Comparative analysis of different TaqMan real-time RT-PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control

Aims:  The aim of this study was to compare the performance of four TaqMan RT-PCR assays with a commonly used nested RT-PCR and to include the Feline calicivirus (FCV) as an internal control.
Methods and Results:  RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3·2 × 10³ PFU of FCV. Detection results obtained on faecal and plasma samples were 35·6% and 4·9% with the nested RT-PCR assay, 8·0% and 0%, 0% and 0%, 13·8% and 0% and 36·8% and 3·9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30·11 and 30·43 for the detection of FCV with HEV contaminated samples and negative samples.
Conclusions:  The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control.
Significance and Impact of the Study:  FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.     

Polyaluminum chloride-enhanced concentration efficiency of poliovirus and f2 phage from sewage water

Aims:  To enhance the recovery of f₂ bacteriophage and poliovirus by an established method based on the adsorption to and elution from positively-charged Al(OH)₃-treated silica gel.
Methods and Results:  Polyaluminum Chloride (PAC) was added to water samples to neutralize the negatively charged materials, which can reduce virus recovery by providing a competing adsorption mode on the media surface. Using this improved process (PAC 30 mg l⁻¹, pH 6·5, temperature 20∼30°C), the recoveries of Poliovirus I and f₂ from small-volume sewage (100 ml) were 110·76 ± 36·0% and 92·06 ± 8·65%, respectively ( P  < 0·05 vs. traditional methods). Recovery from a 20-L volume of sewage averaged 85·65 ± 4·43% for f₂ and 88·73 ± 9·76% for poliovirus, significantly higher than the recoveries in the traditional methods ( P  < 0·05).
Conclusions:  PAC could enhance concentration efficiency of poliovirus and f₂ phage from sewage water.
Significance and Impact of the Study:  This method should significantly improve the recovery of viruses from sewage.     

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp

Aims:  To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp.
Methods and Results:  Alicyclobacillus acidoterrestris was grown in synthetic medium at 40°C and pH 4·0. The effect on vegetative cells was assayed by adding bovicin HC5 to synthetic medium (40–160 AU ml⁻¹) or to mango pulp (100 AU ml⁻¹) at various pH values and determining the effect on growth (OD_600nm) and viable cell number, respectively. The effect of bovicin HC5 on spore germination and thermal sensitivity of A. acidoterrestris was tested in mango pulp (pH 4·0) containing 80 AU ml⁻¹ of bovicin HC5. Bovicin HC5 was bactericidal against vegetative cells of A. acidoterrestris at different pH values and showed sporicidal activity against endospores of this bacterium. When spores of A. acidoterrestris were heat treated in the presence of bovicin HC5, D -values decreased 77% to 95% compared to untreated controls at temperatures ranging from 80 to 95°C.
Conclusion:  Bovicin HC5 was bactericidal and sporicidal against A. acidoterrestrsi DSMZ 2498.
Significance and Impact of the Study:  These results indicated that bovicin HC5 has potential to prevent spoilage of acidic fruit juices by thermocidophilic spore-forming bacteria.     

Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design

Aims:  The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp . , using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained.
Methods and Results:  Lipase activity values of 9·5 U ml⁻¹ in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l⁻¹) of 20·0, 5·0, 5·0 and of 10·0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4·9 to 5·5 and temperature from 37°C to 42°C. The crude extract maintained its initial activity at freezing temperatures up to 100 days.
Conclusions:  A newly isolated strain of Penicillium sp . used in this work yielded good lipase activities compared to the literature.
Significance and Impact of the Study:  The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).     

Isolation of alkali-tolerant benzene-degrading bacteria from a contaminated aquifer

Aims:  To isolate benzene-degrading strains from neutral and alkaline groundwaters contaminated by benzene, toluene, ethylbenzene, xylenes (BTEX) from the SIReN aquifer, UK, and to test their effective pH range and ability to degrade TEX.
Methods and Results:  The 14 isolates studied had an optimum pH for growth of 8, and could degrade benzene to below detection level (1  μ g l⁻¹). Five Rhodococcus erythropolis strains were able to metabolize benzene up to pH 9, two distinct R. erythropolis strains to pH 10, and one Arthrobacter strain to pH 8·5. These Actinobacteria also degraded benzene at least down to pH 5·5. Six other isolates, a Hydrogenophaga and five Pseudomonas strains, had a narrower pH tolerance for benzene degradation (pH 6 to 8·5), and could metabolize toluene; in addition, the Hydrogenophaga and two Pseudomonas strains utilized o- , m- or p- xylenes. None of these strains degraded ethylbenzene.
Conclusions:  Phylogenetically distinct isolates, able to degrade BTX compounds, were obtained, and some degraded benzene at high pH.
Significance and Impact of the Study:  High pH has previously been found to inhibit in situ degradation of benzene, a widespread, carcinogenic groundwater contaminant. These benzene-degrading organisms therefore have potential applications in the remediation or natural attenuation of alkaline waters.     

Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5

Aims:  The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle.
Methods and Results:  106 bovine-derived faecal/intestinal LAB were tested in vitro for tolerance to pH 2·0, pH 4·0, 0·15% and 0·3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 ( n  = 3) and F5 ( n  = 1). While no LAB grew at pH 2·0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7·6% (8/106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6·6% (7/106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici.
Conclusion:  Lactobacillus plantarum with acid/bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth.
Significance and Impact of the Study:  Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.     

Detection and characterization of hepatitis A virus and Norovirus in estuarine water samples using ultrafiltration – RT-PCR integrated methods

Aims:  Waterborne outbreaks of hepatitis A and Norovirus disease have been reported and associated with contaminated water supply in various countries. However, in Mexico, there are no studies that report HAV and NV presence in water. This study reports the application of ultrafiltration and RT-nested PCR methods to concentrate and identify these viruses.
Methods and Results:  Forty estuarine water samples were collected from the Huizache Caimanero Lagunary Complex. Samples were concentrated by ultrafiltration system (UFS) and RT-nested PCR was performed for HAV and NV identification. These viruses were found in 80% and 70% of the samples collected respectively and both were present in 57·5%. The DNA sequences analysis showed that 21 estuarine water samples were associated with HAV and 13 with NV. Faecal coliforms were isolated in 48·57% of the samples, while Escherichia coli were found in 34·28%.
Conclusions:  DNA sequencing showed that the genotype IB for HAV and GII for NV were predominant in México. No significant relationships were detected between indicators and viruses ( P  < 0·05).
Significance and Impact of the Study:  This study shows that the UFS is adequate for viral concentration. This is the first study analysing the genetic sequence of HAV and NV isolated from Mexican estuarine water.     

Inactivation effect of X-ray treatments on Cronobacter species (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat milk*

Aims:  To determine the inactivation effect of X-ray treatments on Cronobacter ( E. sakazakii ) in tryptic soy broth (TSB), skim milk (0% fat), low-fat milk (1% and 2%) and whole-fat milk (3·5%).
Methods and Results:  X-rays were produced using the RS 2400 generator system (Rad Source Technologies Inc.). Cronobacter (in TSB), inoculated skim milk (0% fat), low-fat milk (1% and 2% fat) and whole-fat milk (3·5% fat) were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 2·0, 3·0, 4·0, 5·0 and 6·0 kGy X-ray doses. Surviving bacteria in the TSB and inoculated milk, before and after treatment, were enumerated using plating method onto trypticase soy agar. Greater than 7·0-log CFU reduction in Cronobacter population was observed with 4·0, 5·0, 6·0, 6·0 and 6·0 kGy X-ray in the TSB, skim milk, 1% fat milk, 2% fat milk and 3·5% fat milk, respectively.
Conclusions:  Treatment with X-rays significantly ( P  <   0·05) reduced Cronobacter to less than detectable limits (<1 log CFU ml⁻¹) in skim milk at 5·0 kGy and milk with 1% fat content and greater at 6·0 kGy dose levels. The D-value for Cronobacter in TSB was significantly ( P  <   0·05) lower than those in milk samples.
Significance and Impact of the Study:  Treatment with X-rays could be an effective and safe alternative technology to control pathogenic bacteria ( Cronobacter ) in the dairy industry.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS

Aims:  To isolate the potential micro-organism for the degradation of textile disperse dye Brown 3 REL and to find out the reaction mechanism.
Methods and Results:  16S rDNA analysis revealed an isolate from textile effluent contaminated soil as Bacillus sp. VUS and was able to degrade (100%) dye Brown 3REL within 8 h at static anoxic condition. A significant increase in the activities of lignin peroxidase, laccase and NADH-DCIP reductase was observed up to complete decolourization of Brown 3REL. The optimum temperature required for degradation was 40°C and pH 6·5–12·0. Phyto-toxicity and chemical oxygen demand revealed nontoxic products of dye degradation. The biodegradation was monitored by UV–VIS, FTIR spectroscopy and HPLC. The final products 6,8-dichloro-quinazoline-4-ol and cyclopentanone were characterized by gas chromatography-mass spectrometry. This Bacillus sp. VUS also decolourized (80%) textile dye effluent within 12 h.
Conclusions:  This study suggests that Bacillus sp. VUS could be a useful tool for textile effluent treatment.
Significance and Impact of the Study:  The newly isolated Bacillus sp. VUS decolourized 16 textile dyes and textile dye effluent also. It achieved complete biodegradation of Brown 3REL. Phytotoxicity study demonstrated no toxicity of the biodegraded products for plants with respect to Triticum aestivum and Sorghum bicolor .     

