Aspergillus parasiticus growth and aflatoxin production on black and white pepper and the inhibitory action of their chemical constituents

Aspergillus parasiticus Speare NRRL 2999 growth and aflatoxin production in black and white pepper and the penetration of the fungus in black pepper corn over various incubation periods were studied. Also, the effects of piperine and pepper oil on growth and aflatoxin production were studied. Under laboratory conditions, black and white pepper supported aflatoxin production (62.5 and 44 ppb (ng/g), respectively) over 30 days of incubation. Fungal growth measured in terms of chitin was considerably less in white pepper than in black pepper. A histological study of black pepper corn showed the fungus penetrating up to the inner mesocarp and establishing itself in the middle mesocarp. Piperine and pepper oil were found to inhibit fungal growth and toxin production in a dose-dependent manner. Thus, both black and white pepper could be considered as poor substrates for fungal growth and aflatoxin production.     

Growth and aflatoxin production ofaspergillus flavus on some spices marketed in Turkey

Growth and aflatoxin production byAspergillus flavus (MAM-268 C₁₇, TNS=98) on autoclaved coconut, black pepper, crushed red pepper, red pepper powder were studied. Coconut were found to be a better substrate for fungal growth, as well as aflatoxin production. Red pepper powder could be considered as a poor substrate for fungal growth. The amount of aflatoxin found in crushed red pepper under laboratory conditions is much less compared with coconut, black pepper and red pepper powder.     

Effect of Corn Steep Liquor on Mycelial Growth and Aflatoxin Production in Aspergillus parasiticus

Total aflatoxin concentrations produced by Aspergillus parasiticus, isolate 64-R8, in Czapek's broth fortified with corn steep liquor increased proportionately as the concentration of corn steep was increased from 0.5 to 8.0% (v/v) until maximal growth, as measured by dry mycelial weight, was reached. Thereafter, aflatoxin concentrations declined more rapidly than the rate of autolysis of mycelial material. Data are presented which indicate that the concentration of corn steep liquor also affects the ratio of production of aflatoxin B(1) and B(2) to that of aflatoxin G(1) and G(2). Further, this ratio also varies with time of incubation. Although both growth of the fungus and aflatoxin production are stimulated by the addition of corn steep to the basic medium, the stimulation of toxin production is much greater than fungus growth.     

An effective formulation for mould- and aflatoxin-free storage of corn

The efficacy of cinnamon oil, individually and in binary mixture with sodium chloride (NaCl), in preserving stored corn from fungal invasion and subsequent deterioration was investigated. It was observed that Aspergillus flavus and A. glaucus , the predominant storage fungi, invade corn during storage with the concomitant production of aflatoxin and the resultant loss of germinability in grains. The treatment with cinnamon oil at the non-phytotoxic level of 5 μl/g corn in combination with 10 μl/g NaCl solution (5%) inhibits synergistically the fungal infection, growth and aflatoxin production together with the deterioration of grains (loss of germinability). However, the test oil, when applied alone, was found to be effective only at the phytotoxic level of 20 μl/g corn.     

Antifungal action and antiaflatoxigenic properties of some essential oil constituents

The effect of 20 essential oil constituents on Aspergillus flavus growth and aflatoxin production was tested at the level of 1000 ppm. Some of the tested oils exhibited inhibitory effects on fungal growth and toxin formation. Five oils, namely geraniol, nerol and citronellol (aliphatic oils), cinnamaldehyde (aromatic aldehyde) and thymol (phenolic ketone), completely suppressed growth and aflatoxin synthesis. Trials for determining the minimum inhibitory concentration (MIC) of these oils revealed that geraniol, nerol and citronellol were effective at 500 ppm, while thymol and cinnamaldehyde were highly effective at doses as low as 250 and 200 ppm, respectively. It was observed that citral, citronellal and eugenol prevented fungal growth and toxin formation for up to 8 d. However, after 15 d of incubation, toxin production was greater than the controls.     

Antifungal activity of different plant extracts against Drechslera bicolor causing leaf blight of bell pepper

Aqueous extracts of 48 plants belonging to six different major groups of the plant kingdom, two commercially available botanicals and different fungicides were screened for antifungal activity against Drechslera bicolor causing leaf blight of bell pepper. The test fungi were isolated from bell pepper leaves collected from Udaipur district, Rajasthan, India. Among several botanicals, maximum inhibition of fungal growth was obtained by marigold, lat jeera, lemon grass, mehandi, onion and neem, respectively. Neem oil was superior over Zatropin against the fungus. Vitavax was also found as the best fungicide followed by Quintal and Saaf against the test fungus. The results revealed that these plants could be exploited for ecofriendly management of the diseases caused by the test fungal pathogen and seed biodeterioration during storage.     

