Biotoxic activity in the Mucorales

The toxigenicity of representatives of 15 species of Mucorales (Absidia glauca, Actinomucor elegans, Cunninghamella elegans, Helicostylum piriforme, Mortierella isabellina, Mortierella (Mucor) rammaniana, Mucor hiemalis, Mucor mucedo, Mucor spinosus, Phycomyces blakesleeanus, Rhizopus oligosporus, Rhizopus stolonifer, Syncephalastrum racemosum, Thamnidium elegans, Zygorhynchus moelleri) towards the larvae of brine shrimp (Artemia salina) and the growth of pea seedlings (Pisum sativum) and tobacco plants (Nicotiana tabacum) was evaluated. The fungi were cultivated on malt extract agar and aqueous solutions of the cultures were tested.Thamnidium elegans showed a marked toxic action towards brine shrimp (mortality: 74.1%) andPhycomyces, Actinomucor andSyncephalastrum were only weakly toxic. Length and weight of stems of pea seedlings were moderately reduced by extracts ofAbsidia, Cunninghamella, Zygorhynchus andThamnidium and to a lesser degree byMucor spinosus. Cunninghamella andMucor spinosus also inhibited the development of pea hypocotyls. The length of tobacco stems was reduced byMortierella ramanniana, Rhizopus stolonifer andCunninghamella elegans. Wilting or other toxic phenomena were never observed with both test plants. Considering the present results and data from literature it is suggested that species of Mucorales have only a weak toxigenicity.     

应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

目的应用反向线点杂交技术(reverse line blot hybridization,RLB)快速鉴定临床常见的曲霉属和毛霉目真菌。方法收集我院真菌和真菌病研究中心保存的5种曲霉菌(烟曲霉、黄曲霉、黑曲霉、土曲霉、构巢曲霉)和7种毛霉目真菌(冻土毛霉菌、总状毛霉菌、卷枝毛霉菌、少根根霉、小孢根霉、微小根毛霉、伞状犁头霉),共计98株菌株。利用真菌通用引物ITS1和ITS4对菌株进行PCR扩增,用12个真菌种特异性探针与扩增后产物进行反向线点杂交。将RLB结果与真菌传统形态学鉴定结果、ITS区DNA测序结果进行比较。结果 RLB可以正确鉴定98株实验菌株,与形态学方法和ITS区测序方法鉴定结果100%一致,种特异性探针之间未见交叉杂交,显示出该方法的高度敏感性和特异性。8株阴性对照菌株(白念珠菌、茄病镰刀菌、尖端赛多孢、马尔尼菲青霉、疣状瓶霉、棒曲霉、日本曲霉以及雅致小克银汉霉),使用RLB方法无法鉴定。通过烟曲霉基因组DNA浓度10倍倍比稀释法验证RLB的敏感性为1.8×10-3 ng/μL。结论 RLB技术为实验室早期快速诊断、鉴定临床常见的曲霉属和毛霉目真菌提供参考。     

Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments

About 34 wild fungal species associated with edible oil mill wastes were isolated by the serial dilution technique. Methods for rapid screening of fungal species against production of extracellular enzymes such as amylase, protease, cellulase, and lipase are reported. Among all the species, Aspergillus versicolor exhibited high amlylolytic and gelatinolytic activity, whereas Penicillium citrinum showed only high amylolytic activity. Maximum cellulolytic activity was recorded for Absidia corymbifera, As. niger, Cunninghamella echinulata, Curvularia lunata, Fusarium solani, Mucor racemosus, Paecilomyces variotii, and Syncephalastrum racemosum. The fungal species Ab. corymbifera, As. fumigatus, As. japonicus, As. nidulans, As. terreus, Cun. verticillata, Cur. pallescens, F. oxysporum, Geotrichum candidum, M. racemosus, Pe. citrinum, Pe. frequentans, Rhizopus stolonifer, and Trichoderma viride exhibited maximum lipase activity. This study confirms the isolated fungi present on a wide range of substrates in the ambient environment, and these results could provide basic data for further investigations on fungal extracellular enzymes.First two authors equally contributed to this work     

Mucor circinelloides a destructive hyperparasite of Rhizopus nigricans

In vitro studies on hyphal interactions between phyllosphere and phylloplane fungi revealed Mucor circinelloides to be a destructive hyperparasite of Rhizopus nigricans. The mycoparasite penetrated the host hypha and formed sporangiophores and sporangia on different parts of Rhizopus nigricans.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

Diversity of mucoralean fungi in soils of papaya (Carica papaya L.) producing regions in Mexico

Mexico is the fifth largest producer of papaya worldwide and has recently reported problems with mucoralean fungi in this crop. These fungi are considered saprophytes in the soil and are ubiquitous in nature. In this work, they were isolated from soil in regions of intensive papaya cultivation in Mexico. Collections were made in the states of Colima, Oaxaca and Veracruz in Apr 2016. A total of 72 mucorales fungal isolates was obtained and morphologically characterized and then molecular characterization (28S ribosomal region) of 25 representative isolates was carried out. Phylogenetic analysis of the sequences confirmed the presence of the species Gilbertella persicaria, Rhizopus oryzae, Rhizopus stolonifer, Mucor circinelloides and Mucor hiemalis, which cause soft rot in papaya fruits, therefore, spores of these fungi found in the orchard soils can be considered as a constant source of contamination that affects healthy fruits. Additionally, Choanephora cucurbitarum, Mucor ellipsoideus, Rhizopus homothallicus, Rhizopus microsporus, Rhizopus schipperae, Lichteimia ramosa, Gongronella butleri, Cunninghamella bertholletiae and Cunninghamella blakesleeana were identified which are considered to have agricultural, biotechnological and medical importance.     

