Osmotic effects on radial growth rate and specific growth rate of three soil fungi

The radial growth rate on osmotically adjusted agar medium and the relative specific growth rate in osmotically adjusted liquid medium were determined for Rhizoctonia solani, Pythium ultimum, and Verticillium dahliae. On basal medium, an isolate of P. ultimum and R. solani had similar radial growth rates of 0.52 and 0.47 mm/h, respectively, whereas V. dahliae grew at a rate of 0.08 mm/h. Radial growth rate was reduced 50% at osmotic potentials of -16, -27, and -32 bars for P. ultimum, R. solani, and V. dahliae, respectively. No growth occurred at -32 bars for P. ultimum, -56.2 bars for R. solani, and -100 bars for V. dahlia. Specific growth rates in liquid culture were 0.011 h-1 for P. ultimum, 0.008 h-1 for V. dahliae, and 0.026 h-1 for R. solani. Ratios of radial growth rate (Kr) to specific growth rate (alphas) were computed for each fungus growing at different osmotic potentials. There was not a constant relationship between Kr on agar and alphas in liquid medium, e.g., Kr/alphas ratios varied from 8-41% from a mean ratio for a particular species. The results indicated that radial growth rate on osmotic agar was not useful as a measure of relative specific growth rate of a fungus in osmotically adjusted liquid medium.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Differentiation of Fusarium oxysporum from Fusarium solani by growth and pigmentation on media containing sugar alcohols

The growth and pigmentation of 99 strains of Fusarium , mainly of Fusarium oxysporum and F. solani , on ammonium salts agar containing either mannitol, sorbitol or xylitol as sole source of carbon is described. After 7–14 d incubation strains of F. oxysporum could be distinguished from F. solani by both their pigmentation and the size and shape of conidiogenous cells. The application of these media to routine screening of isolates is discussed.     

Inhibitory effects of essential oils of medicinal plants from growth of plant pathogenic fungi

Plant cells produce a vast amount of secondary metabolites. Production of some compounds is restricted to a single species. Some compounds are nearly always found only in certain specific plant organs and during a specific developmental period of the plant. Some secondary metabolites of plants serve as defensive compounds against invading microorganisms. Nowadays, it is attempted to substitute the biological and natural agents with chemically synthesized fungicides. In the present research, the antifungal activities of essential oils of seven medicinal plants on mycelial growth of three soilborne plant pathogenic fungi were investigated. The plants consisted of Zataria multiflora, Thymus carmanicus, Mentha pieperata, Satureja hortensis, Lavandual officinolis, Cuminum cyminum and Azadirachta indica. The first five plants are from the family Labiatae. Examined fungi, Fusarium oxysporum f.sp. lycopersici, Fusarium solani and Rhizoctonia solani are the causal agents of tomato root rot. Essential oils of Z. multiflora, T. carmanicus, M. pieperata, S. hortensis and C. cyminum were extracted by hydro-distillation method. Essential oils of L. officinalis and A. indica were extracted by vapor-distillation method. A completely randomized design with five replicates was used to examine the inhibitory impact of each concentration (300, 600 and 900 ppm) of each essential oil. Poisoned food assay using potato dextrose agar (PDA) medium was employed. Results showed that essential oils of A. indica, Z. multiflora, T. carmanicus and S. hortensis in 900 ppm at 12 days post-inoculation, when the control fungi completely covered the plates, prevented about 90% from mycelial growth of each of the fungi. While, the essential oils of M. pieperata, C. cyminum and L. officinalis in the same concentration and time prevented 54.86, 52.77 and 48.84%, respectively, from F. solani growth. These substances did not prevent from F. oxysporum f.sp. lycopersici and R. solani growth. Minimum inhibitory concentration (MIC) of essential oils of T. carmanicus, Z. multiflora and A. indica from R. solani and F. solani growth was 900 and 600 ppm, respectively. In addition, the MIC of essential oils of these plants and essential oil of S. hortensis from F. oxysporum f.sp. lycopersici growth was 900 ppm. The MIC of essential oils of M. pieperata, C. cyminum and L. officinalis from F. solani growth was 900 ppm.     

Colony formations in a halotolerantBrevibacterium sp. JCM 6894 on solid medium with different pH values

Viable cells of a halotolerantBrevibacterium sp. JCM 6894 grown in a liquid medium with pH 7.1 were enumerated as the colony-forming cells on three kinds of agar media with different pH values. Unexpectedly they were lower at neutral pH rather than acidic or alkaline pH. This tendency was invariable regardless of the changes in the concentrations of nutrients in the agar medium as well as in the growth phases of the cells. From the comparison of cell growth between liquid and solid media with different pHs, we notified the importance of the pH changes in liquid medium accompanied with growth. Effects of salts and pH of the liquid medium on protonmotive force (Δp) was estimated from membrane potentials (ΔΨ) and proton gradients (ΔpH) of the strain JCM 6894. In the absence of salts, Δp of the strain JCM 6894 was the largest at neutral pH, which was conflicting with the result of cell viability. The addition of NaCl led to the reduction of Δp at acidic pH, mainly due to the dissipation of ΔΨ, which seems to be consistent with the lower numbers of colony formed at acidic pH in the presence of NaCl.     

