Pathogenicity Determinants of Sclerotinia sclerotiorum and Their Association to Its Aggressiveness on Brassica juncea

White rot or stem rot caused by Sclerotinia sclerotiorum is one of the most destructive fungal diseases that have become a serious threat to the successful cultivation of oilseed Brassicas. The study was designed with an aim to investigate the association between the pathogenic aggressiveness and pathogenicity determinants of this pathogen specifically in Brassica for the first time. For this, a total of 58 isolates of S. sclerotiorum from different geographical regions were collected and purified. These isolates were inoculated on a Brassica juncea cv. RL-1359 and they exhibited high level of variation in their disease progression. The isolates were grouped and then 24 isolates were selected for the biochemical analysis of pathogenicity determinants. The isolates varied significantly with respect to their total organic acids, oxalic acid production and pectin methyl esterase and polygalacturonase activity. The oxalic acid production corresponded to the disease progression of the isolates; the isolates with higher oxalic acid production were the more aggressive ones and vice-versa. This is, in our knowledge, the first study to establish a correlation between oxalic acid production and pathogenic aggressiveness of S. sclerotiorum on B. juncea. However, the pectinases’ enzyme activity did not follow the trend as of disease progression. These suggest an indispensable role of oxalic acid in pathogenicity of the fungus and the potential to be used as biochemical marker for preliminary assessment of pathogenic aggressiveness of various isolates before incorporating them in a breeding program.     

Enzymatic oxalate decarboxylation in isolates of Sclerotinia sclerotiorum

Abstract Sclerotinia sclerotiorum isolates B24 (virulent) and SS41 (hypovirulent) possess oxalate decarboxylase. Production was regulated by composition and pH of culture medium and required the presence of oxalate or its precursor, succinic acid, as inducers. Mycelia of both isolates contain equivalent amounts of enzyme.     

Oxalic acid,versatile peroxidase secretion and chelating ability of <Emphasis Type="Italic">Bjerkandera fumosa</Emphasis> in rich and limited culture conditions

Efficient ligninolytic systems of wood-degrading fungi include not only oxidizing enzymes, but also low-molecular-weight effectors. The ability of Bjerkandera fumosa to secrete oxalic acid and versatile peroxidase (VP) in nitrogen-rich and nitrogen-limited media was studied. Higher activity of VP was determined in the nitrogen-limited media but greater concentration of oxalic acid was observed in the cultures of B. fumosa without nitrogen limitation. Ferric ions chelating ability of Bjerkandera fumosa studied in ferric ions limited media was correlated with the increased level of oxalic acid. The presence of hydroxamate-type siderophores in B. fumosa media were also detected. Oxalate decarboxylase was found to be responsible for regulation of oxalic acid concentration in the tested B. fumosa cultures.     

Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles

Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3-4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos). At the end of culture, there were more (P less than 0.001) morulae (greater than or equal to 16 cells) in B2SS X TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with less than 8 cells (30% vs 15% in B2SS, P greater than 0.05; 70% vs 41% in B2SS + TV, P less than 0.005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf). It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.     

Oxalate-degrading bacteria can protect Arabidopsis thaliana and crop plants against botrytis cinerea

Botrytis cinerea and Sclerotinia sclerotiorum secrete oxalic acid as a pathogenicity factor with a broad action. Consequently, it should be possible to interfere with the infection process by degrading oxalic acid during the interaction of these pathogens with their hosts. We have evaluated the potential of oxalate-degrading bacteria to protect plants against pathogenic fungi. Such bacteria were isolated from agricultural soil and selected on agar plates with Ca-oxalate as the sole carbon source. Four strains were retained with a medium-to-strong protective activity on Arabidopsis thaliana leaves against B. cinerea and S. sclerotiorum. They can provide 30 to 70% protection against fungal infection in different pathosystems, including B. cinerea on A. thaliana, cucumber, grapevine, and tomato. The oxalate-degrading bacteria induced only some marker genes for common plant signaling pathways for defenses, but protective effects were slightly reduced in A. thaliana mutants impaired in the ethylene and jasmonic acid signaling pathways. More detailed studies on the protective mechanism were performed in ox-strain B, identified as Cupriavidus campinensis, by analysis of transposon-tagged mutants that have a reduced ability to degrade oxalic acid.     

