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.     

Observations on the production and dispersal of spores, and infection by Rhynchosporium secalis

The numbers of spores of Rhynchosporium secalis washed from samples of barley plants taken weekly, varied markedly. No consistent association with amount of previous rainfall or length of the preceding dry period was detected. On potted seedlings exposed within a crop most infection occurred during long rain periods or when rain fell late in the evening; fewest lesions usually developed on seedlings prevented from touching adjacent plants. Rotorod traps fitted with a 13 mm diameter disc at the apex of each arm were operated under 24 cm diameter covers. Spores were collected on circular cover slips fixed to each disc with glycerine jelly. At all stages of crop growth more spores were trapped at ground level than at other heights tested up to 1 m. The number of spores trapped was not related to the quantity of or duration of rainfall but was related to the mean rate of fall during brief showers only. Efficiency of droplet retention was assessed in a wind tunnel. It declined rapidly when more than c. 5 pl was presented to the disc surface and was less at a wind speed of 1.0 than 0.5 m s-^l. Spore distribution on discs indicated that spores were washed from the surface during long rain periods.     

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained β-galactosidase activity, and no significant phospho-β-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.     

Isolation and characterization of microsatellite loci from the barley scald pathogen,Rhynchosporium secalis

Fifteen primer pairs were designed for 14 polymorphic microsatellite loci, which were isolated and characterized from genomic libraries of Rhynchosporium secalis. Conditions for multiplexing and simultaneous genotyping of up to eight loci in a single run are described. The number of alleles per polymorphic locus ranged from two to 13 in populations from Switzerland and Australia. Genotypic diversity ranged from 61.5 to 66.7. Gene diversity ranged from 0.08 to 0.89 for individual polymorphic loci, with averages of 0.54 and 0.62 for the populations from Switzerland and Australia, respectively. Variable levels of polymorphism make these ideal markers for population genetic analyses.     

Evolution of resistance to Rhynchosporium secalis (Oud.) Davis in barley composite cross II

Summary Changes in resistance to scald disease which occurred in barley composite Cross II over 45 generations were analyzed genetically. This population, which was synthesized in 1929 by pooling equal numbers of f₁ seeds from 378 pair wise crosses among 28 barley varieties, has subsequently been grown at Davis, California under standard agricultural conditions without conscious selection. Progenies derived from self-pollinated seeds from random plants taken from four generations (F₈, F₁₃, F₂₃, and F₄₅) were tested against four different races of scald (40, 61, 72, and 74), and rated as resistant, susceptible or segregating. Striking increases in the frequency of families resistant to races 40, 61, and 74 occurred in CC II. A test for randomness showed that quadruply susceptible and triply resistant families were more common than expected under the assumption that resistance to different races is independent. Positive correlations were found between resistance to races 40, 61, and 74, but resistance to race 72 was independent of resistance to all other races. Possible reasons for these correlations are discussed.     

Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up.     

Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis.

NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation. In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor. The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture. After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity.     

The origin and colonization history of the barley scald pathogen Rhynchosporium secalis

The origins of pathogens and their past and present migration patterns are often unknown. We used phylogenetic haplotype clustering in conjunction with model-based coalescent approaches to reconstruct the genetic history of the barley leaf pathogen Rhynchosporium secalis using the avirulence gene NIP1 and its flanking regions. Our results falsify the hypothesis that R. secalis emerged in association with its host during the domestication of barley 10,000 to 15,000 years ago in the Fertile Crescent and was introduced into Europe through the migration of Neolithic farmers. Estimates of time since most recent common ancestor (2500-5000 BP) placed the emergence of R. secalis clearly after the domestication of barley. We propose that modern populations of R. secalis originated in northern Europe following a host switch, most probably from a wild grass onto cultivated barley shortly after barley was introduced into northern Europe. R. secalis subsequently spread southwards into already established European barley-growing areas.     

Dispersal of Rhynchosporium secalis conidia from infected barley leaves or straw by simulated rain

Simulated rain (mean drop diameter c. 1 or 3 mm) was allowed to fall for 10 – 15 min on to barley leaves or straw infected by Rhynchosporium secalis (leaf blotch). The leaves were supported on a mesh through which run-off water drained and the straw was supported on a rigid surface on which run-off water collected. The numbers of R. secalis conidia and spore-carrying splash droplets collected by horizontal samplers (microscope slides and pieces of photographic film) decreased rapidly with increasing distance from and increasing height above the sources, with half-distances of 2 – 10 cm. Less than 10% of the spores or droplets reached heights of more than 30 cm. Incident drops 3 mm in diameter produced more spore-carrying droplets and dispersed more conidia than did 1 mm drops. The size category of splash droplets with the greatest proportion of the spore-carrying droplets dispersed by 3 mm drops was 200 – 400 μm, whether the source was infected barley leaves or barley straw. For leaves or straw the greatest proportions of spores were carried in droplets > 1000 μm in diameter. The mean diameter of spore-carrying droplets (478 μm) dispersed from free-draining leaves was less than that of droplets from straw plus run-off water (563 μm). However, the leaf source had more spores cm^-2 and the mean number of spores per droplet was greater (113 as opposed to 6·8) than for the straw source.     

