Selection for acid tolerance inRhizobium meliloti

In the selection of acid-tolerantRhizobium meliloti, procedures for the collection and isolation of rhizobia, and the assessment of acid tolerance, have not been critically evaluated. Such procedures form the basis of this study. Root nodules were collected fromMedicago spp. found growing on acid soil in Sardinia. Their encumbent bacteria were isolated directly onto media adjusted over a range of pH values, and then assessed for acid tolerance in both the laboratory and field. Strains ofRhizobium meliloti isolated onto low pH media were, in general, more acid-tolerant than sister isolates from high pH media, when tested in both the laboratory and field. Dilution (10 or 100 fold) of the inocula used in the laboratory assessment did not greatly influence the rating derived, although there was some effect of bacterial colony type on growth rating. The link between polysaccharide production and acid tolerance was not strong. There was a poor correlation between the growth ratings derived from the laboratory screening and acid tolerance as expressed in the field.     

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.     

An optimum environment for the culturing of cytospora isolates from stone fruits. I. Temperature

Summary Four isolates ofCytospora cincta Fr. and 2 ofC. leucostoma Fr. were obtained from diseased Italian prune, President plum and Bing cherry trees.The minimum temperature for growth of these fungi was found to be 3° C. Temperatures of 45 °C. were lethal to all cultures. The optimum temperature for theC. cincta isolates on solid and liquid media was found to be 30° C.; for theC. leucostoma isolates, nearly 25° C. OneC. cincta isolate produced greatest radial growth on the solid medium at 35° C., but in the liquid medium produced maximum mycelium at 30° C.All factors considered, the conclusion was reached that the best single temperature for laboratory culture of the fungi was 30° C.Approved by the Director of the Idaho Agricultural Experiment Station as Research Paper No. 493.     

An optimum environment for the culturing of Cytospora isolates from stone fruits. III. Nitrogen sources

Summary Four isolates ofCytospora cincta Fr. and 2 ofC. leucostoma Fr. were cultured on media containing 7 different nitrogenous compounds. Maltose served as a constant source of carbon. All experiments were carried out at 25° C.Total growth as determined by mycelial weights, and degree of sporulation as determined by an arbitrary system, revealed that 1) potassium nitrate was most satisfactory as a source of nitrogen, 2) response of the isolates tended to segregate them along species lines, and 3) the pH of the medium appeared to be a factor in the degree of response.Growth-habit experiments emphasized 1) extreme variation in colony characteristics and 2) the need for standardization of laboratory environments for comparative studies of the fungi.Approved by the Director of the Idaho Agricultural Experiment Station as Research Paper No. 518.     

Fungi of the Windmill Islands, continental Antarctica. Effect of temperature, pH and culture media on the growth of selected microfungi

Studies were conducted on microfungi isolated from soils in the Windmill Islands, continental Antarctica. Growth responses of Alternaria alternata, Chrysosporium pannorum, Nectria peziza, Thelebolus microsporus, Mycelia sterile and Phoma cf. herbarum to temperature, pH and culture media were investigated. Maximum growth occurred after 16 days, except in Nectria peziza and Thelebolus microsporus, where maximum growth occurred 12 days after inoculation. All isolates showed poor growth at 0°C. Maximum growth was obtained with temperatures ranging from 15 to 25°C. The optimum temperature for all fungi was 20°C. An acid medium favoured growth. Chrysosporium pannorum, Phoma cf. herbarum and Nectria peziza grew best at pH 3–4, whereas Mycelia sterile, Alternaria alternata and Thelebolus microsporus grew best at pH 5–6. The culture medium had little effect on growth, except for nutrient agar, which showed poor growth against all isolates with the exception of Thelebolus microsporus. Received: 11 September 1996 / Accepted: 13 January 1997     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium

Thermo-acidophilic prokaryotes isolated from geothermal sites in Yellowstone National Park were identified as novel α-Proteobacteria, distantly related (~93% 16S rRNA gene identity) to the mesophilic acidophile Acidisphaera rubrifaciens. One of these isolates (Y008) was shown to be more thermophilic than all previously characterized acidophilic proteobacteria, with a temperature optimum for growth between 50 and 55°C and a temperature maximum of 65°C. Growth was observed in media maintained at pH between 1.75 and 3.0 and was fastest at pH between 2.5 and 3.0. The G + C content of Y008 was 71.8±0.9 mol%. The acidophile was able to grow heterotrophically on a range of organic substrates, including various monosaccharides, alcohols and amino acids and phenol, though growth on single organic compounds required the provision of one or more growth factors. The isolate oxidized sulfur to sulfuric acid in media containing yeast extract, but was not capable of autotrophic growth with sulfur as energy source. Growth occurred under aerobic conditions and also in the absence of oxygen via anaerobic respiration using ferric iron as terminal electron acceptor. Based on these genotypic and phenotypic traits, it is proposed that Y008 represents the type species of Acidicaldus organivorus, gen. nov., sp. nov.     

