Soil Extract Agar Research Articles

Optimization of Screening Media to Improve Antimicrobial Biodiscovery from Soils in Undergraduate/Citizen Science Research-Engaged Initiatives.

Background/Objectives: Research-engaged academic institutions offer the opportunity to couple undergraduate education/citizen science projects with antimicrobial biodiscovery research. Several initiatives reflecting this ethos have been reported internationally (e.g., Small World, Tiny Earth, MicroMundo, Antibiotics Unearthed). These programs target soil habitats due to their high microbial diversity and promote initial screening with non-selective, nutrient media such as tryptic soy agar (TSA). However, evaluation of published outputs to date indicates that isolate recovery on TSA is consistently dominated by the genera Bacillus, Pseudomonas, and Paenibacillus. In this study, we evaluated the potential of soil extract agar to enhance soil isolate diversity and antibiosis induction outcomes in our undergraduate Antibiotics Unearthed research program. Methods: We comparatively screened 229 isolates from woodland and garden soil samples on both tryptic soy agar (TSA) and soil extract agar (SEA) for antimicrobial activity against a panel of clinically relevant microbial pathogens. Results: On one or both media, 15 isolates were found to produce zones of clearing against respective pathogens. 16S rRNA gene sequencing linked the isolates with three genera: Streptomyces (7), Paenibacillus (6), and Pseudomonas (2). Six of the Streptomyces isolates and one Pseudomonas demonstrated antimicrobial activity when screened on SEA, with no activity on TSA. Furthermore, incorporation of the known secondary metabolite inducer N acetyl-glucosamine (20 mM) into SEA media altered the pathogen inhibition profiles of 14 isolates and resulted in broad-spectrum activity of one Streptomyces isolate, not observed on SEA alone. In conclusion, SEA was found to expand the diversity of culturable isolates from soil and specifically enhanced the recovery of members of the genus Streptomyces. SEA was also found to be a superior media for antibiosis induction among Streptomyces isolates when compared to TSA. It was noted that Paenibacillus isolates' antibiosis induction demonstrated a strain-specific response with respect to the growth media used. Conclusions: The authors propose SEA inclusion of in soil screening protocols as a cost-effective, complementary strategy to greatly enhance outcomes in undergraduate/citizen science-engaged antimicrobial biodiscovery initiatives.

Elucidating the impact of environmental factors on the growth of Colletotrichum coccodes strains isolated from potato tubers in Great Britain

Black dot and silver scurf caused by Colletotrichum coccodes and Helminthosporium solani, respectively, are tuber blemish diseases affecting quality in the fresh and pre-pack potato industry. In the last 20 years, the importance of high-quality tuber appearance has increased considerably due to the growing demand for washed and pre-packed potatoes in the UK. Changing climate characterised by rising temperatures and wetter summers is a threat as this will favour the development of pathogens such as C. coccodes in the soil increasing the risk of food spoilage. Moreover, both diseases can develop not only in the field but also after harvest, with postharvest storage temperatures being a crucial factor in controlling fungal growth. Furthermore, anecdotal evidence showed differences on the aggressiveness of black dot depending on its origin (i.e. England and Scotland) on potato tubers. Silver scurf and black dot are difficult to differentiate as they present similar phenotypes characterised by silvery lesions making it challenging for managers to take the necessary corrective action during storage. Hence, the aim of this study was to give a general insight into the ecological conditions affecting the establishment of the causal agent of potato black dot in the field, and black dot and silver scurf during the supply chain. Therefore, invitro experiments were designed to study the growth rate and lag times simulating both scenarios respectively: on soil extract agar (SEA) media at different temperatures (4, 11, 15 °C) and matric potentials (control [unmodified] and − 1.4 MPa [modified]); and on natural potato dextrose agar (NPDA) for different temperatures (4, 11, 15 and 20 °C) at 99 % relative humidity (RH) for 25 days. When simulating the field environment, drier conditions (matric potential = −1.4 MPa) reduced fungal growth for both isolates by 0.1 cm day−1 at the temperature of 15 °C, suggesting temperature as the main limiting factor for the growth of C. coccodes in the soil. The causal agent of black dot exhibited a faster growth rate under retailer-like conditions (i.e., 15 °C) compared to H. solani. Understanding the environmental influence on both the pathogen and the crop is vital for proper disease management to help reduce food loss and waste.

