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We report the first two proven cases of cavitary pulmonary zygomycosis caused by Rhizopus homothallicus. The diagnosis in each case was based on histology, culture of the causal agent, and the nucleotide sequence of the D1/D2 region of the 28S ribosomal DNA.

This study investigated the effectiveness of heat treatment and Pichia guilliermondii, either alone or in combination, to combat postharvest fungal spoilage in cherry tomato fruit. In vitro experiments demonstrated that heat treatment at 38 degrees C significantly inhibited mycelial growth of three different pathogens (Botrytis cinerea, Alternaria alternata and Rhizopus stolonifer Ehrenb). In vivo experiments unveiled that either heat treatment or P. guilliermondii reduced decay caused by these pathogens. Furthermore, a combination of heat treatment followed by the application of P. guilliermondii (H+P) provided the best efficacy in prevention of cherry tomato from fungal spoilage. Following, H+P treatment, electronic nose detected a reduction of volatility in cherry tomato fruit odor, an indicator of preserving fruit's freshness. Scanning electron microscopy unveiled that heat treatment at 38 degrees C for 24h inhibited hyphae growth and spore germination of R. stolonifer Ehrenb while P. guilliermondii multiplied rapidly on fruit wounds, and its cells had a strong capability of adhesion to the hyphae of R. stolonifer Ehrenb. However, heat treatment also seriously injured P. guilliermondii, therefore P. guilliermondii should be applied after heat treatment. A combination of heat treatment and P. guilliermondii is one of the most effective techniques at controlling postharvest fungal spoilage in cherry tomato fruit., (Copyright 2009 Elsevier B.V. All rights reserved.)

Mould fungi are not only well known for food spoilage through toxin formation but also for the production of fermented foods. In Asian countries, the fermentation of soy beans and tofu for tempe and sufu production with various Rhizopus strains is widespread. Here we report the finding of toxinogenic bacteria in a starter culture used for sufu production. By means of metabolic profiling of the fungus under standard conditions for tempe and sufu production, we found that toxins of the rhizoxin complex are produced in critical amounts. Considering that rhizoxins are severe toxins with strong antimitotic activity it is important to notice that our findings uncover a health-threatening symbiosis in food processing. A simple PCR method for detecting toxinogenic endofungal bacteria in starter cultures is proposed., (2009 Elsevier B.V. All rights reserved.)

The effect of cultivation conditions on the optical purity of L(+)-lactic acid produced by Rhizopus oryzae HZS6 was investigated. The isomeric composition of lactic acid was influenced by the supplementation of L(+)-lactic acid to fermentation medium. L(+)-isomer increased with the dosage, no D(-)-lactic acid was observed when the concentration of supplemented L(+)-lactic acid in matrix was > or = 1.5 g/L. However, the L(+)-lactic acid yield, biomass and glucose conversion rate decreased with the dosage. With the same method, the supplementation of L(+)-lactic to substrate had no influence on isomeric composition of lactic acid by Lactobacillus and Escherichia coli.

The microbial growth and simultaneous bioaccumulation of Cu(II) and Ni(II) ions during the growth of Rhizopus delemar in molasses medium was investigated in a batch system. The level of Cu(II) and Ni(II) bioaccumulation and microbial growth was dependent on pH, molasses sucrose concentration and initial Cu(II) and Ni(II) ion concentrations. An increase in initial total metal ion concentration inhibited both the growth rate of fungus and the bioaccumulation capacity. Lineweaver-Burk plot of Monod equation was used to study the inhibition kinetics of bioaccumulation of Cu(II) and Ni(II) and the intrinsic and apparent model parameters were evaluated in metals-free and metals-contaminated fermentation media. The maximum specific growth rate (micro(m)) and the Monod constant (K(s)) of microorganism in metals-free media were found as 0.405 L/h and 3.977 g/L, respectively. As micro(m) remained constant in the presence of increasing concentrations of Cu(II) and Ni(II) ions, the combined inhibition of Cu(II) and Ni(II) ions on the growth rate of R. delemar was found to be a competitive inhibition. The inhibition constants for Cu(II) and Ni(II) ions were determined as 56.71 mg Cu(II)/L and 47.44 mg Ni(II)/L. As the bioaccumulation of Cu(II) and Ni(II) ions was reduced by the presence of increasing concentrations of the other metal ion, compared with the single-metal systems, the individual action of Cu(II) and Ni(II) ions on the bioaccumulation of R. delemar was generally found to be antagonistic. On the other hand, the total combined effects of Cu(II) and Ni(II) ions on the bioaccumulation of R. delemar are thought to be synergistic since the total bioaccumulated metal ion quantities per unit mass of biomass were higher than those obtained in the growth media containing Cu(II) and Ni(II) ions alone at the same concentrations.

