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.     

Utilization of Carbohydrates by Rhynchosporium secalis. I. Growth and Sporulation on Glucose, Galactose, and Galacturonic acid

The fungus Rhynchosporium secalis (Oud.) Davis grew and speculated in liquid nutrient media that contained glucose, galactose or galacturonic acid, or a pair of those substances, as the sole carbon source. Sporulation was inhibited by high concentrations of glucose and galacturonic acid. Growth and sporulation were greatest on glucose, and least on galactose. Growth was increased when glucose and galacturonic acid were mixed. Nitrogen concentration affected sporulation but not growth.     

The Carbohydrate Nutrition of Tomato Roots: VI. The Inhibition of Excised Root Growth by Galactose and Mannose and its Reversal by Dextrose and Xylose

The addition of 0.01–0.015 per cent. galactose or 0.005–0.01per cent, mannose reduces by 50 per cent, the linear growthof excised tomato roots cultured in a I per cent, sucrose medium.An addition of 0.03–0.04 per cent, of either sugar causesnot less than a 90 per cent, inhibition of growth. The survivalof meriste-matic activity is higher in presence of fully inhibitoryconcentrations of mannose than of galactose. Roots inhibitedby galactose are distinguishable from those inhibited by mannose. The inhibitory effect of concentrations of galactose up to 0.15per cent, and of mannose up to 0.4 per cent, can be fully antagonizedby the simultaneous addition to the culture medium of dextrose.The minimum ratio of dextrose: inhibitory sugar for maximumantagonism of the growth inhibition is with galactose 5: 1 andwith mannose 3.5: 1. Growth of roots in a dextrose-containingmedium does not protect them from subsequent inhibition by eithergalactose or mannose. d-xylose has significant activity as an antagonist of mannoseinhibition and even more so of galactose inhibition. However,the restoration of growth achievable from the addition of xyloseis not comparable with that resulting from the addition of dextrose.The inhibition of growth by xylose is not alleviated by thesimultaneous addition of dextrose. Maltose has low activityas an antagonist of galactose and mannose inhibition. All othersugars tested and the sugar alcohols corresponding to galactoseand mannose were quite inactive as antagonists of the growthinhibition by these two sugars. Mixtures of partially inhibitory concentrations of galactoseand mannose were less inhibitory than their more inhibitorycomponent. The concentration of dextrose required to reversethe inhibitory activity of such mixtures was not greater thanthe minimum concentration required to antagonize the actionof the more inhibitory component. The antagonism of galactose inhibition by dextrose is not dueto dextrose impeding galactose uptake.     

Mutual interactions between an invasive bark beetle and its associated fungi

Interactions between invasive insects and their fungal associates have important effects on the behavior, reproductive success, population dynamics and evolution of the organisms involved. The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Scolytinae), an invasive forest pest in China, is closely associated with fungi. By carrying fungi on specialized structures in the exoskeleton, RTB inoculates fungi in the phloem of pines (when females dig galleries for egg laying and when males join them for mating). After eggs hatch, larvae gregariously feed on the phloem colonized by the fungi. We examined the effects of five isolates of RTB associated fungi (two from North America, Leptographium terebrantis and L. procerum, and three from China, Ophiostoma minus, L. sinoprocerum and L. procerum) on larval feeding activity, development and mortality. We also studied the effects of volatile chemicals produced in the beetle hindgut on fungal growth. Ophiostoma minus impaired feeding activity and reduced weight in RTB larvae. Leptographium sinoprocerum, L. terebrantis and L. procerum did not dramatically influence larval feeding and development compared to fungi-free controls. Larval mortality was not influenced by any of the tested fungi. Hindgut volatiles of RTB larvae, verbenol, myrtenol and myrtenal, inhibited growth rate of all the fungi. Our results not only show that D. valens associated fungus, O. minus, can be detrimental to its larvae; but, most importantly, they also show that these notorious beetles have an outstanding adaptive response evidenced by the ability to produce volatiles that inhibit growth of harmful fungus.     

Effects of Some Pesticides on the Growth of ARF18 and Its Pathogenicity to Heterodera glycines

The effects of 22 pesticides on the mycelial growth and pathogenicity of the biocontrol fungus ARFI8 to Heterodera glycines were tested in vitro. The chemicals were added to agar at 10, 100, and 1,000 ppm a.i.; a block of agar containing the fungus was added to each test concentration; and fungal growth was measured. Subsequently, a block of the fungus on the pesticide-containing agar was used to determine the ability of the fungus to parasitize eggs of H. glycines. Aldicarb, bentazone, and chlorothalonil had little or no effect on fungal growth, whereas benomyl and thiophanate methyl completely inhibited growth of the fungus at 10 ppm. The relative insensitivity of ARF18 to certain pesticides would permit selected use of those pesticides with ARF18 in an integrated control program if the effects on the fungus in the field are similar to results from petri dish studies.     

