Endophytic Actinobacteria Research Articles

Biodiversity and plant growth promoting traits of culturable endophytic actinobacteria associated with Jatropha curcas L. growing in Panxi dry-hot valley soil

One of the proposed mechanisms through which plant growth-promoting endophyte (PGPE) enhances plant growth is the production of 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD). However, information about the endophytic actinobacteria with ACC deaminase activity associated with native plants is still very scarce. In this study, a total of 257 endophytic actinobacterial isolates were obtained using actinobacteria-selective media from surface sterilized roots, stems, leaves and seeds of the oil-seed plant Jatropha curcas L. collected from dry-hot valley soil. Morphological and the 16S rRNA sequence analysis showed that most of the isolates belong to the Streptomyces genus and other non-Streptomyces strains distributed onto 13 genera, with several new species. 19 strains were found to have ACC deaminase activity and they belong to the genera Streptomyces, Nonomuraea, Micrococcus and Kibdelosporangium. The functional ability of the ACC deaminase producing isolates to produce indole-3-acetic acid (IAA), siderophores, mineral phosphate solubilization and growth on nitrogen free semi-solid medium was also determined. Seven strains were selected to inoculate the axenically grown seedlings and they resulted in a significant increase in the seedling fresh weight, the seedling length, the root length and the leaf area of the endophytes-treated seedlings compared to the control. This is the first report on the diversity and characterization of endophytic actinobacteria associated with important oil-seed plant J. curcas L. Our results demonstrate that some endophytic actinobacterial strains have the promising PGP attributes to be developed as biofertilizers to enhance soil fertility and promote the plant growth.

Arthrobacter endophyticus sp. nov., an endophytic actinobacterium isolated from root of Salsola affinis C. A. Mey.

A Gram-staining-positive, white-coloured, aerobic, non-motile, catalase-positive and oxidase-negative, endophytic actinobacterium, designated strain EGI 6500322(T), was isolated from the surface-sterilized root of the halophyte Salsola affinis C. A. Mey collected from Urumqi, Xinjiang province, north-west China. Growth occurred at 5-35 °C (optimum 25-30 °C), at pH 5-10 (optimum pH 7-8) and with 0-13% NaCl (w/v) (optimum 0-5%). The predominant menaquinone was MK-9 (93.1%). The major cellular fatty acids were anteiso-C15:0 (49.5%) and iso-C15:0 (15.1%). The cell-wall peptidoglycan contained lysine, alanine and glutamic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two unknown phospholipids and one unknown glycolipid. The DNA G+C content of strain EGI 6500322(T) was 62.0 mol%. Based on 16S rRNA gene sequence analysis, the nearest phylogenetic neighbours of strain EGI 6500322(T) were identified as Arthrobacter ardleyensis DSM 17432(T) (98.38%) and Arthrobacter bergerei DSM 16367(T (98.37%). The DNA-DNA relatedness between strain EGI 6500322(T) and Arthrobacter ardleyensis DSM 17432(T) and Arthrobacter bergerei DSM 16367(T) was 53.4 ± 4.1% and 30.5 ± 1.7%, respectively. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics and DNA-DNA hybridization data, strain EGI 6500322(T) should represent a novel species of the genus Arthrobacter, for which the name Arthrobacter endophyticus sp. nov. is proposed. The type strain is EGI 6500322(T) ( = CTC 29490(T) = JCM 30091(T)).

Prauserella endophytica sp. nov., an endophytic actinobacterium isolated from Tamarix taklamakanensis.

