Transcriptional regulation contributes more to Monascus pigments diversity in different strains than to DNA sequence variation.

Monascus azaphilone pigments, including red, orange, and yellow, are world-famous food colorants. However, the pigments produced by different Monascus species vary in yields and compositions. The underlying mechanism is unclear. In this study, four wild-type Monascus strains, namely M. anka M7, M. purpureus M9, M. ruber C100, and M. aurantiacus M15, were selected as research objects according to the diversification of their pigments fermented in the same mediums and conditions. Twenty-three 3 kbp segments (300 bp overlap with adjacent segments) of the pigment gene cluster were amplified, sequenced, and assembled into the DNA sequences of the clusters. The DNA sequences of pigment biosynthetic gene clusters of the four strains showed 99.94% similarity according to the results of multiple alignment. The expression levels of 17 pigment biosynthetic genes of four strains were determined by using real-time quantitative PCR. The transcriptional regulation contributed more than the DNA sequence variation in Monascus pigments metabolism. Our result gives insight into the study of Monascus pigment biosynthesis.

Cellulase-assisted extraction and anti-ultraviolet activity of polysaccharides from the root of Flammulina velutipes on Caenorhabditis elegans.

We investigated the cellulase-assisted extraction and anti-ultraviolet activity of water-soluble polysaccharides from the root of Flammulina velutipes on Caenorhabditis elegans. A Box-Behnken design experiment with three factors and three levels, including enzymolysis temperature, microwave time, and microwave power, was designed on the basis of the results of single-factor experiments. For improving the polysaccharide yield of F. velutipes root, the following optimal extraction conditions were used: 52.67°C enzymolysis temperature, 80s microwave time, and 144 W microwave power. Under optimal conditions, the actual measured value of the yield was 2.01% (w/w) and the predicted value was 2.06% (w/w). One fraction (FRP-2) was isolated and purified, and its characteristics were analyzed. The average mean molecular weight of FRP-2 was measured to be 2.60×105 Da, and its monosaccharide composition is mainly glucose. The sugar units are present both in the α-configuration and ß-configuration. Moreover, FRP-2 exhibited certain anti-ultraviolet activity to C. elegans when the polysaccharide concentration ranged between 0.05mg/mL and 0.20mg/mL.

Effect of Temperature-Shift and Temperature-Constant Cultivation on the Monacolin K Biosynthetic Gene Cluster Expression in Monascus sp.

In this study, the effects of temperature-shift (from 30 to 25 °C) and temperature-constant (at 30 °C) cultivation on the mass of Monascus fuliginosus CG-6 mycelia and concentration of the produced monacolin K (MK) were monitored. The expression levels of the MK biosynthetic genes of M. fuliginosus CG-6 at constant and variable culture temperatures were analysed by real-time quantitative polymerase chain reaction (RT-qPCR). The total protein was collected and determined by liquid chromatography-electrospray ionisation with tandem mass spectrometry (LC-ESI-MS/MS). Results showed that the maximum mycelial mass in temperature-shift cultivation was only 0.477 g of dry cell mass per dish, which was lower than that in temperature-constant cultivation (0.581 g of dry cell mass per dish); however, the maximum concentration of MK in temperature-shift cultivation (34.5 µg/mL) was 16 times higher than that in temperature-constant cultivation at 30 °C (2.11 µg/mL). Gene expression analysis showed that the expression of the MK biosynthetic gene cluster at culture temperature of 25 °C was higher than that at 30 °C, which was similar to the trend of the MK concentration, except for individual MK B and MK C genes. Analysis of differential protein expression revealed that 2016 proteins were detected by LC-ESI-MS/MS. The expression level of efflux pump protein coded by the MK I gene exhibited the same upregulated trend as the expression of MK I in temperature-shift cultivation. Temperature-shift cultivation enhanced the expression of proteins in the secondary metabolite production pathway, but suppressed the expression of proteins involved in the mycelial growth.

Optimization of submerged fermentation medium for citrinin-free monascin production by Monascus.

