Humic Acid Vitamin Research Articles

Isolation and potency of Actinomycetes from rhizosphere of nutmeg (Myristica fragrans Houtt)

Nutmeg (Myristica fragrans Houtt) is commonly cultivated by people in the forests of Moluccas Islands. This plant grows well on relatively infertile soil types. This is presumably due to the presence of symbiotic microbes in the root of nutmeg. The research aimed to isolate, characterize and test the potential of Actinomycetes from rhizosphere of nutmeg. Soil sample were taken from the nutmeg forest in Ambon Island. The Actinomycetes isolation using humic acid vitamin, continued with yeast malt agar (YMA) media. The testing of antibacterial and antifungal activities using YMA media, while cellulolytic activity, phosphate solubilizing, and xylanolytic activity using carboxyl methyl cellulose, Picovskaya agar, and birchwood agar or oat spelt xylan agar. A total of 12 isolates of Actinomycetes were isolated and dominated by Streptomyces with various types of aerial mycelia. The substrate mycelium looks brown and cream, while the aerial mycelium looks white and gray. These isolates had the highest inhibitory power against Escherichia coli and Fusarium oxysporum with indexes of 16.5 mm and 16.0 mm, respectively. The other isolates have the ability of cellulolytic, phosphate solubilizing, and xylanolytic with indexes 3.26, 3.87, and 1.2, respectively. The Actinomycetes isolates that were found can be used as starter to improve the biofertilizer formula for nutmeg.

Isolation and Celulolytic Activity Assay of Actinobacteria Isolated from Palm Oil Wastewater

Cellulose plays an important role in the carbon cycle in nature and is the largest compound. This study aimed to isolate, to characterize and to determine actinobacteria that capable of producing cellulases. The sampling method in this study was carried out by purposive sampling at the outlet point of aeration pond of the wastewater treatment plant (IPAL) from palm oil waste station of PT. Teupin Lada. Isolation of actinobacteria was carried out on Humic Acid Vitamin b Agar (HVA), morphological characterization was carried out on Yeast Malt Agar (YMA), Yeast Starch Agar (YSA), Oatmeal Agar (OA), and microscopic characterization of actinobacteria and measuring the diameter of the clear zone formed on Carboxymethyl Cellulose (CMC) medium using the indicator Congo Red. Eight isolates were obtained from the isolation. Of the 8 isolates obtained, 7 of them were able to produce cellulase enzymes which were measured based on the clear zone formed in the test Congo Red on Carboxymethil Cellulose (CMC), and one isolate did not show any clear zones. The highest value of Cellulolytic Index (IS) was obtained from isolate ATLS-05, namely 8.38 mm. HIGHLIGHTS Cellulose plays an important role in the carbon cycle in nature and is the largest compound The high waste load especially palm oil mill effluent (Elaeis guineensis) or known as Palm Oil Mill Effluent(POME) could cause various problems for the environment and society. POME is wastewater from the palm oil industry, which is one of the most polluting agro-industrial wastes Actinobacteria are one of the soil microbes which have the greatest abundance and play an important role in the decomposition process. One of the roles of soil microbes is too degrading cellulose GRAPHICAL ABSTRACT

TAXONOMIC CHARACTERIZATION, ANTIMICROBIAL ACTIVITY AND ANTIOXIDANT POTENTIAL OF RARE ACTINOBACTERIUM ISOLATED FROM MANGROVE ECOSYSTEM OF MACHILIPATNAM, ANDHRAPRADESH, INDIA