Comparison of growth, viability, sporulation, and virulence of Phytophthora capsici isolated from black pepper (Piper nigrum L.) on various media.

Black pepper (Piper nigrum L.) is an important agricultural crop in Indonesia. However, black pepper production in Indonesia is limited by plant pathogen infection, especially Phytophthora capsici, which causes Foot Rot in pepper plants. This pathogen contributes to a high decline in black pepper productivity. This study aims to determine the proper media composition for P. capsici growth based on the mycelial growth, viability, sporulation, and virulence of P. capsici on pepper leaves. The media used in this study were potato dextrose agar (PDA), martin agar (MA), carrot agar (CA), water agar (WA), corn meal agar (CMA), soil extract agar (SEA), and vegetable 8 (V8). The results showed that CMA media was the most optimal medium for P. capsici growth, as indicated by the size of the mycelium with a diameter of 8.00 cm on the fifth day after inoculation. The viability of mycelium of P. capsici was best found on PDA, CMA, and V8 media. Meanwhile, the optimum sporulation and virulence of P. capsici occurred on SEA media, characterized by zoospore production of 5.42x106/ml and symptom areas on leaves of 18.61 cm2 and 16.69 cm2.

Survey of Rice Genotypes of Southern India for Seed Zinc Concentration to Explore its Seed Endophytic Microbial Diversity

Background: Rice is a vital staple food for a large part of the global population and zinc deficiency poses health risks, particularly in rice-dependent regions. Studying genotypic differences in rice seed zinc content is vital for breeding zinc enriched varieties to address malnutrition and enhance food security. In the plant microbe partnership exploring the role of seed bacterial endophytes in influencing the plant physiology holds promise for Zn accumulation contributing to the broader goals of enhancing food quality, nutrition and crop production in a world facing increasing agricultural demands and challenges. Methods: Rice genotypes were surveyed and their seed zinc content was analysed using non-destructive X-ray fluorescence spectrometry. Surface sterilization of rice seed samples was performed to isolate endophytic bacteria, involving a series of treatments with distilled water, ethanol and sodium hypochlorite to remove surface microflora. The sterilized seeds were then macerated and diluted to obtain a countable number of colonies, which were subsequently spread-plated on nutrient agar, tryptic soy agar and soil extract agar to enumerate the different nutritional types of seed endophytic microflora. Result: In this study, 34 different rice genotypes, comprising traditional landraces and modern cultivars, were collected and analysed for their inherent zinc content. One variety in each category of low, medium and high seed Zn content was selected. The occurrence of all nutritional types of bacterial and their population was highest in the genotype Karuppunel followed by CO51 and ADT 39. Nutrient agar was found to promote a higher count of culturable bacterial endophytes compared to Tryptic soy agar and Soil extract agar.

Isolation and screening of Thermoactinomycetaceae family members as an extremophilic poor investigated and promising natural source of antimicrobial substances.

Recent evidences have shown that methicillin-resistant Staphylococcus aureus (MRSA) can cause severe infections and is resistant to almost all commercially available antibiotics. Therefore, screening unknown sources of biological compounds such as the Thermoactinomycetaceae family as extremophilic bacteria may be helpful to find new antimicrobial agents. Various samples were collected from different ecosystems, including desert, volcano, compost, and forest. They were cultured on Soil extract agar and Water agar. The antimicrobial activity of the isolates was evaluated using agar overlay and well diffusion methods. Members of the Thermoactinomycetaceae family were selected for further study: Their ability to grow at different temperatures, NaCl concentrations, and pH values, enzyme production ability, antimicrobial secondary screening, fractionation of their supernatants and so on. According to molecular identification of active isolates against MRSA, three strains, including Laceyella sacchari UTMC 2705, Thermoactinomyces sp. UTMC 2721, and Laceyella sp. UTMC 2731, belonged to Thermoactinomycetaceae were identified. The minimum inhibitory concentrations of their extracts were tested against some pathogenic bacteria, showing their antimicrobial activity with a broad spectrum. The results of TLC bioautography of the extracts showed that the most active fractions were semi-polar. Also, the results of HPLC analysis showed the existence of several UV-active compounds in their extracts. The present study highlighted the importance and potential of Thermoactinomycetaceae members as a less-known source of antibiotics against pathogenic bacteria.

