Optimization of the decolorization conditions of Rose Bengal by using Aspergillus niger TF05 and a decolorization mechanism

Aspergillus niger TF05 was applied to decolorize Rose Bengal dye. The effects of carbon source, nitrogen source, metal ion and spore concentration on Rose Bengal treatment with A. niger TF05 were studied. A Plackett–Burman design (PBD) and a uniform design (UD) were used to optimize the decolorization conditions of A. niger TF05 and enhance its decolorization effect. The mechanism of Rose Bengal decolorization by A. niger TF05 was examined by analysing degradation products via UV–visible light spectroscopy, IR spectroscopy and GC-MS. The best decolorization effect was achieved in the single factor test with glucose and ammonium chloride as carbon and nitrogen sources, respectively. Mg2+ was an essential ion that could improve the mould ball state and adsorption efficiency if the spore concentration was maintained at 106 spores ml–1. The optimal decolorization conditions obtained using the PBD and UD methods were 11.5 g l−1 glucose, 6.5 g l−1 ammonium chloride, 0.4 g l−1 magnesium sulphate, pH 5.8, 28 °C, 140 r.p.m. rotational speed, 0.18 g l−1 dye concentration, 0.5 ml of inocula and 120 h decolorization time. Under these conditions, the maximum decolorization rate was 106%. Spectral analysis suggested that the absorption peak of the product changed clearly after decolorization; GC-MS analysis revealed that the intermediate product tetrachlorophthalic anhydride formed after decolorization. The combined use of the PBD and UD methods can optimize multi-factor experiments. A. niger TF05 decolorized Rose Bengal during intracellular enzymatic degradation after adsorption.

Theoretical and Experimental Substantiation of Alternative Methods for Quality Control of Live Anthrax Vaccine

Preventive immunisation against anthrax is carried out in accordance with the national Immunisation Schedule for Epidemic Settings. The vaccination is performed using a live vaccine—a freeze-dried suspension of Bacillus anthracis STI-1 vaccine strain spores in a stabilizing media. Improvement of the quality control of immunobiological medicines is a pressing issue and an integral part of the quality management system.The aim of study was to streamline quality control of live anthrax vaccine in terms of the following test parameters: identification and specific activity (total spore concentration).Materials and methods: identification and specific activity (total spore concentration) tests were performed for samples of live anthrax vaccine, batch 266, produced by the 48 Central Scientific Research Institute. The identification test was performed using the B. anthracis immunochromatography test kit for express detection and identification of anthrax pathogen spores produced by the State Research Center for Applied Microbiology and Biotechnology (Obolensk). The specific activity (total spore concentration) was assessed by the visual method and calculated in the Goryaev chamber using the industry reference standard of bacterial suspension turbidity equivalent to 10 IU—OSO 42-28-85 (by the Scientific Centre for Expert Evaluation of Medicinal Products). The number of live spores in live anthrax vaccine was determined by the microbiological method (by inoculating media). The statistical processing of the results was performed using Excel and Statistica 10.0.Results: the authors provided theoretical and experimental substantiation to support the feasibility of using immunochromatography as an alternative identification test method for live anthrax vaccine. Test samples dilutions of 108 microbial cells per millilitre and 109 microbial cells per millilitre are used in the test. The authors developed a test procedure for determination of the total spore concentration (specific activity) in live anthrax vaccine using an industry reference standard of turbidity equivalent to 10 IU, and proposed a formula for calculation of the total spore concentration.Conclusions: the developed test procedures could be recommended for inclusion in the live anthrax vaccine specification files as alternative methods of quality control.

Comparative evaluation of hydrogen peroxide sporicidal efficacy by different standard test methods

Background and Objectives: There are different sporicidal standard tests with various specifications to deal with products that are claimed for sporicidal activity. The aim of this study was to compare the 7% H O sporicidal efficacy against Bacillus 2 2 subtilis spores using different standard test methods. Materials and Methods: The 7% H O sporicidal efficacy against Bacillus subtilis spores was determined according to the 2 2 AOAC MB-15-04 standard of carrier test and two standard suspension tests (BS EN 13704, AFNOR NF 72-230) in both clean and dirty conditions and by using different interfering substances including bovine serum albumin, yeast extract and skimmed milk. Results: The results of suspension tests with 3 × 105 and 2 × 107 CFU/ml of B. subtilis spore concentration demonstrated that the higher spore counts lead to lower efficacy of 7% H O . Also, the sporicidal activity of 7% H O was reduced in the 2 2 2 2 presence of interfering substances. Bovine serum albumin, yeast, and skimmed milk showed similar interfering effects in suspension test with 3 × 105 CFU/ml. While, in suspension tests with higher initial spore count (2 × 107 CFU/ml) severity of interfering effects were intensified and distinct. Our results indicated that the carrier sporicidal test in comparison with suspension tests required more contact time to kill B. subtilis spores. Conclusion: The results of this study showed that it is reasonable to use interfering substances and inoculated carriers in accordance with actual conditions of product usage in a sporicidal test. Interfering substances may reduce the contact surface between H O and test spores; therefore, the sporicidal efficacy of H O was diminished. So applying suspension test in clean 2 2 2 2 condition to verify the claim of sporicidal activity is strongly discouraged

Quantification of Plasmodiophora brassicae Resting Spores in Soils Using Droplet Digital PCR (ddPCR)

