Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their abilities for the production of β-fructofuranosidase. Three of them, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyltransferase activity higher than 12,500 units l⁻¹. In addition, the β-fructofuranosidases of those strains have a high fructosyltransferase activity-to-hydrolytic activity ratio. The temperature and pH effects on the sucrose-β-fructofuranosidase reaction rate were studied using a 2² factorial experimental design. The comparative analysis of the tested variable coefficients shows that the variable pH contributes mostly to the changes in the fructosyltransferase and hydrolytic rates and in the V _t/V _h ratio. At 40 and 50°C, there were no significant differences between the fructosyltransferase and hydrolytic velocities of these enzymes.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Identification, cloning and analysis of theAspergillus niger genepacC, a wide domain regulatory gene responsive to ambient pH

A wide domain regulatory gene implicated in modulating gene expression in response to ambient pH has been cloned and sequenced from the industrially useful filamentous fungusAspergillus niger. This gene,pacC, is able to restore apacC ⁺ phenotype toA. nidulans pacC ^c 11 andpacC ^c 14 mutants with respect to extent of conidiation, conidial pigment intensity and acid phosphatase regulation. ThepacC gene ofA. niger comprises three exons, encodes a three-zinc-finger protein of 677 amino acids, and shows pH-dependent regulation of expression: mRNA levels are elevated under alkaline conditions and considerably reduced under acidic conditions. The occurrence of PacC consensus binding targets within the sequences upstream ofpacC may indicate autoregulation.     

Characteristics of an ideotype acid tolerant pasture legume symbiosis in Mediterranean agriculture

A series of experiments has led to the following concept of eight characteristics being required in an acid-tolerant pasture legume symbiosis for use in ley-farming: In the bulk soil i -microsymbiont (preferably of the Bradyrhizobium genus) capable of maintaining high numbers into autumn, through processes which allow saprophytic function at low pH such as regulation of its internal pH, ii -microsymbiont with a carboxylated cell surface electrochemistry stable under the influence of ambient pH in its interactions with soil colloids, minerals and root surfaces. In the rhizosphere iii -microsymbiont capable of appreciable growth in response to substrate availability, iv icrosymbiont able to recognise root exudates allowing interaction with its nodD gene protein, v -microsymbiont whose nodABC gene products (nod metabolites) are pH stable and which induce cortical cell division and root-hair curling in the host, vi -microsymbiont whose surface polysaccharides and proteins are pH stable to allow attenuation of the cell at the root surface, At the root surface vii-host apoplast function unaffected by low pH such that it may (a) produce pH stable exudates capable of interacting with the rhizobial nodD gene protein and (b) receive rhizobial nod metabolites and respond physiologically, viii-root able to produce pH stable organic acids for linkage with rhizobial cell surface structures.These ideotype characteristics reflect our current understanding of the mechanisms of acid tolerance in the nodulation phase of species such as Medicago polymorpha and M. murex, and acid soil tolerance in rhizobial inoculant strains such as WSM540.     

Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

Optimization of cellulase production in batch fermentation byTrichoderma reesei

Maximum cellulase production was sought by comparing the activities of the cellulases produced by differentTrichoderma reesei strains andAspergillus niger. Trichoderma reesei Rut-C30 showed higher cellulase activity than otherTrichoderma reesei strains andAspergillus niger that was isolated from soil. By optimizing the cultivation condition during shake flask culture, higher cellulase production could be achieved. The FP (filter paper) activity of 3.7 U/ml and CMCase (Carboxymethylcellulase) activity of 60 U/ml were obtained from shake flask culture. When it was grown in 2.5L fermentor, where pH and DO levels are controlled, the Enzyme activities were 133.35 U/ml (CMCase) and 11.67 U./ml (FP), respectively. Ammonium sulfate precipitation method was used to recover enzymes from fermentation broth. The dried cellulase powder showed 3074.9 U/g of CMCase activity and 166.7 U/g of FP activity with 83.5% CMCase recovery.     

Isolation and Sequencing of a New Glucoamylase Gene from an Aspergillus niger Aggregate Strain (DSM 823) Molecularly Classified as Aspergillus tubingensis

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92% An erratum to this article is available at .     