High-yield production of oxalic acid for metal leaching processes by Aspergillus niger

Abstract The complex-forming compound oxalic acid can effectively solubilise metals such as aluminium, iron, lithium, and manganese. In order to produce high amounts of oxalic acid for biohydrometallurgical processes, it was the aim of this work to optimise oxalic acid production by Aspergillus niger , a fungus well known for its ability to produce oxalic acid. A. niger excreted 427 mmol oxalic acid 1⁻¹ if it was cultivated in a pH-controlled (pH 6.0) fed-batch run in a 2-1 stirred tank reactor. Sucrose and lactose permeate were suitable carbon sources for oxalic acid production. In sucrose medium, A. niger produced high amounts of gluconic and oxalic acids, whereas in lactose permeate medium only oxalic acid was produced. Cultivation in green syrup and molasses media lead to high yields of biomass, but low oxalic acid production (<20 mmol 1⁻¹).     

Competitiveness of different polysaccharide utilization mutants of Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice

Bacteroides thetaiotaomicron, an obligate anaerobe found in high numbers in human colons, can utilize a variety of polysaccharides. To determine which type of polysaccharide contributes most to the nutrition of B. thetaiotaomicron in vivo, we isolated and characterized transposon-generated mutants deficient in the ability to use different polysaccharides. Some mutants were deficient in polysaccharide utilization because of the inability to utilize a component monosaccharide. These mutants included a mutant that was unable to utilize L-fucose (a component of goblet cell mucin), a mutant that was unable to utilize D-galactose (a component of raffinose, stachyose, arabinogalactan, and goblet cell mucin), and a mutant that was unable to utilize either glucuronic acid (a component of mucopolysaccharides) or galacturonic acid (a component of polygalacturonic acid or pectin). Other mutants were unable to use the polysaccharide but could use the component sugars. These included four mutants that were unable to utilize starch and one mutant that was unable to utilize polygalacturonic acid. The mutants were tested for the ability to compete with the wild type for colonization of the intestinal tracts of germfree mice. The only mutants against which the wild type competed successfully in the intestinal tracts of germfree mice were a galactose-negative mutant and a uronic acid-negative mutant. These mutations differed from the others tested in that they affected utilization of more than one type of polysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitiveness of different polysaccharide utilization mutants of Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice.

Bacteroides thetaiotaomicron, an obligate anaerobe found in high numbers in human colons, can utilize a variety of polysaccharides. To determine which type of polysaccharide contributes most to the nutrition of B. thetaiotaomicron in vivo, we isolated and characterized transposon-generated mutants deficient in the ability to use different polysaccharides. Some mutants were deficient in polysaccharide utilization because of the inability to utilize a component monosaccharide. These mutants included a mutant that was unable to utilize L-fucose (a component of goblet cell mucin), a mutant that was unable to utilize D-galactose (a component of raffinose, stachyose, arabinogalactan, and goblet cell mucin), and a mutant that was unable to utilize either glucuronic acid (a component of mucopolysaccharides) or galacturonic acid (a component of polygalacturonic acid or pectin). Other mutants were unable to use the polysaccharide but could use the component sugars. These included four mutants that were unable to utilize starch and one mutant that was unable to utilize polygalacturonic acid. The mutants were tested for the ability to compete with the wild type for colonization of the intestinal tracts of germfree mice. The only mutants against which the wild type competed successfully in the intestinal tracts of germfree mice were a galactose-negative mutant and a uronic acid-negative mutant. These mutations differed from the others tested in that they affected utilization of more than one type of polysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of 1,4-dioxane and cyclic ethers by an isolated fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d8 and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

A type III effector protease NleC from enteropathogenic Escherichia coli targets NF-κB for degradation

Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP-NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His(6) -NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation.     

