Enhancement of anthocyanin production in callus cultures ofDaucus canota L. under the influence of fungal elicitors

Aqueous extracts of fungal biomass ofAspergillus niger, A. flavus, Penicillium notatum, Fusarium oxysporum and the filtrates of their culture media were analysed for elicitation capability to enhance anthocyanin production in callus cultures ofDaucus carota. The mycelial extract ofA. flavus at the 2.5% level gave maximum elicitation, which resulted in a two-fold increase in anthocyanin with maximal productivity of 23.7% on a dry weight basis. Whereas the media filtrates ofA. flavus induced a 1.25-fold increase in anthocyanin production with a yield of 20.6% on a dry weight basis,P. notatum andF. oxysporum were not as effective asA. flavus orA. niger. The contact time and the concentration required for maximal elicitation of anthocyanin differed with the type of elicitor used. Qualitative analysis of anthocyanins revealed the presence of cyanidin glycosides in control and elicited cultures.     

Studies on antagonism betweenFusarium udum Butler and root region microflora of pigeon-pea

Antagonism betweenFusarium udum Butler causing wilt of pigeon-pea (Cajanus cajan (L.) Millsp.) and the saprophytic microflora of the root region of the host was studied with reference to colony interaction, hyphal interference, volatile and non-volatile metabolites and staling growth products. Studies were extended to screen potential antagonists against the wilt pathogen in soil. Aspergillus flavus, A. niger, A. terreus, Penicillium citrinum andMicromonospora globosa (an actinomycete) were antagonistic againstF. udum, whereas the pathogen parasitized and killedAspergillus luchuensis, Cunninghamella echinulata, Curvularia lunata, Mortierella subtilissima andSyncephalastrum racemosum. The pattern of growth of microorganisms on nutrient agar staled by rhizosphere soil inocula of healthy or wilted pigeon-pea plants was found to be different.F. udum colonized and grew on nutrient agar staled by the rhizosphere inoculum of the wilted plants upto 120h of incubation. However, it could not colonise and grow on the nutrient agar staled by rhizosphere microflora of healthy plants after 48h of incubation because of the presence of antagonists likeA. niger, A. flavus, A. terreus and a few species ofPenicillium in the soil inoculum. When pure cultures in soil ofF. udum was mixed with those of antagonists in different ratios,A. niger, A. flavus andM. globosa significantly suppressed the population ofF. udum, whereasA. terreus markedly reduced the population. When inoculated in soil, the antagonists exhibited a high fungistatic activity againstF. udum.     

Peanut-pod mycoflora and kernel infection

Summary The mycoflora in soil clinging to dry pods of peanuts of the Spanish variety Argentine was sampled in 2 experiments by serially washing pods for increasing periods in changes of sterile water. Of the 9 principal fungi found,Aspergillus niger, A. flavus, A. terreus, Rhizopus spp. andSclerotium bataticola were present initially in relatively small numbers and decreased rapidly in subsequent dilutions. This decrease paralleled a decrease in weight of suspended material and in percentage of soil and organic particles greater than 0.016 mm in size.Penicillium funiculosum, P. rubrum, P. citrinum, andFusarium spp. were found in large numbers and increased or slowly decreased in numbers in subsequent dilutions. In some instances variations in numbers followed trends of percentages of soil and organic particles less than 0.016 mm in size.When dry pods with this known mycoflora were allowed to hydrate over a 6-day period at 26°, 32°, or 38°C, there was extensive pod penetration and kernel infection byA. niger, A. flavus, S. bataticola andRhizopus spp. but not by other fungi. The degree ofA. flavus andA. niger infection increased with increasing temperatures.Approved by the Director as Journal Series Paper No. 135.     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Antagonistic interactions between fungal pathogens and phylloplane fungi of guava