A novel endophytic actinobacterium, designated strain SP28S-3(T), was isolated from a surface-sterilized stem of Tamarix taklamakanensis collected from the southern edge of Taklamakan desert, Xinjiang, China. Strain SP28S-3(T) was found to show chemotaxonomic and morphological properties consistent with its classification in the genus Prauserella. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphoglycolipid, phosphatidylcholine, phosphatidylinositol, a glycolipid, an aminolipid and unidentified phospholipids. The major fatty acids (>10%) were identified as iso-C16:0 and C16:0. The genomic DNA G+C content was determined to be 69.7mol%. Phylogenetic analysis of strain SP28S-3(T) clearly showed that the strain had the highest similarity of 16S rRNA gene sequence with Prauserella coralliicola SCSIO 11529(T) (99.9%), followed by Prauserella marina DSM 45268(T) (97.0%) and is affiliated with the genus Prauserella. The low level (47.8±5.5%) of DNA-DNA relatedness between strain SP28S-3(T) and P. coralliicola SCSIO 11529(T) combined with other polyphasic taxonomic evidence clearly support the conclusion that strain SP28S-3(T) represents a novel Prauserella species, for which the name Prauserella endophytica sp. nov. is proposed. The type strain is SP28S-3(T) (=DSM 46655(T)=CGMCC 4.7182 (T)).

Stackebrandtia endophytica sp. nov., an actinobacterium isolated from Tripterygium wilfordii.

A novel endophytic actinobacterium, designated strain YIM 64602T, was isolated from healthy stems of Tripterygium wilfordii. It grew at 15-40 °C, pH 6.0-9.0 and in the presence of 0-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain YIM 64602T belongs to the genus Stackebrandtia. Whole-cell hydrolysates of strain YIM 64602T contained the amino acid meso-diaminopimelic acid with the sugars mannose, rhamnose and glucose, and a trace of ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine and phosphatidylethanolamine. MK-10(H6), MK-10(H4) and MK-11(H4) were the predominant components in the quinone system. The fatty-acid pattern was mainly composed of the saturated branched-chain acids iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and iso-C17 : 0. The DNA G+C content was 72.4 mol%. 16S rRNA gene sequence analysis showed the highest pairwise sequence identity (96.0-98.5 %) with the members of the genus Stackebrandtia. Strain YIM 64602T displayed a DNA-DNA relatedness of 43.9±0.4 % with the type strain Stackebrandtia albiflava YIM 45751T. Based on evidence from this polyphasic study, strain YIM 64602T ( = BCRC 16954T = DSM 45928T) is considered to represent a novel species of the genus Stackebrandtia, for which the name Stackebrandtia endophytica is proposed.

Frigoribacterium endophyticum sp. nov., an endophytic actinobacterium isolated from the root of Anabasis elatior (C. A. Mey.) Schischk.

A novel endophytic actinobacterium, designated EGI 6500707(T), was isolated from the surface-sterilized root of a halophyte Anabasis elatior (C. A. Mey.) Schischk collected from Urumqi, Xinjiang province, north-west China, and characterized using a polyphasic approach. Cells were Gram-stain-positive, non-motile, short rods and produced white colonies. Growth occurred at 10-45 °C (optimum 25-30 °C), at pH 5-10 (optimum pH 8) and in presence of 0-4% (w/v) NaCl (optimum 0-3%). The predominant menaquinone was MK-9. The diagnostic phospholipids were diphosphatidylglycerol and phosphatidylglycerol. The major fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0). The DNA G+C content of strain EGI 6500707(T) was 69.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain EGI 6500707(T) should be placed in the genus Frigoribacterium (family Microbacteriaceae , phylum Actinobacteria ), and that the novel strain exhibited the highest 16S rRNA gene sequence similarity to Frigoribacterium faeni JCM 11265(T) (99.1%) and Frigoribacterium mesophilum MSL-08(T) (96.5%). DNA-DNA relatedness between strain EGI 6500707(T) and F. faeni JCM 11265(T) was 47.2%. On the basis of phenotypic and chemotaxonomic characteristics, phylogenetic analysis and DNA-DNA relatedness data, strain EGI 6500707(T) represents a novel species of the genus Frigoribacterium , for which the name Frigoribacterium endophyticum sp. nov. is proposed. The type strain is EGI 6500707(T) ( = JCM 30093(T) = KCTC 29493(T)).