Growth and reducing capacity of Listeria monocytogenes under different initial redox potential

Aims:  To determine the reducing capacity of Listeria monocytogenes and to highlight the effect of redox potential on its growth parameters.
Methods and Results:  The reducing capacity of L. monocytogenes was monitored in Brain Heart Infusion Broth media at different initial redox potential (Eh) and pH at 37°C. The effect of Eh obtained by gas flushing (air, N₂ and N₂-H₂) or by adding potassium ferricyanide and dithiotreitol in concentration from 1 to 10 mmol l⁻¹on L. monocytogenes growth parameters at pH 6·0, 7·0 and 8·0 was investigated. A total change of 539 mV (±44 mV) from an initial redox value of +330 ± 8 mV to a more negative potential in redox curves was observed resulting from L. monocytogenes growth at pH 7·0 at 37°C. A significant influence of pH and redox potential on L. monocytogenes lag phase of growth was shown ( P  < 0·05).
Conclusions:  Listeria monocytogenes exhibited longer lag phase in reducing conditions and at pH 6·0. The method used to modify the redox potential was shown to have no effect on growth parameters at pH 7·0.
Significance and Impact of the Study:  The provided information on the extending lag time and the possible delayed growth of this major pathogen in reducing conditions might be useful for its control in foods.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro

Aims:  Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures.
Methods and Results:  Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (−1 and −2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC₅₀) against BoHV-1 and PV-1 infection were 8·6 and 19·8 μg ml⁻¹, respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66·5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20·0 μg ml⁻¹, when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66·4% for PV-1 and BoHV-1, respectively, by PRA.
Conclusions:  These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle.
Significance and Impact of the Study:  CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.     

Keratinolytic activity of Bacillus subtilis AMR using human hair

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.
Methods and Results:  The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0·01% yeast extract increased the keratinolytic activity 4·2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0·01% yeast extract (HMY) at pH 8·0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50°C and pH 9·0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800–2600 Da.
Conclusions:  This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides.
Significance and Impact of the Study:  This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair . These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.     

The risk of Listeria monocytogenes infection in beef cattle operations

Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow-calf and feedlot) in central and southern California.
Methods and Results:  A repeated cross-sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf-cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut-grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots.
Conclusions:  Listeria monocytogenes was present at a higher proportion among cow-calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations.
Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow-calf operations.     

An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally-contaminated gelatine

Aims:  Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine.
Methods and Results:  A comprehensive TaqMan^® probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0·94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines ( n  = 162). Relative accuracy, relative sensitivity and relative specificity were 97·5%.
Conclusions:  The real-time PCR assay is an adequate alternative of the current plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.     

Characterization of some culture factors affecting oxalate degradation by the mycoparasite Coniothyrium minitans

Aims:  To find possible approaches to utilize the mechanism of oxalate degradation by Coniothyrium minitans (Cm) in controlling the plant pathogen Sclerotinia sclerotiorum (Ss).
Methods and Results:  Differences in oxalate degradation by different Cm strains and effects of the initial oxalate concentration, ambient pH and nutrient factors on mycelial growth and oxalate degradation by Cm were studied in shaken cultures. Results showed that two wild-type Cm strains, Chy-1 and ZS-1, did not differ in oxalate degradation in modified potato dextrose broth (mPDB) amended with oxalic acid (OA). Cm could grow in mPDB amended with sodium oxalate (SO-mPDB) at pH 6·5 or with ammonium oxalate (AO-PDB) at pH 6·2, but oxalate degradation was very low; oxalate degradation was greatly enhanced in SO- or AO-mPDB with pH being lowered to 2·8–2·9. Similarly, oxalate degradation was higher than 90% in OA-amended mPDB at pH 4·4 but was reduced to be <22% at pH 7·0. Five carbon sources and three nitrogen sources investigated and nutrients from mycelia and sclerotia of Ss were favorable for the growth of Cm and OA degradation by Cm.
Conclusions:  Cm can degrade oxalate under acidic pH. Exudates from mycelia or sclerotia of Ss may serve as nutrients for Cm mycelial growth and degradation of oxalate secreted by Ss.
Significance and Impact of the Study:  The finding of oxalate degradation laid a foundation for mining-related genes in Cm for engineering plant resistance against Ss. Elucidation of the importance of acidic pH and nutrients from Ss in oxalate degradation by Cm will help to understand the interaction between Cm and Ss.     

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology

Aim:  To evaluate the influence of water activity ( a _w), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising.
Methods and Results:  Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a _w (0·998, 0·982, 0·955, 0·928, 0·892) were combined with three incubation temperatures (10, 15, 20°C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a _w and temperature ( P <  0·0001) and a significant effect of pH ( P <  0·05). The radial growth rate and lag phase of P. vitreus were very sensitive to a _w reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a _w and growth became inhibited at a _w = 0·955. Optimal conditions for growth of P. vitreus were a _w = 0·998, 20°C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a _w than on temperature or pH under in vitro conditions.
Conclusions:  Low levels of a _w can prevent growth of P. vitreus , so wood moisture content should be adjusted accordingly.
Significance and Impact of the Study:  Implementation of these results should contribute towards the optimization and efficiency of bioincising.