Oligounsaturated fatty acid production by selected strains of micromycetes

Fifteen strains of filamentous fungi from theCulture Collection of Fungi (Charles University, Prague) were tested for their lipid production, fatty acid composition with emphasis on accumulation of oligounsaturated fatty acids. All cultures contained palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and γ-linolenic (18:3) acid (GLA). The mycelium ofCunninghamella elegans, Rhizopus arrhizus, Mortierella parvispora, M. elongata andM. alpina contained arachidonic acid (ARA) in the range of 2.3–33.5% of the total fatty acids. The strains used in our experiment were capable to accumulate a relatively high amount of intracellular lipid (9.6–20.1% in dry biomass). The highest content of GLA (22.3 mg/g) was found inMucor circinelloides. The strain ofM. alpina containing 47.1 mg/g of ARA could be considered as the best producer of ARA.     

Formation of antigenic extracellular polysaccharides by selected strains ofMucor spp.,Rhizopus spp.,Rhizomucor spp.,Absidia corymbifera andSyncephalastrum racemosum

In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) ofMucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeastPichia membranaefaciens. EPS were isolated from various cultures ofM. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains includingMucor spp.,Rhizopus spp.,Rhizomucor spp.,Absidia corymbifera andSyncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.     

Outbreaks of Mucorales and the Species Involved

The order Mucorales is an ancient group of fungi classified in the subphylum Mucoromycotina. Mucorales are mainly fast-growing saprotrophs that belong to the first colonizers of diverse organic materials and represent a permanent part of the human environment. Several species are able to cause human infections (mucormycoses) predominantly in patients with impaired immune system, diabetes, or deep trauma. In this review, we compiled 32 reports on community- and hospital-acquired outbreaks caused by Mucorales. The most common source of mucoralean outbreaks was contaminated medical devices that are responsible for 40.7% of the outbreaks followed by contaminated air (31.3%), traumatic inoculation of soil or foreign bodies (9.4%), and the contact (6.2%) or the ingestion (6.2%) of contaminated plant material. The most prevalent species were Rhizopus arrhizus and R. microsporus causing 57% of the outbreaks. The genus Rhizomucor was dominating in outbreaks related to contaminated air while outbreaks of Lichtheimia species and Mucor circinelloides were transmitted by direct contact. Outbreaks with the involvement of several species are reported. Subtyping of strains revealed clonality in two outbreaks and no close relation in two other outbreaks. Based on the existing data, outbreaks of Mucorales can be caused by heterogeneous sources consisting of different strains or different species. Person-to-person transmission cannot be excluded because Mucorales can sporulate on wounds. For a better understanding and prevention of outbreaks, we need to increase our knowledge on the physiology, ecology, and population structure of outbreak causing species and more subtyping data.

     

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.     

Fungal Treatment of an Edible-oil-containing Industrial Effluent

Summary A typical edible oil-processing plant discharges large volumes of effluent high in Chemical oxygen demand (COD). This leads to fines from the municipality and the consequent loss of profit. In this study the effluent composition of an oil-processing plant was monitored for 1 year. The COD ranged between 16,000 and 250,000 mg/l and the conductivity between 88.2 and 268 mS/m. It is known that edible oil is a good carbon source and Mucoralean fungi have the ability to accumulate large quantities of oil. Therefore, the reduction of the COD by 12 fungal strains (from culture collections) was determined. The most promising organisms were Rhizopus stolonifer (CBS 263.28), reducing the COD by 91.3%, a Penicillium species (TUTC 077) by 85.3%, Mucor circinelloides f. circinelloides (CBS 108.16) by 84.0% and Aspergillus niger (TUTC 120) by 83.8%. Although the reduction of the effluent COD was significant, the lowest COD of 1625.08 mg/l was still higher than the legal limits. This led to the isolation of fungi from soil, using selective media. Fifty-eight fungal strains were isolated, of which seven isolates could reduce the COD to below 760 mg/l (legal limit). The best isolate reduced the COD by 98.2% from 16,000 to 286.96 mg/l and was identified as Cunninghamella echinulata. The best COD reducers were tested for their ability to produce gammalinolenic acid (GLA) as a high value by-product. All these isolates were able to produce GLA and the best production was obtained from Emerisella nidulans, namely 408.70 mg GLA/l.     