N-carboxymethylchitosan inhibition of aflatoxin production: Role of zinc

Aqueous Solutions of N-carboxymethylchitosan (NCMC) suppressed both growth and aflatoxin production byAspergillus flavus andA. parasiticus in submerged culture (Adye and Mateles A&M). Test media were amended with various concentrations of zinc (15, 30, 45, 60 uM), and NCMC solution (0.62 uM). After 8 days incubation NCMC-treated cultures showed marked reduction of aflatoxin production and fungal growth. Enhanced levels of zinc did not overcome the NCMC-mediated inhibition of fungal growth or aflatoxin production.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

脂肪酸及其氧合物对曲霉属真菌菌丝生长、产孢和黄曲霉毒素合成的影响

黄曲霉毒素是由黄曲霉菌合成的一类毒性极高、致癌性极强的次生代谢物。一般认为,高油脂含量的作物种子被曲霉属真菌感染后容易产生黄曲霉毒素,但是,脂肪酸的处理实验结果表明不同类型的脂肪酸对曲霉属真菌毒素合成的作用不同,有的促进合成,有的抑制合成。最近研究结果显示所有脂肪酸都促进黄曲霉毒素合成,但是多不饱和脂肪酸在暴露空气之后对毒素合成有抑制作用。这种抑制产毒的作用似乎是由多不饱和脂肪酸氧化所产生的脂氧合物所介导。本文结合我们的研究结果,综合评述了脂肪酸和脂氧合物调控曲霉属真菌菌丝生长、产孢和毒素合成研究的最新进展。     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

The influences of fungitoxicants on growth and aflatoxin production by Aspergillus flavus

The influence of six fungitoxicants on growth and aflatoxin production by Aspergillus flavus was tested in liquid SMKY medium at two concentrations, viz . 0.1 and 0.5%. Thiram completely inhibited the aflatoxin production at 0.5% concentration. Other fungitoxicants showing more than 60% inhibition were bavistin and daconil. Vitavax (0.1%) and agrosan GN (0.1 and 0.5%) stimulated the growth of fungus and aflatoxin elaboration after 7 d of incubation. Dithane M-45 moderately inhibited aflatoxin synthesis. Treatment with fungitoxicants also alters the ratio of B₁ and G₁.     

Suppression of spore germination and aflatoxin biosynthesis in Aspergillus parasiticus during and after exposure to high levels of phosphine

Agar cultures of toxigenic Aspergillus parasiticus NRRL 2999 were exposed to phosphine (PH3), in levels ranging from 0 to 2000 ppm (vol/vol). It was found that with PH3 concentrations of 400 ppm or higher the growth of the fungus was totally arrested. When PH3 was vented and the agar plates were exposed to open air, 100% of the initial CFU developed into fully grown colonies after PH3 levels below 300 ppm, but at higher PH3 concentrations only 50% of the colonies developed. The same strain of A. parasiticus was inoculated into high moisture corn under conditions highly favorable for aflatoxin production, and it was exposed to a range of PH3 levels. After exposure to 500 ppm PH3, both fungal growth and aflatoxin synthesis resumed shortly after elimination of the toxic gas, but after exposure to PH3 levels of 1000 ppm and higher, the physical appearance of the contaminated corn was remarkably changed, showing reduced mycelial growth and almost complete absence of green pigmentation. In addition, aflatoxin synthesis was totally absent for the remainder of the experiment (20 days). These results strongly suggest that exposure to PH3 levels of 1000 ppm or higher could bring about persistent metabolic changes in surviving Aspergillus organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

Isolation, Growth Characteristics, and Long-Term Storage of Fungi Cultivated by Attine Ants

Seven pure-culture strains of fungi cultivated by attine ants (ant-garden fungi) were isolated from locally maintained leaf-cutting ant colonies. An ant-garden fungus strain obtained from an Atta cephalotes colony, when offered to ants of the colony from which the fungus was isolated, was accepted as their own. Young fungus cultures were harvested and incorporated into the fungus garden, and cultures of intermediate age were used to begin a new fungus garden; old cultures were simply harvested. To facilitate further research on this fungus, growth characteristics of the different isolates were studied under a variety of conditions. They grew better at 24°C than at 30°C, and growth did not occur at an incubation temperature of 37°C. In a broth culture medium, growth was enhanced by aeration of the culture and by addition of yeast extract, olive oil, sesame oil, peanut oil, soybean oil, corn oil, sunflower oil, cottonseed oil, walnut oil, safflower oil, or mineral oil. Glycerol did not noticeably affect growth, but Tween 80 inhibited growth. These fungi were extremely sensitive to cycloheximide, growth being totally inhibited at cycloheximide concentrations ranging from 0.4 to 4.0 μg/ml. To date, the ant-garden fungus isolates have remained viable in long-term mineral oil-overlay storage cultures for up to 4 years.     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/1) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