Dominant phylloplane fungi of guava (Psidium guajava L.) were screened for their antagonistic activities against the two pathogens,Colletotrichum gloeosporioides andPestalotia psidii, bothin vitro andin vivo. Culture filtrates ofAspergillus niger, Fusarium oxysporum andPenicillium citrinum caused more than 50% growth inhibition ofC. gloeosporioides. Filtrates ofCephalosporium roseo-griseum andF. oxysporum were most effective in reducing the growth ofP. psidii. Volatiles produced from the cultures ofA. niger, F. oxysporum, P. citrinum andP. oxalicum inhibited the growth ofC. gloeosporioides, whereas volatiles fromC. roseo-griseum, F. oxysporum andTrichoderma harzianum inhibited the growth ofP. psidii. The inhibitory effect of volatiles decreased with increase in incubation time. In general, the maximum effect of volatiles was noticed after 48 h incubation. Different grades of colony interactions in dual cultures were recognised between the two pathogens and the phylloplane fungi examined. Maximum inhibition ofC. gloeosporioides was caused byAureobasidium pullulans, Cladosporium cladosporioides, epicoccum purpurascens, F. oxysporum andMyrothecium roridum, whereasAspergillus terreus, C. roseo-griseum andP. oxalicum significantly reduced the growth ofP. psidii. Application of a spore suspension of each test fungus inhibited lesion development of guava leaves caused by the test pathogensin vitro. Inhibition was more pronounced when the spore concentration was increased.A. pullulans, C. cladosporioides, E. purpurascens, F. oxysporum, andT. harzianum were found to be strongly antagonistic toC. gloeosporioides. A. niger, A. terreus, C. roseo-grisem andT. harzianum were strongly antagonistic toP. psidii.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Keratinophilic fungi and other moulds associated with air-dust particles from Egypt

One-hundred and eleven species and three species varieties belonging to 39 genera were collected from 50 dust samples on the five media used at 28°C. Using the hair-baiting technique with horse hair, 10 species ofChrysosporium were isolated:C. asperatum, C. state ofArthroderma tuberculatum, C. indicum, C. inops, C. keratinophilum, C. merdarium, C. pannorum, C. queenslandicum, C. tropicum andC. xerophilum. True dermatophytes were isolated:Trichophyton verrucosum andTrichophyton sp. Also, numerous fungi tolerating high levels of cycloheximide were encountered, such as members ofAcremonium, Aspergillus andPenicillium. On plates of glucose or cellulose Czapek-Dox agar (free from sucrose) the most frequent fungi were:Alternaria alternata, Aspergillus flavus, A. flavus var.columnaris, A. fumigatus, A. niger, A. ochraceus, A. sydowii, A. terreus, Chaetomium globosum, Cladosporium herbarum, Emericella nidulans, Fusarium oxysporum, Mucor hiemalis, Penicillium chrysogenum, P. oxalicum, Scopulariopsis brevicaulis andUlocladium atrum. On plates of 50% sucrose or 10 and 20% NaCl-Czapek's agar, some interesting species were frequently encountered:Eurotium amstelodami, E. chevalieri, E. halophilicum, E. montevidensis, E. repens, E. rubrum andScopulariopsis halophilica. The isolated, fungi have been tested for osmophilicity and halophilicity, they showed different rates of growth on sucrose and sodium chloride-Czapek's medium of various osmotic potential.     

Fungicide inhibition of aflatoxins,diacetoxyscirpenol and zearalenone production

The effect of fungicides on the production of aflatoxin byAspergillus flavus IMI 89717, diacetoxyscirpenol and zearalenone byFusarium graminearum was studied. In a yeast extract - sucrose medium, dicloran, iprodione and vinclozolin fungicides significantly inhibited mycelial growth ofA. flavus at 250 ppm and significantly decreased aflatoxin production at 100, 250 and 500 ppm, respectively. In potato — dextrose broth, these fungicides diminished the mycelial growth ofF. graminearum and production of diacetoxyscirpenol and zearalenone at 100 ppm. Sensitivity of toxigenic mycelia to fungicides increased approximately five-fold in a yeast extract-starch medium with an appreciable reduction in sugar uptake andα-amylase activity.     

Aflatoxin: A metabolic product of several fungi

Summary Culture extracts produced by 107 fungi isolated from corn grains were assayed by thin layer chromatography for aflatoxin. Certain isolates ofAspergillus flavus, A. niger, A. parasiticus, A. ruber, A. wentii, Penicillium citrinum, andP. variabile produced aflatoxin.Penicillium frequentans andP. puberulum elaborated this toxin only in trace amounts. Bioassays of extracts from 4 of these fungi showed that only the extract fromA. parasiticus was highly toxic.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Colonization ofRetama raetam seeds by fungi and their significance in seed germination

Examination by scanning electron microscopy and incubation on potato-dextrose agar medium showed that dry seeds ofRetama raetam were externally free of fungi. When planted in sandy loam soil, the seeds become colonized with eleven soil-borne fungal species. The fungi were isolated on cellulose agar, pectin agar and lignin agar media.Aspergillus flavus, A. niger, A. fumigatus, Penicillium capsolatum andFusarium oxysporum had broad occurrence and were recovered on all the three media. The production of hydrolytic enzymes by the isolated fungi depends on the substrate and species.Penicillium capsolatum, P. spinulosum andA. niger had wide enzymatic amplitude and they were able to produce cellulolytic, pectolytic and lignolytic activities on corresponding substrates as well as on seed-coat-containing media. The lignolytic activities of the isolated species exceptChaetomium bostrychodes andTrichoderma viride were enhanced by applying the seed-coat materials as C- source rather than lignin. SoakingR. raetam seeds in culture filtrates of most of the fungi grown on seed-coat-supplemented media induced a pronounced and distinct stimulating effect on seed germination. The most effective filtrates were those ofP. capsolatum, P. spinulosum andSporotrichum pulverulentum.     