Objective: The objective of the present study is to isolate, characterize the potent actinomycete strain from mangrove habitats of South coast of Andhra Pradesh (Machilipatnam), India. Methods: The samples collected from mangrove niches were pretreated with calcium carbonate, diluted, Plated on Starch casein agar, Humic acid vitamin agar and yeast extract malt extract dextrose (YMD) agar media to isolate actinomycetes. The three selective media were supplemented with clotrimazole (25 μg/mL) and streptomycin (25 μg/mL) to inhibit the growth of fungi and bacteria. Results: A total of ten actinobacterial strains were isolated and designated as VLCH-1 to VLCH-10. All of them were screened and evaluated for their antimicrobial and antioxidant properties. Among them, one strain VLCH-6 was found to be potent as it exhibited high antimicrobial potential and it was identified as Pseudonocardia parietis VLCH-6 through polyphasic taxonomy (GenBank accession number: MZ951166). The growth profile of the strain was studied by culturing it in YMD broth. The culture broth extracted with ethyl acetate was tested for antimicrobial assay and found to be active against Escherichia coli, Xanthomonas campestris and Aspergillus niger. The antioxidant potential of the strain VLCH-6 was evaluated by 2,2- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. In the screening, the culture extract exhibited strong antioxidant activity against DPPH free radicals. Conclusion: Over all, it was evident from the present study that mangrove habitats of Machilipatnam serve as a good source for potent actinomycetes possessing broad spectrum of antimicrobial and antioxidant metabolites.

Antibiotic Activity of Actinomycetes Isolated from Young Tectona Grandis (L.) Wood and Pith

Actinomycetes are a source for novel bioactive compounds and justify obtaining new species from various sources. Hardwoods such as Tectona grandis (L.) have not been studied for actinomycete isolation. We aim to isolate endophytic actinomycetes from young Tectona grandis wood and pith and screen for antibiotic activity. Five young wood were cut and surface sterilized using ethanol and hypochlorous acid. The wood and pith of each sample are placed in eight plates of Humic acid-vitamin B (HV), Tap water Yeast extract (TWYE), and Yeast Extract Casein Digest (YECD) medium and incubated at 27°C for four weeks. Actinomycetes were isolated from such medium, observed every week, and transferred to an International Streptomyces Project-2 (ISP-2) medium for identification and antibiotic production tested against Staphylococcus aureus, Helicobacter pylori, and Escherichia coli using the Kirby-Bauer method. Seven actinomycetes were isolated from the wood, primarily from YECD and TWYE media, with varying morphological characteristics. One isolate having maroon-colored aerial and vegetative mycelium with grey spores showed moderate antibacterial activity against S. aureus and H. pylori (13.49±1.03 mm and 14.9±0.7 mm, respectively), while two other actinomycetes showed weak activity against these bacteria. However, none of the actinomycetes show any activity against E. coli. Tectona grandis (L.) is a potential source for novel actinomycetes with an antibiotic activity which warrants further exploration

POTENSI STREPTOMYCES ENDOFIT RIMPANG KENCUR DAN DAUN SIRIH MERAH SEBAGAI ANTITUBERKULOSIS PADA MYCOBACTERIUM TUBERCULOSIS GALUR SENSITIF

The objective of this research was to obtain Endophitic Streptomyces from some medical plants which have antituberculosis effect and also to test its antitubercullosis activity againts sensitive stain of Mycobacterium tuberculosis. Medicinal plants used in this study were Kaemferia galanga, Hibiscus sabdariffa and Piper cronatum. Streptomyces was isolated using two different media namely Humic Acid Vitamin (HV) agar and HV with plant extract, and purified on Yeast Malt Agar (YMA) medium. Morphological characterization of Streptomyces obtained was observed including aerial mycelium and substrate mycelium, pigmentation, and colony surface. A total of 5 endophitic Streptomyces genus were obtained from medicinal plant. Isolates K.J3A, K.J3B, K.J4 and K.J11 from Kaemferia galanga radix, S.MJ1 from Piper cronatum folium. Streptomyces was not found from Hibiscus sabdariffa calyx. Each isolate had variety of characteristics which could be seen in aerial mass color, substrate mass color, and pigmentation. Antitubercullosis activity showed that total of 5 endophitic Streptomyces were not capable to inhibit the growth sensitive stain of M. tubercullosis.Keywods: Antituberculosis, endophitic actinomycetes, Kaemferia galanga, Hibiscus sabdariffa

Antifungal strains and gene mapping of secondary metabolites in mangrove sediments from Semarang city and Karimunjawa islands, Indonesia.