Nocardiopsis sp. 5 Endophytic to Tulsi Leaves – Isolation and Antimicrobial Activity

Aims: To isolate and identify the endophytic Nocardiopsis sp.5 from Tulsi (Ocimum sanctum) leaves and estimates its antimicrobial activity against various human pathogens and tea pathogens. Study Design: Rare endophytic actinobacteria isolation, identification and estimation of antimicrobial activity. Place and Duration of Study: Department of Botany and Department of Bioinformatics, Nirmala College for Women, Coimbatore – between December 2011 and December 2012. Methodology: Pre-sterilization of Ocimum sanctum leaves and isolation of rare endophytic actinomycetes using selective media. Estimation of antimicrobial activity of these isolates by primary and secondary streaking methods. Morphological and Molecular Identification of the most bioactive isolate. Results: Out of 11 endophytes, four isolates (2, 3, 5, and 9) showed antagonistic activity against tea and human pathogens. Among these four isolates, isolate no.5 showed maximum activity against all the human pathogenic microorganisms such as Staphylococcus aureus (21mm), Candida albicans (18 mm), Enterococcus faecalis (19mm), Vibrio cholera (18mm) and tea pathogens such as Glomerella cingulata (14mm) Hypoxylon serpens (17mm) and Pestalopsis theae (18mm). Hence, morphological and phylogenetic studies show that isolate no.5 belongs to Nocardiopsis group, showing 99% similarity to both Nocardiopsis synnemataformans and Nocardiopsis dassonvillei. Conclusion: Endophytic Nocardiopsis strain 5 was shown to produce good antimicrobial activity Short Communication Singh and Padmavathy; BMRJ, 5(3): 194-202, 2015; Article no.BMRJ.2015.021 195 against plant pathogens and human pathogens. Further purification, structure elucidation and characterization of the antimicrobial compound from Nocardiopsis 5 strain are recommended. There is definite scope for bioprospecting of antagonistic actinomycetes from Western Ghats once appropriate consistent studies are undertaken.

METABOLIC SCREENING FOR PKS AND NRPS IN ENDOPHYTIC ACTINOBACTERIA FROMCitrus reticulata

Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains.

Labedella endophytica sp. nov., a novel endophytic actinobacterium isolated from stem of Anabasis elatior (C. A. Mey.) Schischk.

A Gram-stain positive, yellow-coloured, aerobic, non-motile, catalase-positive and oxidase-negative, endophytic actinobacterium, designated strain EGI 6500705(T), was isolated from the surface-sterilized stem of a halophyte Anabasis elatior (C. A. Mey.) Schischk collected from Urumqi, Xinjiang province, north-west China. The organism had ornithine as the diagnostic cell-wall diamino acid. The major fatty acids were anteiso-C15:0, anteiso-C17:0 and iso-C16:0. The predominant menaquinones were MK-9, MK-10 and MK-11. The DNA G+C content of strain EGI 6500705(T) was 69.2mol%. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain EGI 6500705(T) is clearly affiliated with the genus Labedella and most closely related to Labedella gwakjiensis KCTC 19176(T), with 99.0% sequence similarity. DNA-DNA relatedness between strain EGI 6500705(T) and L. gwakjiensis KCTC 19176(T) was 27.4%. On the basis of phenophytic, chemotaxonomic and phylogenetic analysis, the strain EGI 6500705(T) represents a novel species of the genus Labedella, for which the name Labedella endophytica sp. nov. is proposed. The type strain is EGI 6500705(T) (=KCTC 29494(T)=CPCC 203961(T)=JCM 30092(T)).

Genome features of the endophytic actinobacterium Micromonospora lupini strain Lupac 08: on the process of adaptation to an endophytic life style?

