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.     

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.     

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.     

Aflatoxin production by Aspergillus flavus isolates pathogenic to coconut insect pests

Aspergillus flavus isolated from naturally infected leaf-eating caterpillar (Opisina arenosella W.), lace bug (Stephanitis typica D.) and plant hopper (Proutista moesta Westwood), insect pests of the coconut palm, were tested for aflatoxin (AT) production by employing various media formulations. These A. flavus isolates were earlier found to be entomopathogenic in laboratory bioassays. A laboratory contaminant and four standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. All A. flavus isolates were tested on seven AT detection media: coconut extract agar, coconut extract-sodium desoxycholate agar, coconut extract-ascorbic acid agar, coconut extract-Czapek Dox agar, coconut extract-milk powder agar, 10% commercial coconut milk powder agar (CCMPA) and 20% CCMPA. Only two isolates of A. flavus, originally isolated from O. arenosella and P. moesta, produced ATs. AT production was detected within 48 h of incubation and was detected continually up to 1 month. These AT-producing A. flavus isolates also produced bright yellow pigmentation in the medium. Of all the seven media used for AT detection, CCMPA (10%) was found to be the best one, followed by 20% CCMPA, for direct and rapid AT detection. AT production was not necessary for pathogenicity in the insects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibitory Effects of Ephedra major Host on Aspergillus parasiticus Growth and Aflatoxin Production

This study was undertaken to evaluate the effect of Ephedra major Host, an important medicinal plant with various biological activities, on growth and aflatoxin (AF) production by Aspergillus parasiticus NRRL 2999. The fungus was cultured in yeast extract-sucrose (YES) broth, a conductive medium that supports AF production, in the presence of various concentrations of essential oil (EO), hexanic and methanolic extracts of plant aerial parts, fruits, and roots using microbioassay technique. After incubating for 96 h at 28°C in static conditions, mycelial dry weight was determined as an index of fungal growth, and aflatoxin B₁ (AFB₁) was measured using HPLC technique. Based on the obtained results, EO of plant aerial parts significantly inhibited fungal growth at the highest concentration of 1000 μg/ml without any obvious effect on AFB₁ production at all concentrations used. Among plant extracts tested, only methanolic extract of aerial parts and roots were found to inhibit fungal growth and AFB₁ production dose-dependently with an IC₅₀ value of 559.74 and 3.98 μg/ml for AFB₁, respectively. Based on the GC/MS data, the major components of E. major EO were bis (2-ethylhexyl) phthalate (42.48%), pentacosane (20.94%), docosane (14.64%), citronellol (5.15%), heptadecan (4.41%), cis-3-Hexen-1-ol benzoate (4.07%), and 7-Octen-2-ol (3.25%). With respect to the potent inhibition of fungal growth and AF production by E. major, this plant may be useful in protecting crops from both toxigenic fungal growth and AF contamination.     

Contamination of Red Chilli with Aflatoxin B1 in Pakistan

A survey of red chilli (Capsicum indicum) for contamination with aflatoxins was performed on different samples comprising whole, crushed and powdered red chilli collected from various stores located in the city of Karachi, Pakistan. Red chilli required rather rigorous clean-up procedure for removal of adulterants and interference resulting from various types of compounds. A modified Romer method followed by bi-directional thin layer chromatography (TLC) was used for the detection of aflatoxins and confirmatory tests were performed by spraying the TLC plates with 50% sulphuric acid and making the derivative with trifluoroacetic acid. Of all the 176 samples of red chilli examined, 66% were found to be contaminated with aflatoxin B₁. Generally, samples of red chilli exammined were found to be fairly low in aflatoxin B₁ content, whereas only seven samples were found to contain concentrations greater than 25 μg/kg of aflatoxin B₁.     

Dillapiol and Apiol as Specific Inhibitors of the Biosynthesis of Aflatoxin G1 in Aspergillus parasiticus

Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G₁ production. It inhibited aflatoxin G₁ production by Aspergillus parasiticus with an IC₅₀ value of 0.15 μM without inhibiting aflatoxin B₁ production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC₅₀ values of 0.24 and 3.5 μM, respectively.     