Infection caused by pathogenic fungal species is one of the most challenging disease to be tackled today. The antifungal bacteria candidate can be found in terrestrial as well as aquatic ecosystems, with mangrove forests being one of them. The purpose of this study is to obtain candidate isolates of antifungal strains with a detection approach and gene mapping simulation of bioactive compounds producers and screening to determine qualitative antifungal activity. The research will be carried out by collecting sediment samples from the mangrove ecosystems of Karimunjawa and Mangkang sub-district of Semarang city, isolating and purifying bacteria with Humic Acid Vitamin Agar (HVA), International Streptomyces Project 1 (ISP 1) and Zobell (Marine Agar). added with antibiotics, qualitative antifungal ability screening of each isolate obtained, detection of the presence of PKS gene and NRPS using special primers using the Polymerase Chain Reaction (PCR) method, and molecular identification of each isolate by 16s rRNA sequencing method. Of the total 59 isolates produced from the sample isolation process, 31 isolates from Karimunjawa sediments and 8 isolates from Semarang sediments showed activity against test pathogenic bacteria, namely Candida albicans, Trichoderma sp., and Aspergillus niger. Detection of Biosynthethic Gene Cluster (BGC) showed that the genes encoding secondary metabolites (NRPS, PKS 1 and PKS 2) were detected in KI 2-2 isolates from Karimunjawa. NRPS were detected only in isolates SP 3-9, SH 3-4, KI 1-6, KI 2-2, KI 2-4. The secondary metabolite-encoding gene, PKS1, was detected in isolates SP 3-5, SP 3-8, KI 2-2. PKS II genes were detected only on isolates SP 2-4, SH 3-8, KI 1-6, KI 2-2, and KI 2-4. Isolate SP 3-5 was revealed as Pseudomonas aeruginosa (93.14%), isolate SP 2-4 was Zhouia amylolytica strain HN-181 (100%) and isolate SP 3-8 was P. aeruginosa strain QK -2 (100%).

Isolation and Antimicrobial Screening of Actinomycetes from the Soil of Enggano Island, Indonesia

Actinomycetes are filamentous Gram-positive bacteria that potential sources of antimicrobial substances. The aim of this study was to isolate actinomycetes from different soil types in Enggano Island and screened their antimicrobial activity. The phenol method with 802 medium and humic acid vitamin (HVA) medium was used to isolate the actinomycetes. Furthermore, molecular identifications were performed based on 16s rRNA gene sequence. Antimicrobial activity screening was performed against Bacillus subtilis BTCC B.612, Escherichia coli BTCC B.612, Micrococcus luteus BTCC B.552, Staphylococcus aureus BTCC B.611, and Candida albicans BTCC Y.33 by using the agar plug diffusion method. Fifty-six actinomycetes isolates were obtained from 11 soil types. Most of the isolates were Streptomyces genus with species S. hydrogenans, S. coelicolor, and S. koyangensis, which have similarities in the 16S rRNA gene sequence ranging from 98 -100%. Among isolates screened for their antimicrobial activity, five isolates, i.e. SMP 6, SMP 7b, SMP 18, SHP 9-3, and SHP 22-1a showed a potential activity against test microorganisms selected. All of isolates were more active against C. albicans compared with the tested bacteria. Further study will be conducted to isolate active substances against C. albicans.

Short Communication: Isolation and characterization of thermophilic actinobacteria as proteolytic enzyme producer from Ie Seuum Hot Spring, Aceh Besar, Indonesia

Abstract. Fitri L, Putri KA, Suhartono, Ismail YS. 2019. Short Communication: Isolation and characterization of thermophilic actinobacteria as proteolytic enzyme producer from Ie Seuum Hot Spring, Aceh Besar, Indonesia. Biodiversitas 20: 2802-2808. Proteases are enzymes who catalyze the breakdown of peptide bonds in proteins. This enzyme could be produced from thermophilic bacteria that are able to grow at temperatures of 45-80ºC and are stable to heat. The samples were collected at Ie Seu'um hot spring, Aceh Besar. This study aimed to isolate, to characterize and to determine actinobacteria that were capable of producing protease enzymes. The sampling method in this study was conducted by purposive sampling at temperatures of 50, 60 and 70ºC. Isolation of thermophilic actinobacteria was carried out in Humic Acid Vitamin B (HV) Agar medium and morphological characterization was carried out in Yeast Malt Agar (YMA), Yeast Starch Agar (YSA), Oatmeal Agar (OA) media. Microscopic characterization and measurement of clear zone diameter formed were carried out in skim milk medium. The results showed that one actinobacteria isolate was obtained at a temperature of 50ºC and five isolates at a temperature of 60ºC, meanwhile, no actinobacteria could be obtained at a temperature of 70ºC. A total of 4 isolates obtained were able to produce protease enzymes. The highest Proteolytic Index (IP) value was obtained from IS01 which was 3.8.