Isolation and Characterization of Effective Microorganism from Oil Palm Rhizhopheric Soil and Evaluation of Their Potential as Biofertiliser

Plant nutrients are vital components of sustainable agriculture. Soil microorganisms play a significant role in regulating dynamics of organic matter decomposition and availability of plant nutrients such as nitrogen (N), phosphate (P) and sulphur (S), as well as stimulating plant growth. Soil microbial inoculants associated with rhizosphere region of plants work as biofertilisers. In this study, beneficial bacteria were isolated from rhizospheric soils collected from an oil palm plantation in Temerloh, Pahang, Malaysia. Rhizospheric soils (n = 6) were sampled from oil palm plants with heavy production of fresh fruit bunches, due to supplementation of biofertiliser. Isolation of bacteria was carried out on soil extract agar using a dilution plate method. Total bacterial count from isolation plates was in the range of 6.0 × 107 to 7.3 × 107 cfu per gram of soil. All bacterial isolates (n = 150) were purified and characterised for production of plant beneficial enzymes and hormones such as indole-3-acetic acid (IAA), siderophore, nitrogenase and phosphatase. Two bacterial isolates that were positive in phosphate solubilising assay, nitrogen fixers and IAA producers assay were identified as Brevibacillus sp., and Burkholderia sp. respectively, based on 16S rRNA gene sequences.

Soil Microorganisms Quantitative Dynamic Characterizing the Overall Biological State in Rhizosphere of Tobacco and Tomato Plants Infected by Broomrape

The quantitative dynamics of soil microorganisms in the rhizosphere of tobacco and tomato plants infected by broomrape /Phelipanche ramosa L./ were investigated. Parallel pot experiments with the two hosts of the parasite - Oriental type of tobacco and tomato were presented. The variants were: by introducing contamination into the soil - seeds by broomrape and control pots - without the seeds by broomrape. Experiments were carried out under controlled conditions. Soil samples for microbiological analyzes were taken from the rhizosphere, in dynamics. The microbiological analyses were executed according to Koh’s, in three replications (MPN/g a.d.s), with confidence level 0.05. The surveyed set of soil microorganisms has been comprised two indicators groups characterizing the overall biological state of the microbial communities: Autochthonous (on soil extract agar) and Oligotrophic (on diluted soil extract agar). Diversity indexes Shannon (H) and Simpson (D) and the distribution evenness (EH) in the microbial communities were determined. A statistical analysis has been made. The obtained results showed that the quantities of the two groups of microorganisms begun to change visibly around and after 20th day, compared to the amount at the rhizosphere of uninfected by broomrape plants. There was an increase at the population density of autochthonous microorganisms between 20 and 60 days and reduced significantly after this period. The trend was reversed at oligotrophic microorganisms, but the quantities were higher throughout the study period. These dynamics coincide with the phases of broomrape development - germination, formation of a haustorium (about 10-20th day) and attachment to the root of the host (up to about 40-60 days) and the above-ground phases (80-100 days). The analysis showed that microbial communities formed in rhizospheres in the presence of a broomrape had been an oligotrophic character. The relative share of oligotrophic microorganisms in these microbial communities was over 50% at both host rhizospheres.

