Antifungal Activity of Bacillus thuringiensis Chitinase and Its Potential for the Biocontrol of Phytopathogenic Fungi in Soybean Seeds

ABSTRACT: Bacillus thuringiensis var israelensis was used to produce chitinase on shrimp wastes by fermentation at 30°C and 250 rpm for 120 h. The enzyme was concentrated by ultrafiltration and was adjusted to pH 5.8. Antifungal chitinase activity on phytopathogenic fungi was investigated in growing cultures and on soybean seeds infested with Sclerotium rolfsii . Fungal inhibition was found to be 100% for S. rolfsii ; 55% to 82% for A. terreus , A. flavus, Nigrospora sp, Rhizopus sp, A. niger , Fusarium sp, A. candidus, Absidia sp, and Helminthosporium sp; 45% for Curvularia sp; and 10% for A. fumigatus (P < 0.05). When soybean seeds were infected with S. rolfsii , germination was reduced from 93% to 25%; the addition of chitinase (0.8 U/mg protein) increased germination to 90%. B. thuringiensis chitinase may contribute to the biocontrol of S. rolfsii and other phytopathogenic fungi in soybean seeds in Integrated Pest Management programs.     

Efficacy of Artabotrys odoratissimus oil as a plant based antimicrobial against storage fungi and aflatoxin B1 secretion

The essential oil of Artabotrys odoratissimus R.Br. was evaluated for antifungal activity against some storage fungi causing contamination of food stuffs. Minimum inhibitory concentration of the oil was found to be 750 μL L⁻¹ against Aspergillus flavus Link. It was found superior over different prevalent synthetic fungicides which inhibited the growth of A. flavus between 1000–5000 μL L⁻¹. The oil exhibited broad fungitoxic spectrum against fourteen different storage fungi. Aspergillus fumigatus was inhibited at 1000 μL L⁻¹ whereas Cladosporium cladosporioides , Curvularia lunata , Fusarium oxysporum , Helminthosporium oryzae , Macrophomina phaseolina , Microsporum gypseum , Mucor racemosus , Penicillium italicum , Pythium debaryanum , Rhizoctonia solani , Sclerotium rolfsii and Trichoderma viride at 500 μL L⁻¹. Aspergillus niger was found to be inhibited only 84.9% at 1000 μL L⁻¹. In addition, the oil showed significant efficacy in arresting aflatoxin B₁ secretion by the toxigenic strain (Navjot 4NSt) of A. flavus at 750 μL L⁻¹. The efficacy of A. odoratissimus oil as aflatoxin suppressor is being reported for the first time.     

Evaluation of Chenopodium ambrosioides oil as a potential source of antifungal, antiaflatoxigenic and antioxidant activity

Essential oil extracted from the leaves of Chenopodium ambrosioides Linn. (Chenopodiaceae) was tested against the aflatoxigenic strain of test fungus Aspergillus flavus Link. The oil completely inhibited the mycelial growth at 100 microg/ml. The oil exhibited broad fungitoxic spectrum against Aspergillus niger, Aspergillus fumigatus, Botryodiplodia theobromae, Fusarium oxysporum, Sclerotium rolfsii, Macrophomina phaseolina, Cladosporium cladosporioides, Helminthosporium oryzae and Pythium debaryanum at 100 microg/ml. The oil showed significant efficacy in inhibiting the aflatoxin B1 production by the aflatoxigenic strain of A. flavus. During in vivo investigation it protected stored wheat from different storage fungi for one year. Chenopodium oil also exhibited potent antioxidant activity when tested by ABTS method. All these observations suggest the possible exploitation of the Chenopodium oil as potential botanical fungitoxicant in ecofriendly control of post harvest biodeterioration of food commodities from storage fungi.     