Plant Growth Promoting Activity of Actinomycetes Isolated from Soybean Rhizosphere

Plant Growth Promoting Rhizobacteria (PGPR) is considered as the biological agent for improving plant growth. One Group of PGPR that have an important role in growth promoting of plant is Actinomycetes. The objective of this study was to isolate and screen Actinomycetes isolated from soybean rhizosphere as growth promoter of soybean in vitro. Fifty-three Actinomycetes isolates have successfully been isolated from soybean rhizosphere using two media, mainly Humic acid Vitamin Agar (HVA) and starch casein agar (SCA). Among 53 isolates, 18 (34%) isolates were able to produce IAA in range of 2.08 ppm to 16.70 ppm. Growth promotion test of soybean in vitro using Ragdoll method resulted 7 Actinomycetes isolates that significantly enhanced 3 plant growth parameters, including hypocotyl and radicular length as well as the number of lateral roots. Of those 7 isolates of Actinomycetes, 5 isolates were able to grow on nitrogen-free medium and solubilize phosphate. Those 5 isolates also were found as non-pathogenic, based on the negative reaction in hypersensitivity test. Based on 16S rRNA sequence analysis, 5 selected Actinomycetes isolates were highly homolog with Streptomyces genera in different taxa of species and strains (similarity ≥99%). Our finding reveals a potent application of 5 Actinomycetes isolates as plant growth promoter in soybean agriculture.

Isolation of actinomycetes from maize rhizosphere from Kupang, East Nusa Tenggara Province, and evaluation of their antibacterial, antifungal, and extracellular enzyme activity

Actinomycetes are the one of the components of the rhizospheric microbial population and useful for producing secondary metabolites such as lytic enzymes, antibiotics, and antifungal. The aim of the study was to isolate the actinomycetes from maize rhizosphere collected from Kupang, East Nusa Tenggara. The screening was focused on the actinomycetes that showed the ability to produce antibacterial, antifungal, and extracellular enzymes such as amylase, cellulase, and protease. The actinomycetes were isolated using Humic-Acid Vitamin B (HV) agar media. The antagonistic assay was tested against Escherichia coli, Staphylococcus aureus, Sclerotium rolfsii and Fusarium oxysporum. Isolate JKP-8 was an isolate that showed the highest activity in inhibiting the growth of E. coli and S. aureus bacteria. Isolate JKP-5 showed the highest activity in inhibiting the growth of F.oxysporum. There were no actinomycetes isolates that showed an ability to inhibit the growth of S. rolfsii fungus based on dual culture assay. JKP-3 and JKP-4 isolates exhibited the highest ability to hydrolyze amylum, while JKP-5 and JKP-8 isolates exhibited the highest ability to hydrolyze CMC. The results of the amplification of 16S rRNA gene in selected potential isolates JKP 5 and JKP 8 indicated that both isolates belong to the genus Streptomyces.

Comparison of Various Methods for Isolation of Nocardia from Soil

Background: The genus Nocardia is Gram-positive, aerobic filamentous bacilli and saprophytic micro-organisms that can be isolated from freshwater, salt water, dust and decaying vegetation especially the soil. This study aimed to investigate the several media for to determine a suitable culture media with the ability to better for the isolation of Nocardia from soil. Methods: In this study, 400 soil samples were collected from different areas from Iran. The soil samples were then cultured on the four culture media such as Humic acid vitamin B agar, Paraffin agar, Sabouraud dextrose agar supplemented whit cycloheximide and carbon-free broth containing paraffin rods and incubated at 35°C. All of culture media investigated every 3 days for a month. Colonies suspicious to Nocardia were stained with Gram-stain, acid-fast and partially acid-fast and evaluated for resistance to lysozyme. Results: From 400 soil samples, the number of 62, 10, 28 and 19 strains of Nocardia were isolated by paraffin rods, Humic Acid Vitamin B agar, Paraffin agar and Sabouraud dextrose agar whit cycloheximide, respectively. Most Nocardia strains were isolated using paraffin bait technique. Conclusions: Isolation of Nocardia spp. is enhanced by using the paraffin baiting technique that relies on the selective ability of this micro-organism to metabolize paraffin.

