Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

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Effects of diverse materials-based methods on DNA extraction for Clostridium difficle from stool samples

Materials Express ◽

10.1166/mex.2019.1520 ◽

2019 ◽

Vol 9 (5) ◽

pp. 509-516 ◽

Cited By ~ 3

Author(s):

Ziqi Xiao ◽

Gaojian Yang ◽

Deng Yan ◽

Song Li ◽

Zhu Chen ◽

...

Keyword(s):

Nucleic Acid ◽

Dna Extraction ◽

Magnetic Beads ◽

Diagnostic Technique ◽

Proteinase K ◽

Molecular Diagnostic ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Spin Column ◽

Stool Samples

Nosocomial infections, including Clostridium difficile infection (CDI), and their fatality rates have increased in the past few decades. Despite emerging molecular diagnostic technologies with rapid, accurate outcomes, nucleic acid extraction from stool samples remains the first limiting step before downstream applications. Commercial nucleic acid extraction kits greatly decrease labor and time requirements, and also provide nucleic acid preparations with higher quality and purity for enzyme digestion analysis or genotyping. The magnetic bead based technique is a novel method compared with the conventional spin-column method, and currently has widespread use in nucleic acid extraction. We evaluated five DNA extraction kits with magnetic beads using materials with various properties (particle size, concentration of magnetic beads, grinding beads) and reagents (proteinase K, lysozyme, isopropanol, and absolute ethanol) to determine the cost, hands-on time, number of essential operations, and quality and purity of the DNA preparations, compared with those obtained using the QIAamp Fast DNA Stool Mini Kit. The six DNA extraction kits yielded A260/280 ratios ranging from 0.85 to 1.9 (average 1.57), and concentrations from 3.70 to 108.09 ng/μL (average 34.64 ng/μL). All the DNA samples had acceptable downstream application effects, except for those obtained using the TIANGEN Magnetic Soil and Stool DNA Kit. However, gel electrophoresis analysis of the DNA samples resulted in a light strip on the gel, indicating that the proteinaceous contaminant may not have been removed completely. A rapid and accurate molecular diagnostic technique could allow for more suitable treatment and prognosis outcomes for inpatients, depending, in large part, on the quality and purity of DNA preparations, which are frequently neglected. Our study focused on the quality of commercial kits with a primary focus on the treatment of stool samples and molecular diagnostic applications.

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Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽

10.3390/genes12020146 ◽

2021 ◽

Vol 12 (2) ◽

pp. 146

Author(s):

Catarina Xavier ◽

Mayra Eduardoff ◽

Barbara Bertoglio ◽

Christina Amory ◽

Cordula Berger ◽

...

Keyword(s):

Dna Extraction ◽

Ancient Dna ◽

Extraction Method ◽

Fragment Size ◽

Molecular Genetic ◽

Extraction Methods ◽

Degraded Dna ◽

Size Analysis ◽

Dna Fragments ◽

Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

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Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11353-4 ◽

2021 ◽

Author(s):

Annemarie Siebert ◽

Katharina Hofmann ◽

Lena Staib ◽

Etienne V. Doll ◽

Siegfried Scherer ◽

...

Keyword(s):

Sample Preparation ◽

Dna Extraction ◽

Raw Milk ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Bacterial Dna ◽

Microbiome Analysis ◽

Eukaryotic Dna ◽

Selective Lysis ◽

The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

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Methodological Approaches to DNA Authentication of Foods, Wines and Raw Materials for Their Production

Foods ◽

10.3390/foods10030595 ◽

2021 ◽

Vol 10 (3) ◽

pp. 595

Author(s):

Aram G. Galstyan ◽

Vladislav K. Semipyatniy ◽

Irina Yu. Mikhailova ◽

Khamid Kh. Gilmanov ◽

Alana V. Bigaeva ◽

...

Keyword(s):

Sample Preparation ◽

Dna Extraction ◽

Raw Materials ◽

Direct Sequencing ◽

Genetic Identification ◽

Proteinase K ◽

Vitis Vinifera L ◽

Acid Extraction ◽

Plant Component ◽

Sensitivity Specificity

DNA authentication of wines is a process of verifying their authenticity by genetic identification of the main plant component. The sample preparation of experimental and commercial wines was carried out by precipitation of wine debris by centrifugation with preliminary exposure with precipitators and co-precipitators, including developed macro- and micro- volume methods applicable to white or red wines, using polyvinylpyrrolidone as a co-precipitator. Addition of 2-mercaptoethanol and proteinase K to the lysing solution made it possible to adapt the technology for DNA extraction from the precipitated wine debris. The additionally tested technique of DNA extraction from wine debris by dimethyl sulfoxide (DMSO) lysis had fewer stages and, consequently, a lower risk of contamination. The results of further testing of one of the designed primer pairs (UFGT-F1 and UFGT-R1) in conjunction with the tested methods of wine material sample preparation and nucleic acid extraction, showed the advantage in the given set of oligonucleotides over previously used ones in terms of sensitivity, specificity and reproducibility. The developing strategy for genetic identification of grape varieties and DNA authentication of wines produced from them based on direct sequencing of polymerase chain reaction (PCR) products is implemented by interpreting the detected polymorphic positions of variable Vitis vinifera L. UFGT gene locus with distribution and split into 13 UFGT gene-associated groups.