Effects of Moisture Content and Temperature on Aflatoxin Production in Corn

Samples of freshly harvested and remoistened corn, of various moisture contents, were stored at different temperatures; analyses for aflatoxin content were made periodically. At moisture levels above 17.5% and at temperatures of 24 C or warmer, aflatoxins were formed by Aspergillus flavus present in the original epiphytic mycoflora. Remoistened dried corn was subject to more rapid fungal deterioration and aflatoxin formation than freshly harvested corn. Screening of the fungi present in the corn revealed aflatoxin production only by A. flavus. The toxigenic strains produced only aflatoxins B(1) and B(2).     

Bright Greenish-Yellow Fluorescence and Aflatoxin in Agricultural Commodities

The corn milling industry has widely accepted the presence of bright greenish-yellow fluorescence under a black light as a presumptive indicator of aflatoxin (a poison produced by the mold Aspergillus flavus). This test was applied to wheat, oats, barley, rice, coconut, white corn, yellow corn, peanuts, sorghum, and soybeans, and evaluated in the laboratory. Our study supported the use of bright greenish-yellow fluorescence as a presumptive test for aflatoxin in wheat, oats, barley, corn, and sorghum.     

Aflatoxin contamination of developing corn kernels

Preharvest of corn and its contamination with aflatoxin is a serious problem. Some environmental and cultural factors responsible for infection and subsequent aflatoxin production were investigated in this study. Stage of growth and location of kernels on corn ears were found to be one of the important factors in the process of kernel infection with A. flavus & A. parasiticus. The results showed positive correlation between the stage of growth and kernel infection. Treatment of corn with aflatoxin reduced germination, protein and total nitrogen contents. Total and reducing soluble sugar was increase in corn kernels as response to infection. Sucrose and protein content were reduced in case of both pathogens. Shoot system length, seeding fresh weigh and seedling dry weigh was also affected. Both pathogens induced reduction of starch content. Healthy corn seedlings treated with aflatoxin solution were badly affected. Their leaves became yellow then, turned brown with further incubation. Moreover, their total chlorophyll and protein contents showed pronounced decrease. On the other hand, total phenolic compounds were increased. Histopathological studies indicated that A. flavus & A. parasiticus could colonize corn silks and invade developing kernels. Germination of A. flavus spores was occurred and hyphae spread rapidly across the silk, producing extensive growth and lateral branching. Conidiophores and conidia had formed in and on the corn silk. Temperature and relative humidity greatly influenced the growth of A. flavus & A. parasiticus and aflatoxin production.     

Fungal Contamination of Raw Materials of Some Herbal Drugs and Recommendation of <Emphasis Type="Italic">Cinnamomum camphora</Emphasis> Oil as Herbal Fungitoxicant

The paper explores fungal infection and aflatoxin B(1) contamination of six medicinal plant samples viz. Adhatoda vasica Nees, Asparagus racemosus Linn., Evolvulus alsinoides Linn., Glycyrrhiza glabra Linn., Plumbago zeylanica Linn. and Terminalia chebula Retz. A total of 858 fungal isolates were detected from the raw materials. Maximum number of fungal isolates was detected from A. racemosus (228). The genus Aspergillus was found to be the most dominant genus causing infection to most of the raw materials. Among the 32 isolates of A. flavus tested, 13 isolates were found to be toxigenic elaborating aflatoxin B(1). The highest elaboration of aflatoxin B(1) was 394.95 ppb by the isolates of A. flavus from G. glabra. The essential oil of Cinnamomum camphora (L.) Presl showed efficacy in arresting aflatoxin B(1) by the toxigenic strain. The growth of a toxigenic strain of A. flavus decreased progressively with increasing concentration of essential oil from leaves of C. camphora. The oil completely inhibited aflatoxin B(1) production even at 750 ppm. Hence, the oil of C. camphora is recommended as herbal fungitoxicant against the fungal contamination of the raw materials.