Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Starch industry wastewater as a substrate for antagonist, Trichoderma viride production

Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia ( approximately 3.02 and 4.2 x 10(10)CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6 x 10(8) in control to 3.0 x 10(7)CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO(3) were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43 IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/microL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone.     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Production Factors Involved in the Formulation of Erynia neoaphidis as Alginate Granules

Granular formulations of the aphid-pathogenic fungus Erynia neoaphidis were produced by entrapping mycelia in alginate polysaccharide polymers. Four Swiss isolates were compared for the numbers of conidia discharged from the surface of alginate granules in standardized laboratory assays and two were considered to be suitable for further development. Conidiation was achieved from granules produced using nozzle diameters of 2.0. 1.0 and 0.5 mm from glass burettes or a novel vibrating tip apparatus. The mean diameters of dried granules varied from 0.5 to 1.8 mm. The addition of sucrose, potato starch or chitin in alginate solutions significantly improved the numbers of discharged conidia. W ith freshly produced granules, there was a 14.2- fold increase in sporulation from 6.3 to 89.7 conidia mm - 2 using 2% (w/v) sucrose. Increases of 1.6-to 2.3-fold, from 11.0 to 17.7 and 25.2 conidia mm - 2, were observed using 5% (w/v) starch or chitin respectively. The overnight drying of granules in a laminar flow hood and storage for 4 days at 4 C made differences in sporulation more obvious. There was a 15.5-fold difference in conidial numbers of 12.4 and 0.8 conidia mm - 2 from granules with and without sucrose respectively. For starch and chitin, there were 76.0-and 46.5-fold increases from 0.4 to 30.4 and 18.6 conidia mm - 2respectively. Fresh or dried alginate granules containing 2% sucrose and 5% starch gave 8.6-26.6% infection in laboratory bioassays with nymphs of pea aphid, Acyrthosiphon pisum , which were not significantly different when compared with infections of 6.7-22.9% using agar cultures or unsupplemented granules. Further studies on desiccation and storage regimes are required in order to improve the short-term shelf-life of E. neoaphidis alginate granules.     

Pathogenesis of barley leaves by Helminthosporium teres II: Carbohydrate involvement

Conidia of Helminthosporium teres had negligible difference in germination and germ tube length between the decolorized and non-decolorized host leaves. Appressoria, penetration and colonization were less on decolorized host leaves, but addition of exogenous nutrients stimulated these stages. Leached conidia had reduced germination on decolorized host leaves, while appressoria formation, penetration and colonization were negligible. The addition of nutrients in the external environment, however, enabled some of the leached conidia to penetrate and colonize. Stimulation by the exogenous nutrients in decreasing order were: sucrose > glucose > yeast extract > leaf leachates. Optimum levels for various nutrients tested were 2% (w/v) each of sucrose and glucose, and 0.1% (w/v) yeast extract. Higher concentrations inhibited these stages of infection. Leached conidia and decolorized leaves had smaller amounts of carbohydrates than non-leached conidia and non-decolorized leaves, respectively. Depletion of host carbohydrates reduced appressoria formation, penetration and colonization and loss of carbohydrates from spores reduced germination.     

Growth and metabolism of mannitol by strains of Saccharomyces cerevisiae

Of 40 polyploid strains of Saccharomyces cerevisiae screened for growth on D-mannitol (5%, w/v), half grew well (5-20 mg dry biomass ml-1). Certain of these strains were unable to grow on low concentrations of mannitol (1-2%, w/v) and others, initially unable to grow on mannitol, exhibited long-term adaptation to growth. An NAD+-dependent D-mannitol dehydrogenase (EC 1.1.1.67) was detected in mannitol-grown yeast. Growth was dependent on mitochondrial function and was obligately aerobic. Measurement of products of metabolism and respiratory activity indicated that growth on mannitol allows catabolite derepression.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes.

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.     

Synthesis of alkaline protease by catabolite repression- resistant Thermoactinomyces sp. E79 mutant

The production of alkaline protease from Thermoactinomyces sp. E79 was repressed by 0.2% (w/v) glucose in the medium. Catabolite repression-resistant mutant M1 was obtained by combined treatment of UV light and N-methyl- N-nitro-N-nitrosoguanidine. The glucose uptake studies accomplished by [14C]glucose showed that the mutant has lost its ability for glucose uptake. The protease production by mutant M1 in the enzyme production medium was 62 U/mg, which was twice that of the wild-type strain.     

Mass Production of Conidia of Penicillium frequentans,a Biocontrol Agent Against Brown Rot of Stone Fruits

Conidial production of Penicillium frequentans , a biocontrol agent of the fungal pathogen Monilinia laxa , was tested in liquid and solid-state fermentation. Conidial production of P. frequentans in solid-state fermentation was higher than in liquid-state fermentation. Solidstate fermentation was made in specially designed plastic bags (VALMIC ® ) containing peat:vermiculite (1:1 w/w). Addition of nutrients to the peat:vermiculite increased conidial production of P. frequentans , especially when lentil meal was added. The number of conidia obtained in this solid-state fermentation was maintained in the range of 10 8 -10 9 conidia g -1 from 5 to 120 days after inoculation. Germinability of these conidia was > 90% until 90 days of incubation and declined at 120 days. Optimal initial moisture content in the substrate was 30-40% (v/w). At lower moisture contents, significant reductions in conidial production and germinability were observed, particularly at 10% (v/w). Conidial production was similar when the substrate was inoculated with 10 5 , 10 6 or 10 7 conidia g -1 dry substrate. Fresh conidia produced by solid-state fermentation reduced the incidence of brown rot on plums by 75%.     