Identification and Antimicrobial Activity of Marine Streptomyces from Geographically Different Regions of Indonesia

Identification and antimicrobial assay of indegenous marine Streptomyces have been conducted. Samples were obtained from culture collection of Biotech Center, Agency for the Assessment and Application of Technology (BPPT). They were originated from several Indonesian seashores including Pelabuhan Ratu, Pangandaran, Manokwari, Pulau Seribu, Garut, Bangka, Banjarmasin, Belitung, Cirebon, and Palu. Isolates stored as glycerol stocks were inoculated onto HV (Humic acid vitamin) agar and incubated for 5 days at 30 °C. Each colony was cultivated using yeast-peptone medium and then extracted by butanol. Antimicrobial activities were monitored by the agar diffusion paper-disc method against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 66923, Pseudomonas aeruginosa ATCC 27853, Candida albicans BIOMCC 00122 and Aspergillus niger BIOMCC 0013. Molecular identification of Streptomyces was carried out based on 16S rRNA gene analysis. Our research results showed that 71 isolates obtained from several Indonesian seashore were identified as 57 different Streptomyces species. Fifty of them showed antimicrobial activity. Twenty three isolates inhibited B. subtilis ATCC 66923, 14 isolates inhibited S. aureus ATCC 25923, 24 isolates inhibited C. albicans BIOMCC 00122 and 26 isolates inhibited A. niger BIOMCC 0013 and there was no active isolates inhibited the growth of E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Phylogenetic analysis showed that Streptomyces isolates originated from the same geographically region was not necessarily grouped into the same cluster. Likewise a phylogenetic cluster may contain isolates of the same Streptomyces species, but from geographically different locations.

Actinomycetes from solitary wasp mud nest and swallow bird mud nest: isolation and screening for their antibacterial activity

The aim of this study was to isolate and screen actinomycetes from solitary wasp and swallow bird mud nests for antimicrobial activity. The actinomycetes were isolated from soil of nests of solitary wasp and swallow bird, and identified on the basis of morphological characteristics and molecular biological methods. A total of 109 actinomycetal isolates were obtained from 12 soil samples (6 from each habitat) using two media. The highest number of actinomycetes were recovered on Humic acid vitamin agar media (65.13%, n = 71) as compared to actinomycetes isolation agar media (34.86%, n = 38). The antimicrobial activity of actinomycetes isolates was determined using the agar plug method. Among 109 isolates, 51 isolates (46.78%) showed antibacterial activity by agar plug assay. The morphological and molecular characteristics confirmed that the most of active isolates in both sample belonged to the genus Streptomyces, the other potential genera like Streptosporangium, Actinomadura, Saccharopolyspora, Thermoactinomycetes and Nocardia were also recovered, but in a low frequency. The isolates designated as 8(1), BN-6, MN 2(6), MN 2(7) and MN 9(V) showed most promising activity against various drug resistant bacterial pathogens. It seems that the promising isolates from these unusual/unexplored habitats may prove to be an important step in development of drug for treating multi-drug resistant bacterial pathogens.

Isolation and characterization of potent antifungal strains of the Streptomyces violaceusniger clade active against Candida albicans