Biodegradation of chlorinated organic pesticides endosulfan and chlorpyrifos in soil extract broth using fungi

AbstractThe present study was designed to screen 20 fungi for their potential to degrade the chlorinated organic pesticides endosulfan and chlorpyrifos. Fungi were first screened for their tolerance to various concentrations of target pesticides using soil extract agar and subsequent degradation studies were performed in soil extract broth containing 25 mg/L of the individual pesticide. Pesticide degradation was evaluated using gas chromatography. Other parameters, such as pH and mycelial weight, were also determined. Based on percent growth inhibition of test fungi and subsequent analysis of EC50values, the overall results revealed that chlorpyrifos showed significantly more growth inhibition in all tested fungi compared with endosulfan.Trametes hirsutashowed complete degradation of both α‐ and β‐endosulfan isomers andCladosporium cladosporioidesdisplayed maximum degradation of chlorpyrifos. All test fungi degraded endosulfan more efficiently than chlorpyrifos, exceptPhanerochaete chrysosporium, Trichoderma harzianum, andTrichoderma virenswhich showed higher degradation of chlorpyrifos than endosulfan. It was also found that all tested fungi degraded α‐endosulfan more efficiently than β‐endosulfan. Endosulfan sulfate was found to be the major degradation product with all tested fungi. Fungi which showed more endosulfan degradation also produced more endosulfan sulfate. However, less endosulfan sulfate was detected withT. hirsutaandTrametes versicolor, although they degraded endosulfan more efficiently.

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects.

Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition.

Effect of nutrient status, temperature and PH on mycelial growth, sclerotial production and germination of Rhizoctonia Solani Isolated from paddy fields

Rhizoctonia solani Kuhn, is one of the most important pathogen of rice causing sheath blight disease, which accounts for heavy yield losses in all the major rice growing areas of the world including India. In the present investigation the effect of different culture media, carbon and nitrogen sources, temperature and pH on mycelial growth, sclerotia production and germination of Rhizoctonia solani (anastomosis groups AG1-IA from rice and AG3 from potato) were studied. Among the five culture media including potato dextrose agar (PDA), malt yeast extract agar (MYA), water agar (WA), oat meal agar (OMA) and soil extract agar (SEA), PDA medium supported maximum mycelial growth and sclerotium yield. The maximum mycelial growth and sclerotia formation was observed in D-glucose as carbon source while the maximum mycelial growth and sclerotia formation was recorded in potassium nitrate as nitrogen source. Different range of temperatures i.e. 10, 15, 20, 25, 30 and 35°C were tested and the maximum hyphal growth was measured at 25°C temperature. Mycelial growth of all isolates occurred between pH 4.0 and 9.0, with an optimum of pH 5.6. Sclerotium germination for all the AG1-IA isolates was optimum between 20 and 30°C on all the media tested, with a pH optimum between pH 5.0 and 6.0.

Transcriptional Responses of the Bacterium Burkholderia terrae BS001 to the Fungal Host Lyophyllum sp. Strain Karsten under Soil-Mimicking Conditions

In this study, the mycosphere isolate Burkholderia terrae BS001 was confronted with the soil fungus Lyophyllum sp. strain Karsten on soil extract agar plates in order to examine its transcriptional responses over time. At the initial stages of the experiment (T1—day 3; T2—day 5), contact between both partner organisms was absent, whereas in the final stage (T3—day 8), the two populations made intimate physical contact. Overall, a strong modulation of the strain BS001 gene expression patterns was found. First, the stationary-phase sigma factor RpoS, and numerous genes under its control, were strongly expressed as a response to the soil extract agar, and this extended over the whole temporal regime. In the system, B. terrae BS001 apparently perceived the presence of the fungal hyphae already at the early experimental stages (T1, T2), by strongly upregulating a suite of chemotaxis and flagellar motility genes. With respect to specific metabolism and energy generation, a picture of differential involvement in different metabolic routes was obtained. Initial (T1, T2) up- or downregulation of ethanolamine and mandelate uptake and utilization pathways was substituted by a strong investment, in the presence of the fungus, in the expression of putative metabolic gene clusters (T3). Specifically at T3, five clustered genes that are potentially involved in energy generation coupled to an oxidative stress response, and two genes encoding short-chain dehydrogenases/oxidoreductases (SDR), were highly upregulated. In contrast, the dnaE2 gene (related to general stress response; encoding error-prone DNA polymerase) was transcriptionally downregulated at this stage. This study revealed that B. terrae BS001, from a stress-induced state, resulting from the soil extract agar milieu, responds positively to fungal hyphae that encroach upon it, in a temporally dynamic manner. The response is characterized by phases in which the modulation of (1) chemotaxis, (2) metabolic activity, and (3) oxidative stress responses are key mechanisms.