The crude ethanolic extract of Nauclea latifolia yielded five known compounds, strictosamide (1), naucleamides A (2), naucleamide F (3), quinovic acid-3-O-beta-rhamnosylpyranoside (4), and quinovic acid 3-O-beta-fucosylpyranoside (5). Microbial reactions, using a whole cell culture of Rhizopus circinans on compound (1), yielded three analogues, 10-hydroxystrictosamide (6), 10-beta-glucosyloxyvincoside lactam (7) and 16,17-dihydro-10-beta-glucosyloxyvincoside lactam (8). Compounds 1-8 were identified with the aid of extensive NMR spectral studies. Compound 8 was found to be a new metabolite. Compounds 1-8 exhibited different levels of anti-GST and anti-fungal activities.

Toxinogenic endobacteria were isolated from a collection of Rhizopus spp. representing highly diverse geographic origins and ecological niches. All endosymbionts belonged to the Burkholderia rhizoxinica complex according to matrix-assisted laser desorption ionization-time of flight biotyping and multilocus sequence typing, suggesting a common ancestor. Comparison of host and symbiont phylogenies provides insights into possible cospeciation and horizontal-transmission events.

Rhizopus microsporus was grown in an attached growth system using corn wet-milling effluent as a growth medium. This strain was chosen due to its ability to effectively degrade organic matter in corn wet-milling effluent and for its properties to produce significant levels of protein, chitin and chitosan. Fungal growth and organic removal efficiency were examined under both aseptic and non-aseptic conditions with and without nutrient supplementation. Plastic composite support (PCS) tubes, composed of 50% (w/w) polypropylene (PP) and 50% (w/w) agricultural products were used as support media. Significantly higher organic removal measured as chemical oxygen demand (COD) and biomass yield were observed in the bioreactor with PCS tubes than in two control bioreactors; that is with PP tubes alone and suspended growth (without support media). This confirmed that the PCS support medium with agricultural components enhanced fungal growth and organic removal. The results showed that supplementation of nutrients (e.g., mineral salts) under aseptic conditions enhanced the COD removal from 50% to 55% and observed biomass yield from 0.11 to 0.16 g (dry-weight)/g COD(removed) (i.e., from 0.10 to 0.14 g volatile solids (VS)/g COD(removed) approximately). Non-aseptic operation without nutrient supplementation resulted in an observed biomass yield of 0.32 g volatile suspended solids (VSS)/g COD(removed) with no significant improvement in COD removal ( approximately 53%); whereas with nutrient supplementation, the observed biomass yield increased to 0.56 g VSS/g COD(removed) and COD removal improved to 85%. The fungal system was able to degrade the organic matter with concomitant production of high-value fungal biomass. This is the first study that examined the conversion of corn milling waste stream into high value fungal protein.

Acid-producing Rhizopus fungi from loog-pang, a traditional Thai fermented food, was screened to investigate its potential for use in industrial lactic acid production from starch. A thermotolerant strain, TISTR 3518, was isolated and characterized by its morphological, physiological, genetic and fermentation properties, and compared with its mesophilic isolates, TISTR 3514 and TISTR 3523. TISTR 3518 was characterized by shorter sporangiophores and smaller sporangia than the other isolates; however, apparent differences between the mesophilic isolates and the strain could not be clarified. Moreover, TISTR 3518 grew at 45 degrees C, whereas the others did not. The three isolates showed different profiles of oligosaccharide assimilation and organic acid production. Their rDNA ITS sequences indicated that TISTR 3518 is a strain of Rhizopus microsporus, and TISTR 3514 and TISTR 3523 are strains of Rhizopus oryzae. TISTR 3523 and TISTR 3518 mainly formed L-lactic acid from glucose, while TISTR 3514 primarily formed fumaric acid. Under thermotolerant conditions, R. microsporus TISTR 3518 showed higher glucoamylase activity than the others, suggesting this enzyme from TISTR 3518 is more thermostable than that from TISTR 3523. The strain formed higher amounts of L-lactic acid from starch at 40 degrees C compared to R. oryzae TISTR 3523. This is the first report on the production of optically active L-lactic acid from starch by a thermotolerant fungus and could potentially provide a good tool for transforming biomass resources to chemical materials.