Endophytic microorganisms live inside plants for at least part of their life cycle. According to their life strategies, bacterial endophytes can be classified as “obligate” or “facultative”. Reports that members of the genus Micromonospora, Gram-positive Actinobacteria, are normal occupants of nitrogen-fixing nodules has opened up a question as to what is the ecological role of these bacteria in interactions with nitrogen-fixing plants and whether it is in a process of adaptation from a terrestrial to a facultative endophytic life. The aim of this work was to analyse the genome sequence of Micromonospora lupini Lupac 08 isolated from a nitrogen fixing nodule of the legume Lupinus angustifolius and to identify genomic traits that provide information on this new plant-microbe interaction. The genome of M. lupini contains a diverse array of genes that may help its survival in soil or in plant tissues, while the high number of putative plant degrading enzyme genes identified is quite surprising since this bacterium is not considered a plant-pathogen. Functionality of several of these genes was demonstrated in vitro, showing that Lupac 08 degraded carboxymethylcellulose, starch and xylan. In addition, the production of chitinases detected in vitro, indicates that strain Lupac 08 may also confer protection to the plant. Micromonospora species appears as new candidates in plant-microbe interactions with an important potential in agriculture and biotechnology. The current data strongly suggests that a beneficial effect is produced on the host-plant.

Description of Micrococcus aloeverae sp. nov., an endophytic actinobacterium isolated from Aloe vera.

A yellow Gram-stain-positive, non-motile, non-endospore -forming, spherical endophytic actinobacterium, designated strain AE-6(T), was isolated from the inner fleshy leaf tissues of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. Strain AE-6(T) grew at high salt concentrations [10% (w/v) NaCl], temperatures of 15-41 °C and a pH range of 5-12. It showed highest (99.7%) 16S rRNA gene sequence similarity with Micrococcus yunnanensis YIM 65004(T) followed by Micrococcus luteus NCTC 2665(T) (99.6%) and Micrococcus endophyticus YIM 56238(T) (99.0%). Ribosomal protein profiling by MALDI-TOF/MS also showed it was most closely related to M. yunnanensis YIM 65004(T) and M. luteus NCTC 2665(T). Like other members of the genus Micrococcus, strain AE-6(T) had a high content of branched chain fatty acids (iso-C15:0 and anteiso-C15:0). MK-8(H2) and MK-8 were the predominant isoprenoid quinones. Cell wall analysis showed an 'A2 L-Lys-peptide subunit' type of peptidoglycan and ribose to be the major cell wall sugar. The DNA G+C content was 70 mol%. Results of DNA-DNA hybridization of AE-6(T) with its closest relatives from the genus Micrococcus produced a value of less than 70%. Based on the results of this study, strain AE-6(T) could be clearly differentiated from other members of the genus Micrococcus. We propose that it represents a novel species of the genus Micrococcus and suggest the name Micrococcus aloeverae sp. nov., with strain AE-6(T) ( = MCC 2184(T) = DSM 27472(T)) as the type strain of the species.

Insights into grisorixin biosynthesis, an ionophore polyether from endophytic strain Streptomyces platensis RTd22

The endophytic actinobacteria S. platensis RTd22 produces the antibiotic ionophore polyether grisorixin (1), an analogue of nigericin (2), and the siderophore nocardamine (3). Grisorixin is the major compound produced in different culture media by this endophyte. Whole genome sequencing of this strain was performed and presented 11 million of base pairs, 103 contigs and 439 subsystems. The antiSMASH platform allowed identify several clusters related to production of secondary metabolites, among them t1-PKS, NRPS, terpene, siderophore, butyrolactone, bacteriocin, latipeptide and mixed pathways. The gene cluster involved in grisorixin production was determined. Deletion strategies in this gene are being carried out to reconstruct the pathway of these polyethers and eventually lead to the production of new compounds. These results will be presented.

Mechanism of phosphate solubilization and antifungal activity of Streptomyces spp. isolated from wheat roots and rhizosphere and their application in improving plant growth