A Survey on Distribution of Aspergillus Section Flavi in Corn Field Soils in Iran: Population Patterns Based on Aflatoxins, Cyclopiazonic Acid and Sclerotia Production

Soil isolates of Aspergillus section Flavi from Mazandaran and Semnan provinces with totally different climatic conditions in Iran were examined for aflatoxins (AFs; B and G types), cyclopiazonic acid (CPA) and sclerotia production. A total of 66 Aspergillus flavus group strains were identified from three species viz. Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius in both locations. A. flavus (87.9%) was found to be the prominent species followed by A. nomius (9.1%) and A. parasiticus (3.0%). Only 27.5% of A. flavus isolates were aflatoxigenic (B₁ or B₁ and B₂), out of which approximately 75% were capable to producing CPA. All the A. parasiticus and A. nomius isolates produced AFs of both B (B₁ and B₂) and G (G₁ and G₂) types, but did not produce CPA. Sclerotia production was observed in only 4 isolates of A. flavus among all 66 isolates from three identified species. A. flavus isolates were classified into various chemotypes based on the ability to produce aflatoxins and CPA. In this study, a new naturally occurring toxigenic A. flavus chemotype comprising of two strains capable of producing more AFB₂ than AFB₁ has been identified. A relatively larger proportion of aflatoxigenic A. flavus strains were isolated from corn field soils of Mazandaran province which indicate a possible relationship between high levels of relative humidity and the incidence of aflatoxin-producing fungi. The importance of incidence of Aspergillus section Flavi in corn field soils regard to their mycotoxin production profiles and crop contamination with special reference to climatic conditions is discussed.     

Gene pacA+ codes for the multiple active forms of Pi-repressible acid phosphatase in the mould Aspergillus nidulans

The acid phosphatase secreted by the biA1 strain of the mould Aspergillus nidulans was separated into at least nine isoforms by isoelectric focusing (IEF). The components visualized by activity were predominantly acidic proteins with isoelectric points ranging from pH 4.0 to 6.5. Almost the same isoforms were secreted by strains pabaA1 and palD8 biA1. Furthermore, the isoforms secreted by strain pacA1 biA1 were not visualized by staining after IEF, indicating that these isoforms are encoded by gene pacA. Treatment of the secreted enzyme with endoglycosidase H also reduced the number of isoforms visualized by staining after IEF and enhanced the R_f (electrophoretic mobility) value of this enzyme visualized after PAGE.     

The Aspergillus nidulans phsB4 mutation alters colonial growth and development of the mould at acidic pH

The phsB4 mutant of the mould Aspergillus nidulans, identified as showing increased sensitivity to acid pH, is mitotically unstable and its conidia swell and lyse, forming protoplasts during germination and early development in shaken liquid cultures. On solid medium, we observed balloon-shaped hyphal swellings, a phenotype also exhibited by the chitin synthase gene (chsD) disruptants. We also observed that lysis was osmotically remediable with 0.5 M NaCl, but the balloon-shaped hyphal swelling was remedied in a pH-dependent way i.e., this phenotype was remedied only at pH values above 6.5. Based on the nature of our mutant selection, the pH sensitive phenotype of the selected strains, the known occurrence of hyphal swelling in cell wall mutants of A. nidulans, and the transformation with cosmids that hybridize to chsD gene, the phsB and chsD genes are possibly alleles.     

Microbiology of pozol,a Mexican fermented maize dough

Mexican fermented maize dough, pozol, including traditional banana leaf-wrapped samples and material in plastic bags, was purchased. All samples were pH 4.7 to 5.7 approx. 12 h after preparation, pH declining to 3.6 to 3.9 after 6 to 9 days storage at ambient temperature. These latter samples had dry matter contents of 31% to 48% (w/w), 0.35% to 0.75% titratable acidity as lactic acid and lactic acid bacteria as predominant microbial flora at about 10⁸ c.f.u./ml. The lactic acid bacteria included strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus confusus, Lactococcus lactis and Lactococcus raffinolactis. Fungi were not found in the samples stored in plastic bags. The samples wrapped in banana leaf, however, developed a large surface mycoflora within 2 days. This included Geotrichum candidum, yeasts and moulds. The majority of the lactic acid bacteria and approx. 50% of yeasts hydrolysed starch to some extent. No Geotrichum isolate hydrolysed starch. Lactate was assimilated by all the Geotrichum isolates and by 17 of 39 yeast strains.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Evaluation of biological control formulations to reduce aflatoxin contamination in peanuts

