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.

Caracterização de isolados de Rhizoctonia associados à queima foliar em Roraima.

O objetivo desse trabalho foi caracterizar isolados do fungo Rhizoctonia associados à queima foliar, obtidos de hospedeiros de importância econômica no estado de Roraima. Os isolados foram obtidos de plantas de feijão-caupi (Vigna unguiculata), soja (Glycine max), seringueira (Hevea brasiliensis), melancia (Citrullus lanatus), alface (Lactuca sativa) e feijão-guandu (Cajanus cajan). Os parâmetros utilizados foram números de núcleos, grupo de anastomose e as características culturais da colônia, taxa de crescimento micelial e a formação de escleródios nos meios de cultura: batata dextrose agar (BDA), BDA+asparagina, BDA+extrato de levedura, Czapek Agar, maltose-peptona-agar, soil extract agar, sacarose-yeast-asparagina e V-8. Todos os 10 isolados estudados foram caracterizados como multinucleados e pertencentes à espécie Rhizoctonia solani. Três isolados de feijão-caupi, um de soja e o isolado de melancia foram identificados como AGI-1A e um isolado de feijãocaupi, um de soja e o isolado de feijão-guandu como AGI-1B. O isolado de seringueira não foi identificado como nenhum dos padrões de anastomose utilizado. Para a maioria dos isolados as maiores taxas de crescimento micelial foram obtidas no meio de cultura Soil Extract Agar. Dois tipos de escleródios, característicos do grupo AGI, foram observados: formação de 2-20 tufos.placa-1 coloração variável, 1-2 mm e formação de 38-611 microescleródios placa-1, de coloração marrom, medindo 100 μm. A produção e o tipo de escleródio variaram com o isolado e o meio de cultura utilizado.

Accuracy and precision of population estimates of Verticillium dahliae on growth media in quantitative soil assays

Verticillium dahliae Kleb. is a serious pathogen of many plant species. Growth media used to measure population density of V. dahliae in soil were evaluated for high recovery of the pathogen, as well as accuracy and precision of population estimates from naturally and artificially infested sandy loam soil using the soil dilution method. Recovery of V. dahliae from naturally infested field soil was highest on soil pectate Tergitol agar (SPT), soil extract agar + sodium polypectate (SEAP), modified pectate agar (MPA), potato dextrose agar + streptomycin sulphate (PDAS), and Talboys' prune lactose agar (TPA); however, PDAS and TPA were overgrown with contaminating fungi, making enumeration difficult. Use of SPT medium resulted in the most precise population estimate with a standard error (SE) of 12.6% of the mean followed by use of pectate agar (PA) (SE = 14.8%) and SEAP (SE = 19.1%). Ethanol, biotin, and Dox salts enhanced recovery of V. dahliae from naturally infested soil, but combining Dox salts with ethanol and biotin significantly reduced population density. Soil extract had no significant effects on population density. Accuracy of V. dahliae population estimates from sterile artificially inoculated soil was highest on modified soil extract agar (MSEA) (64%) followed by SPT (58%). However, accuracy of V. dahliae population estimates from nonsterile artificially inoculated soil was highest on SPT (36%). Soil extract is not an essential ingredient and biotin may increase recovery of V. dahliae from soil.Key words: Verticillium dahliae, verticillium wilt, population density, recovery, accuracy, precision.

Common Characteristics of the Swiss and Argentine Strains of Clostridium botulinum Type G1

Five strains of Clostridium botulinum type G of human origin, from Switzerland, were compared with two strains isolated from soil in Argentina. The Swiss and Argentine strains are the only type G strains isolated to date. Characteristics compared were toxigenicity, sporulation, proteolysis and carbohydrate fermentation. High toxin titers were produced in trypticase-peptone-glucose-yeast extract broth incubated anaerobically at 30°C for 10 d. Sporulation occurred in three strains incubated anaerobically on soil extract agar at 35°C for 15 d. Different concentrations of soil extract in the medium promoted sporulation of different strains. Toxins of the Swiss and Argentine strains showed identical patterns for trypsin activation, reaction to A-F antitoxin and neutralization by antitoxin prepared from strain 89G. All seven strains showed delayed proteolytic activity but failed to ferment any of the sugars tested.

