Monitoring the infection process of Rhizopus stolonifer on strawberry fruit during storage using films based on chitosan/polyvinyl alcohol/polyvinylpyrrolidone and plant extracts.

Different formulations based on nanoparticles of chitosan-plant extracts were evaluated to detect the infection process from the earliest stage of the fungus Rhizopus stolonifer on strawberry fruit during storage. Chitosan/polyvinyl alcohol (Ch/PVA) and chitosan/polyvinylpyrrolidone (Ch/PVP) films enriched with nanoparticles (NPs) of chitosan blended with plant extracts were prepared. They were placed inside a plastic package containing inoculated fruits and stored at 25 °C for 72 h. The thickness values of the films were in the range of 0.10 to 0.25 mm. All samples showed a maximum absorbance peak of about 300-320 nm; however, the Ch/PVP films enriched with NPs of chitosan and 10% of radish extract had an evident decrease in the optical absorbance as the fungal infection progressed. Additionally, as observed by scanning electron microscopy, the cross-section and surface morphology of films were not modified during storage, and the growth of R. stolonifer was evident after 48 h. Therefore, the Ch/PVP films enriched with chitosan NPs blended with 10% radish extract could be a reliable indicator of this fungus's growth.

Improvement of the Culture Medium for the Dichlorvos-Ammonia (DV-AM) Method to Selectively Detect Aflatoxigenic Fungi in Soil.

The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5⁻10% sucrose concentrations and 0.1⁻0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments.

Effect on tomato plant and fruit of the application of biopolymer-oregano essential oil coatings.

BACKGROUND: Oregano essential oil (EO) was incorporated into film-forming dispersions (FFDs) based on biopolymers (chitosan and/or methylcellulose) at two different concentrations. The effect of the application of the FFDs was evaluated on tomato plants (cultivar Micro-Tom) at three different stages of development, and on pre-harvest and postharvest applications on tomato fruit. RESULTS: The application of the FFDs at '3 Leaves' stage caused phytotoxic problems, which were lethal when the EO was applied without biopolymers. Even though plant growth and development were delayed, the total biomass and the crop yield were not affected by biopolymer-EO treatments. When the FFDs were applied in the 'Fruit' stage the pre-harvest application of FFDs had no negative effects. All FFDs containing EO significantly reduced the respiration rate of tomato fruit and diminished weight loss during storage. Moreover, biopolymer-EO FFDs led to a decrease in the fungal decay of tomato fruit inoculated with Rhizopus stolonifer spores, as compared with non-treated tomato fruit and those coated with FFDs without EO. CONCLUSION: The application of biopolymer-oregano essential oil coatings has been proven to be an effective treatment to control R. stolonifer in tomato fruit. © 2016 Society of Chemical Industry.

Necrotising ventriculitis due to combined infection with Rhizopus microsporus var. chinensis and Candida krusei in an eclectus parrot (Eclectus roratus).

Acute necrosis of the ventriculus is a very uncommon lesion in birds. We describe a fatal case of acute necrotising ventriculitis caused by Rhizopus microsporus var. chinensis in a mature female eclectus parrot (Eclectus roratus). The bird presented acutely dull and lethargic, was vomiting and had bright green droppings, suggestive of acute heavy metal poisoning. It was treated with fluids and chelation therapy, but died within 12 h. Necropsy, cytology, histopathology and culture results demonstrated fungal invasion of the ventriculus associated with transmural necrosis, haemorrhage, acute inflammation and abundant R. microsporus var. chinensis and lesser numbers of Candida krusei.

In vitro and in vivo activities of posaconazole against zygomycetes with various degrees of susceptibility.

OBJECTIVES: We analysed the in vitro and in vivo effects of posaconazole and amphotericin B against three clinical isolates of zygomycetes: Lichtheimia corymbifera, F1; and Rhizopus oryzae, F5 and F6. METHODS: In vitro activities of both drugs were assessed by determining MICs, minimum fungicidal concentrations (MFCs) and fungal damage measured by the XTT assay against either the spores or the hyphal forms. Additionally, the survival curves of neutropenic mice systemically infected with the zygomycete isolates were used as the marker of antifungal response to amphotericin B (1 mg/kg/day) or posaconazole (2.5, 10 and 50 mg/kg/day). RESULTS: In terms of MICs, posaconazole proved to be active against the three isolates (MICs ranging from 0.125 to 1.0 mg/L). The median posaconazole MFCs were 0.25, 0.5 and >16 mg/L for F1, F5 and F6, respectively. The XTT assay showed that posaconazole was active against spores of all three isolates, but only partially effective against the hyphae. The survival studies showed that amphotericin B at 1 mg/kg/day and posaconazole at 10 mg/kg/day prolonged the survival of the animals infected with L. corymbifera F1. In mice infected with R. oryzae F5, only posaconazole at 50 mg/kg/day significantly prolonged survival, whereas amphotericin B at 1 mg/kg/day was the only regimen active against R. oryzae F6. CONCLUSIONS: Our findings showed that posaconazole could be useful in the treatment of zygomycosis. Also, we report that an isolate of R. oryzae with low MFC responded to posaconazole, while another isolate with high MFC did not.