Fungi associated with the spoilage of stored yam chips and flour in Nigeria

A study was carried out to investigate the organisms associated with the spoilage of yam chips and flour in many locations in 5 states of Nigeria. Monilinia fructicola, Aspergillus flavus, A. fumigatus, A. niger, Penicillium expansum, P. wortmanni, P. duponti, P. funiculosum, Rhizopus sexualis, Piricularia sp., Cladosporium cucumerinum, Helminthosporium sp. and Mucor sp. were isolated and found to cause spoilage of the yam products. Araecerus fasciculatus, Tribolium castaneum and Ahasverus advena were found on the yam chips as infesting insects. The yam chips or flour were either contained in exposed baskets or sealed in fibre bags in the market stalls. A. fumigatus had the highest occurrence. Most of the fungi were found not to sporulate heavily at a temperature of 10–15 °C while only spores of A. fumigatus, A. flavus and C. cucumerinum germinated at 32.5% relative humidity.     

山茱萸中有益菌的分离和鉴定

将药用植物山茱萸作为有益菌的分离源,以获得常见有害菌的抗性菌,提高山茱萸的利用价值。以马铃薯葡萄糖琼脂和营养琼脂平板分离、纯化其体外真菌和细菌,对获得的菌株进行黑曲霉、黄曲霉和大肠杆菌等有害菌的对峙实验,并对抗性菌进行分子生物学和生理生化的种属鉴定。实验结果表明：从山茱萸中分离纯化28株菌（12株细菌、16株真菌）,其中2株菌对黑曲霉、黄曲霉有一定的抑菌效果,经分子生物学和生理生化实验鉴定,菌株S136是肠杆菌属（Enterobacter sp.）、菌株S138是芽孢杆菌属（Bacillus sp.）的细菌。     

荔枝干微生物菌落分析及其霉菌的分离鉴定

为了研究荔枝干贮藏销售期内普遍存在的微生物超标及霉变问题,对市场零售的散装荔枝干样品进行了微生物数量的测定,并对散装和真空包装的荔枝干样品中的霉菌进行了分离、纯化、鉴定。结果表明:20%样品菌落总数>750cfu/g,80%样品霉菌总数>50cfu/g,抽样合格率为20%;从荔枝干样品中分离得到5株霉菌,通过形态学观察鉴定为黄曲霉、黑曲霉、烟曲霉、红曲霉和黑绿青霉,并确定曲霉属菌株为荔枝干表面霉菌的优势菌群。     

河南烟叶仓储期优势霉菌的分离鉴定及防霉剂的筛选

对来自河南产区仓储烟叶中的主要霉菌进行了分离、鉴定,并测定了防霉剂对优势霉菌的抑制作用.结果表明,河南烟叶样品中霉菌数量以曲霉最多,其次是青霉.山梨酸钾、丙酸钙、双乙酸钠、脱氢醋酸钠、对羟基苯甲酸乙酯和富马酸二甲酯对黄曲霉、黑曲霉和桔青霉这3种优势霉菌均有明显的抑霉作用.     

INFLUENCE OF OTHER FUNGI ON AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS IN MAIZE KERNELS

Experiments were designed to determine whether certain nontoxigenic fungi commonly isolated from maize kernels can affect aflatoxin B₁ development when inoculated with A. flavus onto individual unsterilized, and autoclaved maize kernels . Trichoderma viride and Aspergillus niger were found to be strongly antagonist inhibiting the growth of A. flavus by 87 and 66% respectively, whereas Aspergillus versicolor, Fusarium moniliforme, Paecilomyces variotii and Emericella quadrillineata inhibited the growth of A. flavus by less than 51%. Less aflatoxin B₁ was detected when A. flavus was paired with A. niger or T. viride than with the other test fungi. When A. niger or T. viride was introduced onto the kernels 72 h before inoculation with A. flavus, no aflatoxin B₁ was detected in unsterilized kernels and the levels of aflatoxin B₁ were greatly reduced from 700 ppb to 160 and 140 ppb in autoclaved kernels, respectively. When inoculation of A. flavus followed 72 h of incubation of either A. niger and T. viride, no aflatoxin B₁ was detected. However, when both A. niger and T. viride were introduced 72 h after inoculation with A. flavus, the levels of aflatoxin B₁ were reduced to 385 and 560 ppb, respectively in unsterilized and autoclaved maize kernels . Trichoderma viride and Aspergillus niger may be useful in biological control of aflatoxin contamination of maize kernels .; Accepted for Publication June 11, 1997     