A two-year study was conducted to evaluate the efficacy of three formulations of nontoxigenic strains of Aspergillus flavus and Aspergillus parasiticus to reduce preharvest aflatoxin contamination of peanuts. Formulations included: (1) solid-state fermented rice; (2) fungal conidia encapsulated in an extrusion product termed Pesta; (3) conidia encapsulated in pregelatinized corn flour granules. Formulations were applied to peanut plots in 1996 and reapplied to the same plots in 1997 in a randomized design with four replications, including untreated controls. Analysis of soils for A. flavus and A. parasiticus showed that a large soil population of the nontoxigenic strains resulted from all formulations. In the first year, the percentage of kernels infected by wild-type A. flavus and A. parasiticus was significantly reduced in plots treated with rice and corn flour granules, but it was reduced only in the rice-treated plots in year two. There were no significant differences in total infection of kernels by all strains of A. flavus and A. parasiticus in either year. Aflatoxin concentrations in peanuts were significantly reduced in year two by all formulation treatments with an average reduction of 92%. Reductions were also noted for all formulation treatments in year one (average 86%), but they were not statistically significant because of wide variation in the aflatoxin concentrations in the untreated controls. Each of the formulations tested, therefore, was effective in delivering competitive levels of nontoxigenic strains of A. flavus and A. parasiticus to soil and in reducing subsequent aflatoxin contamination of peanuts.     

A mycological and bacteriological survey on feed ingredients and mixed poultry feeds in Reunion island

A survey was carried out in Reunion island to obtain data on the occurrence of fungi, aflatoxigenic strains of Aspergillus flavus, aflatoxins, total aerobic bacteria and salmonellae of 150 samples of mixed poultry feeds and raw materials. These were collected at five farms over a 3-month period during the warm rainy season.White corn and Brazilian soybean meal seemed to present a better microbiological quality than yellow corn and US soybean meal.Mixed poultry feeds presented a high total mold count reflecting the mold flora of raw materials. The most frequent and abundant fungi were Aspergillus flavus, A. glaucus group, Fusarium spp., Penicillium spp., A. candidus, Mucor spp., A. restrictus, Scopulariopsis spp., Cladosporium spp. and A. versicolor. Of the 118 A. flavus strains screened, 42 (35.6%) were aflatoxigenic. Yellow corn samples were the most frequently contamined with aflatoxigenic strains (54.5%), followed by mixed feeds (44%).Of the 66 samples tested, 24 (36%) contained aflatoxins (traces to 22 ng/g). A good correlation seemed to exist between presence of at least one aflatoxigenic strain per sample and presence of aflatoxins.     

Degradation and enzymatic activities of three Paecilomyces inflatus strains grown on diverse lignocellulosic substrates

This paper describes for the first time in detail the lignocellulose degradation system in Paecilomyces inflatus. The fungal genus Paecilomyces contributes the carbon turnover from lignin and carbohydrate plant residues, particularly in compost and soil environment, where basidiomycetes appear very seldom. We studied three different strains of P. inflatus, obtained from different ecophysiological and geographical origin. Various cultivation conditions were employed, and the chemical analysis of decayed straw, compost, birch and spruce wood chips indicated variable responses. Endoglucanase, xylanase and laccase were assayed. All strains of P. inflatus, regardless of their origin, altered the ambient pH in a similar manner in all investigated substrates, suggesting that all P. inflatus isolates may share the common regulatory system to control their environmental pH. The variability among strains of P. inflatus in their ability to remove lignocellulose components often was related to the nature of the substrate and the production of specific enzymes although it was not strictly correlated. This may implicate that other enzymes and/or even other parameters needed for lignocellulosics degradation in P. inflatus should be evaluated. Indications for specific adaptation strategies that may operate in P. inflatus were found.     

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity – comparable to that obtained with the common preservative calcium propionate (0.3% w/v) – of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.