Chemotaxis and adherence to fungal surfaces are key components of the behavioral response of Burkholderia terrae BS001 to two selected soil fungi.

Burkholderia terrae BS001 has previously been proposed to be a 'generalist' associate of soil fungi, but its strategies of interaction have been largely ignored. Here, we studied the chemotactic behavior of B. terrae BS001 towards Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 and the role of fungal surface molecules in their physical interaction with the bacteria. To assess the involvement of the type 3 secretion system (T3SS), wild-type strain BS001 and T3SS mutant strain BS001-ΔsctD were used in the experiments. First, the two fungi showed divergent behavior when confronted with B. terrae BS001 on soil extract agar medium. Lyophyllum sp. strain Karsten revealed slow growth towards the bacterium, whereas T. asperellum 302 grew avidly over it. Both on soil extract and M9 agar, B. terrae BS001 and BS001-ΔsctD moved chemotactically towards the hyphae of both fungi, with a stronger response to Lyophyllum sp. strain Karsten than to T. asperellum 302. The presence of a progressively increasing glycerol level in the M9 agar enhanced the level of movement. Different oxalic acid concentrations exerted varied effects, with a significantly raised chemotactic response at lower, and a subdued response at higher concentrations. Testing of the adherence of B. terrae BS001 and BS001-ΔsctD to Lyophyllum sp. strain Karsten and to cell envelope-extracted ceramide monohexosides (CMHs) revealed that CMHs in both conidia and hyphae could bind strain BS001 cells. As BS001-ΔsctD adhered significantly less to the CMHs than BS001, the T3SS was presumed to have a role in the interaction. In contrast, such avid adherence was not detected with T. asperellum 302. Thus, B. terrae BS001 shows a behavior characterized by swimming towards Lyophyllum sp. strain Karsten and T. asperellum 302 and attachment to the CMH moiety in the cell envelope, in particular of the former.

Effect of Growth Media and Incubation Time on the Culturability of Soil Bacteria

The effect of growth medium and incubation time were investigated by determining the number of colonies that were visible on triplicate plates of three different media on daily intervals. The viable counts were significantly different between the three different media even after the first day and this continued for seven days. Colony counts on the three media reached their maximum on the third day of incubation. The use of Nutrient agar (NA) and Plate count agar (PCA) resulted in increasing counts with increasing incubation time respectively. Counts obtained from the Nutrient agar were higher than that of Plate count agar followed by the soil extract agar (SEA) which had the lowest count and a longer bacteria lag phase due to the low nutrient content in the soil extract agar. The Plate count agar formed more distinct colonies than the Nutrient agar and Soil extract agar. During the incubation period, colonies appeared on the Nutrient agar even on the seventh day. These characteristics of the Nutrient agar to support a higher number of colonies for a long period of time could be attributed to its nutrient composition. The colony counts upon incubation for seven days ranged from a mean of 3.0x10 5 cfu/g on Soil extract agar, 6.1x10 5 cfu/g on Plate count agar and 7.6x10 5 cfu/g on Nutrient agar for example A which was a cultivated loamy soil and 1.3x10 5 cfu/g also on Soil extract agar, 3.9x10 5 cfu/g on Plate agar and 5.6x10 5 cfu/g Nutrient agar for soil sample B which was a non-cultivated sandy soil. Longer incubation time increased colony count on the different growth media because it was so obvious on the Soil extract agar. No visible colonies were observed from the first to the third day on the Soil extract agar, visible colonies appeared on the fourth day of incubation. Also in the other growth media, there was an increase in colony count on the third day upon incubation.