Effect of Macerase, Oxalic Acid, and EGTA on Deep Supercooling and Pit Membrane Structure of Xylem Parenchyma of Peach

The object of this study was to determine if calcium cross-linking of pectin in the pit membrane of xylem parenchyma restricts water movement which results in deep supercooling. Current year shoots of `Loring' peach (Prunus persica) were infiltrated with oxalic acid or EGTA solutions for 24 or 48 hours and then either prepared for ultrastructural analysis or subjected to differential thermal analysis. The effect of 0.25 to 1.0% pectinase (weight/volume) on deep supercooling was also investigated. The use of 5 to 50 millimolar oxalic acid and pectinase resulted in a significant reduction (flattening) of the low temperature exotherm and a distinct swelling and partial degradation of the pit membrane. EGTA (10 millimolar) for 24 or 48 hours shifted the low temperature exotherm to warmer temperatures and effected the outermost layer of the pit membrane. A hypothesis is presented on pectin-mediated regulation of deep supercooling of xylem parenchyma.     

Degradation of 1,4-Dioxane and Cyclic Ethers by an Isolated Fungus

By using 1,4-dioxane as the sole source of carbon, a 1,4-dioxane-degrading microorganism was isolated from soil. The fungus, termed strain A, was able to utilize 1,4-dioxane and many kinds of cyclic ethers as the sole source of carbon and was identified as Cordyceps sinensis from its 18S rRNA gene sequence. Ethylene glycol was identified as a degradation product of 1,4-dioxane by the use of deuterated 1,4-dioxane-d₈ and gas chromatography-mass spectrometry analysis. A degradation pathway involving ethylene glycol, glycolic acid, and oxalic acid was proposed, followed by incorporation of the glycolic acid and/or oxalic acid via glyoxylic acid into the tricarboxylic acid cycle.     

Transketolase mutation in riboflavin-synthesizing strains of Bacillus subtilis

Unlike its predecessors B. subtilis rosR and 41, riboflavin producing B. subtilis 24 strain does not utilize pentose and gluconate and poorly assimilates glucose. Simultaneous addition of glutamic and shikimic acid restored its capacity to grow and produce riboflavin in medium with pentose and gluconate. This strain lacks the activity of transketolase, the key enzyme of the pentose phosphate cycle, and possesses normal ribulose-5-phosphate-epimerase and glucose phosphate isomerase activities. Like enterobacteria, B. subtilis has two different transport systems for glucose and mannose. The data are discussed from the viewpoint of increasing riboflavin production by transketolase mutants. Probable consequences of cell wall and cytoplasmatic membrane damage in B. subtilis with this mutation are discussed.     

The effect of acetylsalicylic acid and oxalic acid on Myzus persicae and Aphidius colemani

Plants can respond to damage by pests with both induced direct defences and indirect defences by the attraction of their natural enemies. Foliar application of several plant-derived chemicals, such as salicylic acid and oxalic acid, can induce these defence mechanisms. The effect of acetylsalicylic acid and oxalic acid on the aphid Myzus persicae Sulzer (Homoptera: Aphididae) and its parasitoid Aphidius colemani Viereck (Hymenoptera: Aphidiidae) was investigated. Experiments were carried out with direct application of acetylsalicylic and oxalic acids on these insects, as well as choice and no-choice tests using foliar application of both chemicals on Brussels sprouts plants, Brassica oleracea var. gemmifera L. (Brassicaceae). Parasitoids were given a choice between treated and untreated plants for oviposition, and the effects of the chemicals on aphid and parasitoid development were determined. Although direct application of both chemicals increased aphid mortality, their foliar application did not induce resistance against aphids. The foliar application of such compounds, even in low concentration as shown in the choice tests, has the potential to induce indirect plant defences against aphids by encouraging aphid parasitisation. Although the direct application of both chemicals reduced parasitoid emergence from their hosts, the foliar application of acetylsalicylic acid and low concentrations of oxalic acid did not have a negative effect on parasitoid emergence ability. However, 10 m m oxalic acid reduced the number of emerged parasitoids in no-choice experiments. This study shows that foliar application of acetylsalicylic and oxalic acids has the potential to encourage aphid parasitisation, but care is needed as high concentrations of oxalic acid can have a negative effect on these beneficial organisms.     

Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, Gi function, and somatostatin receptors in rat exocrine pancreas.