Streptomyces strains were isolated from a sagebrush rhizosphere soil sample on humic acid vitamin (HV) agar and water yeast extract (WYE) agar supplemented with 1.5% (w/w) phenol as a selective medium. Acidic, neutral and alkaline pH conditions were also used in the isolation procedures. The phenol treatment reduced the numbers of both actinomycetes and non-actinomycetes on plates under all three pH conditions. From phenol-amended HV and WYE agar, 16 strains were isolated in pure culture; 14 from the HV agar and two from the WYE agar. All the isolates were tested for their antifungal activities against Pythium ultimum P8 and five yeast strains, including two antifungal drug-resistant Candida albicans strains. HV isolates that showed broad-spectrum antifungal antibiotic activities were all found to be members of the Streptomyces violaceusniger clade, while those that did not were non-clade members. The phenol treatment was not selective for S. violaceusniger clade members. Therefore, we tested the spores of both S. violaceusniger clade and non-clade members using two biocides, phenol and hydrogen peroxide, as selection agents. Spores of non-clade members, such as S. coelicolor M145 and S. lividans TK 21, survived these two biocides just as well as S. violaceusniger clade members. Thus, in our hands, biocide resistance was not S. violaceusniger clade specific as previously reported. However, isolates showing broad-spectrum antifungal and antiyeast activity were all members of the clade. We conclude that screening of isolates for broad-spectrum antifungal/antiyeast activity is the preferred method of isolating S. violaceusniger clade strains rather than biocide-based selection. Phylogenetic analysis of the phenol-resistant isolates revealed that the HV isolates that exhibited broad-spectrum antifungal antibiotic activity were all clustered and closely related to the S. violaceusniger clade, while the isolates that did not exhibit antifungal antibiotic activity were all non-clade members.

A New Enrichment Method for the Selective Isolation of Streptomycetes from the Root Surfaces of Herbaceous Plants

Diverse rhizoplane streptomycetes with high levels of anti-phytopathogenic activity were efficiently isolated from healthy herbaceous plants using a new enrichment method, which we designated as the moist incubation and desiccation (MI&D) method. The MI&D method involves incubating root tissues on humic acid-vitamin (HV) agar, desiccating roots bearing arthrospore chains of colonized streptomycetes in dried soil particles, then liberating spores by agitation in water. Dilutions of the liquid enriched with streptomycete spores are then plated and incubated on HV agar. The desiccation stage drastically reduces bacterial contamination, thereby achieving selective isolation of streptomycetes.

Selective isolation of bioactive soil actinomycetes belonging to the Streptomyces violaceusniger phenotypic cluster.

To devise and evaluate a strategy for isolating members of the Streptomyces violaceusniger phenotypic cluster, which are known to be a promising source of bioactive metabolites. The treatment of four soil samples with 1.5% phenol (30 degrees C, 30 min) prior to inoculation on humic acid-vitamin (HV) agar eliminated most of the streptomycetes and other bacterial populations. The surviving streptomycetes on the HV isolation plates were subcultured, and species-group identification was made according to the probabilistic identification system of Williams et al. (1989). Of the 133 streptomycetes subcultured, 102 (77%), were assigned to the S. violaceusniger cluster. A test with an overlay technique revealed that all of these S. violaceusniger-cluster isolates had broad antimicrobial spectra, as they inhibited the growth of all test Gram-positive bacteria, yeasts and filamentous fungi. Antitumour activity against colon carcinoma cells was found among 68 or 67%, of these S. violaceusniger-cluster isolates, following growth in submerged culture. Chemical pretreatment of soil samples with phenol reduces the growth of ubiquitous Streptomyces species, thereby facilitating the recovery of less-abundant S. violaceusniger-cluster strains that are characterized by high antimicrobial and antitumour activities. The development and application of new methodologies with which to selectively isolate rare, bioactive streptomycete groups is important for discovering novel secondary metabolites with bioactive properties.

Isolation of endophytic actinomycetes from selected plants and their antifungal activity

The isolation of endophytic actinomycetes from surface-sterilized tissues of 36 plant species was made using humic acid–vitamin (HV) agar as a selection medium. Of the 330 isolates recovered, 212 were from roots, 97 from leaves and 21 isolates from stems with a prevalence of 3.9, 1.7 and 0.3%, respectively. Identification of endophytic actinomycetes was based on their morphology and the amino acid composition of the whole-cell extract. Most isolates were classified as Streptomyces sp. (n = 277); with the remainder belonging to Microbispora sp. (n = 14), Nocardia sp. (n = 8) and Micromonospora sp. (n = 4). Four isolates were unclassified and 23 were lost during subculture. The most prevalent group of isolates were the Streptomyces sp. occurring in 6.4% of the tissue samples of Zingiber officinale. Scanning electron microscopy investigation of this plant revealed that 7.5% of the root and 5% of the leaf samples contained endophytes. Three of the Streptomyces sp. isolates strongly inhibited Colletotrichum musae, five were very active against Fusarium oxysporum and two strongly inhibited growth of both test fungi.

