Hideo Nonomura

Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes

Abstract A new medium, designated HV agar, containing soil humic acid as the sole source of carbon and nitrogen was developed. The HV agar was superior to other currently used media, including colloidal chitin agar, glycerol-arginine agar and starch-casein-nitrate agar, for the isolation and enumeration of soil actinomycetes: It allowed the growth of the largest numbers of actinomycete colonies belonging to each genus of Streptomyces, Micromonospora, Microbispora , Streptosporangium, Nocardia, Dactylosporangium, Microtetraspora and Thermomonospora on the plate, while restricting the development of true bacteria. The HV agar supported adequate growth and good sporulation for these actinomycetes. Even when spore suspensions were used as the inoculum, the HV agar produced remarkably larger numbers of actinomycetes, especially strains of the genera Micromonospora, Microbispora, Streptosporangium, Dactylosporangium and Saccharomonospora , than did glycerol-arginine agar. It was found that the spores of these actinomycetes were activated upon germination by treatment at 20°C for 30 min with a O.2% solution of humic acid prior to incubation.

New methods for the highly selective isolation of Micromonospora and Microbispora from soil

Abstract Two plate culture methods for the highly selective isolation of the soil actinomycetes Micromonospora and Microbispora, are described, which utilize the differential antibiotic resistance of these microbes and the superior ability of their spores to withstand deleterious agents. (i) To isolate micromonosporae, a water suspension of air-dried soil is first treated with 1.5% phenol, diluted with water and then cultured on HV agar supplemented with both tunicamycin and nalidixic acid. (ii) To isolate microbisporae, air-dried soil is heated in a hot-air oven at 120°C for 1 h. A water suspension of the heated sample is then treated with a solution of 1.5% phenol and 0.03% chlorhexidine gluconate (CG), diluted and cultured on HV agar supplemented with nalidixic acid. The phenol pretreatment of the soil killed bacteria and streptomycetes in the samples, while keeping micromonosporae and microbisporae alive. The use of tunicamycin suppressed the growth of the remaining microbisporae on HV agar isolation plates, thereby facilitating the intensive isolation of micromonosporae. On the other hand, the dry heat treatment of the soil drastically reduced the number of bacteria in the sample and significantly reduced the numbers of streptomycetes and micromonosporae, without reducing the microbisporae content. Further treatment with a mixture of phenol and CG eliminated dry heat-resisting actinomycetes apart from microbisporae. Dilution plating of samples subjected to heat and the phenol-CG treatment resulted in the intensive isolation of microbisporae on HV agar. From various field soils (vegetable, orchard, corn, and rice paddy), the methods described above always enabled the highly selective isolation of micromonosporae and microbisporae, respectively, as each group accounted for more than 90% of the total number of colonies recovered.

Selective isolation of Actinoplanes and Dactylosporangium from soil by using γ-collidine as the chemoattractant

Abstract Zoospores of the actinoplanetes Actinoplanes missouriensis and Dactylosporangium thailandense were attracted to a number of amino acids, aromatic compounds and sugars. Among these compounds, d -xylose and γ-collidine served as universal chemoattractants for most or all of other 9 species belonging to the genera, and moreover, γ-collidine always elicited an extremely strong response. In contrast, potassium chloride was a weak to moderate attractant only for a half of the strains tested. Palleronis chemotactic method (Palleroni, N.J., Arch. Microbiol., 128, 53–55, 1980) for the isolation of motile actinoplanetes was complemented by using γ-collidine as a substitute for the originally used chemoattractant, potassium chloride, and by using humic acid-vitamin (HV) agar in place of the isolation medium, starch-casein agar. The improved method not only enabled the increased recovery of Actinoplanes spp. but also the consistent recovery of Dactylosporangium spp., which have only incidentally been isolated by the original method and by conventional dilution plating techniques, from various field soils (16 samples). The recovery of motile actinoplanetes was more successfully achieved by using HV agar supplemented with nalidixic acid to reduce associated bacterial contaminants.