Degradation of Coal by the Fungi Polyporus versicolor and Poria monticola

We report that two species of basidiomycete fungi (Polyporus versicolor and Poria monticola) grow in minimal liquid or solid medium when supplemented with crushed lignite coal. The fungi also grow directly on crushed lignite coal. The growth of both fungi was observed qualitatively as the production and extension of hyphae. No fungal growth occurred in minimal agar medium without coal. The fungi degraded solid lignite coal to a black liquid product which never appeared in cultures unless fungi and coal were present together. Apparently, lignite coal can serve as the principal substrate for the growth of the fungi. Infrared analyses of the liquid products of lignite degradation showed both similarities to and differences from the original lignite.     

Compatibility of <Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> as dual inoculants in black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

The compatibility of two biological inoculants, Trichoderma harzianum, a mycoparasitic biological control fungus and Piriformospora indica, a root colonizing plant-growth promoting endophytic fungus was evaluated using tissue cultured black pepper plantlets. We report, for the first time, the ability of P. indica to colonize black pepper, a perennial climber. T. harzianum inhibited the growth of P. indica in an in vitro dual culture plate assay. Simultaneous inoculation with both biological inoculants of tissue cultured black pepper plantlets negatively influenced root colonization by P. indica. However, when P. indica was applied initially followed 30 days later by T. harzianum, there was increased root colonization by the root endophyte P. indica and beneficial effects were found on the growth of the black pepper plants. The present study also showed that the efficacy of inoculation of the two fungal biological agents can be increased by sequential application of P. indica at the hardening stage followed by T. harzianum during transplanting into a soil-sand mixture.     

Fungi associated with black pepper plants in Quang Tri province (Vietnam), and interaction between Meloidogyne incognita and Fusarium solani

During a survey of nurseries and plantations of black pepper plants in Quang Tri province in Vietnam during the rainy season of 2007, nine fungal taxa were isolated from the roots of the black pepper plants. Fusarium solani was found in about one out of four black pepper root samples examined but not in the nurseries and also not from black pepper plants younger than five years growing in plantations. Since in these nurseries about one out of two black pepper plants examined had yellow leaves, this observation suggests that another pathogen must be the initial cause of the yellowing of the leaves. A likely pathogenic candidate is M. incognita which was extracted from every single black pepper plant examined in the nurseries. During the same survey, we also observed that F. solani was not isolated from the roots of black pepper plants that did not had yellow leaves and that the percentage of black pepper plants with yellow leaves increased with increased frequency of occurrence of F. solani. This observation indicates that F. solani plays a role in the yellowing of the leaves of black pepper plants in a later stage of the development of the plants. The results of a greenhouse experiment showed the negative effects inoculation with M. incognita alone or in combination with F. solani may have on the percentage of black pepper plants with yellow leaves and on plant growth. No effect of inoculation with F. solani before, at the same time, or two weeks after inoculation with M. incognita on root galling and nematode reproduction was observed.     

Mixed Herbs Drugs: Inhibitory Effect on Growth of the Endogenous Mycoflora and Aflatoxin Production

Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log₁₀ CFU and 2.4 log₁₀ CFU per gram of sample. When the drugs were incubated in 85% humidity at 25°C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B₁ and aflatoxin G₁ production by 62–97%. All but two of the extracts reduced aflatoxin B₂ and aflatoxin G₂ production by 39–95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.     

Coconut as a Medium for the Experimental Production of Aflatoxin

Fresh, grated coconut has been found to be an excellent medium for aflatoxin production by Aspergillus flavus. Under optimal conditions, yields of 8 mg of total aflatoxin per g of substrate were obtained. Continuous agitation of the growth medium under moist conditions at 24 C produced highest yields. Aflatoxin was assayed both biologically and chromatographically. The aflatoxin content of cultures varied biphasically with the duration of incubation. It is suggested that this pattern could result from the sequential operation of factors promoting aflatoxin formation on the one hand and a detoxifying mechanism on the other.     

Inhibition of Aflatoxin Accumulation in Smoked Substrates

The pattern of fungal colonization on processed (smoked) copra kernels and the levels of aflatoxin detected in copra and in culture of Aspergillus flavus on fresh coconut suggested that aflatoxin accumulation was inhibited by the smoking process. This paper describes the study of the smoking process under laboratory conditions with fresh coconut and potato dextrose broth; aflatoxin accumulation was found to be significantly or totally inhibited in both smoked coconut and in smoked broth. Mycelial growth was inhibited to a lesser degree.
On account of the urgent need for simple, cheap and efficent techniques for the processing and safe storage of food substrates especially in poor tropical countries, we suggest that the smoking process deserves study under industrial conditions for the protection of other agricultural food crops (e.g. groundnut) as well.     