Utilization of Carbohydrates by Rhynchosporium secalis II. Absorption and Metabolism of Glucose, Galactose and Galacturonic acid

Glucose, galactose and galacturonic acid were taken up at different rates by the fungus Rhynchosporium secalis and were intracellularly converted to other forms of carbohydrate at different rates. These differences explain why, when there is only a single source of nutrient carbon in the growth medium, development of the fungus is greatest when glucose is present and least when galactose is present. Glucose and galactose were taken up by the same mechanism for their uptake showed a reciprocal competitive inhibition. Uptake mechanisms had a high affinity for glucose (apparent K_m 2.76 mM) and galacturonic acid (apparent K_m 3.10 mM) and a low affinity for galactose (apparent K_m 29.67 mM). After uptake, galactose accumulated in the mycelium, whereas glucose and galacturonic acid were rapidly converted to other soluble carbohydrates, principally trehalose and mannitol. The insoluble carbohydrates within the mycelium were little affected by the type of carbohydrate that was supplied to the fungus.     

Concurrent Production and Consumption of Ethanol by Cultures of Pachysolen tannophilus Growing on d-Xylose

Growing cultures of Pachysolen tannophilus concurrently consumed and produced ethanol in the presence of substantial concentrations of d-xylose. Ethanol was also assimilated in the presence of other sugars, the amount depending on the sugar. Less ethanol assimilation occurred with d-glucose than with d-xylose. The rate of ethanol consumption decreased as the concentration of glucose was increased, but some consumption still occurred when 2% glucose was present. The rate increased with the amount of oxygen available to the culture when d-xylose or ethanol was the carbon source. In most instances, estimates of consumption were based on the extent of incorporation of C from [1-C]ethanol into trichloroacetic acid-insoluble material. The results are pertinent to the use of P. tannophilus for the production of ethanol from d-xylose.     

Ophiostoma breviusculum sp. nov. (Ophiostomatales, Ascomycota) is a new species in the Ophiostoma piceae complex associated with bark beetles infesting larch in Japan

A new species of the Ophiostoma piceae-complex was isolated from bark beetles and the insect galleries of Larix kaempferi in Japan. This species was described as Ophiostoma breviusculum. The fungus was morphologically similar to O. piceae and O. quercus. However the average length of the perithecial necks and synnemata were shorter than for O. piceae and O. quercus. Synnemata morphological characteristics also differentiated O. breviusculum from the other species of the Ophiostoma piceae-complex isolated from conifers. Mating tests demonstrated that this fungus did not produce perithecia with O. floccosum, O. piceae and O. quercus. In phylogenetic trees using rDNA ITS O. breviusculum was placed in a clade with O. canum, O. piceae and O. subalpinum, but when using beta-tubulin it was placed into a separate clade.     

Extracellular lipase production by a sapwood-staining fungus,Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH₄)₂SO₄ gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH₄)₂SO₄and peptone as nitrogen source.The authors are with Forest Products Biotechnology, Department of Wood Science, Facully of Forestry, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada     

The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.     

FURTHER NOTES ON THE GROWTH AND CHLOROPHYLL FORMATION OF CHLORELLA PROTOTHECOIDES

1. Using area of a fixed concentration as a nitrogen source,cells of Chlorella protothecoides were grown in the presenceof various carbon compounds. Magnitudes of growth of the cellswere widely different depending on the carbon sources used;glucose and fructose being most favourable substrates and galactose,glycerol and acetate coming next. But the amounts of chlorophyllformed in the cells during the experimental period were almost,the same irrespective of the different carbon sources, withsome exceptions. The similarity of the chlorophyll level observedin these experiments seemed to indicate that the formation ofchlorophyll was limited largely by the nitrogen source but notby the carbon source. 2. Strong bleaching effect was recognized with glucose and fructoseat their high concentration, which produced totally chlorophyll-lesscells. On the other hand, a stimulating effect on chlorophyllformation was observed with galactose at the different concentrationsexamined. 3. Effects of glycine and ammonium carbonate as the nitrogensource on the algal growth and pigmentation were studied insome details. The results were similar to those previously obtainedwith urea, confirming our previous conclusion that the algalpigmentation is profoundly affected by the concentration balancebetween glucose and nitrogen source. ¹Present address: Tokyo Research Laboratories, Tanabe SeiyakuCo., Toda-machi, Saitama.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

Kinetic characterization of d-xylose isomerases by enzymatic assays using d-sorbitol dehydrogenase

The enzymatic and coupled d-xylose isomerase/d-sorbitol dehydrogenase assay is a rapid and specific method, permitting accurate quantification of d-xylose isomerization and of d-xylose. The method is based on the isomerization of d-xylose to d-xylulose, followed by reduction of the latter to xylitol by commercially available d-sorbitol dehydrogenase and NADH. The application of this one-step method cannot be extended to d-glucose isomerization since the conditions for a valid coupled assay are not fulfilled. For quantification of d-glucose isomerization, the two-step procedure with d-sorbitol dehydrogenase is recommended. Kinetic parameters for d-xylose and d-glucose using d-xylose isomerase from Streptomyces violaceoruber are reported. The results are compared with the widely used colorimetric cysteine-carbazole method.     