Rhizopus microsporus var. rhizopodiformis sinus-orbital zygomycosis in an immunosuppressed patient: successful treatment with posaconazole after a complicated clinical course.

A case of sinus-orbital Rhizopus microsporus var. rhizopodiformis infection in a patient with graft versus host disease following allogeneic blood stem cell transplantation is reported. Commercially available pea straw compost used for gardening was suspected to be the source of the infection. After an initial relapse, treatment with surgical debridement, liposomal amphotericin B and posaconazole was successful.

Deferiprone iron chelation as a novel therapy for experimental mucormycosis.

OBJECTIVES: Patients treated with the iron chelator deferoxamine are known to be more susceptible to mucormycosis. However, while deferoxamine is an iron chelator from the perspective of the human host, deferoxamine actually serves as a siderophore, delivering free iron to Rhizopus oryzae, the major cause of mucormycosis. Other iron chelators, including deferiprone, which do not deliver iron to R. oryzae have been described. We therefore sought to determine whether iron-chelation therapy with deferiprone would effectively treat mucormycosis. METHODS: In vitro MIC and minimum fungicidal concentration (MFC) of the iron chelator, deferiprone, for R. oryzae were determined by microdilution assay. In addition, we compared the efficacy of deferiprone with that of liposomal amphotericin B (LAmB) in treating mucormycosis in diabetic ketoacidotic mice. RESULTS: Deferiprone demonstrated static activity against R. oryzae at 24 h, but showed cidality at 48 h of incubation. Deferiprone was as effective as LAmB at improving survival and decreasing brain fungal burden, and both drugs were more effective than placebo in non-iron-overloaded animals. Administration of free iron with deferiprone reversed protection, confirming that the mechanism of protection was iron chelation. CONCLUSIONS: Iron chelation is a promising, novel therapeutic strategy for refractory mucormycosis infections. Further studies are warranted to evaluate combination antifungal/iron chelation therapy and to evaluate the efficacy of other iron-chelating agents.

Periorbital zygomycosis (mucormycosis) treated with posaconazole.

PURPOSE: To report on the successful treatment of periorbital zygomycosis (mucormycosis) with posaconazole, a broad-spectrum oral antifungal available for compassionate use. DESIGN: Interventional case report. METHODS: Review of a medical record. RESULTS: A 22-year-old male undergoing induction chemotherapy for acute lymphoblastic leukemia presented with periorbital cellulitis attributable to Rhizopus. The patient was initially treated with liposomal amphotericin B, surgical debridement, and reversal of immune compromise. The patient was switched to posaconazole because of amphotericin side effects and lack of improvement. He took posaconazole for five months while undergoing additional cycles of chemotherapy. Despite recurrent profound neutropenia, the periorbital infection resolved, he tolerated reconstructive procedures, and he did not develop orbital invasion. His Rhizopus isolate was highly susceptible to posaconazole in vitro. CONCLUSIONS: Drug toxicities can limit the use of amphotericin in some patients with zygomycosis. Posaconazole shows promise as an alternative antifungal agent in the treatment of periorbital zygomycosis.

[Testing of seed-borne fungi of Glycyrrihiza seed and disinfection effect of several fungicides on seed-borne fungi].

OBJECTIVE: To study the dominant seed-borne fungi of Glycyrrihiza seeds which were from different producing area and compare the disinfection effect of several fungicides on seed-borne fungi of Glycyrrihiza seed. METHOD: Petri-dish testing was used to determine the external and internal seed-borne fungi and the disinfection effect of fungicides. RESULT AND CONCLUSION: The result showed that the amount of spore on the surface of one Glycyrrihiza seed varied from 0.3% to 37.0% among samples. Penicillium spp. and Aspergillus spp. were the two major dominant fungi and there was few differences in the type of the fungi among producing areas but differences in the isolation frequency of the furgi; Penicillium spp. , Rhizopus spp. , Aspergillus spp. and Alternaria spp. were the internally dominant seed-borne fungi, including seed capsule and the internal tissue of seed and their fungi-carrying percentage was 8.0%-48.3% and 3.5%-42.0% respectively. There were differences in fungi percentage and dominant seed borne fungi among different producing areas. The disinfection effect of both mancozeb and thriam was up to 89.0%, so it is suggested to use them to disinfect Glycyrrihiza seed.