The application of plant-growth-promoting rhizobacteria (PGPR) at field scale has been hindered by an inadequate understanding of the mechanisms that enhance plant growth, rhizosphere incompetence and the inability of bacterial strains to thrive in different soil types and environmental conditions. Actinobacteria with their sporulation, nutrient cycling, root colonization, bio-control and other plant-growth-promoting activities could be potential field bio-inoculants. We report the isolation of five rhizospheric and two root endophytic actinobacteria from Triticum aestivum (wheat) plants. The cultures exhibited plant-growth-promoting activities, namely phosphate solubilization (1916 mg l(-1)), phytase (0.68 U ml(-1)), chitinase (6.2 U ml(-1)), indole-3-acetic acid (136.5 mg l(-1)) and siderophore (47.4 mg l(-1)) production, as well as utilizing all the rhizospheric sugars under test. Malate (50-55 mmol l(-1)) was estimated in the culture supernatant of the highest phosphate solublizer, Streptomyces mhcr0816. The mechanism of malate overproduction was studied by gene expression and assays of key glyoxalate cycle enzymes - isocitrate dehydrogenase (IDH), isocitrate lyase (ICL) and malate synthase (MS). The significant increase in gene expression (ICL fourfold, MS sixfold) and enzyme activity (ICL fourfold, MS tenfold) of ICL and MS during stationary phase resulted in malate production as indicated by lowered pH (2.9) and HPLC analysis (retention time 13.1 min). Similarly, the secondary metabolites for chitinase-independent biocontrol activity of Streptomyces mhcr0817, as identified by GC-MS and (1)H-NMR spectra, were isoforms of pyrrole derivatives. The inoculation of actinobacterial isolate mhce0811 in T. aestivum (wheat) significantly improved plant growth, biomass (33%) and mineral (Fe, Mn, P) content in non-axenic conditions. Thus the actinobacterial isolates reported here were efficient PGPR possessing significant antifungal activity and may have potential field applications.

A new prenylated indole derivative from endophytic actinobacteria Streptomyces sp. neau-D50

A new prenylated indole derivative 3-acetonylidene-7-prenylindolin-2-one (1) was isolated from the endophytic actinobacterium Streptomyces sp. neau-D50, together with four known hybrid isoprenoids, 7-isoprenylindole-3-carboxylic acid (2), 3-cyanomethyl-6-prenylindole (3), 6-isoprenylindole-3-carboxylic acid (4) and 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (5). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with data from the literature. Compounds 1 and 2 demonstrated strong cytotoxic activities against human lung adenocarcinoma cell line A549 with an IC50 of 3.3 and 5.1 μg mL− 1, respectively, which are comparable to that of the positive control doxorubicin (4.2 μg mL− 1). Furthermore, compounds 1–4 exhibited potent antifungal activity against phytopathogenic fungi Colletotrichum orbiculare, Phytophthora capsici, Corynespora cassiicola and Fusarium oxysporum.

Rothia endophytica sp. nov., an actinobacterium isolated from Dysophylla stellata (Lour.) Benth

A novel endophytic actinobacterium, designated strain YIM 67072(T), was isolated from healthy roots of Dysophylla stellata (Lour.) Benth. Cells of this aerobic, cream-yellow-coloured strain occurred singly, in pairs or in tetrads, were Gram-stain-positive and ovoid- to spherical-shaped. Strain YIM 67072(T) grew at 4-45 °C, pH 5.0-10.0 and in the presence of 0-7 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 67072(T) belonged to the genus Rothia. The isolate contained MK-7 as the major component of the quinone system. The peptidoglycan type was A3α. The polar lipid profile consisted predominantly of diphosphatidylglycerol and glycolipids. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The DNA G+C content was 53.2 mol%. However, strain YIM 67072(T) differed from its closest relatives Rothia nasimurium CCUG 35957(T) (98.5 % 16S rRNA gene sequence similarity), Rothia amarae JCM 11375(T) (97.6 %) and Rothia terrae L-143(T) (97.3 %) in many phenotypic characteristics. Moreover, the levels of DNA-DNA relatedness between the novel isolate and the three above-mentioned type strains were 28.7±1.3 %, 36.5±1.2 %, 46.8±1.5 %, respectively. Based on comparative analysis of physiological and chemotaxonomic data, strain YIM 67072(T) represents a novel species of the genus Rothia, for which the name Rothia endophytica sp. nov. is proposed. The type strain is YIM 67072(T) ( = DSM 26247(T) = JCM 18541(T)).