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Degradation of zearalenone in swine feed and feed ingredients by Bacillus subtilis ANSB01G

World Mycotoxin Journal ◽

10.3920/wmj2013.1623 ◽

2014 ◽

Vol 7 (2) ◽

pp. 143-151 ◽

Cited By ~ 18

Author(s):

Y.P. Lei ◽

L.H. Zhao ◽

Q.G. Ma ◽

J.Y. Zhang ◽

T. Zhou ◽

...

Keyword(s):

Bacillus Subtilis ◽

Culture Supernatant ◽

Antimicrobial Activities ◽

Proteinase K ◽

Rrna Gene ◽

Distillers Grains With Solubles ◽

Dried Distillers Grains ◽

Cell Extracts ◽

Gene Sequence Analysis ◽

And Staphylococcus Aureus

Zearalenone (ZEA) and its derivatives are mycotoxins that can cause oestrogenic effects and impair the reproductive physiology of animals, especially in female swine. Strategies to reduce or eliminate ZEA contamination in foods and feeds are very much needed. Among 36 bacterial isolates obtained from a variety of animal intestinal chyme, mouldy foods and feeds, soils, etc., five isolates demonstrated the ability to reduce more than 50% of ZEA in a liquid medium; ANSB01G isolate taken from normal broiler intestinal chyme reduced ZEA the most, by 88.65%. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, the ANSB01G isolate was identified as Bacillus subtilis. Under simulated intestinal tract conditions, the ANSB01G B. subtilis isolate degraded 84.58, 66.34 and 83.04% of ZEA in naturally contaminated maize, dried distillers’ grains with solubles, and swine complete feed, respectively. The highest degradation of ZEA occurred when the mycotoxin was co-incubated with the whole bacterial culture, resulting in a reduction of 88.65%, followed by 75.60% using culture supernatant, 26.11% using cell extracts, and 15.06% using viable cells. Treatments consisting of both heating and addition of proteinase K significantly reduced the rate of ZEA degradation in the culture supernatant, indicating that the ZEA degradation might be enzymatic. B. subtilis ANSB01G displayed resistance to simulated gastrointestinal tract environments and antimicrobial activities against several common bacterial pathogens, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. These properties of B. subtilis ANSB01G suggest the possibility of its potential to effectively degrade ZEA in feed and to develop functional feed products for livestock industries.

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Genome Insight and Description of Carotenoids-producing Croceicoccus Gelatinilyticus Sp. Nov., Isolated From the Tidal Flat Sediment

10.21203/rs.3.rs-687094/v1 ◽

2021 ◽

Author(s):

Tao Pei ◽

Yang Liu ◽

Juan Du ◽

Kun peng Huang ◽

Ming rong Deng ◽

...

Keyword(s):

Tidal Flat ◽

Biosynthetic Gene Cluster ◽

Phylogenomic Analysis ◽

Rrna Gene ◽

Species Definition ◽

Gram Staining ◽

Taxonomic Approach ◽

Pigment Absorption ◽

Genotypic Characteristics ◽

Type Strains

Abstract A novel Gram-staining-negative and short-rod-shaped bacterial strain designated as 1NDH52T was isolated from tidal flat sediments and characterized by using a polyphasic taxonomic approach. The predominant cellular fatty acids of strain 1NDH52T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and C14:0 2-OH; the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid; the major respiratory quinones were Q-10 and Q-9. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 1NDH52T belonged to the genus Croceicoccus with high similarities to the close type strains Croceicoccus pelagius Ery9T, Croceicoccus sediminis S2-4-2T and Croceicoccus bisphenolivorans H4T. Phylogenomic analysis indicated that strain 1NDH52T formed an independent branch distinct from the known type strains of this genus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain 1NDH52T and the three type strains above were well below thresholds of 70% DDH and 95-96% ANI for species definition, implying that strain 1NDH52T should represent a novel genospecies. The genomic DNA G + C content was 62.6%. The carotenoids production of the novel strain was determined by the detection of the pigment absorption spectrum and the identification of the complete biosynthetic gene cluster in its genome. Based on the phenotypic and genotypic characteristics, strain 1NDH52T is concluded to represent a novel species of the genus Croceicoccus, for which the name Croceicoccus gelatinilyticus sp. nov., is proposed. The type strain of the species is 1NDH52T (= GDMCC 1.2381T = KCTC 82668T). The description of the genus Croceicoccus has also been emended.