Effect of delayed harvest on contamination of pearl millet grain with mycotoxin-producing fungi and mycotoxins

The response to delayed harvest of fungal and mycotoxin contamination of grain of the pearl millet hybrid HGM 100 was examined in 1992 and 1993. Samples of grain were assayed from seven plantings at locations near Tifton, Georgia, USA. Grain was harvested at 30, 40, and 50 days after anthesis and evaluated for infection byFusarium species andAspergillus flavus, and mycotoxin contamination. Mean isolation frequencies ofF. semitectum (35.6%) andF. chlamydosporum (17.2%) increased linearly with delayed harvest.Fusarium moniliforme andF. equisiti were infrequently isolated (<0.5%) and did not increase in the grain when harvest was delayed. Low mean concentrations of zearalenone (0.17 ppm), nivalenol (0.42 ppm), and deoxynivalenol (0.01 ppm) were detected but were not affected by delayed harvest. Isolation frequencies ofF. chlamydosporum andF. equiseti were correlated (P=0.07) with levels of nivalenol.Aspergillus flavus was not isolated from the grain, and aflatoxin concentrations averaged 1.9 ppb.     

The effects of some commonly-used foliar fungicides on Collembola in winter barley: laboratory and field studies

Four commonly-used cereal foliar fungicides were screened for their laboratory toxicity against the symphypleone collembolan, Sminthurinus aureus. A proportional hazards analysis of time-survival curves following the fungicide treatments showed that carbendazim, propiconazole, pyrazophos and triadimenol significantly increased the laboratory mortality of S. aureus. The organophosphorus fungicide pyrazophos caused high levels of mortality of S. aureus in the laboratory so a field evaluation of the effects of this fungicide on a wider range of Collembola was undertaken in winter barley. Comparison of the effects of pyrazophos with those of the broad-spectrum insecticide dimethoate in the field revealed both compounds to have similar activity against some Collembola. Of the 11 species caught only the four symphypleone species exhibited these effects but the numbers of three symphypleone species were reduced to zero 4 wk after treatment with pyrazophos. The effects of pyrazophos and dimethoate were, however, not detectable in individual species after 11 wk.     

Inhibitory effects of condiments and herbal drugs on the growth and toxin production of toxigenic fungi

The effects of thirteen kinds of powdered herbal drugs and seven kinds of commercial dry condiments on the growth and toxin production ofAspergillus parasiticus, A. flavus,A. ochraceus, andA. versicolor were observed by introducing these substances into culture media for mycotoxin production.Of the twenty samples tested, cinnamon bark completely inhibited the fungal growth, while the others only inhibited the toxin production.The inhibitors were easily extracted from the samples with solvents such as hot water, chloroform, or ethanol.The extracts from coptis, philodendron bark, mustard, green tea leaves, and zanthoxylum completely inhibited the aflatoxin production ofA. parasiticus, however, they had little or no inhibitory effect againstA. flavus.     

Survey of Aspergillus species associated with the human respiratory tract

A comprehensive survey has been carried out on the occurrence ofAspergillus species in the respiratory tract of patients of bronchopulmonary diseases in Delhi. In all, 1238 clinical specimens, which included 1082 sputa, 143 bronchial aspirates and 13 throat swabs obtained from 812 patients, were examined. Of these 61.7 per cent patients were culturally positive yielding 29 different species ofAspergillus. The prevalence of aspergilli in sputa was significantly higher than in the bronchial aspirates.Aspergillus niger was the commonest species isolated showing a prevalence of 36.7 per cent. It was followed byA. flavus, A. nidulans, A. terreus, A. versicolor, A. sydowi, A. japonicus andA. oryzae. None of theAspergillus species showed a significant correlation with any of the diseases, or the type of treatment the patients had received. Of the 8 broad occupational groups investigated farmers and labourers showed higher prevalence ofA. niger andA. flavus. The prevalence ofAspergillus species in the throats of healthy persons was 16 per cent withA. versicolor being the commonest species followed byA. flavus, A. amstelodami, A. sydowi andA. terreus. A comparison of the prevalence ofAspergillus species in the patients, healthy individuals and atmosphere of Delhi appears to support the view that the aspergilli are transient residents in the human respiratory tract following their inhalation from the environment.     