Change in medium components and colony morphology due to mycelial growth of ectomycorrhizal fungusTricholoma bakamatsutake

Mycelia ofTricholoma bakamatsutake isolate No. 4 grew at temperatures ranging from 10 to 30°C, and the optimum was around 25°C. In well-buffered media of initial pH 5.0 and 6.0, No. 4 mycelia secreted gluconic acid and lowered medium pH. Mycelial growth then accelerated slightly; and with the exhaustion of glucose, growth and secretion of gluconic acid stopped. In 10 different media of initial pH 4.0–7.0, No. 4 mycelia showed higher gluconic acid secretion with higher initial pH. No. 4 mycelial grew best in pH 5.0 media, in which gluconic acid secretion was low. Mycelia of 29 isolates including No. 4 grew better in the media in which less glucose, total carbon and total nitrogen remained, and almost all isolates secreted gluconic acid. Most of the 29 isolates showed irregular colony shapes with rough mycelial fronts, brown pigmentation and aerial hypha on colony surfaces, and brown pigmentation of media under colonies. Dissimilarities were calculated with coded morphological characters on colonies, and similarity between isolates was found not to correlate with proximity of origin. Chlamydospores were observed on every colony of the 29 isolates. Chlamydospores were present on colonies of No. 4, reaching to 2 mm from the mycelial front, where brown pigmentation had not yet developed, and the numbers of chlamydospores incresed with mycelial aging.     

An optimum environment for the culturing of cytospora isolates from stone fruits. II. Carbon sources

Summary Six isolates (Cytospora cincta Fr. andC. leucostoma Fr.) were innoculated to liquid and solid synthetic laboratory media containing nine different sources of carbon.One of the isolates did not grow. The remaining five grew at rates, and in relationships, which segregated them in accordance with their species grouping. These relationships were more nearly constant than any other relationships previously noted in Idaho for these or otherCytopora isolates.Two conclusions were reached: 1) For best growth and sporulation, maltose should be incorporated in the laboratory culture medium and not the commonly used dextrose, which was demonstrated to be an inferior source of carbon; and 2) separate investigations, which utilize different carbon sources in the laboratory media, are likely to yield sufficiently different results that co-identity of species strains used by the separate workers may not be evident.Approved by the Director of the Idaho Agricultural Experiment Station as Research Paper No. 489.     

Natronobacillus azotifigens gen. nov., sp. nov., an anaerobic diazotrophic haloalkaliphile from soda-rich habitats

Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5–10, and within a salt range from 0.2 to 4 M Na⁺, with an optimum at 0.5–1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the 16S rRNA gene of the novel strains are EU143681-EU143690 and EU850814-EU850816; for the nifH gene the accession numbers are EU542601, EU563380-EU563386 and EU850817-EU850819.     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

COPPER TOLERANCE OF CHLAMYDOMONAS ACIDOPHILA (CHLOROPHYCEAE) ISOLATED FROM ACIDIC,COPPER-CONTAMINATED SOILS1

Cells of Chlamydomonas acidophila Negoro, isolated from three soils with different available copper contents (74, 80, and 87 μg·g^?1), were assayed for their responses to copper. Soil pH ranged from 3.3–3.9. Responses were evaluated using algistatic assays involving five day exposure to copper concentrations from 0.1–100 mg·L^?1 at pH 3.8 and 6.6 in defined liquid media. Interspecies and intraspecies comparisons were made between the soil isolates and laboratory strains of C. reinhardtii and C. acidophila, respectively. Algistatic copper concentrations of soil isolates were 20–125 times greater than those of the laboratory strain of C. reinhardtii. Concentrations of 0.1 mg Cu·L^?1, or greater, killed the laboratory strain of C. acidophila. Soil isolates of C. acidophila appeared to be copper tolerant; however, there was no conclusive evidence to indicate that the level of copper tolerance in the soil isolates was positively correlated with the level of available copper in the soil.     