Selective isolation and study on the global distribution of the genus Planobispora in soils

Planobispora strains, including the type strain, produced abundant sporangia on humic acid – vitamin gellan gum medium when more than 50 colonies grew on a plate. However, these strains did not produce sporangia on the aerial mycelium, a diagnostic characteristic of this genus, when fewer than 20 colonies grew on a plate. Trace salts, such as FeSO4·7H2O, MnCl2·4H2O, ZnSO4·7H2O, and NiSO4·6H2O, stimulated sporangium formation of Planobispora strains. Better sporangium formation of Planobispora strains was observed on the humic acid – trace salts gellan gum medium at pH 9.0 than at neutral pH. Moreover, an alkaline medium repressed the growth of three out of six Streptomyces strains so that this condition was effective for selective isolation of Planobispora strains. An alkaline flooding solution of skim milk, five antimicrobial agents, and dry heat treatment at 90°C for 60 min were effective for selective isolation. Using these techniques, we examined the distribution of Planobispora strains by using 1467 soil samples collected from Japan and other parts of the world. One hundred and nineteen Planobispora strains were isolated from 51 soil samples (3.5% of the samples tested) that were collected in Ecuador, Egypt, French Guiana, India, and Madagascar. Planobispora strains were recovered only in tropical and subtropical soils. To our knowledge, this is the first record that Planobispora strains have been isolated from locations other than Venezuela, Namibia, or South Africa.Key words: Planobispora, gellan gum, selective isolation, actinomycete, distribution.

An integrated method for the enrichment and selective isolation of Actinokineospora spp. in soil and plant litter.

To devise and evaluate a method for isolating the rare, zoosporic actinomycetes, Actinokineospora spp. in soil and plant litter. The newly developed method consists of two enrichment stages followed by plating on a selective medium. The source material is initially incubated with calcium carbonate to multiply the population of Actinokineospora spp., and is then air-dried. The second stage consists of rehydration-centrifugation, in which the amended substrate is immersed in phosphate buffer-soil extract to liberate actinomycete zoospores, and nonmotile microbial associates are then eliminated by centrifugation. Portions of the supernatant enriched with zoospores are plated on humic-acid vitamin agar supplemented with fradiomycin, kanamycin, nalidixic acid and trimethoprim. We examined 39 soil and plant-litter samples taken from fields, forests and stream banks. The proposed method consistently enriched and selectively isolated Actinokineospora spp. in 17 samples. Evidence for antimicrobial activity was found in most of the isolates. A combination of enrichment and a medium containing selective antibiotics can be used successfully for efficient isolation of certain rare actinomycete taxa. The development of new methodologies with which to isolate rare actinomycetes is of great importance to extend our understanding of their ecology, taxonomy and bioactivity.

Combined Use of Trimethoprim with Nalidixic Acid for the Selective Isolation and Enumeration of Actinomycetes from Soil.

The use of synthetic antibacterial compounds as selective supplements in agar media for the isolation and enumeration of soil actinomycetes is described. A preliminary survey revealed that the combined application of trimethoprim (TP) and nalidixic acid (NA) had no deleterious effect on most test actinomycetes (35 genera and 159 species), while the growth of most test non-filamentous soil bacteria (10 genera and 20 species) was inhibited or discouraged. A mixture of TP (20 mg/l) and NA (10 mg/l) was incorporated into humic acid-vitamin (HV) agar and four other isolation media. The efficiency of isolating and enumerating actinomycetes cultivated under these conditions was evaluated using 13 different natural soil samples. The TP-NA combination substantially reduced the occurrence of competing non-filamentous bacteria on the isolation plates without adversely affecting actinomycete growth, thus greatly facilitating the recognition, enumeration and pure isolation of actinomycete colonies. A detailed taxonomic examination showed that the antimicrobial combination allowed the recovery of varieties of actinomycete genera. At least 11 different genera were isolated from a garden soil sample on HV plates containing TP and NA. The most frequently isolated Streptomyces strains were further categorized into various species-groups. The results suggest that the TP-NA combination may prove to be a useful tool for autecological and enumeration studies of actinomycetes, and for the isolation of wide varieties of actinomycete strains.