Antifungal Activities of Four Fatty Acids against Plant Pathogenic Fungi

The effect of the fatty acids linolenic acid, linoleic acid, erucic acid and oleic acid on the growth of the plant pathogenic fungi Rhizoctonia solani, Pythium ultimum, Pyrenophora avenae and Crinipellis perniciosa were examined in in vitro studies. Linolenic and linoleic acids exhibited activity against all of the fungi. However, whereas linolenic acid reduced mycelial growth of R. solani and C. perniciosa at 100 microM, the concentration had to be increased to 1000 microM before any effect on mycelial growth of P. ultimum and P. avenae was observed. Linoleic acid only reduced mycelial growth of R. solani, P. ultimum and P. avenae at 1000 microM, but led to a significant reduction in growth of C. perniciosa at 100 microM. In contrast, oleic acid had no significant effect on growth of R. solani or P. avenae, but gave significant reductions in mycelial growth of P. ultimum at 100 microM and reduced growth of C. perniciosa significantly at 1000 microM. All of the fatty acids reduced biomass production by all of the fungi significantly in liquid culture when added to the media at 100 microM. Erucic acid had no effect on fungal growth at any concentration examined. The antifungal activities exhibited by linolenic, linoleic and oleic acids may be useful in the search for alternative approaches to controlling important plant pathogens, such as those examined in this study.     

Antimycotic Compounds from the Plant Pathogen Rhizoctonia solani and its Antagonist Trichoderma harzianum

The soilborne rhizosphere-competent fungal biocontrol agent Trichoderma harzianum isolate Th008 secreted trichodermin (MW = 292) and a small peptide (MW = 876) in culture. These compounds were antagonistic in culture to the mycelial growth of the soilborne fungal pathogen Rhizoctonia solani isolate 2B-12, which is highly virulent to soybean ( Glycine max )seedlings. When 100mg of dried autoclaved mycelial mat of R. solani was added to 200 ml liquid cultures of T. harzianum , the quantity of antimycotic compounds secreted by the latter was 3.5 times greater than that of the antagonist alone. R. solani secreted a coumarin derivative (MW = 313) in liquid culture, which inhibited the mycelial growth of T. harzianum ; however, inhibition of the growth of the antagonist required a greater concentration than that for the antimycotic compounds produced by the antagonist against the pathogen. The inclusion of 100 mg of dried autoclaved mycelial mat of T. harzianum in a 200 ml liquid culture of R. solani did not affect the quantity of the antimycotic compound produced by the pathogen.     

Assessing the pathogenic effect ofFusarium, Geosmithia andOphiostoma fungi from broad-leaved trees

Phytopathogenic effect of Geosmithia pallida, G. langdonii, Ophiostoma grandicarpum, O. querci, two isolates of O. piceae, and two isolates of Fusarium solani was compared using plant growth test (stem and root length of garden cress plants seeded on mycelium-covered potato carrot agar); Ophiostoma spp. and F. solani were isolated from oak, Geosmithia spp. from galleries of Scolytus intricatus on beech. All fungi inhibited more the root elongation than that of stems. F. solani led to plant collapse after briefly stimulating the growth of stem and in one case also root. G. langdonii inhibited stem and root growth to 20% and led to plant collapse. G. pallida inhibited root growth to 25% whereas stem growth was almost unimpaired. Ophiostoma spp. reduced stem growth to approximately 60-80% and root growth to 25-60%. O. piceae and O. querci caused plant collapse after 15-20 d.     

Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans

TPK1 and TPK2 encode both isoforms of protein kinase A (PKA) catalytic subunits in Candida albicans. Mutants lacking both TPK1 alleles showed defective hyphal morphogenesis on solid inducing media, whereas in liquid hypha, formation was affected slightly. In contrast, tpk2 mutants were only partially morphogenesis defective on solid media, whereas a strong block was observed in liquid. In addition, the yeast forms of tpk2-- but not tpk1-- mutants were completely deficient in invading agar. Because Tpk1p and Tpk2p differ in their N-terminal domains of approximately 80--90 amino acids, while the catalytic portions are highly homologous, the functions of hybrid Tpk proteins with exchanged N-terminal domains were tested. The results demonstrate that the catalytic portions mediate Tpk protein specificities with regard to filamentation, whereas agar invasion is mediated by the N-terminal domain of Tpk2p. Homozygous tpk1 and tpk2 mutants grew normally; however, a tpk2 mutant strain containing a single regulatable TPK1 allele (PCK1p-TPK1) at low expression levels was severely growth defective. It was completely blocked in hyphal morphogenesis and was stress resistant to high osmolarities or temperatures. Thus, both Tpk isoforms in C. albicans share growth functions but, unlike Saccharomyces cerevisiae isoforms, they have positive, specific roles in filament formation in different environments.     