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in yemen

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera wereAspergillus, Alternaria, Penicillium, andCladosporium followed byFusarium, Drechslera, Chœtomium, andMucor. The most prevalent species in above genera wereAspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, andFusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic.A. alternata produced alternariol and alternariol monomethyl ether whereasA. flavus produced aflatoxins B₁ and B₂. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced byA. ochraceus, A. versicolor, P. citrinum andF. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.     

Aspergillus flavus and other mycoflora of groundnut kernels in Israel and the absence of aflatoxin

More than 300 groundnut (peanut) samples collected from different regions of Israel were examined by ELISA for aflatoxin contamination. Samples were designated for export, local consumption or for sowing. None of the samples were contaminated with the toxin. However, when kernels were kept at high humidity (RH?99%), aflatoxin could be frequently detected seven days after incubation and the toxin was not uniformly distributed among kernels.Aspergillus niger, A flavus, Penicillium citrinum andP pinophilum were the dominant fungi and no differences were observed among cultivars. Almost half of the commercial samples examined were devoid ofA flavus. Other fungi identified wereA tamaril, A amstelodami, P rubrum, Rhizoctonia solani, Macrophomina phaseolina, Rhizopus spp., Sclerotium rolfsll, Fusarium andAlternaria spp; the two last ones comprising a group of low incidence. Although groundnut samples that containA flavus—infected kernels are moderately common, the local climate and agrotechniques In use in Israel are not conducive to aflatoxin accumulation. Nevertheless infected kernels may become a threat to health if stored under inadequate conditions.     

Fungal succession and decay of herbicide-treated wheat straw

The effects of alachlor and haloxyfop on wheat straw decay and fungal succession were studied in laboratory trials. Wheat straw segments were immersed in (100 and 1 000 mg/L) herbicides, buried in soil and incubated at 28±1°C for 8 weeks. Both herbicides significantly stimulated the mass loss and N-mineralization of wheat straw. CO₂ evolution was not significantly affected while C-mineralization was inhibited. The pattern of succession of fungi was slightly affected in herbicidetreated wheat straw. However, the frequency of occurrence ofRhizopus stolonifer was lowered throughout all experimental periods. In a pure culture experiment, both alachlor and haloxyfop at 100, 500 and 1 000 mg/L reduced the radial growth ofAlternaria alternata, Aspergillus niger, A. flavus, Penicillium funiculosum, R. stolonifer andTrichoderma harzianum.     

Colonization capacity of the soil micro-organisms in relation to fungistasis

Summary Evans' tube and cellophane agar-disc techniques were used to evaluate the colonization capacity of the soil microbes and fungistasis, respectively. Spore germination ofAlternaria tenuis, Curvularia geniculata, Helminthosporium rostratum andPestalotia sp. was studied on a natural soil. Aspergillus fumigatus was the pioneer colonizer, followed in the order of decreasing colonizing capacity by,A. flavus, A. niger, A. terreus andPenicillium nigricans. Sensitivity of test spores to soil fungistasis declined in the order,C. geniculata → Pestalotia sp. →A. tenuis → H. rostratum. The fungistatic level of the soil samples decreased gradually from side arms 1 to 7 of the Evans tube, inoculated with unsterilized soil from one end. Studies conducted with an inoculum of four soil fungi (A. fumigatus, A. flavus, A. niger, andP. nigricans) as well as those involving fumigation of Evans tube by CS₂ also proved the greater colonizing capacity ofA. fumigatus. The level of fungistasis followed closely the colonization pattern of this fungus. When added individually to the soil, all fungi were able to inhibit spore germination of test fungi to some degree or other.     

Relationships betweenAspergillus flavus,A. niger and some other fungi in the mycoflora of groundnut kernels

Summary The relationships betweenAspergillus flavus, A. niger, Penicillium funiculosum, P. rubrum, andFusarium solani have been studied in 234 samples and 5850 plates from fresh and stored kernels derived from 2 years' groundnut crops in Israel.Numerical relations between these fungi, as obtained by totalling the number of colonies developing in all the 25 platings made for each sample, did not always give reliable indications of potential antagonism between the species. But consideration of the number of colonies developing in individual plates showed pronounced antagonism betweenA. flavus andA. niger, and slightly less but still marked antagonism between each of these species andP. funiculosum, P. rubrum andF. solani.This research is supported by Grant Number FG-Is-161 of the United States Department of Agriculture to whom the author is indebted.