Citric acid production using immobilized conidia of Aspergillus niger TMB 2022

Conidia of Aspergillus niger TMB 2022 were immobilized in calcium alginate for the production of citric acid. A 1-mL conidia suspension containing ca. 2.32 x 10(8) conidia were entrapped into sodium alginate solution in order to prepare 3% Ca-alginate (w/v) gel bead. Immobilized conidia were inoculated into productive medium containing 14% sucrose, 0.25% (NH(4))(2)CO(3), 0.25% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O with addition of 0.06 mg/L CuSO(4).5H(2)O, 0.25 mg/L ZnCl(2), 1.3 mg/L FeCl(3).6H(2)O, pH 3.8, and incubated at 35 degrees C for 13 days by surface culture to produce 61.53 g/L anhydrous citric acid. Under the same conditions with a batchwise culture, it was found that immobilized conidia could maintain a longer period for citric acid production (31 days): over 70 g/L anhydrous citric acid from runs No. 2-4, with the maximum yield for anhydrous citric acid reaching 77.02 g/L for run No. 2. In contrast, free conidia maintained a shorter acid-producing phase, ca. 17 days; the maximum yield for anhydrous citric acid was 71.07 g/L for run No. 2 but dropped quickly as the run number increased.     

Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities

Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml⁻¹, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Characterization and control of thread mould in cheese

AIMS: The origin of a mould responsible for the contamination of an Argentinian cheese factory was identified and several antifungal treatments were assessed. METHODS AND RESULTS: Moulds were isolated and identified from vacuum-packed hard cheeses, from the environment and from the surfaces of the factory. A suspension conidia test containing different fungicides was performed; another assay involved the fumigation with p-OH fenilsalicidamide. Only Phoma glomerata was found in all of the mouldy cheeses, and was also obtained from different environments and machine surfaces. The most effective treatments against P. glomerata isolates were 0.5% (w/v) natamycin and 2% (v/v) parabens. Fumigation with p-OH fenilsalicidamide showed no satisfactory results. CONCLUSION: P. glomerata is an important thread mould-contaminating agent in vacuum-packed hard cheeses. Significance and Impact of the Study: Taking into account the survival of the conidia of the P. glomerata isolates to different antifungal treatments, the sources of contamination need to be controlled by designing a good factory layout.     

Isoelectric characterization of extracellular polygalacturanases of various Aspergillus alliaceus strains

The composition of pectin hydrolase complexes produced by various Aspergillus alliaceus strains was studied under the conditions of induction, catabolite repression, or constitutive synthesis. The strains were found similar in terms of the polygalacturonase spectrum and different with regard to the levels of endo- and exoenzyme activities. The analysis of the zymograms of inducible polygalacturonases revealed that all tested cultures contained at least 24 molecular forms of polygalacturonase. Taking into account only the three molecular forms typical of all analyzed strains of A. alliaceus with pI values of 5.7, 5.9, and 6.3, one can use the spectrum of constitutive, catabolite repression-resistant polygalacturonases as an additional taxonomic species criterion.     

Isoelectrophoretic characterization of extracellular polygalacturonases of variousAspergillus alliaceus strains

The composition of pectin hydrolase complexes produced by variousAspergillus alliaceus strains was studied under the conditions of induction, catabolite repression, or constitutive synthesis. The strains were found similar in terms of the polygalacturonase spectrum and different with regard to the levels of endo- and exoenzyme activities. The analysis of the zymograms of inducible polygalacturonases revealed that all tested cultures contained at least 24 molecular forms of polygalacturonase. Taking into account only the three molecular forms typical of all analyzed strains ofA. alliaceus with pI values of 5.7, 5.9, and 6.3, one can use the spectrum of constitutive, catabolite repression-resistant polygalacturonases as an additional taxonomic species criterion.     

Multinucleation in the conidia of polyploids derived fromTrichoderma reesei QM 9414 by colchicine treatment

Conidia ofTrichoderma reesei QM 9414 were treated with colchicine in order to obtain polyploids (diploids; tetraploids). Cellulase production by diploids (mononucleate conidia) was almost twice as great as that of the original strain, but that of tetraploids (binucleate conidia) was not increased. When these latter conidia were re-treated with 2.0% (w/v) colchicine, multiple nuclei were produced in each conidium, and their diameter was almost the same as that of the original nucleus. Cellulase production of the diploid was almost the same in either mononucleate or multinucleate nature. However, cellulase production by the tetraploid which produced multinucleate conidia was greater than that of the binucleate tetraploid and that of the diploid. The multinucleation technique can contribute to enhancing cellulase production.     

Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.