Microbial metabolism. Part 5. Dihydrokawain.

Preparative scale fermentation of (6S)-dihydrokawain (1) with Rhizopus arrhizus (ATCC 11145) gave 3'-hydroxydihydrokawain (2) and (8S)-hydroxydihydrokawain (3). Structure elucidation of the metabolites was based on spectroscopic data. The C-8 absolute configuration of (3) was assessed via its Mosher's esters.

An optimization study of solid-state fermentation: xanthophylls extraction from marigold flowers.

Marigold flowers are the main natural source of xanthophylls, and marigold saponified extract is used as an additive in several food and pharmaceutical industries. In this work, the use of a solid-state fermentation (ensilage) process for increasing the yield of xanthophylls extracted from fermented marigold flowers was examined. The process consisted of a mixed culture of three microorganisms (Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans), part of the normal microbiota associated with the marigold flower. These microorganisms had been previously isolated, and were identified as relevant for the ensilage process due to their capacity to produce cellulolytic enzymes. Based on experimental design strategies, optimum operation values were determined for aeration, moisture, agitation, and marigold-to-inoculum ratio in the proposed solid-state fermentation equipment, leading to a xanthophylls yield of 17.8-g/kg dry weight. The optimum achieved represents a 65% increase with respect to the control. HPLC analysis indicated conservation of extracted oleoresin. Based on the experimental results, interactions were identified that could be associated with the heat and mass-transfer reactions taking place within the bioreactor. The insight gained allows conditions that limit growth and metabolic activity to be avoided.

Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae.

Thirty-eight strains of the fungus Rhizopus oryzae were grown on potato pulp, an agricultural by-product of the starch industry. Either lactic acid or fumaric acid and ethanol were formed, and the ratio differed among the strains tested. The highest amount of L(+)-lactic acid (10 mg/g fresh matter) was observed in the pulp fermented for six days by Rhizopus oryzae IFO 4707. The IFO 4707 strain rapidly reduced the hardness and pH of potato pulp within one day followed by the gradual synthesis of lactic acid. A composition analysis showed that the enzymes secreted from the fungal cells hydrolyzed starch efficiently with partial degradation of the cell wall. Rhizopus oryzae may be used as an inoculant for ensiling potato pulp and other agricultural by-products containing starch.

Selection of pectinesterase overproducing fungal strains for the low degree methoxylated pectin preparation.

The selection of the novel pectinesterase (PE) producing strains is an important step in their biotechnological application. The present work displays the purposeful multistage selection of strains with the increased ability to synthesize extracellular PE among 700 filamentous fungal collection cultures belonging to 45 genera and 93 species. A scheme of targeted multistage screening of PE producers has been developed. At the first stage of the screening 23 potentially active strains belonging to the genera Penicillium, Fusarium, Aspergillus, Rhizopus, Gliocladium, etc., were selected. Strains of the Trichoderma, Cochliobolus, Cladosporium and Thielavia genera not yet described as PE-active ones were selected. The P. funiculosum species (0.28-0.56 units/ml) possessed higher ability to synthesize PE. High level of the PE activity was revealed in P. rubrum (0.37 units/ml). No correlation between the level of fungal biomass accumulation on pectin-containing medium and the level of their PE activity was observed. A principal chance of the fungal PE substitution for some chemical reagents at the stage of demethoxylation of the pectin-containing raw materials should be noted. The technology of the fungal PE application for production of the low-degree methoxylated pectin is under development. Such pectin is necessary for the low-calorie jellfying products in the food industry and in medicine as a preventive agent removing the toxic substances from the human body.

[Fungal detection of American ginseng seeds from Beijing and northeast area in China].