Generic and functional diversity in endophytic actinomycetes from wild Compositae plant species at South Sinai – Egypt

The diversity of culturable endophytic actinomycetes associated with wild Compositae plants is scantily explored. In this study, one hundred and thirty one endophytic actinobacteria were isolated from ten Compositae plant species collected from South Sinai in Egypt. Microscopic and chemotaxonomic investigation of the isolates indicated fourteen genera. Rare genera, such as Microtetraspora, and Intrasporangium, which have never been previously reported to be endophytic, were identified. Each plant species accommodated between three to eight genera of actinobacteria and unidentified strains were recovered from seven plant species.The generic diversity analysis of endophytic assemblages grouped the plant species into three main clusters, representing high, moderate and low endophytic diversity. The endophytes showed high functional diversity, based on forty four catabolic and plant growth promotion traits; providing some evidence that such traits could represent key criteria for successful residence of endophytes in the endosphere. Stress-tolerance traits were more predictive measure of functional diversity differences between the endophyte assemblages (Shannon's index, p = 0.01). The results indicate a potential prominent role of endophytes for their hosts and emphasize the potency of plant endosphere as a habitat for actinobacteria with promising future applications.

Indole-3-Acetic Acid Production by Endophytic Streptomyces sp. En-1 Isolated from Medicinal Plants

Plant-associated actinobacteria are rich sources of bioactive compounds including indole-derived molecules such as phytohormone indole-3-acetic acid (IAA). In view of few investigations concerning the biosynthesis of IAA by endophytic actinobacteria, this study evaluated the potential of IAA production in endophytic streptomycete isolates sourced from medicinal plant species Taxus chinensis and Artemisia annua. By HPLC analysis of IAA combined with molecular screening approach of iaaM, a genetic determinant of streptomycete IAA synthesis via indole-3-acetamide (IAM), our data showed the putative operation of IAM-mediated IAA biosynthesis in Streptomyces sp. En-1 endophytic to Taxus chinensis. Furthermore, using the co-cultivation system of model plant Arabidopsis thaliana and streptomycete, En-1 was found to be colonized intercellularly in the tissues of Arabidopsis, an alternative host, and the effects of endophytic En-1 inoculation on the model plant were also assayed. The phytostimulatory effects of En-1 inoculation suggest that IAA-producing Streptomyces sp. En-1 of endophytic origin could be a promising candidate for utilization in growth improvement of plants of economic and agricultural value.

Blastococcus endophyticus sp. nov., an actinobacterium isolated from Camptotheca acuminata

A novel endophytic actinobacterium, designated strain YIM 68236(T), was isolated from healthy leaves of Camptotheca acuminata. and characterized by using a polyphasic approach. Cells of this strain occurred singly, in pairs or in tetrads. It grew at 10-45 °C, at pH 5.0-8.0 (optimum pH 7.0) and in the presence of 0-3% (w/v) NaCl. The DNA G+C content was 71.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 68236(T) belongs to the genus Blastococcus. However, it differed from its closest relatives, Blastococcus aggregatus DSM 4725(T), Blastococcus saxobsidens DSM 44509(T) and Blastococcus jejuensis DSM 19597(T) in many phenotypic characteristics. Moreover, the DNA-DNA relatedness values between the novel isolate and the three above-mentioned type strains were 49.0 ± 1.6%, 46.1 ± 3.2% and 39.8 ± 1.5%, respectively. Based on comparative analysis of physiological and chemotaxonomic data, strain YIM 68236(T) represents a novel species of the genus Blastococcus, for which the name Blastococcus endophyticus sp. nov. is proposed. The type strain is YIM 68236(T) ( =CCTCC AA 209045(T) =DSM 45413(T) =KCTC 19998(T)).

