Algorithm of automatic identification of diabetic retinopathy foci based on ultra-widefield scanning laser ophthalmoscopy.

AIM: To propose an algorithm for automatic detection of diabetic retinopathy (DR) lesions based on ultra-widefield scanning laser ophthalmoscopy (SLO). METHODS: The algorithm utilized the FasterRCNN (Faster Regions with CNN features)+ResNet50 (Residua Network 50)+FPN (Feature Pyramid Networks) method for detecting hemorrhagic spots, cotton wool spots, exudates, and microaneurysms in DR ultra-widefield SLO. Subimage segmentation combined with a deeper residual network FasterRCNN+ResNet50 was employed for feature extraction to enhance intelligent learning rate. Feature fusion was carried out by the feature pyramid network FPN, which significantly improved lesion detection rates in SLO fundus images. RESULTS: By analyzing 1076 ultra-widefield SLO images provided by our hospital, with a resolution of 2600×2048 dpi, the accuracy rates for hemorrhagic spots, cotton wool spots, exudates, and microaneurysms were found to be 87.23%, 83.57%, 86.75%, and 54.94%, respectively. CONCLUSION: The proposed algorithm demonstrates intelligent detection of DR lesions in ultra-widefield SLO, providing significant advantages over traditional fundus color imaging intelligent diagnosis algorithms.

Streptomyces endophyticus sp. nov., an endophytic actinomycete isolated from Artemisia annua L.

Three filamentous actinomycetes, strains YIM 65594(T), YIM 65638 and YIM 65642, were isolated from the surface-sterilized roots of Artemisia annua L. collected from Yunnan province, south-west China. These strains were found to have morphological and chemotaxonomic characteristics typical of the genus Streptomyces. The organisms formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into spores. The cell wall of the isolates contained ll-diaminopimelic acid and the menaquinones were MK-9(H(8)) and MK-9(H(6)). The major fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0). Phylogenetic analysis of the 16S rRNA gene sequences of these strains revealed that the strains clustered together and were most closely related to Streptomyces kunmingensis NBRC 14463(T), with 98.5-98.6 % 16S rRNA gene sequence similarity. The results of DNA-DNA hybridization and physiological tests allowed the genotypic and phenotypic differentiation of strains YIM 65594(T), YIM 65638 and YIM 65642 from related species. However, the high level of DNA-DNA relatedness between them showed that these three strains belong to the same species. Strain YIM 65594(T) (= DSM 41984(T) = CCTCC AA 209036(T)) was selected as the type strain to represent this novel species, for which the name Streptomyces endophyticus sp. nov. is proposed.

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.

Isolation and characterization of culturable endophytic actinobacteria associated with Artemisia annua L.

Endophytic actinobacteria isolated from Artemisia annua were characterized and evaluated for their bioactivities. A total of 228 isolates representing at least 19 different genera of actinobacteria were obtained and several of them seemed to be novel taxa. An evaluation of antimicrobial activity showed that more isolates possessed activity towards plant pathogens than activity against other pathogenic bacteria or yeasts. High frequencies of PCR amplification were obtained for type I polyketide synthases (PKS-I, 21.1%), type II polyketide synthases (PKS-II, 45.2%) and nonribosomal peptide synthetases (NRPS, 32.5%). The results of herbicidal activity screening indicated that 19 out of 117 samples of fermentation broths completely inhibited the germination of Echinochloa crusgalli. This study indicated that endophytic actinobacteria associated with A. annua are abundant and have potentially beneficial and diverse bioactivities which should be pursued for their biotechnical promise.

Sphingomonas endophytica sp. nov., isolated from Artemisia annua L.