Increased culturability of soil bacteria from Marcellus shale temperate forest in Pennsylvania

Soil bacteria comprise a largely untapped resource with only 1-10% of bacterial species predicted to live in soil being culturable in the laboratory. Establishing culture-dependent protocols that identify unique operational taxonomic units (OTUs) is an important research topic in soil bacterial ecology. The culturability of soil bacteria may be improved by employing different culture media due to inherent preferences of growth substrate utilization. Soil-extract agar, R-2A agar, and 1% nutrient agar were used in this study to isolate bacteria obtained from soil samples collected in winter months to increase the understanding of bacterial diversity in Abernathy Field Station, a Marcellus shale temperate forest in Washington, Pennsylvania. Changes in bacterial diversity can be used to assess the early impact of anthropological factors, such as hydraulic fracturing in the Marcellus shale region, which may lead to severe environmental problems. For the purpose of long term ecological monitoring, data obtained from this year’s sample collection were analyzed in conjunction with previous years’ assessments. Bacterial isolates were analyzed taxonomically and phylogenetically. Unique OTUs were identified through comparative analysis of 16S rDNA. The Shannon-Weaver and Simpson’s diversity indices ranked isolates on soil-extract agar highest for species richness, and rarefaction analysis suggests that sampling saturation of OTUs identified on soil-extract agar has not yet been reached. Each culture medium studied supported isolates of four common phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Soil-extract agar supported the greatest proportion of pigmented colonies including a Cyanobacterium which exhibited intra-16S rDNA polymorphism. Each culture medium supported the growth of unique OTUs and genera with Bacillus, Flavobacterium, Pseudomonas, Rhizobium, and Streptomyces found on each. This study suggests that utilizing different culture media can increase the culturability of soil bacteria.

Studies on salinization in Haryana soils on free‐living nitrogen‐fixing bacterial populations and their activity

A total of 26 soil samples from saline soils of Haryana were collected. Based on their electrical conductivity (EC) values, which varied from 1.04 to 21.00 dS m(-1), the soils were categorized into non-saline soils (EC 0-2 dS m(-1)), weakly saline soils (EC 2-4 dS m(-1)), saline soils (EC 4-8 dS m(-1)), strongly saline soils (EC 8-16 dS m(-1)), and very strongly saline soils (EC >16 dS m(-1)). The pH values of these soil samples ranged from 6.03 to 8.62, while organic C, total N, and available P were in the range of 0.06-0.94%, 0.07-0.15%, and 0.11-0.29 μg g(-1) soil, respectively. As a measure of the impact of salinity on free-living N(2) fixers and their activity, the total bacterial populations on four media (Jensen's nitrogen-free medium, malate medium, Burk's medium, and soil extract agar medium) decreased from 6.12 to 3.70 log CFU g(-1) soil with increasing salinity level. PCR amplification of the nifH region of the DNA from 234 selected morphotypes from all the media showed the presence of nifH in 71 isolates. Out of these, 37% of the isolates were obtained using Jensen's medium; 35, 28, and 21% of the isolates were obtained using soil extract medium, Burk's medium, and malate medium, respectively. The majority of the free-living N(2) fixers (67%) were Gram negative. Apart from the acetylene reduction assay (ARA) activity in these isolates, other beneficial traits like ammonia excretion and indole acetic acid (IAA) production were also present. A decreasing trend in the activities was observed with increasing salinity levels. Isolates JN6, BP8, and MJ4 showed the highest ARA activity, ammonia excretion, and IAA production. The performance of isolates like BNC2 with good ARA activity, ammonia excretion, and IAA production and isolated from a very strongly saline soil should be further evaluated under high-saline conditions.