A simple plate test for the determination of protocatechuic acid utilization by soil bacteria

The method described makes use of the color reaction between protocatechuic acid and ferric ions. Separately sterilized protocatechuic acid is added to soil extract agar before plates are poured. After suitable incubation the plates are flooded with ferric chloride solution. Colorless zones around growth contrasting with the green color of the protocatechuic acid containing agar indicate utilization of protocatechuic acid. With the aid of this method the frequency of strains that use protocatechuic acid, among random bacterial isolates of soil origin, was found to vary between 26 and 44% according to the soil investigated.

THE EFFECT OF YEAST EXTRACT AND CASITONE ON THE RESPONSE TO SALT OF THE MICROFLORA OF A HIGHLY SALINE SOIL

Salt-dependent, salt-resistant, and salt-sensitive bacteria were found at depths of 0–50 cm in a hydrohalomorphic soil near the shore of the Red Sea. Highest counts were obtained on soil-extract agar supplemented with 5% sodium chloride, 0.02% yeast extract, and 0.04% Casitone. Most of the bacteria, upon initial isolation, failed to grow on counting media containing 5% or 10% sodium chloride unless yeast extract and Casitone were present. However, all isolates, randomly selected from the counting medium containing 10% sodium chloride and supplemented with yeast extract and Casitone, did not need these supplements when transferred onto a new medium of the same salt concentration.Bacterial counts were significantly affected by the salt concentration of both the diluting solution and the growth media. It was concluded that the addition of yeast extract and Casitone to the growth media resulted in a partial recovery and protection of the cells from damage occurring during the initial dilution and plating procedure.

EFFECT OF PLATING MEDIUM AND INCUBATION TEMPERATURE ON GROWTH OF FUNGI IN SOIL-DILUTION PLATES

The effects of 20, 25, or 30 °C incubation temperatures for dilution-plates and of five plating media (Ohio Agricultural Experiment Station Medium (OAES), Liftman"s crystal-violet agar, dextrose-peptone agar, soil-extract agar, and glucose-nitrate soil-extract agar) on total numbers and types of fungi isolated from two soils (sugar beet or corn cropped) by a soil-dilution plate method were determined. The data revealed that significant differences existed among the three variables as well as their interactions. Although numbers of colonies of fungi isolated were not affected significantly, the types of fungi (number of individual genera) isolated were significantly greater in sugar beet than in corn cropped soils. In nearly all instances, the greatest total number and types of fungi were isolated at temperatures of 20 or 25 °C. Although each medium appeared to favor one or more groups of fungi, the overall frequency and distribution of fungi was essentially the same on all media. On the basis of its transparency, total number and types of fungi isolated, the elimination of bacteria and actinomycetes, and its restriction of rapidly growing fungi, the OAES medium was deemed the most suitable for use in the soil-dilution plate method.

PLATE COUNTS OF BACTERIA AND FUNGI IN A SALINE SOIL

Numbers of bacteria and of fungi in a saline soil were about one-fifth of numbers in a Red River clay soil. Bacterial counts on two soil-extract agar media, one prepared from the saline soil and the other from Red River soil, were the same, and the populations on the two were the same as shown by the replica plating technique. They were larger than counts on sodium albuminate agar or on asparagin–mannitol agar. Likewise, fungal counts on either soil-extract agar, with 0.02% dipotassmm phosphate, 0.1% glucose, and 0.1% peptone, were higher than counts on Waksman's acidified medium, or on Martin's medium, or on Smith and Dawson's medium. Interestingly, fungal counts on a medium with the same three chemicals as the soil-extract media but with the soil extract replaced by water were as high as those on the soil-extract media. Different levels of potassium phosphate were tested in each of the above media. In each medium for bacteria, and in each for fungi, counts varied inversely as the amount of potassium phosphate. The same held true when sodium phosphate was used instead of potassium phosphate in each medium.