Galactose transport in Streptococcus thermophilus

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Energetic irrelevance of aerobiosis for S. cerevisiae growing on sugars

Summary The net benefit that Saccharomyces cerevisiae obtains from aerobiosis as compared to anaerobiosis has been studied. For this purpose yeasts with different respiratory capacities have been obtained by growing them in batch cultures on different substrates. Even with sugars with low catabolite repression effect, as is the case of galactose, aerobiosis increased the growth rate and the growth yield by less than two-fold. These variations, which are much lower than the expected considering the actual oxygen utilization, indicate that either the amount of ATP produced in respiration is much lower than the theoretically expected or a much greater expenditure of ATP occurs in aerobic than in anaerobic growth. The results show that S. cerevisiae obtains only a slight benefit from aerobiosis when growing on sugars at the relatively high concentration prevailing in its natural habitats.The inhibition of sugar consumption rate by aerobiosis (Pasteur effect) has also been studied, Pasteur effect was almost unnoticeable during growth on any tested sugar and very low during ammonia starvation. These results contrast with the general belief that Pasteur effect is a quantitatively important phenomenon in yeast. It is concluded that the relevant observations of Louis Pasteur have little relationship with the phenomenon that bears his name.     

Fungicidal activity of Ranunculus asiaticus and other weeds against Fusarium oxysporum f.sp. lycopersici

The effects of aqueous extracts of some common weed species against Fusarium oxysporum Schlecht f.sp. lycopersici (the causal agent of tomato wilt disease) were investigated under laboratory conditions. Anagallis foemina L., Cerastium dicotomum L., Falcaria vulgaris L., Ranunculus asiaticus L., Scorpiurus mur-icatus L. and Solanum nigrum L. extracts were the most toxic to the fungus. Further studies on buttercup (Ranunculus asiaticus L.) showed that fresh shoot extract of this species prevented growth of F. oxysporum when incorporated into agar medium. Extracts of different parts of the plant inhibited fungus growth and sporulation, but the fungitoxicity decreased with incubation period with only slight changes in the toxicity of fresh shoot extract. The shoot and fresh parts extracts were more toxic than root and dried tissue extracts. Addition of 0.5 ml fresh shoot or 1 ml fresh root extract to the growing medium significantly reduced fungal colony growth, and the effect was extract concentration dependent. Fresh shoot extract of R. asiaticus added to a liquid medium significantly reduced mycelial dry weight compared with the control, and incorporation of 0.1 g dried shoot or 0.2 g dried roots in the media strongly inhibited fungus growth. Results of a pot experiment showed no harmful effects of R. asiaticus extracts on tomato growth.     

Observations on orchid seed mycorrhizae

Summary In the natural germination of orchid seeds, the fungus undoubtedly contributes sugars, auxins, and growth factors in stimulating the growth of the embryo. However, none of these factors alone are as successful in stimulating germination as they are when combined. No single factor can be identified as the specific germination agent in the mycorrhizal relationship. Seeds can germinate, although poorly and slowly, without added sugars or auxins. Rather, a combination of factors promoting vigorous aerobic respiration provides the cellular environment necessary for germination. Fungus infection in nature, or artificial media in the lab, can provide these conditions: either rapidly by providing exogenous substrates and growth factors for respiration; or slowly by stimulating the utilization of endogenous lipid seed reserves. In either case, cell division and synthesis of chlorophyll follow. A speculatory metabolic pathway is outlined.     

Direct Fermentation of d-Xylose to Ethanol by Kluyveromyces marxianus Strains

Eight strains of Kluyveromyces marxianus were screened, and all of them were found to ferment the aldopentose d-xylose directly to ethanol under aerobic conditions. One of these strains, K. marxianus SUB-80-S, was grown in a medium containing 20 g of d-xylose per liter, and the following results were obtained: maximum ethanol concentration, 5.6 g/liter; ethanol yield, 0.28 g of ethanol per g of d-xylose (55% of theoretical); maximum specific growth rate, 0.12 h; 100% d-xylose utilization was completed in 48 h.