Microbial fermentation of citrinin-free Monascus pigments is in favor in the development of food industry. This study investigated the influences of carbon source, nitrogen source, and mineral salts on the cell growth, monascin (MS), and citrinin (CT) production in Monascus M9. A culture medium composition was established for maximizing the production of citrinin-free MS in submerged culture, as follows: 50 g/L Japonica rice powder, 20 g/L NH4NO3, 3 g/L NaNO3, 1.5 g/L KH2PO4, 1 g/L MgSO4 · 7H2O, 0.2 g/L MnSO4. Under these conditions, no CT was detectable by high performance liquid chromatography. The yield of MS reached 14.11 mg/g, improving approximately 30% compared with before optimization.

Stimulatory effects of blue light on the growth, monascin and ankaflavin production in Monascus.

Light is an important signal for fungi. We analyzed the influence of blue light of various intensities and illumination times on growth, monascin (MS) and ankaflavin (AK) biosyntheses in Monascus strain M9. Blue light changed the color of colonies. The colonies grown in the dark were orange, but turned pale when exposed to continuous blue light. MS production increased by 12.5, 27, and 14.5 % under blue light of 100 lux for 15 min/day, 100 lux for 30 min/day, and 200 lux for 15 min/day, respectively, compared to growth in the dark. AK production increased by 14.4, 22, and 13 % under the same condition. MS and AK production decreased when exposed to blue light of 300 and 450 lux. The expression of pigment biosynthetic genes were analyzed by real-time quantitative PCR and correlated with phenotypic production of MS and AK.

A Zn(II)(2)Cys(6) transcription factor MPsGeI suppresses pigment biosynthesis in Monascus.

High-quality natural edible pigments known as monascus pigments (MPs) are widely used in food, medicine, and chemical industries as active functional ingredients. At the transcriptional level, the expression of MPs genes are tightly controlled, limiting their productivity and color value. Hitherto our understanding of the regulation of expression of MPs genes has been rather limited. Here, we describe a pathway-specific Zn(II)(2)Cys(6) transcription factor involved in the MPs biosynthetic cluster named MPsGeI, which encodes a 813-amino-acid protein with six introns. Expression of all MPs biosynthetic genes and accumulation of MPs were remarkably increased in ΔMPsGeI strain, and MPs production was significantly reduced in MPsGeI over-expressing strain. Results clearly demonstrated that MPsGeI negatively regulates MPs accumulation via transcriptional regulation of MPs biosynthetic genes, and plays a central repressive role in MPs' biosynthesis. Transcriptomic analyses revealed that MPsGeI disruptant regulated higher concentrations of precursors flowing to pigment and resulted in accumulation of a large amount of red MPs in hyphae. This work offers an efficient method for increasing MPs's productivity and color value and provides novel insights into the regulatory mechanisms of fungal cellular processes, which will assist to enhance MPs production and application.

Binding of ankaflavin with bovine serum albumin (BSA) in the presence of carrageenan and protective effects of Monascus yellow pigments against oxidative damage to BSA after forming a complex with carrageenan.

Ankaflavin (AK) is a typical yellow pigment extracted from Monascus-fermented rice with several biological effects; however, its solubility is poor. Thus, research studies of the delivery systems of AK, especially those constructed from protein-polysaccharide complexes, have attracted considerable attention. However, the interactions that exist in the system have rarely been investigated. This work focused on the interactions between AK and bovine serum albumin (BSA) as well as the influence of carrageenan (Car) on the binding of AK to BSA. Results revealed that the quenching of BSA by AK involved the static quenching mechanism. The formed BSA-AK complexes were mainly maintained by hydrophobic forces and AK was located within the hydrophobic cavity of BSA. Compared to free AK or AK only complexed with BSA, a higher absorption intensity of AK was observed for the formed BSA-AK-Car complexes, indicating changes in the microenvironment of AK. This was confirmed by the increase in the α-helix content of BSA after the formation of BSA-AK-Car complexes. Hydrogen bond, van der Waals, and electrostatic interactions were verified to be the primary forces preserving the BSA-AK-Car complexes. Moreover, the antioxidant potential of Monascus-fermented products rich in AK (denoted as Mps), namely BSA-Mps and BSA-Mps-Car was evaluated. The antioxidant activity of Mps was negatively impacted by BSA, while the addition of Car could enhance the antioxidant capacity of BSA-Mps-Car complexes. Meanwhile, Mps showed a protective effect against free radical-induced oxidation damage to BSA, and Car could further improve this effect.