Metabolism of Carbohydrates by Pasteurella pseudotuberculosis

Cell-free extracts of Pasteurella pseudotuberculosis and P. pestis catalyzed a rapid and reversible exchange of electrons between pyridine nucleotides. Although the extent of this exchange approximated that promoted by the soluble nicotinamide adenine dinucleotide (phosphate) transhydrogenase of Pseudomonas fluorescens, the reaction in the pasteurellae was associated with a particulate fraction and was not influenced by adenosine-2'-monophosphate. The ability of P. pseudotuberculosis to utilize this system for the maintenance of a large pool of nicotinamide adenine dinucleotide phosphate could not be correlated with significant participation of the Entner-Doudoroff path or catabolic use of the hexose-monophosphate path during metabolism of glucose. As judged by the distribution of radioactivity in metabolic pyruvate, glucose and gluconate were fermented via the Embden-Meyerhof and Entner-Doudoroff paths, respectively. With the exception of hexosediphosphatase, all enzymes of the three paths were detected, although little or no gluconokinase or phosphogluconate dehydrase was present unless the organisms were cultivated with gluconate. The significance of these findings is discussed with respect to the regulation of carbohydrate metabolism in the pasteurellae, related enteric bacteria, and P. fluorescens.     

Cooperative Anti-Diabetic Effects of Deoxynojirimycin-Polysaccharide by Inhibiting Glucose Absorption and Modulating Glucose Metabolism in Streptozotocin-Induced Diabetic Mice

We had previously shown that deoxynojirimycin-polysaccharide mixture (DPM) not only decreased blood glucose but also reversed the damage to pancreatic β-cells in diabetic mice, and that the anti-hyperglycemic efficacy of this combination was better than that of 1-deoxynojirimycin (DNJ) or polysachharide alone. However, the mechanisms behind these effects were not fully understood. The present study aimed to evaluate the therapeutic effects of DPM on streptozotocin (STZ)-induced diabetic symptoms and their potential mechanisms. Diabetic mice were treated with DPM (150 mg/kg body weight) for 90 days and continued to be fed without DPM for an additional 30 days. Strikingly, decrease of blood glucose levels was observed in all DPM treated diabetic mice, which persisted 30 days after cessation of DPM administration. Significant decrease of glycosylated hemoglobin and hepatic pyruvate concentrations, along with marked increase of serum insulin and hepatic glycogen levels were detected in DPM treated diabetic mice. Results of a labeled ¹³C₆-glucose uptake assay indicated that DPM can restrain glucose absorption. Additionally, DPM down-regulated the mRNA and protein expression of jejunal Na⁺/glucose cotransporter, Na⁺/K⁺-ATPase and glucose transporter 2, and enhanced the activities as well as mRNA and protein levels of hepatic glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylas E1). Activity and expression of hepatic gluconeogenesis enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) were also found to be attenuated in diabetic mice treated with DPM. Purified enzyme activity assays verified that the increased activities of glucose glycolysis enzymes resulted not from their direct activation, but from the relative increase in protein expression. Importantly, our histopathological observations support the results of our biochemical analyses and validate the protective effects of DPM on STZ-induced damage to the pancreas. Thus, DPM has significant potential as a therapeutic agent against diabetes.     

Remodeling of Oxidative Energy Metabolism by Galactose Improves Glucose Handling and Metabolic Switching in Human Skeletal Muscle Cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [¹⁴C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [¹⁴C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.     

The relationship between leaf blotch caused by Rhynchosporium secalis and losses in grain yield of spring barley

Two methods were used to investigate the loss in grain yield associated with specific levels of leaf blotch. Yields from plots sprayed with fungicide were compared with those from unsprayed plots and yields of varieties of different susceptibility to the disease were compared with one another. A disease assessment key is presented, which was used to assess the percentage laminar area of the top two leaves affected by the disease. A linear relationship between disease on the upper two leaves and yield was established. Results from nine trials showed a consistent relationship between the disease level, at growth stage 11·1 (Feekes scale), and loss in yield. The loss in yield expressed as a percentage of the yield of an uninfected crop was equivalent to approximately two-thirds of the percentage of the flag-leaf area visibly infected, or one-half of the infected area on the second leaf. The predicted loss in yield is the average of these two estimates.