To analyze the effect of bombesin on the somatostatin (SS) mechanism of action in the exocrine pancreas, male Wistar rats (250-270 g) were injected intraperitoneally with bombesin (10 microg/kg) three times daily at 8-h intervals for 7 or 14 days. Bombesin attenuated the ability of SS to inhibit forskolin-stimulated adenylyl cyclase activity in pancreatic acinar membranes. However, it did not decrease the ability of forskolin to stimulate the adenylyl cyclase catalytic subunit. The ability of 5'-guanylylimidodiphosphate [Gpp(NH)p] (a nonhydrolyzable GTP analog) to inhibit forskolin-stimulated adenylyl cyclase activity was diminished in pancreatic acinar cell membranes from bombesin-treated rats. Bombesin administration did not affect the ADP-ribosylation of a 41-kDa G protein catalyzed by pertussis toxin. The maximal SS binding capacity of pancreatic acinar membranes from bombesin-treated rats was decreased when compared with controls at the two time periods studied. The bombesin/gastrin-releasing peptide antagonist [D-Tpi6,Leu13psi(CH2NH)Leu14]bombesin (6-14) (RC-3095) (10 microg/kg i.p.), injected three times daily at 8-h intervals for 7 or 14 days, had a similar effect to that of bombesin on the SS mechanism of action. The combined administration of bombesin and its antagonist RC-3095 had a greater effect on the SS receptor-effector system than when administered separately. The present study indicates that the pancreatic SS receptor-effector system may be regulated by bombesin in vivo.     

Colonization and bioweathering of monazite by Aspergillus niger: solubilization and precipitation of rare earth elements

Geoactive fungi play a significant role in bioweathering of rock and mineral substrates. Monazite is a phosphate mineral containing the rare earth elements (REE) cerium, lanthanum and neodymium. Little is known about geomicrobial transformations of REE-bearing minerals which are also relevant to REE biorecovery from terrestrial and extra-terrestrial reserves. The geoactive soil fungus Aspergillus niger colonized monazite in solid and liquid growth media without any apparent growth inhibition. In a glucose-minerals salts medium, monazite enhanced growth and mycelium extensively covered rock particle surfaces, probably due to the provision of phosphate and essential trace metals. Teeth-like and pagoda-like etching patterns indicated monazite dissolution, with extensive precipitation of secondary oxalate minerals. Biomechanical forces ensued causing aggressive bioweathering effects by tunnelling, penetration and splitting of the ore particles. High amounts of oxalic acid (~46 mM) and moderate amounts of citric acid (~5 mM) were produced in liquid media containing 2% (wt./vol.) monazite, and REE and phosphate were released. Correlation analysis suggested that citric acid was more effective than oxalic acid in REE mobilization, although the higher concentration of oxalic acid also implied complexant activity, as well as the prime role in REE-oxalate precipitation.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Determination of oxalic acid in urine by high-performance liquid chromatography

A new method for determination of oxalic acid in urine is described. The method encloses sample purification prior to the treatment by High Performance Liquid Chromatography (HPLC). The purification step consists in the passage of acidified urine through Sep-pak C18 cartridge (Waters), followed by the precipitation of the oxalic acid eluted with CaCl2, new dilution of the calcium oxalate precipitate, oxalic acid extraction with diethyl ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm, with a retention time of 3.20 minutes. The method shows a mean recovery value of 98.25%, with an intra-run and between-run values of 5.13 and 8.06 respectively. The oxalic acid measured in urine by this method is 35.52 +/- 9.42 mg/24 h in normal subjects.     

Organic Acid Production by Basidiomycetes: I. Screening of Acid-Producing Strains

Sixty-seven strains belonging to 47 species of Basidiomycetes were examined for their acid-producing abilities in glucose media, in both the presence and absence of CaCO₃, in stationary and shake cultures. Some strains were found to produce large quantities of oxalic acid. The oxalic acid-producing strains could be separated into two groups. Strains of one group (mostly brown-rot fungi) were able to produce oxalic acid, regardless of whether CaCO₃ was present in the medium. Strains of the other group (mostly white-rot fungi) were characterized by their ability to produce oxalic acid only when CaCO₃ was added to the medium. With the latter group, shake-culturing was generally more effective than stationary culturing in respect to acid production. In the CaCO₃-containing media, Schizophyllum commune, Merulius tremellosus, and Porodisculus pendulus were found to produce substantial amounts of L-malic acid as a main metabolic product, along with small quantities of oxalic and other acids in shake cultures. Especially, S. commune and M. tremellosus may be employed as malic acid-producing species.