Real-time quantitative analysis of the influence of blue light on citrinin biosynthetic gene cluster expression in Monascus.

When Monascus MX was grown under blue light instead of in the dark, citrinin production increased from 478 mg l(-1) to 698 mg l(-1). To explain this, the expression of the pksCT gene, which encodes citrinin polyketide synthase, and of 5 ORFs around it, were monitored. Blue light enhanced citrinin production by upregulating the expression of orf1, orf3, and orf4, indicating that pksCT was not the key gene responsible for the quantity of citrinin production in blue light.

Ammonium nitrate regulated the color characteristic changes of pigments in Monascus purpureus M9.

Monascus pigments (MPs) with different color characteristics, produced by submerged fermentation of Monascus purpureus M9, have potential application in food industry. In the present study, the effects and regulatory mechanisms of ammonium nitrate (AN) on the color characteristics of MPs were investigated. The concentration of intracellular pigments was significantly decreased when subjected to AN. The hue and lightness value indicated AN altered the total pigments appearance from original red to orange. The HPLC analysis for six major components of MPs showed that the production of rubropunctatin or monascorubrin, was significantly reduced to the undetectable level, whereas the yields of monascin, ankaflavin, rubropunctamine and monascorubramine, were apparently increased with AN supplement. To be noted, via real-time quantitative PCR strategy, the expression level of mppG, closely relative to orange pigments biosynthesis, was significantly down-regulated. However, the expression of mppE, involved in yellow pigments pathway, was up-regulated. Moreover, the broth pH value was dropped to 2.5-3.5 in the fermentation process resulted from AN treatment, along with the increased extracellular polysaccharide biosynthesis. Taken together, the change of MPs categories and amounts by AN might be the driving force for the color characteristics variation in M. purpureus M9. The present study provided useful data for producing MPs with different compositions and modified color characteristics.

The noncovalent conjugations of human serum albumin (HSA) with MS/AK and the effect on anti-oxidant capacity as well as anti-glycation activity of Monascus yellow pigments.

Monascin (MS) and ankaflavin (AK), as typical yellow lipid-soluble pigments identified from Monascus-fermented products, have been confirmed to possess diverse biological activities such as anti-oxidation, reversing diabetes, and anti-atherosclerosis, and have received increasing attention in recent years. Certainly Monascus-fermented product with a high content of MS/AK is also a concern. The current work explored interactions between MS/AK and human serum albumin (HSA) as well as their influence on the anti-oxidant properties of MS/AK. Moreover, the anti-glycation potential of Monascus-fermented products rich in MS and AK (denoted as Mps) was assessed. The results showed that the fluorescence emission of HSA was quenched by MS/AK through a static quenching mechanism, and MS-HSA and AK-HSA complexes were mainly formed by van der Waals forces and hydrophobic interactions, but AK showed a higher binding affinity than MS. Although the DPPH radical-scavenging abilities of MS-HSA and AK-HSA complexes declined, Mps significantly reduced the formation of fructosamine, α-dicarbonyl compounds and advanced glycation end products (AGEs) in the in vitro glycation model (HSA-glucose). Notably, approximately 80% of fluorescent-AGEs were suppressed by Mps at a concentration of 0.95 mg mL-1, while aminoguanidine (AG, a reference standard) caused only 65% decrease at the same concentration. Although radical scavenging and metal chelating activities could justify the observed anti-glycation activity of Mps, in-depth research on the structures of other functional compounds present in Mps except MS/AK and reaction mechanisms should be performed. Overall, the present study proved that Mps would be promising sources of food-based anti-glycation agents because of their superior inhibitory effect on AGEs.

Molecular insight on the binding of monascin to bovine serum albumin (BSA) and its effect on antioxidant characteristics of monascin.