A novel bacterium (strain YIM 65583(T)) belonging to the genus Sphingomonas was isolated from surface-sterilized tissue of Artemisia annua L., which was collected from Yunnan province, south-west China. Its morphology, physiology and biochemical features were consistent with those of members of the genus Sphingomonas. Analysis of the 16S rRNA gene sequence of strain YIM 65583(T) further confirmed that it should be classified as a member of the genus Sphingomonas and was most closely related to Sphingomonas phyllosphaerae FA2(T) (99.7%) and Sphingomonas yunnanensis YIM 003(T) (98.3%). The isolate was Gram-negative and formed yellow-pigmented colonies on ISP 2 medium. It grew optimally at pH 6.0-8.0, at 20-37 °C and in the presence of 0-1% (w/v) NaCl. The major respiratory lipoquinone was ubiquinone-10; C(18:1)ω7c, anteiso-C(16:1), C(14:0)-2OH, C(17:1)ω6c, C(16:0) and C(15:0) were the major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and sphingoglycolipid. The G+C content of the genomic DNA was 63.3 mol%. The DNA-DNA relatedness values of the isolate YIM 65583(T) with S. phyllosphaerae FA2(T) and S. yunnanensis YIM 003(T) were 43.1% and 37.9%, respectively. Based on these features, it is concluded that the strain represents a novel species of the genus Sphingomonas, for which the name Sphingomonas endophytica sp. nov. is proposed, with YIM 65583(T) (=CCTCC AA 209035(T)=JCM 17394(T)) as the type strain.

Phytomonospora endophytica gen. nov., sp. nov., isolated from the roots of Artemisia annua L.

A novel endophytic actinomycete, strain YIM 65646(T), was isolated from the roots of Artemisia annua L. collected from Yunnan Province, south-west China. Growth was observed in the temperature range 10-40 °C (optimum 20-28 °C) and at pH 6.0-9.0 (optimum pH 7.0). The organism formed well-developed, branched substrate mycelia, but aerial mycelium was not produced. Phenotypic characterization and 16S rRNA gene sequence analysis indicated that strain YIM 65646(T) belonged to the family Micromonosporaceae (sharing ≤93.6% sequence similarity with members of this family) and formed a distinct clade in the Micromonosporaceae phylogenetic tree. The strain contained meso-diaminopimelic acid in the cell wall and mannose, ribose, galactose and glucose in whole-cell hydrolysates. The major menaquinones were MK-10(H(4)), MK-10(H(2)), MK-8(H(2)), MK-9(H(2)) and MK-10(H(6)). The major fatty acids were iso-C(15:0), anteiso-C(15:0), anteiso-C(17:0), iso-C(17:0) and iso-C(16:0). The DNA G+C content of strain YIM 65646(T) was 70.0 mol%. On the basis of morphological and chemotaxonomic properties and phylogenetic analysis based on 16S rRNA gene sequence data, it is proposed that this strain should be classified in a novel genus and species, Phytomonospora endophytica gen. nov., sp. nov., in the family Micromonosporaceae. The type strain is YIM 65646(T) (=CCTCC AA 209041(T) =DSM 45386(T)).

Pseudonocardia kunmingensis sp. nov., an actinobacterium isolated from surface-sterilized roots of Artemisia annua L.

A Gram-positive, aerobic, actinobacterial strain with rod-shaped spores, designated YIM 63158(T), was isolated from the surface-sterilized roots of Artemisia annua L. collected from Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 63158(T) belonged to the genus Pseudonocardia. The closest neighbours were 'Pseudonocardia sichuanensis' KLBMP 1115 (99.9 % 16S rRNA gene sequence similarity), Pseudonocardia adelaidensis EUM 221(T) (99.1 %) and Pseudonocardia zijingensis DSM 44774(T) (98.8 %); sequence similarities to other members of the genus Pseudonocardia ranged from 98.6 to 94.4 %. The chemotaxonomic characteristics, such as the cell-wall diaminopimelic acid, whole-cell sugars, fatty acid components and major menaquinones, suggested that the isolate belonged to the genus Pseudonocardia. The G+C content of the genomic DNA was 73.3 mol%. On the basis of physiological, biochemical and chemotaxonomic data, including low DNA-DNA relatedness between the isolate and other members of the genus Pseudonocardia, it is proposed that strain YIM 63158(T) represents a novel species in this genus, with the name Pseudonocardia kunmingensis sp. nov. The type strain is YIM 63158(T) ( = DSM 45301(T)  = CCTCC AA 208078(T)). [corrected].

Pseudonocardia artemisiae sp. nov., isolated from surface-sterilized Artemisia annua L.