Potential plant growth-promoting activity of <Emphasis Type="Italic">Serratia nematodiphila</Emphasis> NII-0928 on black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

A potential bacterial strain designated as NII-0928 isolated from Western ghat forest soil with multiple plant growth promoting attributes, and it has been identified and characterized. Plant growth promoting traits were analyzed by determining the P-solubilization efficiency, Indole acetic acid production, HCN, siderophore production and growth in nitrogen free medium. It was able to solubilize phosphate (76.6 μg ml⁻¹), and produce indole acetic acid (58.9 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). 16S rRNA gene sequencing reveals the identity of the isolate as Serratia nematodiphila with which it shares highest sequence similarity (99.4%). Seed bacterization with black pepper cuttings in greenhouse trials using Sand: Soil: FYM with three individual experimental sets with their respective control showed clearly the growth promoting activity. Hence, Serratia nematodiphila NII-0928 is a promising plant growth promoting isolate showing multiple PGPR attributes that can significantly influence black pepper cuttings. The result of this study provides a strong basis for further development of this strain as a bioinoculants to attain the desired plant growth promoting activity in black pepper growing fields.     

Development of Aspergillus parasiticus and formation of aflatoxin B1 under the influence of conidiogenesis affecting compounds

The influence of various inhibitors of hyphal growth, sporulation and biosynthesis of aflatoxin B₁ in Aspergillus parasiticus NRRL 2999 was studied. 6-Thioguanine, dl-ethionine, fluoroacetic acid and phenylboric acid, inhibitors of maturation of fungal conidiophores and of conidiogenesis, were added at various concentrations to malt extract agar. Lower concentrations of 6-thioguanine and dl-ethionine did not inhibit the growth of hyphae and the sporulation. Phenylboric acid reduced conidiogenesis more than hyphal growth. The yields of aflatoxin B₁ were significantly reduced. Additions of fluoroacetic acid did not greatly affect the growth of hyphae but totally inhibited the production of conidia and concurrently significantly reduced the formation of aflatoxin B₁. An interrelation between conidiogenesis and onset of secondary metabolism in A. parasiticus is evident.     

Microbe‐mediated control of Aspergillus flavus in stored rice grains with a focus on aflatoxin inhibition and biodegradation

Biological control of mycotoxigenic fungi using antagonistic microbes is a promising alternative to agricultural chemicals for postharvest storage. In this study, we evaluated rice‐derived bacterial strains to identify biocontrol agents to inhibit Aspergillus flavus in stored rice grains. Consequently, we obtained three potential biocontrol strains (Microbacterium testaceum KU313, Bacillus megaterium KU143 and Pseudomonas protegens AS15) from 26 tested strains that were prescreened from the 460 strains isolated from rice grains. The three selected strains proved to be effective biocontrol agents showing antifungal activity against A. flavus and good colonisation ability on rice grains, along with inhibition of the fungal growth and aflatoxin production. In particular, P. protegens AS15 greatly inhibited the aflatoxins produced by A. flavus on rice grains to 8.68 (percent aflatoxin reduction relative to control = 82.9%) and 18.05 (68.3 %) ng g^?1 dry weight of rice grains, compared with the 50.89 and 56.97 ng g^?1 dry weight of rice grains of the MgSO₄ control at 1 and 2 weeks after inoculation, respectively. In addition, strain AS15 had a significant ability to not only degrade aflatoxin B₁ (the most harmful aflatoxin), but also utilise the toxin for bacterial growth in a nutrient‐deficient medium. Therefore, the selected bacterial strains could be environmentally sound alternatives for the management of A. flavus and aflatoxin production by reducing the fungal damage to stored rice grains. This would also reduce the human and animal health hazards associated with the consumption of fungus‐contaminated rice grains. To our knowledge, this is the first report of the potential of the bacterial species M. testaceum and P. protegens as biocontrol agents for controlling aflatoxigenic A. flavus on stored rice grains.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.