Polygalacturonase,biomass and ascospore production by Byssochlamys fulva. II. Effects of sugars found in fruits

Polygalacturonase, biomass, and ascospore production by four strains of Byssochlamys fulva cultured in laboratory media supplemented with glucose, sucrose, or fructose was studied over a 20-day incubation period at 30° C. The production of polygalacturonase was variable, but most activity was detected between 4 and 8 days in 1% sugar media at an initial pH of 4 or 5. The rate of biomass production was retarded early in the incubation period in media initially at pH 3 or 4 as compared to pH 5, but the amount of growth was about the same in media containing the test sugars after 20 days. Large numbers of ascospores were produced between 8 and 10 days in media containing 5% sugar initially at pH 5 and 4. Production of ascospores was retarded at pH 3 in media containing 5% sugar as compared to media initially at pH 5 and 4.     

On the sensitivity of isolates of Rhizobium spp. and Azotobacter chroococcum to TMTD and its degradation product NaDDC

Summary The in-vitro effect of the seed dressing fungicide tetramethyl thiuram disulphide (TMTD, Thiram) and its degradation product sodium dimethyl dithiocarbamate (NaDDC) on the sensitivity of Rhizobium spp. and Azotobacter chroococcum isolates was studied at different pH values. The Azotobacter chroococcum isolates were more sensitive than Rhizobium spp. isolates at all pH values. The sensitivity of these isolates to TMTD and NaDDC had a maximum at pH 7, and it decreased on either side of this value. The Rhizobium isolates from urid (Phaseolus radiatus) were resistant to TMTD at pH 7 but proved sensitive to its degradation product. A difference between the power of the sensitive and resistant isolates to reduce TMTD was revealed.     

Isolation and characterization of a novel facultatively alkaliphilic Nitrobacter species, N. alkalicus sp. nov.

Five strains of lithotrophic, nitrite-oxidizing bacteria (AN1-AN5) were isolated from sediments of three soda lakes (Kunkur Steppe, Siberia; Crater Lake and Lake Nakuru, Kenya) and from a soda soil (Kunkur Steppe, Siberia) after enrichment at pH 10 with nitrite as sole electron source. Morphologically, the isolates resembled representatives of the genus Nitrobacter. However, they differed from recognized species of this genus by the presence of an additional S-layer in their cell wall and by their unique capacity to grow and oxidize nitrite under highly alkaline conditions. The influence of pH on growth of one of the strains (AN1) was investigated in detail by using nitrite-limited continuous cultivation. Under such conditions, strain AN1 was able to grow at a broad pH range from 6.5 to 10.2, with an optimum at 9.5. Cells grown at pH higher than 9 exhibited a clear shift in the optimal operation of the nitrite-oxidizing system towards the alkaline pH region with respect to both reaction rates and the affinity. Cells grown at neutral pH values behaved more like neutrophilic Nitrobacter species. These data demonstrated the remarkable potential of the new nitrite-oxidizing bacteria for adaptation to varying alkaline conditions. The 16S rRNA gene sequences of isolates AN1, AN2, and AN4 showed high similarity (≥ 99.8%) to each other, and to sequences of Nitrobacter strain R6 and of Nitrobacter winogradskyi. However, the DNA-DNA homology in hybridization studies was too low to consider these isolates as new strains. Therefore, the new isolates from the alkaline habitats are described as a new species of the genus Nitrobacter, N. alkalicus, on the basis of their substantial morphological, physiological, and genetic differences from the recognized neutrophilic representatives of this genus. Received: 3 April 1998 / Accepted: 2 July 1998     

Studies on the distribution of alkalophilic and alkali-tolerant soil fungi II: Fungal flora in two limestone caves in Japan

In a series of studies on the distribution of alkalophilic and alkali-tolerant fungi, soil fungi were isolated from five alkaline calcareous soil samples in two closely located limestone caves (stalactite caves) in Japan using slightly acidic and alkaline media. Some common soil fungi that can grow in alkaline conditions were obtained in high frequencies. The growth response to pH of the isolates revealed that approximately one third (30.8%) of the isolates had the optimum pH in the alkaline range. All isolates of fourAcremonium species and twoChrysosporium species grew well in alkaline conditions, of whichAcremonium sp. andChrysosporium sp. were pronounced alkalophiles. These fungi were thought to be indigenous species in this alkaline environment. The fungal flora in the Japanese alkaline soils was considerably different from the flora reported in alkaline environments in other countries.     