Distribution of rare actinomycetes in Japanese soils

The distribution of rare actinomycetes in 237 soil samples from various locations throughout Japan was investigated using a special isolation medium, HV agar. The populations (colony forming units) of these actinomycetes per gram of dried soil were Microtetraspora 6 × 103, Saccharomonospora 1.7 × 104, Dactylosporangium 5.4 × 104, Streptosporangium 1.2 × 105, Microbispora 1.4 × 105, Nocardioforms 1.9 × 105, and Micromonospora 6.8 × 105. Streptomycetes 2.2 × 106, and Unidentified actinomycetes 0.9 × 106 were also observed. Their distributions seemed to be associated with environmental factors such as soil type (Land Use Classification), soil pH, humus content, and the characteristics of the humic acid. In general, the largest populations were found in soils of cultivated fields, which were rich in humus and had pH values between 6.5–7.0. However, the distribution of some genera in cultivated field soils (154 samples) was remarkable. The numbers of Microbispora and Streptosporangium were the largest in humus-rich acidic (pH 5.0–6.05) soils with low humic acid Δ log K values (black colored humic acid). Saccharomonospora was found most frequently in relatively humus-poor alkaline (pH 7.0–7.5) soils having higher Δ log K values (brown humic acid). Dactylosporangium and Microtetraspora, Saccharomonospora, and Micromonospora were most frequently isolated from mountainous forest soils, level-land forest or cultivated field soils, and pasture soils, respectively.

Efficacy of artificial humic acid as a selective nutrient in HV agar used for the isolation of soil actinomycetes

Abstract The efficacies of various kinds of humic acid, as the source of carbon and nitrogen in HV agar reported in the previous paper, were compared to the selective isolation of soil actinomycetes. The 4 types of natural humic acid, A , B , P and Rp were prepared from different soils, and 3 kinds of artificial humic acid were made from carbohydrates and urea in our laboratory. Among the natural humic acids, type Rp , which has been reported to be associated with an initial state of humification in natural conditions, showed the greatest efficacy. However, one of the artificial humic acids, which was prepared from glucose and urea, was considered to be superior to the Rp natural humic acid: 1) The HV agar containing this artificial humic acid (HV-glucose HA agar) produced the same large number of actinomycete colonies on the plate as that of the HV agar with type Rp soil humic acid (HV- Rp agar). 2) The HV-glucose HA agar restricted the number of bacterial colonies on the plates to one-half of that on HV- Rp agar plates. 3) The quality of natural himic acids varies, whereas artificial ones are more constant and can be made in any laboratory.

New methods for the highly selective isolation of Streptosporangium and Dactylosporangium from soil

Abstract Two methods for the isolation of the rare actinomycetes Streptosporangium and Dactylosporangium in soil, are described. The methods use the ability of both the sporangiospores of Streptosporangium and the globose bodies (aleuriospore) of Dactylosporangium to withstand dry heating and treatment with benzethonium chloride (BC). In addition, the differential antibiotic resistance of these actinomycetes is also utilized. (i) To isolate streptosporangia, an air-dried soil sample is first subjected to dry heat treatment (120°C, 1 h). A water suspension of the heated sample is then treated with 0.01% BC, diluted and cultured on HV agar supplemented with nalidixic acid and leucomycin. (ii) To isolate dactylosporangia, a water suspension of a soil sample given dry heat and 0.03% BC treatment is cultured on HV agar supplemented with nalidixic acid and tunicamycin. The dry heat and BC treatment drastically eliminated bacteria and unwanted actinomycetes contaminants, including streptomycetes, from the isolation plates, thereby facilitating the selective isolation of streptosporangia and dactylosporangia. Nalidixic acid in HV agar was useful to suppress the growth of dry heat- and BC-resistant bacteria. The use of leucomycin and tunicamycin increased the selectivity of HV agar for streptosporangia and dactylosporangia, respectively, through elimination of the growth of unwanted actinomycetes which remained after the employment of pretreatments. From various field soils (vegetable, orchard, corn, and rice paddy), the methods described above consistently achieved the highly selective isolation of streplosporangia and dactylosporangia, respectively, as each group accounted for more than 20% of the total number of colonies recovered.