Monascin (MS) is a yellow lipid-soluble azaphilonoid pigment identified from Monascus-fermented products with promising biological activities. This work studied interactions between MS and bovine serum albumin (BSA) as well as their influences on the antioxidant activity of MS. Experimental results demonstrated that the fluorescence emission of BSA was quenched by MS via static quenching mechanism and the formed BSA-MS complex was mainly maintained by hydrophobic and hydrogen bond interactions. Meanwhile, the probable binding pocket of MS located near site I of BSA and the corresponding conformational and structural alterations of BSA were determined. Furthermore, the molecular modeling approach was performed to understand the visual representation of binding mode between BSA and MS. It was noticeable that the BSA-MS complex exhibited reduced DPPH radical-scavenging ability, which might be attributed to the restraining effect of BSA on the relevant reaction pathways involved in antioxidation by MS.

The molecular mechanisms of Monascus purpureus M9 responses to blue light based on the transcriptome analysis.

Light is an important environmental factor that regulates various physiological processes of fungi. To thoroughly study the responses of Monascus to blue light, transcriptome sequencing was performed on mRNAs isolated from samples of Monascus purpureus M9 cultured under three conditions: darkness (D); exposure to blue light for 15 min/d (B15); and exposure to blue light for 60 min/d over 8 days (B60). The number of differentially expressed genes between the three pairs of samples-B15 vs D, B60 vs B15, and B60 vs D-was 1167, 1172, and 220, respectively. KEGG analysis showed the genes involved in primary metabolism including carbon and nitrogen metabolism were downregulated by B15 light treatment, whereas B15 upregulated expression of genes involved with aromatic amino acid metabolism, which associated with development, and branched chain amino acid metabolism, and fatty acid degradation, which can produce the biosynthetic precursors of pigments. When exposed to B60 conditions, genes with roles in carbohydrate metabolism and protein synthesis were upregulated as part of a stress response to blue light. Based on this study, we propose a predicted light-stimulated signal transduction pathway in Monascus. Our work is the first comprehensive investigation concerning the mechanism of Monascus responses to blue light.

Effects of blue light on pigment biosynthesis of Monascus.

The influence of different illumination levels of blue light on the growth and intracellular pigment yields of Monascus strain M9 was investigated. Compared with darkness, constant exposure to blue light of 100 lux reduced the yields of six pigments, namely, rubropunctatamine (RUM), monascorubramine (MOM), rubropunctatin (RUN), monascorubrin (MON), monascin (MS), and ankaflavin (AK). However, exposure to varying levels of blue light had different effects on pigment production. Exposure to 100 lux of blue light once for 30 min/day and to 100 lux of blue light once and twice for 15 min/day could enhance RUM, MOM, MS, and AK production and reduce RUN and MON compared with non-exposure. Exposure to 100 lux twice for 30 min/day and to 200 lux once for 45 min/day decreased the RUM, MOM, MS, and AK yields and increased the RUN and MON. Meanwhile, the expression levels of pigment biosynthetic genes were analyzed by real-time quantitative PCR. Results indicated that gene MpPKS5, mppR1, mppA, mppB, mmpC, mppD, MpFasA, MpFasB, and mppF were positively correlated with the yields of RUN and MON, whereas mppE and mppR2 were associated with RUM, MOM, MS, and AK production.

Polysaccharide from Pleurotus nebrodensis induces apoptosis via a mitochondrial pathway in HepG2 cells.

A novel alkali extractable polysaccharide (designated as PNA-2) was purified from Pleurotus nebrodensis and the effects of purified PNA-2 on the proliferation and apoptosis of human hepatic cancer cells (HepG2) were investigated in this study. The results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that PNA-2 inhibited the proliferation of HepG2 cells by apoptosis induction, which was also characterized using scanning electron microscopy (SEM). Moreover, the expression of apoptosis-associated mRNA, proteins and the cell-cycle arrest at the G0/G1 phase was determined using RT-qPCR, Western blot and flow cytometry, respectively. A notable inhibition of the migration rate of PNA-2-treated HepG2 cells was observed using a cell scratch assay. DNA damage was observed using a comet assay and AO/EB staining in HepG2 cells, which were exposed to PNA-2. Induction of the mitochondria-mediated intrinsic apoptotic pathway by PNA-2 was indicated by the loss of mitochondrial membrane potential (ΔΨm), Bcl-2 dysregulation and cytochrome c release. All the results suggested that the mitochondria-mediated intrinsic apoptotic pathway could be involved in PNA-2-mediated apoptosis of human liver carcinoma cells HepG2. Finally, the results indicated that PNA-2 significantly suppressed tumor growth in HepG2 tumor-bearing mice, indicating that PNA-2 may be developed as a candidate drug or functional food factor to prevent or treat liver cancer.