In vitro cloning of tumor stem cells in semi-solid media containing agar and agarose

Summary The formation of tumor stem cell colonies in vitro has been studied by comparing the growth of three mouse teratocarcinoma derived cell lines and one human teratocarcinoma derived cell line in semi-solid media containing either agar or agarose. We show that agaroses should be used in higher concentrations than agar to obtain comparable results. The maximum number of colonies were obtained in agarose over a broader range of concentrations (1%–4% for SeaPrep and 0.5%–2% for SeaPlaque agarose) than in agar, which allowed anchorage-independent growth of tumor cells only over a narrow concentration range (0.3%–0.5%). Overall, the preparation of media containing agarose was less cumbersome than preparation of agar-containing media, primarily because agaroses gelled more slowly and remained liquid in the physiological temperature range. Furthermore, the transfer of colonies from semi-solid media containing agarose to solid surface tissue culture dishes was much more efficient than the transfer of colonies from agar. The stock solutions of SeaPrep agarose could be kept ready for use for extended periods of time. All these features show that the low melting point agarose has considerable advantages over agar for preparation of semi-solid media for anchorage-independent tumor cell growth.     

Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots

AIMS: To isolate endophytic Streptomyces strains from tomato and examine their antimicrobial activity. METHODS: Endophytic Streptomyces strains were isolated using surface-sterilization methods and identified by morphological characteristics. Antimicrobial activities were measured by the agar plate sensitivity method. Antifungal activity in vivo was measured by seedling mortality in infested soils. RESULTS: Twenty-one per cent of endophytic streptomycete isolates produced antibacterial metabolites and 41% produced antifungal metabolites in S medium. Sixty-five per cent of the most frequently isolated strains inhibited the growth of Rhizoctonia solani by the antibiosis assay but only 32% produced metabolites active against R. solani in S medium. Growth promotion and enhanced disease resistance of seedlings inoculated with Streptomyces sp. strain S30 were observed in tomato but not in cucumber seedlings. CONCLUSIONS: Endophytic Streptomyces spp. strains were successfully isolated using stringent methods and strain S30 promoted growth and enhanced resistance to R. solani in tomato seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic streptomycetes showing antifungal activity in vitro and in vivo may indicate the potential for their use as biocontrol agents particularly of R. solani disease of tomato seedlings.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

Structure, production characteristics and fungal antagonism of tensin - a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

AIM: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. METHODS AND RESULTS: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or purified tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. CONCLUSION: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. SIGNIFICANCE AND IMPACT OF THE STUDY: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of significant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.     

Role of chitinase and beta-1,3-glucanase activities produced by a fluorescent pseudomonad and in vitro inhibition of Phytophthora capsici and Rhizoctonia solani

A study was conducted to investigate the possibility of involvement of chitinase and beta-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani. Fluorescent Pseudomonas isolates GRC(3) and GRC(4) were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC(3). The involvement of chitinases, beta-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and beta-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.     

Roles of low pH, carbon and inorganic nitrogen source use in chlamydospore formation by Fusarium solani.

Citrate and malate were poorer sources of exogenous carbon than several hexose, pentose, or disaccharide sugars for supporting macroconidial germination by Fusarium solani at high conidial density (1 X 10(5) condia/ml). Only citrate, however, failed to block chlamydospore morphogenesis to a degree comparable to glucose or other readily used sugars. Mostly immature chlamydospores were formed in the presence of citrate. At low conidial density (5 X 10(3) conidia/ml), exogenous carbon-independent macroconidial germination and subsequent rapid chalmydospore formation on germ tubes was not inhibited by ammonium or nitrate nitrogen. The citrate-phosphate buffered, low pH (4.0) medium of Cochrane induced more immature chlamydospore formation by F. solani than a pH 6.0 medium, but few mature chlamydospores were formed in either medium. Condensation of hyphal cytoplasm into developing chlamydospores, a character typical of chlamydospore formation, did not occur extensively and macroconidia, hyphae, and immature chlamydospores stained deeply with Sudan III, suggesting lipid biosynthesis. This inhibition of chlamydospore maturation may be due partly to nitrogen deficiency imposed by the high C:N ratio of the medium and to the presence of citrate. Only vesiculate hyphal cells were formed by F. solani f. sp. phaseoli in both media. Field soils to which the clone of F. solani used is indigenous had mean pH values ranging from 5.2 to 6.0.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.     

New selective agent for isolation of Pseudomonas aeruginosa.

Results of minimal inhibitory concentration tests with a diversity of bacterial strains showed that 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) inhibited the growth of all microorganisms tested (other than Pseudomonas aeruginosa) at 25 microgram/ml or less, whereas MICs obtained for P. aeruginosa ranged from to to greater than 100 microgram/ml. Therefore, C-390 was evaluated as a potential selective agent for P. aeruginosa in pseudomonas agar F. Recovery tests were conducted on this medium with 53 strains o P. aeruginosa, and the results were compared to those obtained in similar tests on commercially available selective media, i.e., pseudomonas isolation agar and Pseudosel agar. The results of these comparisons indicated that pseudomonas agar F with C-390 was significantly less inhibitory than Pseudosel agar and pseudomonas isolation agar and more selective than pseudomonas isolation agar. The incorporation of C-390 in pseudomonas agar F also provided a medium that was both selective and differential. Preliminary evidence also suggested that C-390 may be added to other basal media with comparable results.