The Influence of pH and Temperature on Cellulolytic and Pectolytic Activity of Cylindrocarpon destructans (Zins.) Scholt.

In studies on the effect of pH and temperature on cellulolytic and pectolytic activity of C. destructans, it was found that the isolates used produced only endoglucanases. The temperature and pH affected the synthesis of these enzymes. Fungi cultured at 26°C produced more of these enzymes than those grown at the two other temperatures. At 10°C, only one isolate produced minute amounts of endoglucanases. None of fungi studied exhibited cellulolytic activity in cultures grown at 20°C. Cellulolytic activity was found only in acidic media (pH 5.0). The fungi studied exhibited higher pectolytic than cellulolytic activity. In the post culture liquids of these organisms, both types of pectolytic enzymes (exo- and endo-PMG) were detected. Different temperature and pH values affected the production of these enzymes differently in various isolates.     

Alkaliflexus imshenetskii gen. nov. sp. nov., a new alkaliphilic gliding carbohydrate-fermenting bacterium with propionate formation from a soda lake

Anaerobic saccharolytic bacteria thriving at high pH values were studied in a cellulose-degrading enrichment culture originating from the alkaline lake, Verkhneye Beloye (Central Asia). In situ hybridization of the enrichment culture with 16S rRNA-targeted probes revealed that abundant, long, thin, rod-shaped cells were related to Cytophaga. Bacteria of this type were isolated with cellobiose and five isolates were characterized. Isolates were thin, flexible, gliding rods. They formed a spherical cyst-like structure at one cell end during the late growth phase. The pH range for growth was 7.5–10.2, with an optimum around pH 8.5. Cultures produced a pinkish pigment tentatively identified as a carotenoid. Isolates did not degrade cellulose, indicating that they utilized soluble products formed by so far uncultured hydrolytic cellulose degraders. Besides cellobiose, the isolates utilized other carbohydrates, including xylose, maltose, xylan, starch, and pectin. The main organic fermentation products were propionate, acetate, and succinate. Oxygen, which was not used as electron acceptor, impaired growth. A representative isolate, strain Z-7010, with Marinilabilia salmonicolor as the closest relative, is described as a new genus and species, Alkaliflexus imshenetskii. This is the first cultivated alkaliphilic anaerobic member of the Cytophaga/Flavobacterium/Bacteroides phylum.Dedicated to Prof. Dr. Hans Günter Schlegel on the occasion of his 80th birthday.     

Influence of pH on copper and zinc sensitivity of ericoid mycobionts in vitro

The effect of pH on growth, metal uptake and toxicity in four isolates of ericoid mycobionts (two Hymenoscyphus ericae from unpolluted heathland sites and two H. ericae-type mycobionts from metal-contaminated mine spoil) was assessed in vitro. These isolates were incubated in liquid medium (10% Rorisons medium, glucose at 10 g l^–1) containing either 0.25 mM Cu or 2.0 mM Zn and adjusted to pH 2, 3, 4, 5 or 6. After 30 days incubation, dry mass and mycelial metal content were determined and growth was expressed as tolerance index, i.e. dry mass in the presence of metal as a percentage of dry mass in the absence of metal. Initial medium pH had a significant effect on both tolerance index and metal accumulation. Tolerance indices were highest at pH 2, with several isolates showing a stimulation of growth (i.e. tolerance index >100%) at this pH. Tolerance index decreased at higher initial pH values and growth of two mycobionts was completely inhibited (tolerance index=0) in the Cu-supplemented media at pH 6. Reduction in tolerance index coincided with an increase in mycelial accumulation of Cu and Zn. Practical and environmental implications of these results are discussed.     

Polygalacturonase,biomass, and ascospore production by byssochlamys fulva. I. Effects of acids found in fruits

Polygalacturonase, biomass, and ascospore production by four strains of Byssochlamys fulva cultured in laboratory media supplemented with citric, malic, or tartaric acids was determined over a 20-day incubation period at 30°C. Polygalacturonase activity was higher in tartaric acid media than in malic or citric acid media, with 1 % acid supporting the greatest activity in media initially at pH 4 or 5; at 0.1 % acid, highest activity was noted in media initially at pH 3. Most activity was produced between 4 and 8 days of incubation. Malic acid supported greater biomass production than did citric or tartaric acids. Media containing tartaric acid was the best for the production of ascospores whereas citric acid was the poorest. Higher numbers of ascospores generally were produced in media initially at pH 3 as compared to pH 4 or 5 after 10 days of incubation.