Pleurotus nebrodensis polysaccharide(PN50G) evokes A549 cell apoptosis by the ROS/AMPK/PI3K/AKT/mTOR pathway to suppress tumor growth.

Since the strong antineoplastic potential against A549 cells of Pleurotus nebrodensis polysaccharide (PN50G) in vitro has been proven previously, the definitive mechanism of PN50G-induced apoptosis in A549 cells in vivo was further investigated. All the results indicated that PN50G significantly suppressed tumor growth in A549 tumor-bearing mice. Tumor cells treated with PN50G were arrested in the G0/G1 phase, and marked changes in the expression of cell cycle-related proteins, including cyclin D1, cyclin A and cyclin B1, were observed. Moreover, western blotting analysis indicated that PN50G triggered the mitochondrial apoptotic pathway, for an increased Bax/Bcl-2 ratio, release of cytochrome c, cleavage of caspase-3 and PRPP in A549 tumor cells were observed. And the decrease in the expression of the translation related protein P70S6K was observed, because PN50G activated AMPK phosphorylation, but inhibited PI3K/AKT phosphorylation and suppressed the activation of the mammalian target of rapamycin (mTOR) induced by PN50G. In vivo imaging was performed on tumor-bearing mice, and the results indicated that PN50G significantly increased the intracellular levels of reactive oxygen species (ROS). Furthermore, it indicated that PN50G promoted the protein expression of Beclin 1 and LC-3 in a dose-dependent manner. All the results suggested that PN50G-mediated apoptosis and autophagy of A549 tumor cells in vivo mainly involved in the mitochondrial pathway and the AMPK/PI3K/mTOR pathway.

Cytotoxicity, DNA damage, and apoptosis induced by titanium dioxide nanoparticles in human non-small cell lung cancer A549 cells.

Concerns about the risk of titanium dioxide nanoparticles (TiO2 NPs) to human health and environment are gradually increasing due to their wide range of applications. In this study, cytotoxicity, DNA damage, and apoptosis induced by TiO2 NPs (5 nm) in A549 cells were investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed the time- and concentration-dependent cytotoxic effects of TiO2 NPs in a concentration range of 50 to 200 µg/mL. A statistically significant (p < 0.05) induction in DNA damage was observed by the comet assay in cells exposed to 50 to 200 µg/mL TiO2 NPs for 48 h. A significant (p < 0.05) induction in micronucleus formation determined by 4,6-diamino-2-phenylindole (DAPI) staining was also observed at the above concentrations. Typical apoptotic morphological feature and apoptotic bodies in A549 cells induced by TiO2 NPs at the above concentrations were observed by scanning electron micrographs. Flow cytometric analysis demonstrated that the cells treated with TiO2 NPs at concentrations of 100 and 200 µg/mL showed a significant G2/M phase arrest and a significant increased proportion of apoptotic cells. TiO2 NPs also disrupted the mitochondrial membrane potential evaluated by rhodamine 123 staining. Further analysis by quantitative real-time PCR (qRT-PCR) indicated that the expression of caspase-3 and caspase-9 messenger RNA (mRNA) was increased significantly at the concentrations of 100 and 200 µg/mL TiO2 NPs for 48 h. Taken together, these findings suggest that TiO2 NPs can inhibit A549 cell proliferation, cause DNA damage, and induce apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data provide strong evidence that TiO2 NPs can induce cytotoxicity, significant DNA damage, and apoptosis of A549 cells, suggesting that exposure to TiO2 NPs could cause cell injury and be hazardous to health.

Pleurotus nebrodensis polysaccharide (PN-S) enhances the immunity of immunosuppressed mice.