A novel actinomycete strain, designated YIM 63587(T), was isolated from surface-sterilized roots of Artemisia annua L. collected from Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 63587(T) was affiliated to the genus Pseudonocardia. 16S rRNA gene sequence similarities between strain YIM 63587(T) and type strains of species of the genus Pseudonocardia were 96.6-93.8 %. The diagnostic cell-wall diamino acid in the peptidoglycan layer of strain YIM 63587(T) was meso-diaminopimelic acid and the whole-cell sugars were arabinose, galactose, mannose and ribose. The predominant menaquinone was MK-8(H(4)) (97.7 %). The phospholipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol mannosides, phosphatidylinositol and an unknown phospholipid. The major cellular fatty acids (>5 %) were iso-C(16 : 0) (44.7 %), iso-C(14 : 0) (10.3 %), iso-C(16 : 1) H (9.8 %) and iso-C(15 : 0) (7.7 %). The G+C content of the genomic DNA was 68.2 mol%. On the basis of phylogenetic, physiological and chemotaxonomic data, strain YIM 63587(T) represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia artemisiae sp. nov. is proposed. The type strain is YIM 63587(T) ( = DSM 45313(T) = CCTCC AA 208081(T)).

Nonomuraea rhizophila sp. nov., an actinomycete isolated from rhizosphere soil.

A novel actinomycete, designated strain YIM 67092(T), was isolated from rhizosphere soil of the perennial vine Tripterygium wilfordii Hook. f. collected from Yunnan province, South-west China. The strain formed well differentiated aerial and substrate mycelia and grew in the presence of up to 7 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene showed that strain YIM 67092(T) belonged to the genus Nonomuraea, with highest sequence similarity to Nonomuraea rosea GW 12687(T) (99.0 %). Sequence similarities between strain YIM 67092(T) and other species of the genus Nonomuraea ranged from 97.8 % (Nonomuraea dietziae DSM 44320(T)) to 93.8 % (Nonomuraea kuesteri GW 14-1925(T)). Key morphological, physiological and chemotaxonomic characteristics of strain YIM 67092(T) were congruent with the description of the genus Nonomuraea. The G+C content of the genomic DNA was 69.3 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA-DNA hybridization results, strain YIM 67092(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea rhizophila sp. nov. is proposed. The type strain is YIM 67092(T) ( = CCTCC AA 209044(T)  = DSM 45382(T)).

Nonomuraea endophytica sp. nov., an endophytic actinomycete isolated from Artemisia annua L.

A Gram-stain-positive, aerobic, non-motile actinomycete strain, designated YIM 65601(T), was studied by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain YIM 65601(T) should be assigned to the genus Nonomuraea, with highest 16S rRNA gene sequence similarities to Nonomuraea candida HMC10(T) (98.8 %), N. salmonea DSM 43678(T) (98.7 %), N. turkmeniaca DSM 43926(T) (98.5 %), N. roseola DSM 43767(T) (98.4 %), N. dietziae IFO 14309(T) (98.2 %) and N. kuesteri GW 14-1925(T) (98.1 %). 16S rRNA gene sequence similarities to strains of other Nonomuraea species were below 98.0 %. Morphological and chemotaxonomic properties of the isolate were consistent with those of members of the genus Nonomuraea. The results of DNA-DNA hybridization and physiological and biochemical tests and fatty acid profiles allowed genotypic and phenotypic differentiation of strain YIM 65601(T) from closely related species. Thus, YIM 65601(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea endophytica sp. nov. is proposed, with YIM 65601(T) ( = CCTCC AA 209037(T)  = DSM 45385(T)) as the type strain.

Pseudonocardia rhizophila sp. nov., a novel actinomycete isolated from a rhizosphere soil.

During the course of our research on new actinobacterial sources, a novel actinomycete strain YIM 67013(T) was isolated from rhizosphere soil of Tripterygium wilfordii Hook. f. collected from Yunnan province, south-west China. The strain formed well-differentiated aerial and substrate mycelia, grew in the presence of up to 15% NaCl (optimum, 0-3% NaCl). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 67013(T) belongs to the genus Pseudonocardia, with highest similarity to Pseudonocardia thermophila DSM 43832(T) (97.6%). Sequence similarities between strain YIM 67013(T) and the other Pseudonocardia species ranged from 96.6 to 93.2%. Key morphological and physiological characteristics as well as chemotaxonomic features of strain YIM 67013(T) were congruent with the description of the genus Pseudonocardia. The G+C content of the genomic DNA was 69.7 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA-DNA hybridization results, it is proposed that strain YIM 67013(T) represents a novel species of the genus Pseudonocardia, named Pseudonocardia rhizophila sp. nov. The type strain is YIM 67013(T) (= CCTCC AA 209043(T) = DSM 45381(T)).