Pollen-baiting and drying method for the highly selective isolation of Actinoplanes spp. from soil

Abstract A simplified enrichment method for the highly selective isolation of the zoosporic actinomycetes Actinoplanes spp. from soil is described. The method consists of baiting the species with Pinus pollen grains, desiccating (30°C, 2 h) the baits bearing sporangia in dried soil particles with the aid of silica gel and following the spore liberation upon immersion in water. Portions of the liquid enriched with zoospores are plated out on humic acid-vitamin (HV) agar supplemented with nalidixic acid at a concentration of 10 μg ml−1. The desiccation stage has enabled the almost complete elimination of associated bacteria from colonized baits while allowing the Actinoplanes sporangia to survive and still posses the ability to release many spores. A total of four different soil samples from fields of corn, peach, vegetable and paddy rice were examined. The pollen-baiting and drying method consistently resulted in the highly selective isolation of Actinoplanes spp. which accounted for over 83% of the total number of micro-organisms recovered on HV agar containing nalidixic acid.

A selective isolation method for Actinomadura viridis in soil

Abstract The rare actinomycete Actinomadura viridis was efficiently isolated from a variety of field soils by a new plate culture method in which a water suspension of dry-heated (110°C, 1h) soil was treated with 1.0% phenol and cultured on Humic acid-vitamin agar supplemented with kanamycin, josamycin, lysozyme and nalidixic acid. The method significantly eliminated unicellular bacteria and undesirable actinomycetes contaminants from the isolation plates, thereby achieving the highly selective isolation of A. viridis .

Four new species of the genus Actinokineospora : Actinokineospora inagensis sp. nov., Actinokineospora globicatena sp. nov., Actinokineospora terrae sp. nov., and Actinokineospora diospyrosa sp. nov

The taxonomic positions of motile actinomycetes that were isolated from soil and fallen leaves obtained from around a pond and a lake and from fallen leaves of persimmons were studied. The aerial mycelia of all of the isolates exhibited fragmentation during growth, and motile spores arranged in chains were produced within the mycelia. Sporangia were not observed. These isolates contained menaquinone MK-9(H4), their DNA G+C contents were 69 to 70 mol%, they contained glutamic acid, glycine, alanine, and meso-diaminopimelic acid as cell wall amino acids, and arabinose and galactose were found in their whole-cell hydrolysates. These taxonomic characteristics are the same as those of Actinokineospora riparia. On the basis of morphological, physiological, and chemotaxonomic characteristics and DNA-DNA hybridization data, we propose the following four new species of the genus Actinokineospora for these strains: Actinokineospora inagensis for a single isolate, type strain YU4-1 (= IFO 15663); Actinokineospora globicatena for isolates YU5-1, YU6-1, YU6-2, YU7-1, and YU7-2 (type strain, YU6-1 [= IFO 15664]); Actinokineospora terrae for a single isolate, type strain YU6-3 (= IFO 15668); and Actinokineospora diospyrosa for a single isolate, type strain YU8-1 (= IFO 15665).

Inherent G418-Resistance in Hybridization of Industrial Yeasts

Abstract Eight strains of sake yeast exhibited inherent-resistance to 100 μg/ml of Geneticin (G418). Fourteen wine yeasts and 1 shochu yeast ( Saccharomyces cerevisiae ) and 1 miso yeast ( Zygosaccharomyces rouxii ) were inherent G418-sensitive. The petites converted from inherent G418-resistants by treatment with ethidium bromide retained G418-resistance (ϱ − G418R), and thus were hybridized by electrofusion with the wine yeast W3 (ϱ + G418S, wild type). A lag phase of 12–18 h was required prior to administration of the drug in glycerol medium when selecting G418-resistant hybridization products. Colonies were formed in the regeneration medium at a frequency of about 1 × 10 −5 per used protoplasts. No growth of any parental strain (10 6 /_~10 7 protoplasts) separately subjected to electrofusion and regeneration was observed. The hybridization products were G418-resistant “grande” strains (ϱ − G418R) in which the genetic traits of parental strains had been complemented. Uninucleate cells (DAPI staining) of the hybridization products showed CHEF electrophoretic karyotypes similar to that of wine yeast, but possessed a single chromosome (approx. 320 kb) presumably from sake yeast.