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Salipiger mangrovisoli sp. nov., isolated from mangrove soil and the proposal for the reclassification of Paraphaeobacter pallidus as Salipiger pallidus comb. nov.

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004892 ◽

2021 ◽

Vol 71 (7) ◽

Author(s):

Juan Du ◽

Yang Liu ◽

Tao Pei ◽

Ming-Rong Deng ◽

Honghui Zhu

Keyword(s):

Type Strain ◽

Type Species ◽

Cellular Fatty Acid ◽

Phylogenomic Analysis ◽

Rrna Gene ◽

Respiratory Quinone ◽

Species Definition ◽

Content Type ◽

Link Type ◽

Mangrove Soil

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain designated as 6D45AT was isolated from mangrove soil and characterized using a polyphasic taxonomic approach. Strain 6D45AT was found to grow at 10–37 °C (optimum, 28 °C), at pH 6.0–9.0 (optimum, 7.0) and in 0–5 % (w/v) NaCl (optimum, 2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 6D45AT fell into the genus Salipiger and shared 99.1 % identity with the closest type strain Salipiger pacificus CGMCC 1.3455T and less than 97.2 % identity with other type strains of this genus. The 34.8 % digital DNA–DNA hybridization (dDDH) and 88.3 % average nucleotide identity (ANI) values between strain 6D45AT and the closest relative above were well below recognized thresholds of 70 % DDH and 95–96 % ANI for species definition, implying that strain 6D45AT should represent a novel genospecies. The phylogenomic analysis indicated that strain 6D45AT formed an independent branch distinct from reference strains. The predominant cellular fatty acid of strain 6D45AT was summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c, 66.9 %); the polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, two unidentified glycolipids and an unknown lipid; the respiratory quinone was Q-10. The genomic DNA G+C content was 66.5 mol %. Based on the phenotypic and genotypic characteristics, strain 6D45AT is concluded to represent a novel species of the genus Salipiger , for which the name Salipiger mangrovisoli sp. nov., is proposed. The type strain of the species is 6D45AT (=GDMCC 1.1960T=KCTC 82334T). We also propose the reclassification of Paraphaeobacter pallidus as Salipiger pallidus comb. nov. and ‘ Pelagibaca abyssi ’ as a species of the genus Salipiger .

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Isolation, Phylogenetic and Gephyromycin Metabolites Characterization of New Exopolysaccharides-Bearing Antarctic Actinobacterium from Feces of Emperor Penguin

Marine Drugs ◽

10.3390/md19080458 ◽

2021 ◽

Vol 19 (8) ◽

pp. 458

Author(s):

Hui-Min Gao ◽

Peng-Fei Xie ◽

Xiao-Ling Zhang ◽

Qiao Yang

Keyword(s):

Genome Mining ◽

Pairwise Comparison ◽

Gene Clusters ◽

Amino Acid Identity ◽

Core Gene ◽

Emperor Penguin ◽

Rrna Gene ◽

Active Metabolites ◽

The Antarctic ◽

Gene Similarity

A new versatile actinobacterium designated as strain NJES-13 was isolated from the feces of the Antarctic emperor penguin. This new isolate was found to produce two active gephyromycin analogues and bioflocculanting exopolysaccharides (EPS) metabolites. Phylogenetic analysis based on pairwise comparison of 16S rRNA gene sequences showed that strain NJES-13 was closely related to Mobilicoccus pelagius Aji5-31T with a gene similarity of 95.9%, which was lower than the threshold value (98.65%) for novel species delineation. Additional phylogenomic calculations of the average nucleotide identity (ANI, 75.9–79.1%), average amino acid identity (AAI, 52.4–66.9%) and digital DNA–DNA hybridization (dDDH, 18.6–21.9%), along with the constructed phylogenomic tree based on the up-to-date bacterial core gene (UBCG) set from the bacterial genomes, unequivocally separated strain NJES-13 from its close relatives within the family Dermatophilaceae. Hence, it clearly indicated that strain NJES-13 represented a putative new actinobacterial species isolated from the gut microbiota of mammals inhabiting the Antarctic. The obtained complete genome of strain NJES-13 consisted of a circular 3.45 Mb chromosome with a DNA G+C content of 67.0 mol%. Furthering genome mining of strain NJES-13 showed the presence of five biosynthetic gene clusters (BGCs) including one type III PKS responsible for the biosynthesis of the core of gephyromycins, and a series of genes encoding for bacterial EPS biosynthesis. Thus, based on the combined phylogenetic and active metabolites characterization presented in this study, we confidently conclude that strain NJES-13 is a novel, fresh actinobacterial candidate to produce active gephyromycins and microbial bioflocculanting EPS, with potential pharmaceutical, environmental and biotechnological implications.