OBJECTIVE: The quantity and genus of fungi on 13 samples of American ginseng seed from Beijing and Northeast area in China were detected. METHOD: Washing and surface sterilization were used to isolate the fungi existed on seed surface and inside of the seed, respectively. RESULT: The genera of the fungi in embryo and shell of the seeds mainly belonged to Fusarium spp., Alternaria spp., Penicilium spp., Cephalosporium sp., Rhizopus spp., and a small quantity of Mucor, Aspergillus, Tritroderma, Doratomyces and so on. The isolated fungal frequencies on the shell and in the embryo were 36%-100% and 40%-100% respectively. CONCLUSION: The fungal frequency of American ginseng seed was higher. There were significant differences among different seed samples, the main fungal genera and isolation frequency in the seed embryo were identical with the results from the seed shell.

Resolution of rhinocerebral zygomycosis associated with adjuvant administration of granulocyte-macrophage colony-stimulating factor.

We successfully treated 3 consecutive patients who had nonneutropenic rhinocerebral zygomycosis, by use of subcutaneous granulocyte-macrophage colony-stimulating factor therapy combined with traditional surgical and medical treatment. All patients are currently free of disease. Granulocyte-macrophage colony-stimulating factor should be considered as adjuvant therapy for rhinocerebral zygomycosis; however, optimum dose and length of therapy are unknown.

Incidence of seed-borne fungi and aflatoxins in Sudanese lentil seeds.

Thirteen seed samples of lentil (Lens esculenta) were incubated on agar plate and moist filter papers (Moist Chambers) at 28 +/- 2 degrees C for determination of the incidence of seed-borne fungi. Aflatoxins content of the seeds was measured using the bright greenish- yellow fluorescence test (BGYF) and thin-layer chromatography (TLC). Sixty-nine species and seven varieties, which belong to 24 genera of fungi, were isolated from this crop. Of these fungi, 51 species and two varieties are considered new for this crop, whereas seven genera and 13 species are new to the mycoflora of the Sudan. The genus Aspergillus (13 species and 6 varieties) which comprising 44% of the total colony count was the most prevalent genus followed by Rhizopus (2 species, 19%), Penicillium (6 species) and Fusarium (8 species) (12%), Chaetomium (3 species) and Cladosporium (5 species) (6%), where the 18 genera (1-4 species) showed very low level of incidence (19%). Of the possible pathogens of lentil plants, F. oxysporum the main cause of vascular wilt was recovered from seeds of this crop. Thin layer chromatographic analysis of chloroform extracts of 13 seed samples showed that only one samples was naturally contaminated with aflatoxins B1, B2, G1 and G2 (14.3 micrograms/kg).

Influence of sundrying on the chemical composition, aflatoxin content and fungal counts of two pepper varieties--Capsicum annum and Capsicum frutescens.

Samples of sundried, matured red pepper, Capsicum annum with a moisture content (MC) of 12.7-26.8 percent had on dry weight basis, vitamin C, 5.0-6.4 mg/100 g; crude protein, 0.8-1.2 percent; total soluble solids, 3.3-4.1 percent, and fungal counts of log 4.4-4.5/g. Ordinary matured red C. annum had MC, 75.7-78.2 percent vitamin C, 36.1-38.5 mg/100 g; crude protein, 2.4-2.8 percent; total soluble solids, 9.3-9.9 percent and fungal count of log 3.32-3.39/g. Sundried matured red C. frutescens had corresponding values of 9.4-18.7 percent; 5.8-6.3 mg/100 g; 0.8-1.1 percent; 0.9-2.6 percent and log 3.2-3.4/g. No aflatoxins were detected in sundried, matured red C. frutescens, but aflatoxin B1 values obtained from C. annum varied from non-detectable to 2.2 micrograms/kg. Dominant fungi isolated from C. annum and C. frutescens were Rhizopus oryzaze, Aspergillus niger, A. flavus, Geotrichum candidum and Saccharomyces spp.

Survey of the mycoflora and mycotoxins of cotton seeds and cotton seed products in Egypt.

Thirty-nine species and 16 fungal genera were isolated from Egyptian cotton seeds, cotton seed meal and cotton seed cake on 1% glucose-Czapek's agar medium incubated at 28 degrees C. Aspergillus was the most frequent genus and it emerged in 87-100% of the samples contributing 70-98% of total fungi in the three substrates tested. The most common species were A. niger, A. flavus, A. fumigatus, A. terreus and Rhizopus stolonifer; A. niger, A. fumigatus and Penicillium corylophilum; and A. niger, A. flavus, A. terreus, A. nidulans and Rhizopus stolonifer, respectively. Cotton seeds and cotton seed products were naturally contaminated by aflatoxin B1 and B2. About 16% of the different substrates tested were positive for aflatoxin contamination. No citrinin, ochratoxin A, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin or zearalenone were detected in the samples assayed.