Aims: This paper developed a novel process for lactic acid and chitin co-production of the pelletized Rhzious oryzae NRRL 395 fermentation using underutilized cull potatoes and glucose as nutrient source., Methods and Results: Whole potato hydrolysate medium was first used to produce the highest pelletized biomass yield accompanying the highest chitin content in biomass. An enhanced lactic acid production then followed up using batch, repeated batch and fed batch culture with glucose as carbon source and mixture of ammonia and sodium hydroxide as neutralizer. The lactic acid productivity peaked at 2.8 and 3 g l(-1 )h(-1) in repeated batch culture and batch culture, respectively. The fed batch culture had the highest lactate concentration of 140 g l(-1)., Conclusions: Separation of the biomass cultivation and the lactic acid production is able to not only improve lactic acid production, but also enhance the chitin content. Cull potato hydrolysate used as a nutrient source for biomass cultivation can significantly increase both biomass yield and chitin content., Significance and Impact of the Study: The three-step process using pelletized R. oryzae fermentation innovatively integrates utilization of agricultural residues into the process of co-producing lactic acid and chitin, so as to improve the efficiency, revenues and cost of fungal lactic acid production.

Environmental isolates of the fungus Rhizopus have been shown to harbor a bacterial endosymbiont (Burkholderia) that produces rhixozin, a plant mycotoxin. We sought to define the role of rhizoxin production by endosymbionts in the pathogenesis of mucormycosis. Endosymbiotic bacteria were identified by polymerase chain reaction in 15 (54%) of 28 clinical isolates of Zygomycetes, with 33% of the bacterial strains showing 87% identity to Burkholderia 16S rDNA. The presence of rhizoxin in myclial extracts from fungi harboring bacteria was confirmed by high-performance liquid chromatography analysis. However, fungal strains with or without endosymbionts did not differ in their ability to cause endothelial cell injury in vitro, nor did antibiotic-mediated eradication of endosymbionts and rhizoxin production decrease the virulence of fungal strains in mice or flies. In summary, although bacterial endosymbiosis is widely detected in clinical isolates of Zygomycetes, including Rhizopus oryzae strains, we found no evidence that bacterial endosymbionts and rhizoxin contribute to the pathogenesis of mucormycosis in the models studied.

In order to carry out the process of the repeated intermittent L-lactic acid fermentation by self-immobilized Rhizopus oryzae, we investigated the effect of medium compositions on the morphology of Rhizopus oryzae. In submerged culture, fungi can be grown as broths of freely suspended mycelia and pellets or clumps, the pellets can be immobilized by themselves. The optimum medium composition for the first patch of fermentation was: 120 g/L glucose, 3 g/L NH4NO3, 0.14 g/L KH2PO4, 0.16 g/L NaH2PO4 (the concentration ratio of K+ and Na+ was 1:1). After 72 h fermentation, the physical form of Rhizopus oryzae was mostly uniform pellet with the diameter of 1.0-2.0 mm, the concentration of L-lactic acid was 100.8 g/L, and the conversion rate of glucose was 84%. During 16 batches of repeated fermentation, the L-lactic acid level was above 60.0 g/L and the conversion rate of glucose was 75%. The fermentation time of every batch was 24 h.

Fumaric acid is widely used as a food additive for flavor and preservation. Rhizopus oryzae ATCC 20344 is a fungus known for good fumaric acid production. It also has been reported that the fungal biomass has high chitin content. This study investigated the possibility of producing both fumaric acid and chitin via R. oryzae fermentation of dairy manure. Co-production of valuable bio-based chemicals such as fumaric acid and chitin could make the utilization of manure more efficient and more profitable. A three step fermentation process was developed which effectively utilized the nitrogen as well as the carbohydrate sources within the manure. These steps were: the culturing of pellet seed; biomass cultivation on liquid manure to produce both biomass and chitin; and fumaric acid production on the hydrolysate from the manure fiber. Under the identified optimal conditions, the fermentation system had a fumaric acid yield of 31%, and a biomass concentration of 11.5 g/L that contained 0.21 g chitin/g biomass.

Four phytopathogenic fungi were cultivated up to six days in media containing chitooligosaccharide mixtures differing in average DP and FA. The three different mixtures were named Q3 (which contained oligosaccharides of DP2-DP10, with DP2-DP7 as main components), Q2 (which contained oligosaccharides of DP2-DP12, with DP2-DP10 as main components) and Q1 (which derived from Q2 and contained oligomers of DP5-DP8 with hexamer and a heptamer as the main components). The novel aspect of this work is the description of the effect of mixtures of oligosaccharides with different and known composition on fungal growth rates. The growth rate of Alternaria alternata and Rhizopus stolonifer was initially inhibited by Q3 and Q2 at higher concentrations. Q1 had a growth stimulating effect on these two fungi. Growth of Botrytis cinerea was inhibited by Q3 and Q2, while Q1 had no effect on the growth of this fungus. Growth of Penicillium expansum was only slightly inhibited by higher concentrations of sample Q3, while Q2 and Q1 had no effect. The inhibition of growth rates or their resistance toward chitooligosaccharides correlated with the absence or presence of chitinolytic enzymes in the culture media, respectively.