真菌发酵预处理麻疯树籽高效提油初步研究

利用真菌固液态发酵麻疯树籽,通过测定还原糖和氨态氮含量变化,判断真菌对营养成份利用情况与油脂提取效果关系。实验结果表明,经黑曲霉固液态发酵预处理麻疯树籽在发酵48 h得到最大提油量,分别为32.75%(w/w)和31.95%(w/w),比对照组(27.5%,w/w)分别提高19.1%和16.2%;补充适量葡萄糖后有利于提高提油量,经黑曲霉固液态发酵预处理麻疯树籽都在发酵72 h后达最大提油量,分别为35.75%(w/w)和34.65%(w/w),比对照组(27.5%,w/w)分别提高30%和26%。经实验,黄曲霉不适于麻疯树籽发酵,发酵后提油效果没明显提高。     

Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 microg/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.     

Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak

The essential oil extracted from the fruits of Cicuta virosa L. var. latisecta Celak was tested in vitro and in vivo against four foodborne fungi, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and Alternaria alternata. Forty-five different components accounting for 98.4% of the total oil composition were identified by gas chromatography-mass spectrometry. The major components were γ-terpinene (40.92%), p-cymene (27.93%), and cumin aldehyde (21.20%). Antifungal activity was tested by the poisoned food technique against the four fungi. Minimum inhibitory concentration against the fungi was 5 μL/mL and percentage inhibition of mycelial growth was determined at day 9. The essential oil had a strong inhibitory effect on spore production and germination in all tested fungi proportional to concentration. The oil exhibited noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B? (AFB?) by A. flavus, completely inhibiting AFB(1) production at 4 μL/mL. The effect of the essential oil on inhibition of decay development in cherry tomatoes was tested in vivo by exposing inoculated and control fruit to essential oil vapor at a concentration of 200 μL/mL. Results indicated that the essential oil from C. virosa var. latisecta (CVEO) has potential as a preservative to control food spoilage.     

Mycofloral changes and aflatoxin contamination in stored chickpea seeds.

Chickpea seeds entering store carry a microflora of 'field' and 'storage' fungi. Field fungi gradually disappear and storage fungi then predominate. These fungi especially Aspergillus flavus, A. niger, A. nidulans, A. ochraceus and Penicillium spp. grow vigorously and initiate grain spoilage and aflatoxin elaboration. The shift in mycofloral spectrum was more rapid in seeds stored in jute bags than those stored in metal bins. Moisture content and aflatoxin contamination in seeds of jute bags was maximum during Sept.-Oct. The internal environment of metal bins is comparatively less influenced by the external conditions and initially restricts fungal growth and aflatoxin elaboration. Prolonged storage however, increases seed moisture content and alfatoxin is increased in chickpea seeds after six months storage in metal bins.     

Microbiological survey of retail herbs and spices from Mexican markets

In the present study, 304 samples of herbs and spices (garlic powder, cumin seeds, black pepper, oregano, and bay leaves) widely used in Mexico were analyzed for the presence of Bacillus cereus, Salmonella Typhi, Shigella dysenteriae, Escherichia coli, total and fecal coliforms, total mesophilic aerobic organisms, and fungi. Samples were nonpackaged or packaged in polyethylene bags or glass containers. High levels (10(5) to 10(7) CFU/g) of mesophilic aerobic microorganisms were found in most of the samples of garlic powder, cumin seed, and black pepper. Lower levels (<102 CFU/g) were found in oregano and bay leaves. Total and fecal coliforms counts were dependent on the type of packaging. More than 70% of the polyethylene-packaged samples had less than 10(3) CFU/g of microorganisms. Glass and nonpackaged spices showed lower levels of these microorganisms. B. cereus was present in 32 samples of which most were polyethylene packaged. The other pathogenic bacteria were not detected. Aspergillus niger was detected in 29% of the samples, Rhizopus sp. in 19%, and Penicillum sp. and Cunninghamella in 8%.     