Streptomyces artemisiae sp. nov., isolated from surface-sterilized tissue of Artemisia annua L.

The taxonomic position of an actinomycete strain YIM 63135(T), which was isolated from the surface-sterilized tissue of Artemisia annua L. collected from Yunnan province, south-west China, was determined by using a polyphasic approach. Morphological and chemical characteristics of the novel strain were consistent with those of the genus Streptomyces. It developed a pinkish aerial mycelium and pinkish-brown substrate mycelium on oatmeal agar. The cell wall of the strain contained ll-diaminopimelic acid. The menaquinones comprised MK-9(H(6)) (62.8 %), MK-9(H(8)) (31.4 %) and MK-9(H(4)) (5.9 %). The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, an unknown glucosamine-containing phospholipid (GluNu), phosphatidylinositol mannosides and four unknown ninhydrin-negative phospholipids. The major fatty acids were iso-C(16 : 0) (30.0 %), anteiso-C(17 : 0) (27.3 %) and anteiso-C(15 : 0) (17.0 %). The DNA G+C content of strain YIM 63135(T) was 72.6 mol%. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain YIM 63135(T) is a member of the genus Streptomyces and exhibited 99.9 % gene sequence similarity to Streptomyces armeniacus NBRC 12555(T), while low sequence similarity values (<97.0 %) distinguished strain YIM 63135(T) from all other Streptomyces species. DNA-DNA hybridization studies suggested that strain YIM 63135(T) represents a different genomic species. On the basis of phenotypic and phylogenetic characteristics, strain YIM 63135(T) was considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces artemisiae sp. nov. is proposed, with YIM 63135(T) (=CCTCC AA 208059(T) =DSM 41953(T)) as the type strain.

Kineosporia mesophila sp. nov., isolated from surface-sterilized stems of Tripterygium wilfordii.

An endophytic actinomycete strain, designated YIM 65293(T), was isolated from a surface-sterilized stem sample of Tripterygium wilfordii collected from Yunnan province, south-west China, and its taxonomic position was investigated. The chemical and morphological properties of the organism were consistent with those of the genus Kineosporia. Phylogenetic analysis indicated that the levels of 16S rRNA gene sequence similarity between strain YIM 65293(T) and other type strains of recognized members of the genus Kineosporia were 97.0-98.2 %. However, the DNA-DNA hybridization values, in combination with differences in phenotypic characteristics, revealed that the strain differed from recognized species of the genus Kineosporia. Therefore, strain YIM 65293(T) represents a novel species of the genus Kineosporia, for which the name Kineosporia mesophila sp. nov. is proposed. The type strain is YIM 65293(T) (=CCTCC AA 208061(T)=DSM 45271(T)).

Saccharopolyspora tripterygii sp. nov., an endophytic actinomycete isolated from the stem of Tripterygium hypoglaucum.

An endophytic actinomycete, designated strain YIM 65359(T), was isolated from a surface-sterilized stem sample of Tripterygium hypoglaucum collected from Yunnan province, south-west China. The morphological and chemotaxonomic properties of the new isolate were consistent with those of members of the genus Saccharopolyspora. Analysis of 16S rRNA gene sequences revealed that the new isolate was most closely related to 'Saccharopolyspora endophytica' YIM 61095 (98.6 %), Saccharopolyspora flava AS4.1520(T) (97.6 %) and Saccharopolyspora spinosa DSM 44228(T) (97.0 %). The results of DNA-DNA hybridizations (57.5 %, 44.9 % and 48.5 %, respectively) with the above micro-organisms, in combination with differences in the biochemical and physiological characteristics, suggested that strain YIM 65359(T) should be classified as a novel species of the genus Saccharopolyspora. The name Saccharopolyspora tripterygii sp. nov. is proposed for this novel species, with YIM 65359(T) (=CCTCC AA 208062(T)=DSM 45269(T)) as the type strain.