The fungus Rhizopus oryzae converts both glucose and xylose under aerobic conditions into chirally pure L+-lactic acid with by-products such as xylitol, glycerol, ethanol, carbon dioxide and fungal biomass. In this paper, we demonstrate that the production of lactic acid by R. oryzae CBS 112.07 only occurs under growing conditions. Deprivation of nutrients such as nitrogen, essential for fungal biomass formation, resulted in a cessation of lactic acid production. Complete xylose utilisation required a significantly lower C/N ratio (61/1) compared to glucose (201/1), caused by higher fungal biomass yields that were obtained with xylose as substrate. Decreasing the oxygen transfer rate resulted in decline of xylose consumption rates, whereas the conversion of glucose by R. oryzae was less affected. Both results were linked to the fact that R. oryzae CBS 112.07 utilises xylose via the two-step reduction/oxidation route. The consequences of these effects for R. oryzae as a potential lactic acid producer are discussed.

Quantitative real-time PCR (qPCR) may improve the detection of fungal pathogens. Extraction of DNA from fungal pathogens is fundamental to optimization of qPCR; however, the loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi. We therefore studied representative automated and manual extraction methods for Aspergillus fumigatus and Rhizopus oryzae. Both were analyzed by qPCR for their ability to extract DNA from propagules and germinated hyphal elements (GHE). The limit of detection of A. fumigatus and R. oryzae GHE in bronchoalveolar lavage (BAL) fluid with either extraction method was 1 GHE/ml. Both methods efficiently extracted DNA from A. fumigatus, with a limit of detection of 1 x 10(2) conidia. Extraction of R. oryzae by the manual method resulted in a limit of detection of 1 x 10(3) sporangiospores. However, extraction with the automated method resulted in a limit of detection of 1 x 10(1) sporangiospores. The amount of time to process 24 samples by the automated method was 2.5 h prior to transferring for automation, 1.3 h of automation, and 10 min postautomation, resulting in a total time of 4 h. The total time required for the manual method was 5.25 h. The automated and manual methods were similar in sensitivity for DNA extraction from A. fumigatus conidia and GHE. For R. oryzae, the automated method was more sensitive for DNA extraction of sporangiospores, while the manual method was more sensitive for GHE in BAL fluid.

The frequency of zygomycosis has increased considerably over recent years mainly in immunocompromised and diabetic patients. Little is known about the effects of host innate immunity against different Zygomycetes especially under the influence of antifungal agents. The antifungal activity of human polymorphonuclear leucocytes (PMN) in combination with liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), voriconazole (VRC) and posaconazole (PSC) against Rhizopus oryzae and Rhizopus microsporus, frequently isolated Zygomycetes, were studied and compared with Absidia corymbifera, a less pathogenic Zygomycete. Antifungal activity was evaluated as per cent of hyphal damage using the XTT metabolic assay. While A. corymbifera was more susceptible to PMN than the other two Zygomycetes, R. microsporus appeared to be the most susceptible to combined effects of amphotericin B formulations and VRC with PMN. LAMB exhibited synergistic activity with PMN in inducing hyphal damage to R. microsporus but not to the other fungi. In contrast, ABLC exhibited synergistic or additive activity with PMN against all three fungi. Among triazoles, only VRC exhibited additive effect with PMN against R. microsporus. Lipid formulations of amphotericin B and particularly ABLC interact with PMN predominantly in inducing augmented hyphal damage to three different species of Zygomycetes.

The effect of some plant growth hormones, viz., gibberellic acid, indole-3-acetic acid, indole-3-butyric acid, and kinetin on chitosan production by Rhizopus oryzae in deproteinized whey was studied. Hormones, at different concentrations, increase the mycelial growth by 19-32%. However, increase in chitosan content of the mycelia was relatively small (1.7-14.3%) over the control. Maximum enhancement was observed with gibberellic acid. Fifty percent more chitosan could be obtained from 1L of whey containing 0.1mg/L gibberellic acid. Hormones, at higher dose, instead of stimulation inhibited both growth and mycelial chitosan content. This study showed that hormones have no influence on degree of deacetylation of chitosan but increase the quality of the chitosan by increasing weight average molecular weight and decreasing polydispersity. All the hormones had been found to enhance chitin deacetylase activity of R. oryzae by 1.067-1.267-fold and may be one of the reasons for increased chitosan production.