Putranjiva roxburghii oil—A potential herbal preservative for peanuts during storage

Samples of peanut seeds were collected from stores in eastern Uttar Pradesh, India and examined for their associated mycoflora and insects. Fifteen species of fungi and one insect, Trogoderma granarium, were isolated from the samples. Volatile constituents extracted in the form of essential oils from 32 plant species were evaluated against the dominant fungi, Aspergillus flavus and Aspergillus niger, as well as Trogoderma granarium. The oil of Putranjiva roxburghii exhibited the greatest toxicity. The oil was found to be fungicidal and thermostable at its minimum inhibitory concentration (MIC) of 400 ppm. The oil was characterized by the determination of its various physico-chemical properties.

The oil protected the peanut seeds completely for 6 months at 0.25 and 0.38 mL in containers of 250 mL capacity holding 200 g seeds. It did not exhibit any adverse effect on seed germination, seedling growth and general health and morphology of plants. Thus, the oil of Putranjiva roxburghii showed potential as a preservative for peanut seeds against spoilage by fungi and insects during storage.     

The microbial spoilage of ‘soft’ melon ball snack under tropical conditions

19 microorganisms, mostly hazardous and spoilage species were isolated from ‘soft’ melon ball snack samples in the humid Western Nigeria. Physical biodeterioration was recorded from the 6th day of storage with the early occurring rope (slime) of Bacillus sp. found to be a faster visible objectionable indicator than the commonly used fungal growth. All the 14 isolated fungi grew though, to varying extents on the snack agar and in the melon seeds infusion medium in which they also induced significant changes in the oil (decrease) and free fatty acids (increase) contents after 10 days of incubation at 30 °C. The results implicated Rhizopus arrhizus, R. nigricans, Aspergillus flavus, A. ochraceus, A. tamarii, A. niger, Mucor fragilis and Penicillium citrinum as the major fungal deteriogens. Isolation of the coliform bacterium (Escherichia coli) suggested the need for a more hygienic handling of the snack. Addition of varying amounts of breadfruit nuts mash as adjunct to melon seeds mash in order to enhance mastication of snacks with lower moisture content was investigated. Samples prepared with 20% adjunct recorded mean acceptability rating of 178 and keepability period of 10 days while 200 and 5 days were obtained respectively for the control.     

The occurrence of aflatoxins in mustard and mustard products

Natural occurrence of aflatoxins at concentrations of 750, 87 and 1420 micrograms/kg was recorded in 44, 33 and 80 samples out of 100 samples each of seeds, oil and cakes of mustard respectively. Out of 17 species of fungi isolated from mustard seeds, percentage incidence of the Aspergillus flavus group was maximum during monsoon and summer. Twenty-five per cent of isolates (out of 1143 isolates screened) of the A. flavus group were toxigenic, producing different components of aflatoxins in varying concentrations.     

Fungal population and distribution of aflatoxigenic fungi in commercial almond powder products

The fungal population and distribution of aflatoxin-producing fungi in 30 samples of imported almond powder products purchased from retail markets were examined in this study. Total counts of fungi ranged from under 1.0 x 10 colony-forming units (CFU)/g to 8.5 x 10(3) CFU/g as determined with the dilution plating technique. The predominant fungi in the mould-contaminated almond samples were Aspergillus niger, A. flavus and the related species, Penicillium, Cladosporium and Rhizopus. Aflatoxin-producing ability in the isolates of A. flavus and related fungi were tested by thin layer chromatography using 2% yeast extract and 15% sucrose broth culture. Four different aflatoxigenic fungi were detected in the isolates; aflatoxins B1 and B2 were produced by some strains of A. flavus and A. parvisclerotigenus, and aflatoxins B1, B2, G1, G2 were produced by all tested strains of A. parasiticus and A. nomius. Identification of the strains was based on morphological and metabolic characters.     

Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions

Mixtures of cinnamon and clove oils were tested for inhibitory activity against important spoilage microorganism of intermediate moisture foods. Four fungal species (Aspergillus flavus, Penicillium roqueforti, Mucor plumbeus and Eurotium sp.), four yeasts species (Debaryomyces hansenii, Pichia membranaefaciens, Zygosaccharomyces rouxii and Candida lipolytica), and two bacteria species (Staphylococcus aureus and Pediococcus halophilus) inoculated separately on agar plates were sealed in a barrier pouch and exposed to essential oil volatiles under a modified atmosphere of low O2 (<0.05-10%) and high CO2 (20% or 40%), with the balance being N2. A. flavus and Eurotium sp. proved to be the most resistant microorganisms. Cinnamon and clove oils added between 1000 and 4000 microL at a ratio of 1:1 were tested for minimum inhibitory volume (MIV) against molds and yeasts. The gas phase above 1000 microL of the oil mixture inhibited growth of C. lipolytica and P. membranaefaciens; 2000 microL inhibited growth of A. flavus, P. roqueforti, M. plumbeus, Eurotium sp., D. hansenii, and Z. rouxii, while inhibition of A. flavus required the addition of 4000 microL. Higher ratios of cinnamon oil/clove oil were more effective for inhibiting the growth of A. flavus.     

Occurrence of Aspergillus spp. and aflatoxin B1 in Malaysian foods used for human consumption

Malaysian population widely consumes the cereal-based foods, oilseeds, nuts, and spices in their daily diet. Mycotoxigenic fungi are well known to invade food products under storage conditions and produce mycotoxins that have threat to human and animal health. Therefore, determining toxigenic fungi and aflatoxin B(1) (AFB1) in foods used for human consumption is of prime importance to develop suitable management strategies and to minimize risk. Ninety-five food products marketed in Penang, Malaysia were randomly collected from different supermarkets and were analyzed for presence of Aspergillus spp. by agar plate assay and AFB1 by enzyme-linked immunosorbent assay (ELISA). A. flavus was the dominant fungi in all foods followed by A. niger. Fifty-five A. flavus strains were tested for their ability to produce aflatoxins on rice grain substrate. Thirty-six (65.4%) strains out of 55 produced AFB1 ranging from 1700 to 4400 μg/kg and 17 strains (31%) produced AFB2 ranging from 620 to 1670 μg/kg. Natural occurrence of AFB1 could be detected in 72.6% food products ranging from 0.54 to 15.33 μg/kg with a mean of 1.95 μg/kg. Maximum AFB1 levels were detected in peanut products ranging from 1.47 to 15.33 μg/kg. AFB1 levels detected in all food products were below the Malaysian permissible limits (<35 μg/kg). Aspergillus spp. and AFB1 was not detected in any cookies tested. Although this survey was not comprehensive, it provides valuable information on aflatoxin levels in foods marketed in Malaysia.     

沙棘果皮渣黄酮抑菌活性研究

考查沙棘果皮渣黄酮对于大肠杆菌、金黄色葡萄球菌、酵母菌、变形杆菌、产气杆菌、嗜热链球菌、枯草芽孢杆菌、根霉、青霉和黑曲霉等几种主要危害食品卫生、安全菌种的抑制作用,并对其最低抑菌浓度进行测定。结果表明:沙棘果皮渣黄酮对大肠杆菌、金黄色葡萄球菌、酵母菌、变形杆菌、嗜热链球菌、枯草芽孢杆菌、根霉、青霉、黑曲霉均有明显抑菌效果,对各个菌种的最低抑菌浓度分别为5、20、40、40、80、5、2.5、20、10mg/mL。