Plasmodiophora brassicae, an obligate soilborne pathogen that causes clubroot on Brassica crops, is spreading rapidly in western Canada, threatening canola production in the region. Bioassays and molecular assays have been used to estimate the concentration of P. brassicae resting spores in soil, which can affect clubroot incidence and severity on crops. Droplet digital PCR (ddPCR) is a promising new approach for quantification of pathogen inoculum owing to its low sensitivity to inhibitors and consistency at low target concentrations. The objective of this study was to assess ddPCR against existing quantitative PCR (qPCR) for potential advantage and/or improvement in quantifying P. brassicae resting spores in soil. The new protocol enumerated resting spores accurately in spiked potting mix or soil samples ranging from 102 to 107 spores per gram. At a spore concentration ≥107 spores per gram, however, ddPCR became less accurate, with a tendency of overestimation. The protocol was validated by quantifying the resting spores in spiked brown, dark brown, and black soils using both ddPCR and qPCR simultaneously. These soil types are found commonly on the Canadian Prairies, and they vary in texture, pH, and organic content. ddPCR showed similar results among the different soil types, whereas qPCR often displayed lower counts for the same spore concentration, with the amplification of DNA inhibited completely in black soil samples. The inhibition can be removed by a 10-fold dilution of DNA samples. The results show that ddPCR can be a more versatile tool than qPCR for detection and quantification of P. brassicae resting spores in soil samples.

Effects of Foliar Treatment with a Trichoderma Plant Biostimulant Consortium on Passiflora caerulea L. Yield and Quality

The influence of spore concentration on the ability of a Trichoderma consortium to colonize the Passiflora caerulea phyllosphere was evaluated by determining the effects of foliar treatments with two spore concentrations, in two repeated treatments, on the morphological, physiological, and ultrastructural characteristics, and on the yield and quality of P. caerulea. The studied crop quality features were related to its nutraceutical use: the accumulation of polyphenols and flavonoids, antioxidant activity, and effects on mouse fibroblast L929 cells. The Trichoderma consortium consisted of two strains, T. asperellum T36b and T. harzianum Td50b, and the concentrations used were 106 colony forming units (cfu)/mL and 108 cfu/mL. As a reference treatment, a commercial product that was based on herbs and algal extracts was used. As compared to the negative control, the treatment with the Trichoderma consortium at 108 cfu/mL concentration determines the accumulation of higher level of polyphenols and flavonoids and increased antioxidant activity. This enhancement of P. caerulea quality characteristics after treatment with the higher concentration of Trichoderma consortium was associated with larger leaves, increased number and size of chloroplasts, improved plant physiology characteristics, and an increased yield. The treatment with high concentration of Trichoderma consortium spores promotes phyllosphere colonization and benefits both crop yield and quality.

Synergistic Effect of Fungus Metarhizium anisopliae and Slow Acting Toxicants Fipronil and Imidacloprid against Subterranean Termites

Three experiments were conducted to check Coptotermes heimi termites mortality in different conditions. In first experiment, two termiticides; fipronil and imidacloprid of specific formulation 25 EC and 5% SC, respectively, were used. Different concentration dilutions were prepared and termites’ mortality assay was performed. Both formulations gave 100% termites mortality on their highest ppm concentration after one week. Second experiment was based on different spore concentrations of Metarhizium anisopliae that were applied on worker termites to check the lethal concentration of fungi. It was observed that spore concentration showed direct relationship with termites’ mortality which means, as the spore concentration increases, termite mortality also increases. Last experiment was conducted to check the synergistic effect of M. anisopliae and termiticides. The fungus was used in combination with termiticides, fipronil and imidacloprid, separately. The rate of mortality of termites which exposed to imidacloprid/fipronil and M. anisopliae simultaneously was higher than mortality of termites exposed to either treatment alone.

Prolonged Shipping and Fluctuating Temperatures Promote Gray Mold Development and Leaf Yellowing on Geranium Liners

Postharvest environments during storage and shipping are often conducive to plant stress and disease development. Liners of four cultivars of geraniums (Pelargonium ×hortorum) were evaluated every 2 days for their susceptibility to gray mold (Botrytis cinerea) and leaf yellowing over an 8-day simulated shipping period at either constant air temperature of 15 °C or variable air temperatures cycling every 24 hours between 10 and 30 °C. The latter treatment was created using air temperature logs of commercial liner shipments sent to five locations during Spring 2016 and Fall 2016. We sprayed a spore suspension of 2 × 104 or 2 × 106 to inoculate liners before they were subjected to the two temperature treatments. Disease ratings did not reach significant levels for the dry control until day 6 of storage. Regardless of the spore concentration, ratings were similar for inoculated cuttings. Independent of the storage temperature and spore concentration, liners developed minor lesions by day 2 of storage. Cultivars varied slightly in disease ratings, with Tango Dark Red being the most susceptible, followed by Patriot Bright Red, Patriot Rose Pink, and Americana Red. During the 8-day incubation period, ‘Patriot Rose Pink’ developed the most leaf yellowing compared with the other three cultivars. Liners that experienced variable air temperatures had marginal leaf yellowing by day 2, and this yellowing increased throughout the experiment. Liners placed at 15 °C had ≈50% less leaf yellowing compared with liners exposed to variable air temperatures until day 8, when leaf yellowing was similar between the two air temperature treatments. Disease caused by B. cinerea was avoided when simulated shipping was 2 days or fewer, and a stable air temperature of 15 °C reduced leaf yellowing on geranium liners compared with variable air temperatures.