In the present study, the effects of Pleurotus nebrodensis polysaccharide (PN-S) on the immune functions of immunosuppressed mice were determined. The immunosuppressed mouse model was established by treating the mice with cyclophosphamide (40 mg/kg/2d, CY) through intraperitoneal injection. The results showed that PN-S administration significantly reversed the CY-induced weight loss, increased the thymic and splenic indices, and promoted proliferation of T lymphocyte, B lymphocyte, and macrophages. PN-S also enhanced the activity of natural killer cells and increased the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in the serum. In addition, PN-S treatment significantly increased the phagocytic activity of mouse peritoneal macrophages. PN-S also increased the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), and nitric oxide (NOS) in splenocytes. qRT-PCR results also indicated that PN-S increased the mRNA expression of IL-6, TNF-α, INF-γ, and nitric oxide synthase (iNOS) in the splenocytes. These results suggest that PN-S treatment enhances the immune function of immunosuppressed mice. This study may provide a basis for the application of this fungus in adjacent immunopotentiating therapy against cancer and in the treatment of chemotherapy-induced immunosuppression.

Pleurotus nebrodensis polysaccharide induces apoptosis in human non-small cell lung cancer A549 cells.

Polysaccharides derived from edible fungi inhibit the proliferation of tumor cells. In this study, we investigated the effects of a polysaccharide (PN50G) from Pleurotus nebrodensis on A549 cell proliferation and apoptosis. MTT assay showed that PN50G induced apoptosis in the A549 cells in a dose-dependent. However, PN50G did not affect the proliferation viability of human fetal lung fibroblast cells MRC-5. Scanning electro microscopy (SEM) results indicate that PN50G induced a typical apoptotic morphological feature in A549. DNA accumulation and fragmentation were determined by acridine orange/ethidium bromide (AO/EB) staining. Flow cytometric analysis demonstrated that PN50G caused A549 cell apoptosis via cell arrest at the S phase. PN50G also extended the comet tail length in single-cell gel electrophoresis and disrupted the mitochondrial membrane potential as determined by Rdamine-123 staining. Further analysis by qRT-PCR showed that the expression of caspase-3 and caspase-9 mRNA increased. These findings suggest that PN50G can inhibit A549 cell proliferation and induce apoptosis mainly by activating the intrinsic mitochondrial pathway.

Bidirectional immunomodulatory activities of polysaccharides purified from Pleurotus nebrodensis.

A novel Pleurotus nebrodensis polysaccharide (PN50G) was purified, characterized, and cultured with RAW264.7 macrophages to evaluate its bidirectional immunostimulating characteristics. PN50G was purified by using Sepharose 4B gel filtration; the molecular weight of PN50G was distributed at 2,000 kDa. The total protein and carbohydrate constituent ratios in PN50G were 2.6 ± 0.9 % and 92.4 ± 6.1 % (w/w), respectively, which suggests that PN50G may be a major proteo-polysaccharide component. The phagocytosis of macrophages was improved significantly, and remarkable changes were observed in the morphology of PN50G-treated cells. Compared with the control group, the productions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and inducible nitric oxide synthase (iNOS) in the macrophages as well as the messenger RNA expressions were strongly induced by PN50G. Pro-/anti-inflammatory (IL-6/IL-10, TNF-α/IL-10, NO/IL-10) cytokine secretion ratios by lipopolysaccharide-stimulated RAW264.7 macrophages were significantly decreased by PN50G treatments in a dose-dependent manner under an excessive immune experimental model. This study suggests that purified PN50G can improve immunity and suppress immune overactivity in LPS-induced macrophages in a preventive manner to coordinate innate immunity and inflammatory responses.

Fumigant activity of volatiles from Streptomyces alboflavus TD-1 against Fusarium moniliforme Sheldon.

The fumigant activity of volatiles generated by Streptomyces alboflavus TD-1 against Fusarium moniliforme Sheldon was investigated. The results showed that the mycelial growth, sporulation, and spore germination of F. moniliforme were significantly suppressed, and that membrane permeability was disrupted in the presence of the volatiles. Gas chromatography-mass Spectrometry analysis revealed 31 kinds of volatile organic compound from the volatiles. Among them, two earthy-smelling substances, namely, 2-methylisoborneol (50.97%) and trans-1,10-dimethyl-trans-9-decalinol (3.10%) were found. The most abundant compound, 2-methylisoborneol, exhibited inhibitory activity against F. moniliforme by fumigation. All these results suggested that S. alboflavus TD-1 can be a promising starter for the inhibition of F. moniliforme through fumigant action.