Human polymorphonuclear neutrophils (HPMNs) displayed attenuated hyphal damage associated with impaired O(2)(-) release following exposure to Rhizopus oryzae versus that with Aspergillus fumigatus. Exposure of HPMNs to R. oryzae hyphae resulted in upregulation in Toll-like receptor 2 mRNA and a robust proinflammatory gene expression with rapid (1-h) induction of NF-kappaB pathway-related genes.

In the present study, solid-state fermentation of black bean with various GRAS filamentous fungi including Aspergillus awamori, Aspergillus oryzae, Aspergillus sojae, Rhizopus azygosporus and Rhizopus sp. No. 2 was preformed to prepared koji. Mycelial propagation of starter organisms and antioxidative activity, including alpha-alpha-diphenyl-2-picyl-hydoxyl (DPPH) radicals, Fe2+-chelating ability, and reducing activity, were examined. Depending upon the starter organism, various amounts of mycelial propagation (23.5-67.3 mg/k koji) were found in the prepared black bean kojis. The methanol extracts of all the black bean kojis, except that prepared with Rhizopus sp. No. 2, exhibited higher levels of DPPH free radical-scavenging activity, Fe2+-chelating activity, and reducing power than did the non-fermented black bean. Taking into account methanol extract content, all the prepared kojis showed greater antioxidative activity than non-fermented black bean. Among the various koji extracts examined, extract of A. awamori-koji exhibited the highest antioxidative activity as did the A. awamori-koji when comparing its antioxidative activity with that of other kojis and non-fermented black bean. In general, the total extractable phenolic compounds and anthocyanins content in black beans increased after fermentation. This might lead to the increased antioxidant activities of black bean kojis observed.

Eighty-eight extracts of different polarity obtained from 18 Lamiaceae medicinal and aromatic plants were screened for their antioxidant and antifungal properties. A TLC analysis combined with two bioassays were used to detect these activities. In most cases the highest radical scavenger capacity was detected in methanolic and aqueous extracts and polyphenols may be responsible. The most interesting antioxidant activity was observed in polar extracts obtained from Lycopus europaeus, Melissa officinalis, Origanum vulgare subsp. virens and Lavandula latifolia. On the contrary, the best results for the antifungal test against Rhizopus stolonifer were produced by non-polar herbal extracts. Phlomis lychnitis, Salvia pratensis and Calamintha sylvatica caused the highest inhibition on Rhizopus growth.

Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze-fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100%, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74-97.38%. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA-DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed.

Aims: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation., Methods and Results: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement., Conclusion: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software., Significance and Impact of the Study: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold storage of tempeh (P<0.01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents and compositions, and did not reduce phytate contents. Slight increases of vitamins B(6) and niacinamide, and slight decreases of B(1) and biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh. The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus, real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous substrate such as barley tempeh.

The potential of using heat treatment alone or in combination with an antagonistic yeast for the control of blue mold decay and Rhizopus decay of peaches caused by Penicillium expansum and Rhizopus stolonifer respectively, and in reducing natural decay development of peach fruits, as well as its effects on postharvest quality of fruit was investigated. In vitro tests, spore germination of pathogens in PDB was greatly controlled by the heat treatment of 37 degrees C for 2 d. In vivo test to control blue mold decay of peaches, heat treatment and antagonist yeast, as stand-alone treatments, were capable of reducing the percentage of infected wounds from 92.5% to 52.5% and 62.5%, respectively, when peach fruits stored at 25 degrees C for 6 d. However, in fruit treated with combination of heat treatment and Cryptococcus laurentii, the percentage of infected wounds of blue mold decay was only 22.5%. The test of using heat treatment alone or in combination with C. laurentii to control Rhizopus decay of peaches gave a similar result. The application of heat treatment and C. laurentii resulted in low average natural decay incidences on peaches after storage at 4 degrees C for 30 days and 20 degrees C for 7 days ranging from 40% to 30%, compared with 20% in the control fruit. The combination of heat treatment and C. laurentii was the most effective treatment, and the percentage of decayed fruits was 20%. Heat treatment in combination with C. laurentii had no significant effect on firmness, TSS, ascorbic acid or titratable acidity compared to control fruit. Thus, the combination of heat treatment and C. laurentii could be an alternative to chemicals for the control of postharvest decay on peach fruits.

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this "first mycotoxin from lower fungi" is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.