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Pareuzebyella sediminis gen. nov., sp. nov., a novel marine bacterium in the family Flavobacteriaceae, isolated from a tidal flat sediment

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004606 ◽

2020 ◽

Author(s):

Zhaobin Huang ◽

Xiaomei Wei ◽

Qiliang Lai ◽

Shiyong Chen ◽

Jianjun Yuan

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Tidal Flat ◽

Type Species ◽

Sequence Similarity ◽

Phylogenomic Analysis ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

The Family

Two marine bacterial strains, designated S2-4-21T and MT2-5-19, were isolated from two tidal flat sediments of cordgrass Spartina alterniflora and adjacent oyster culture field in Quanzhou bay, China, respectively. Both strains were Gram-staining-negative, rod-shaped, non-flagellated, non-motile, aerobic, had NaCl requirements, and contained carotenoid and flexirubin pigments. The 16S rRNA gene sequence similarity (99.8%), average nucleotide identity value (99.4%) and average amino acid identity (99.3%) between strain S2-4-21T and strain MT2-5-19 strongly supported that they belonged to a single species. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2-4-21T and strain MT2-5-19 formed a monophyletic branch affiliated to the family Flavobacteriaceae , sharing similarities of 94.6% with Euzebyella marina CY01T and E. saccharophila 7SM30T, and of 94.1 and 92.8% with E. algicola MEBiC 12267T and Pseudozobellia thermophile DSM 19858T, respectively. Phylogenomic analysis based on the whole genome sequences supported that the two strains formed a distinct monophyletic clade within Flavobacteriaceae members, which was phylogenetically different from the clades of Euzebyella and Pseudozobellia . The major respiratory quinone was menaquinone MK-6. The major fatty acids (>10%) consisted of C15 : 0 iso, C16 : 0, summed feature 9 (C17 : 1 iso ω9c/C16 : 0 10-methyl) and C17 : 0 iso 3-OH. The polar lipid profiles of strain S2-4-21T and strain MT2-5-19 are identical, including phosphatidylethanolamine, four unidentified aminolipids, and four unidentified lipids. The genomic size was 4.9–5.0 Mb with genomic DNA G+C content of 41.5 mol%. Based on the above characteristics, strains S2-4-21T and MT2-5-19 represented a novel species of a novel genus in the family Flavobacteriaceae . Thus, Pareuzebyella sediminis gen. nov. sp. nov. is proposed with type strain S2-4-21T (=MCCC 1K03818T=KCTC 72152T), and another strain MT2-5-19 (=KCTC 72539=MCCC 1K03874).

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Agromyces humi sp. nov., actinobacterium isolated from farm soil

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004376 ◽

2020 ◽

Vol 70 (9) ◽

pp. 5032-5039 ◽

Cited By ~ 3

Author(s):

Jae-Chan Lee ◽

Kyung-Sook Whang

Keyword(s):

Type Species ◽

Phylogenomic Analysis ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

Gene Sequence Analysis ◽

Phyletic Line ◽

Phylogenomic Analyses ◽

The 16S Rrna Gene ◽

Sequence Similarities

A Gram-stain-positive actinobacterial strain, designated ANK073T, was isolated from rhizosphere soil sampled at a spinach farming field in Shinan, Republic of Korea. Cells of strain ANK073T were found to be aerobic, non-motile, non-spore-forming rods which could grow at 20–40 °C (optimum, 30 °C), at pH 6.0–10.0 (optimum, pH 6.5–7.5) and at salinities of 0–4 % (w/v) NaCl (optimum, 0 % NaCl). The 16S rRNA gene sequence analysis showed that strain ANK073T belongs to the genus Agromyces with high sequence similarities to Agromyces humatus CD5T (98.8 %), Agromyces tardus SJ-23T (98.5 %) and Agromyces iriomotensis IY07-20T (98.4 %). The phylogenetic analysis indicated that strain ANK073T formed a distinct phyletic line in the genus Agromyces and the results of DNA–DNA relatedness and phylogenomic analysis based on whole genome sequences demonstrated that strain ANK073T could be separated from its closest relatives in the genus Agromyces . The strain contained 2,4-diaminobutylic acid, glycine, d-glutamic acid and d-alanine in the peptidoglycan. The predominant menaquinones were identified as MK-12 and MK-11, and the major fatty acids were anteiso-C17 : 0, anteiso-C15 : 0 and iso-C15:0. The major polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genome was determined to be 70.2 mol%. On the basis of its phenotypic and chemotaxonomic properties and the results of phylogenetic and phylogenomic analyses, strain ANK073T is considered to represent a novel species in the genus Agromyces , for which the name Agromyces humi sp. nov. is proposed. The type strain is ANK073T (=KACC 18683T=NBRC 111825T).

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