An Endophytic Pseudonocardia Species Induces the Production of Artemisinin in Artemisia annua

Endophytic actinobacteria colonize internal tissues of their host plants and are considered as a rich and reliable source of diverse species and functional microorganisms. In this study, endophytic actinobacterial strain YIM 63111 was isolated from surface-sterilized tissue of the medicinal plant Artemisia annua. We identified strain YIM 63111 as a member of the genus Pseudonocardia. A. annua seedlings grown under both sterile and greenhouse conditions were inoculated with strain YIM 63111. The growth of A. annua seedlings was strongly reduced when YIM 63111 was inoculated at higher concentrations under sterile conditions. However, no growth inhibition was observed when A. annua was grown under greenhouse conditions. Using an enhanced green fluorescent protein (EGFP) expressing YIM 63111 strain, we also observed the endophytic colonization of A. annua seedling using confocal laser-scanning microscopy. The transcription levels of the key genes involved in artemisinin biosynthesis were investigated using real time RT-PCR, revealing that cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) expression were up-regulated in A. annua upon inoculation with strain YIM 63111 under certain conditions. The up-regulation of these genes was associated with the increased accumulation of artemisinin. These results suggest that endophytic actinobacteria effectively stimulate certain plant defense responses. Our data also demonstrate the use of Pseudonocardia sp. strain YIM 63111 as a promising means to enhance artemisinin production in plants.

Rational Approaches to Improving the Isolation of Endophytic Actinobacteria from Australian Native Trees

In recent years, new actinobacterial species have been isolated as endophytes of plants and shrubs and are sought after both for their role as potential producers of new drug candidates for the pharmaceutical industry and as biocontrol inoculants for sustainable agriculture. Molecular-based approaches to the study of microbial ecology generally reveal a broader microbial diversity than can be obtained by cultivation methods. This study aimed to improve the success of isolating individual members of the actinobacterial population as pure cultures as well as improving the ability to characterise the large numbers obtained in pure culture. To achieve this objective, our study successfully employed rational and holistic approaches including the use of isolation media with low concentrations of nutrients normally available to the microorganism in the plant, plating larger quantities of plant sample, incubating isolation plates for up to 16 weeks, excising colonies when they are visible and choosing Australian endemic trees as the source of the actinobacteria. A hierarchy of polyphasic methods based on culture morphology, amplified 16S rRNA gene restriction analysis and limited sequencing was used to classify all 576 actinobacterial isolates from leaf, stem and root samples of two eucalypts: a Grey Box and Red Gum, a native apricot tree and a native pine tree. The classification revealed that, in addition to 413 Streptomyces spp., isolates belonged to 16 other actinobacterial genera: Actinomadura (two strains), Actinomycetospora (six), Actinopolymorpha (two), Amycolatopsis (six), Gordonia (one), Kribbella (25), Micromonospora (six), Nocardia (ten), Nocardioides (11), Nocardiopsis (one), Nonomuraea (one), Polymorphospora (two), Promicromonospora (51), Pseudonocardia (36), Williamsia (two) and a novel genus Flindersiella (one). In order to prove novelty, 12 strains were characterised fully to the species level based on polyphasic taxonomy. One strain represented a novel genus in the family Nocardioides, and the other 11 strains were accepted as novel species. In summary, the holistic isolation strategies were successful in obtaining significant culturable actinobacterial diversity within Australian native trees that includes rare and novel species.

Kribbella endophytica sp. nov., an endophytic actinobacterium isolated from the surface-sterilized leaf of a native apricot tree

A new strain of the genus Kribbella, PIP 118(T), was isolated from the leaf of an Australian native apricot tree (Pittosporum angustifolium), or Gumbi Gumbi in the indigenous language. This strain is an aerobic actinobacterium consisting of hyphae that fragment into short to elongated rod-like elements. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this isolate as a member of the family Nocardioidaceae and most closely related to Kribbella antibiotica YIM 31530(T) (98.6 %) and Kribbella koreensis LM 161(T) (98.4 %). Chemotaxonomic data including cell wall components, major menaquinone and major fatty acids confirmed the affiliation of strain PIP 118(T) to the genus Kribbella. The results of the phylogenetic analysis, including physiological and biochemical studies in combination with DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain PIP 118(T) and members of the most closely related species with validly published names. The name proposed for the new species is Kribbella endophytica sp. nov. The type strain is PIP 118(T) ( = DSM 23718(T) = NRRL B-24812(T)).