Nocardia grenadensis sp. nov., isolated from sand of the Caribbean Sea

A Gram-stain-positive, non-spore-forming bacterium (GW5-5797(T)) was isolated on soil extract agar from sand collected at a depth of 5 m in the Caribbean Sea near Grenada. 16S rRNA gene sequence analysis and similarity studies showed that strain GW5-5797(T) belongs to the genus Nocardia, and is most closely related to Nocardia speluncae N2-11(T) (99.2% similarity) and Nocardia jinanensis 04-5195(T) (99.2%) and more distantly related to Nocardia rhamnosiphila 202GMO(T) (98.6%) and other Nocardia species. Strain GW5-5797(T) could be distinguished from all other recognized Nocardia species by sequence similarity values less than 98.5%. The peptidoglycan diamino acid was meso-diaminopimelic acid. Strain GW5-5797(T) exhibited a quinone system with the predominant compounds MK-8(H(4)ω-cyclo) and MK-8(H(2)). The polar lipid profile of GW5-5797(T) consisted of the major compounds diphosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid, moderate amounts of phosphatidylinositol and a phosphatidylinositol mannoside and minor amounts of several lipids including a second phosphatidylinositol mannoside. The polyamine pattern contained the major compound spermine and moderate amounts of spermidine. The major fatty acids were C(16:0,) C(18:1)ω9c and 10-methyl C(18:0). These chemotaxonomic traits are in excellent agreement with those of other Nocardia species. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW5-5797(T) from the most closely related species, showing 16S rRNA gene sequence similarities >98.5%. Strain GW5-5797(T) therefore merits separate species status, and we propose the name Nocardia grenadensis sp. nov., with the type strain GW5-5797(T) (=CCUG 60970(T) =CIP 110294(T)).

The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site

AbstractIn this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences.

Effects of nutrient status, temperature and pH on mycelial growth, sclerotial production and germination of Rhizoctonia solani from potato

SUMMARY The effects of nutrient status, temperature and pH on mycelial growth, sclerotial production and germination of Rhizoctonia solani [anastomosis groups (AGs) 21 and 3] from potato were studied on a range of artificial media including potato dextrose (PDA), malt yeast extract (MYA), water (WA) and soil extract (SEA) agar. Greatest sclerotium yields were obtained on MYA for AG 3 and PDA for AG 2-1. Sclerotium yields were significantly poorer for AG 2-1 isolates compared to AG 3 isolates tested. Sclerotium yields were significantly improved when mycelia grown on nutrient poor (WA) agar were transferred to nutrient rich (PDA) media after 4 d for AG 3 isolates, but not for AG 2-1 isolates. Optimum temperatures for mycelial growth of all isolates were between 20 and 25°C on all media tested. Mycelial growth of all isolates occurred between pH 4 and 9, with an optimum of pH 5.6. Sclerotium formation occurred between pH 4 and 8 and pH 5 and 6 for AG 3 and AG 2-1 isolates, respectively. Sclerotial germination for all AG 3 isolates was optimal between 20 and 30°C on all media tested, with a pH optimum between pH 5 and 6. In soil, AG 2-1 isolates grew significantly slower compared to AG-3. Optimum temperature for mycelial growth in soil was between 20 and 25°C, regardless of anastomosis group. Sclerotial germination in soil by AG 3 isolates occurred between 10°C and 30°C. The significance of these results on the pathogenicity of R. solani is discussed.

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.

Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection

BackgroundParacoccidioides brasiliensis ecology is not completely understood, although several pieces of evidence point to the soil as its most probable habitat. The present study aimed to investigate the fungal growth, conidia production and molecular pathogen detection in different soil conditions.MethodsSoils samples of clayey, sandy and medium textures were collected from ground surface and the interior of armadillo burrows in a hyperendemic area of Paracoccidioidomycosis. P. brasiliensis was inoculated in soil with controlled humidity and in culture medium containing soil extracts. The molecular detection was carried out by Nested PCR, using panfungal and species specific primers from the ITS-5.8S rDNA region.ResultsThe soil texture does not affect fungus development and the growth is more abundant on/in soil saturated with water. Some soil samples inhibited the development of P. brasiliensis, especially those that contain high values of Exchangeable Aluminum (H+Al) in their composition. Some isolates produced a large number of conidia, mainly in soil-extract agar medium. The molecular detection was positive only in samples collected from armadillo burrows, both in sandy and clayey soil.ConclusionP. brasiliensis may grow and produce the infectious conidia in sandy and clayey soil, containing high water content, mainly in wild animal burrows, but without high values of H+Al.