Rhizopus oligosporus Saito can ferment soybeans or cereal grains to tempeh, a sliceable cake with improved nutritional properties. Volatiles produced by different R. oligosporus strains grown on malt extract agar (MEA), barley and soybean were investigated. The effect of co-cultivation with Lactobacillus plantarum on the production of volatiles was also studied. Volatile compounds were collected in situ by headspace diffusion and identified by GC-MS. The ten R. oligosporus strains that had different colony morphologies on MEA produced very similar volatile profiles, except for slight variations among the minor volatile compounds (e.g. sesquiterpenes). Likewise, practically no differences in volatile profiles were observed between three of the strains grown on soybeans. In contrast, the R. oligosporus volatile profile on soybean was different from that on barley from the same strain. Co-cultivation with L. plantarum did not influence volatile production by R. oligosporus. The dominant compounds produced on all three substrates were ethanol, acetone, ethyl acetate, 2-butanone, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Acetaldehyde and 2-methyl-propanal were also produced on MEA and barley, while 2-pentanone, methyl acetate, 2-butanol and 3-methyl-3-buten-1-ol were observed on soybeans. Ethanol, 2-methyl-1-butanol and 3-methyl-1-butanol were the most abundant volatile compounds produced on MEA and barley, while 2-butanone was the dominant volatile metabolite on soybean. The mushroom odour compounds, 3-octanone and 1-octen-3-ol, were only detected from soybean and soybean tempeh.

Three isolates of zygomycetes belonging to two different genera (Rhizopus oryzae and Absidia corymbifera) were used to produce a systemic infection in neutropenic mice. On days -2 and -1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with R. oryzae were all positive, while treated mice challenged with A. corymbifera generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with R. oryzae, both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with A. corymbifera. Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.

Aflatoxin B1 is a toxigenic and carcinogenic compound produced by Aspergillus flavus and Aspergillus parasiticus. An approach to prevent aflatoxin contamination in feed was carried out by using Saccharomyces cerevisiae (Sc) and Rhizopus oligosporus (Ro). Aspergillus flavus was cultured together with Sc, Ro and their combination (ScRo) in chicken feed. The aflatoxin B1 content was observed at day 0, 5, 10 and 15. The result showed that aflatoxin B1 contaminations in feed were reduced by Sc, Ro and ScRo addition. The highest reduction of aflatoxin B1 content was shown at day 5 for all treatments with Sc, Ro and ScRo. The best activity of reducing aflatoxin B1 was shown by Ro. Although the ability of reducing aflatoxin B1 of Sc, Ro or ScRo was not significantly different, Sc or Ro gave the better result than ScRo and they are better used individually.

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

Spores from the co-culture of Aspergillus foetidus and Rhizopus oryzae were subjected to UV, heat and NTG (3-nitro,5-methylguanidine) mutagenesis. A few colonies were screened from the selected media for tannase study. Amongst all, the best mutant isolated from the heat treatment (60 degrees C for 60 min) was SCPR 337. The maximum yield of gallic acid and tannase in case of mutant strain was 95.2% and 53.6 U/ml with an incubation period of 30 h as compared to wild strain where the incubation period was 48 h with an enzyme activity of 44.2 U/ml and gallic acid yield of 94%, respectively. The mutant was sensitive to tetracycline and was also an over-producer of protease and amylase.

Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.

In order to study the feasibility of commercial-scale L-lactic acid production by Rhizopus sp. MK-96-1196 using large scale airlift bioreactors (ALBs), a scale-up study from 0.003 m3 to 5 m3 ALB was carried out using oxygen transfer rate (OTR) as the scale-up criterion. Enhanced L-lactic acid production was achieved at OTRs higher than 0.28 (g-O2/l/h) irrespective of the scale of the bioreactor in question: in the range of 0.003 m3 to 5 m3, more than 90 g/lL-lactic acid was produced with a yield of approximately 80%, based on the initial glucose concentration. In future research, we plan to design an ALB greater than 3000 m3 (working volume: 2000 m3) for further studies on the production of L-lactic acid in large quantities.

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94-0.97 g/g of starch or sugars associated with 4-5 g/l of fungal biomass produced, while 17-19 g/l fungal biomass with a lactic acid yield of 0.65-0.76 g/g was produced by the R. oryzae 2062 in 36-48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8-15% lactic acid yield and 10-20% fungal biomass.

