonged incubation time, and dilution of the moonmilk suspension were found to most effectively improve colony forming units. Taxonomic analyses of the 40 isolates revealed new representatives of the Agromyces, Amycolatopsis, Kocuria, Micrococcus, Micromonospora, Nocardia, and Rhodococcus species, as well as additional new streptomycetes. The applied methodologies allowed the isolation of strains associated with both the least and most abundant moonmilk-dwelling actinobacterial operational taxonomic units. Finally, bioactivity screenings revealed that some isolates displayed high antibacterial activities, and genome mining uncovered a strong potential for the production of natural compounds.
Project description:Endophytic actinobacteria are one of the important pharmaceutical resources and well known for producing different types of bioactive substances. Nevertheless, detection of the novelty, diversity, and bioactivity on endophytic actinobacteria isolated from mangrove plants are scarce. In this study, five different mangrove plants, Avicennia marina, Aegiceras corniculatum, Kandelia obovota, Bruguiera gymnorrhiza, and Thespesia populnea, were collected from Beilun Estuary National Nature Reserve in Guangxi Zhuang Autonomous Region, China. A total of 101 endophytic actinobacteria strains were recovered by culture-based approaches. They distributed in 7 orders, 15 families, and 28 genera including Streptomyces, Curtobacterium, Mycobacterium, Micrococcus, Brevibacterium, Kocuria, Nocardioides, Kineococcus, Kytococcus, Marmoricola, Microbacterium, Micromonospora, Actinoplanes, Agrococcus, Amnibacterium, Brachybacterium, Citricoccus, Dermacoccus, Glutamicibacter, Gordonia, Isoptericola, Janibacter, Leucobacter, Nocardia, Nocardiopsis, Pseudokineococcus, Sanguibacter, and Verrucosispora. Among them, seven strains were potentially new species of genera Nocardioides, Streptomyces, Amnibacterium, Marmoricola, and Mycobacterium. Above all, strain 8BXZ-J1 has already been characterized as a new species of the genus Marmoricola. A total of 63 out of 101 strains were chosen to screen antibacterial activities by paper-disk diffusion method and inhibitors of ribosome and DNA biosynthesis by means of a double fluorescent protein reporter. A total of 31 strains exhibited positive results in at least one antibacterial assay. Notably, strain 8BXZ-J1 and three other potential novel species, 7BMP-1, 5BQP-J3, and 1BXZ-J1, all showed antibacterial bioactivity. In addition, 21 strains showed inhibitory activities against at least one "ESKAPE" resistant pathogens. We also found that Streptomyces strains 2BBP-J2 and 1BBP-1 produce bioactive compound with inhibitory activity on protein biosynthesis as result of translation stalling. Meanwhile, Streptomyces strain 3BQP-1 produces bioactive compound inducing SOS-response due to DNA damage. In conclusion, this study proved mangrove plants harbored a high diversity of cultivable endophytic actinobacteria, which can be a promising source for discovery of novel species and bioactive compounds.