The zygomycete Rhizopus oligosporus is traditionally used to ferment soybean tempeh, but it is also possible to ferment other legumes and cereals to tempeh. The traditional soybean tempeh harbours a multitude of microorganisms with potentially beneficial or detrimental effects on quality. Lactic acid bacteria (LAB) have positive effects on the safety of soybean tempeh, but the effects of LAB on R. oligosporus growth have not been investigated. We have developed a cereal grain tempeh by fermenting pearled barley with R. oligosporus ATCC 64063. Four LAB species, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri and Lactococcus lactis were assessed for their growth abilities and their effects on R. oligosporus growth during barley tempeh fermentation. Growth of LAB was assayed as colony forming units (cfu), while growth of R. oligosporus was measured as ergosterol content and hyphal length. The two fungal measurements highly correlated (r=0.83, P<0.001, n=90). The ergosterol content of fungal mycelia ranged from 11.7 to 30.1 mg/g fungal dry matter. L. plantarum multiplied from 4.8 to 7.4 log cfu/g dry tempeh and L. fermentum increased from 4.4 to 6.8 log cfu/g during 24 h incubation at 35 degrees C. L. reuteri and L. lactis had significantly slower growth, with increases from 4.8 to 5.6 log cfu/g and 5.0 to 5.4 log cfu/g, respectively. The growth of R. oligosporus and the final pH (4.9) in barley tempeh were not significantly influenced by any of the LAB investigated.

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87-0.97 g/g starch associated with 1.5-2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.

Objectives: The effect of different media and composition on the in vitro activity of posaconazole, caspofungin and voriconazole against 59 zygomycetes species was determined., Methods: The media tested were RPMI 1640 medium with and without 2% glucose, antibiotic medium 3 (AM3) with and without 2% glucose, and high resolution (HR) medium., Results: Posaconazole was significantly more active than caspofungin and voriconazole, both in RPMI 1640 medium with 2% glucose and in HR medium. Adding glucose improved the determination of end points, but had only minor influence on the MICs. MICs evaluated in AM3 were lower than in RPMI 1640 medium or HR medium., Conclusions: The in vivo effect of posaconazole in zygomycosis needs further evaluation.

Fast-growing Zygomycetes, most notably Rhizopus oligosporus, are traditionally used in many food fermentations, for example, for soybean tempeh production. R. oligosporus is considered to belong to the Rhizopus microsporus group. Certain R. microsporus strains have been reported to produce either the pharmaceutically active rhizoxins or the highly toxic rhizonins A and B. In this study was investigated the formation of secondary metabolites by R. microsporus, R. oligosporus, and Rhizopus chinensis grown on a wide range of different semisynthetic and natural substrates. Liquid chromatography, combined with photodiode array detection and high-resolution mass spectrometric techniques, was used to identify secondary metabolites. Growth on maize, brown rice, and Pharma agar gave both the highest amounts and the maximum diversity of rhizoxins and rhizonins. Rhizoxins were produced by all four R. microsporus strains, whereas only one strain produced rhizonins. The six R. oligosporus and four R. chinensis strains investigated did not produce any of these two classes of metabolites.

Several steroids, in particular progesterone, are toxic for the filamentous fungus Rhizopus nigricans and, at high concentrations, inhibit its growth. Previous studies on this microorganism revealed progesterone specific receptors coupled to G proteins at the plasma membrane. In this study, the next step of steroid signalling in R. nigricans following G protein activation is investigated, together with the possible impact of this pathway on fungal growth inhibition. The intracellular level of cAMP decreased in the presence of steroids, demonstrating the probable involvement of cAMP signalling in the response of R. nigricans to steroids. Results of the growth analysis in the presence of cAMP increasing agents suggest that the role of cAMP in fungal growth inhibition by steroids cannot be ruled out, but it would appear to be minor and not make a major contribution to growth inhibition.

The alcoholic beverage parakari is a product of cassava (Manihot esculenta Crantz) fermentation by Amerindians of Guyana. While fermented beverage production is nearly universal among indigenous Amazonians, parakari is unique among New World beverages because it involves the use of an amylolytic mold (Rhizopus sp., Mucoraceae, Zygomycota) followed by a solid substratum ethanol fermentation. The mycological significance of this dual fermentation process previously was unrecognized. A detailed study of parakari fermentation was made in the Wapisiana Amerindian village of Aishalton, South Rupununi, Guyana. Thirty steps were involved in parakari manufacture, and these exhibited a high degree of sophistication, including the use of specific cassava varieties, control of culture temperature and boosting of Rhizopus inoculum potential with purified starch additives. During the fermentation process, changes in glucose content, pH, flavor, odor and culture characteristics were concomitant with a desirable finished product. Parakari is the only known example of an indigenous New World fermentation that uses an amylolytic mold, likely resulting from domestication of a wild Rhizopus species in the distant past. Parakari production is remarkably similar to dual fermentations of Asia, yet it was independently derived.

Conidia of Trichoderma harzianum were formulated in invert emulsion (water-in-oil type) at a concentration of 6.0 x 10(7) conidia/ml of the formulation. Treatments with conidia in the formulated emulsion and conidia in sterile distilled water were conducted to assess the effect on Penicillium expansum Link inoculated on apple fruits. Comparisons were made with control treatments of an uninoculated emulsion and uninoculated distilled water. The T. harzianum-containing emulsion significantly (P

Several microbial volatile organic compounds (MVOCs) that can serve as potential chemical markers for microbial contamination in tobacco have been identified. Four different fungal species, Aspergillus niger (AN), A. ornatus (AO), Pencillium chrysogenum (PC) and Rhizopus stolonifer (RS), commonly reported in moldy tobacco were cultured and screened for MVOCs. Because the MVOCs emitted by a microbial species are substrate specific, the fungal strains were separately grown on potato dextrose agar (PDA) and tobacco products. MVOCs from the mold cultures grown on PDA and tobacco products were extracted using closed-loop stripping analysis (CLSA) and identified by gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). Some of the prominent tobacco mold markers identified by this method include: 1-octen-3-ol; 2-octen-1-ol; 2-methyl-1-butanol; 3-methyl-1-butanol; 1-octene and 2-pentanone. In particular, 1-octen-3-ol was detected in all the mold cultures and moldy tobacco samples analyzed. Olfactory evaluation of 1-octen-3-ol indicated a characteristic musty odor and the odor threshold was determined to be approximately 200 ng/ml. The limits of detection for 1-octen-3-ol using GC/TOF-MS and GC/mass selective detector (MSD) in the full-scan mode and selected ion monitoring (SIM) mode were investigated. The CLSA-GC/TOF-MS demonstrates a fast, sensitive and semi-quantitative analytical technique for screening tobacco materials for the presence of mold via chemical markers of microbial contamination.

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.

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.

Rhizopus stolonifer is an important post-harvest pathogenic fungus. Recent taxonomic findings based on morphological and growth characteristics led to a dramatic reduction in the number of accepted species within the genus. The aim of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random amplified polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, but only low or moderate variability was observed within these groups. These results suggest a high genetic heterogeneity in the case of R. stolonifer: isolates of R. stolonifer var. stolonifer, R. stolonifer var. reflexus and R. niveus displayed species-level genetic distances, which gives rise to considerations that they might be separate species.

A new semi-selective medium, acidified weak potato-dextrose agar (AWPDA) with Mertect (active ingredient: thiabendazole), was developed for the isolation and enumeration of Alternaria species from samples of soil and plant debris. The medium was selected based on growth inhibition tests against Alternaria and several other commonly encountered saprobic fungi utilizing three antifungal agents, Botran (active ingredient: dichloran), Bayleton (active ingredient: triadimefon), and Mertect, and two basal media, acidified potato-dextrose agar (APDA) and AWPDA. Botran inhibited growth of Rhizopus stolonifer moderately, but had little effect on Cladosporium cladosporoides, Fusarium oxysporum, Penicillium chrysogenum, or Trichoderma harzianum. Bayleton inhibited growth of R. stolonifer and C. cladosporoides severely, and inhibited growth of F. oxysporum, P. chrysogenum, and T. harzianum moderately. Mertect inhibited growth of C. cladosporoides, F. oxysporum, P. chrysogenum, and T. harzianum completely, but had little or moderate effect on R. stolonifer. All three antifungal agents inhibited growth of Alternaria species slightly or moderately. The combination of Bayleton and Mertect inhibited growth of all fungi severely. A comparison of recovery rates of Alternaria from soil and plant debris samples on AWPDA with Mertect and weak potato-dextrose agar (WPDA) revealed that Alternaria spp. accounted for 63.6%-81.0% of recovered fungal isolates on AWPDA with Mertect as compared to 0.6%-2.7% of recovered isolates on WPDA. The AWPDA medium with Mertect exhibited superior selective growth of Alternaria species from samples of soil and plant debris, and will be useful in studies where the recovery and enumeration of Alternaria species is necessary.

The present study describes the biosynthesis of proteases by a locally isolated mould culture of Rhizopus oligosporus IHS13 in a low-cost medium by solid-state fermentation technique. The fermentation was carried out in a low-cost medium such as sunflower meal, wheat bran and rice bran. Sunflower meal and wheat bran in a ratio of 1:1 and moistened with distilled water was found to be the best substrate for protease synthesis. All the three substrates are very cheap agricultural by-products found in Pakistan. The production of proteases in sunflower meal and rice bran was also investigated but the results were unsatisfactory. Different cultural conditions such as rate of fermentation, effect of incubation temperature, effect of pH and depth of the fermentation medium were also optimized. The maximum enzyme synthesis was found after 72 h of fermentation at a temperature of 30 degrees C. The optimum pH and depth of the medium for protease synthesis were found to be 5.0 and 20 mm respectively. The maximum enzyme biosynthesis found during the course of present studies was 7.0 U ml(-1)., (Copyright 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)