Nachweis von Fusarien und deren Mykotoxinen in Futterkonservaten aus Grünlandbeständen mit differenzierter Bewirtschaftungsintensität

Conservation forage (17 hay and 18 grass silage samples) from 15 farms with different intensities of grassland management in the Federal State of Brandenburg were examined for contamination with fusaria and their mycotoxins. The numbers of culturable filamentous fungi in hay were determined by plate counting andFusarium isolates were classified taxonomically. The mycotoxins Zearalenone (ZEA) and Deoxynivalenol (DON) were extracted from hay as well as silage by different procedures and detected chromatographically (HPLC). The numbers of filamentous fungi in the hay samples were 10² and 10⁶ CFU/g FM independently of intensive or extensive management. Only fourFusarium species were identified.Fusarium culmorum, a potential toxin producing species, was most frequently detected (52% of all isolates). ZEA was found in two hay and four silage samples (6-66 μg/kg), DON in three hay and seven silage samples (63–1290 μg/kg). There were no differences between forage samples of extensive and intensive cultivated grassland of the year 2003 regarding numbers of fusaria and the content of their mycotoxins.

Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004.     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea. Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride. T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10-30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.     

Keratinophilic fungi from soil of Brittany,France

The Present work reports the study of the distribution of keratinophilic fungi isolated from 39 samples of soil collected in Brittany, France. Keratinophilic fungi were isolated from 35 (92.3%) of the samples studied. The most frequently found species wereFusarium moniliforme, Penicillium viridicatum and an unidentified species ofAcremonium. The other fungi isolated were members of the generaChrysosporium, Gliocladium, Mucor, Trichoderma andTrichophyton.     

Mycotoxigenic infestation in samples of cereal straw from Bihar, India

A survey of different types of cereal straw samples viz. paddy, maize and wheat, from Bihar State, India, was conducted in order to examine the mould flora and mycotoxin contamination. Out of 170 samples examined for mould flora,Aspergillus flavus group of fungi had highest level of incidence followed byA niger. Isolates ofA flavus, A ochraceus, Fusarium verticillioides andPenicillium citrinum were screened for their mycotoxins producing abilities. Out of 75, 63 and 68 isolates ofA flavus group obtained from stored straw of paddy, maize and wheat samples, respectively, 27 (36%), 14 (22%) and 24 (35%) were found to be toxigenic which produced different combinations of aflatoxins in different concentrations. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi from all types of samples. Out of 222 samples of straw analysed for natural occurrence of different mycotoxins, besides the aflatoxins present, zearalenone, ochratoxin A and citrinin were also recorded alone or as co-contaminants. A conducive climate together with the socioeconomic conditions of this region are important determinants for the high incidence of mycotoxins in cereal straw samples.     

Mycoflora of mangrove mud

Summary Thirty-nine species and four varieties of fungi are reported from mangrove mud collected from Kagh Island, Port Canning and Diamond Harbour, West Bengal, India. Of these forms twelve species and two varieties belonged to genusAspergillus and three species belonged, each toMucor, Penicillium andPreussia. Two species both ofFusarium andTrichoderma were also isolated. Two new species,Cladosporium indicum andPaecilomyces indicus and five other interesting fungi are described in detail.     

Presence of carotenoids and ergosterol inMucor azygospora and inMucor inaequisporus

The yellow-orange mycelia ofMucor azygospora andM. inaequisporus were analyzed for the presence of carotenoids.Mucor azygospora contained 7 carotenoids with γ-carotene as the principal pigment andMucor inaequisporus the same carotenoids with Β-carotene predominating. The steroid ergosterol was obtained in a crystalline state from the unsaponifiable fraction of both fungi. The author thanks Miss M. Krul for her technical assistance.     

Isolation and characterization of a strain of Lichtheimia corymbifera (ex Absidia corymbifera) from a case of bovine abortion

Background  

Lichtheimia corymbifera (previously Absidia corymbifera) is a filamentous zygomycetes belonging to the order Mucorales and to the family Lichtheimiaceae. Members of genus Lichtheimia spp. are cosmopolitan and ubiquitous in nature. Lichtheimia corymbifera is a recognized agent of diseases in man and animals. In cattle it causes abortion and mastitis. Three cases of bovine abortion occurred in a herd located in the Po Valley. Serological examinations were performed on fetal and mother's blood. One of the aborted fetus was referred to our laboratory. The paper describes the isolation and characterization of Lichtheimia corymbifera from a bovine aborted fetus.     

Antigenic characterization of some potentially pathogenic mucoraceous fungi

The antigenic profiles of 10 mucoraceous fungi--Absidia corymbifera, Mortierella wolfii, Mucor miehei, M. pusillus, M. racemosus, Rhizopus arrhizus, R. microsporus, R. oryzae, R. rhizopodiformis, R. stolonifer,--Candida albicans and Aspergillus fumigatus were compared by crossed immunoelectrophoresis (XIE). Antigen-rich material was obtained from homogenized hyphae (or yeasts in the case of C. albicans), and antisera by multiple subcutaneous innoculation of rabbits with macerated but viable hyphal fragments of Ab. corymbifera, M. pusillus, R. oryzae or Asp. fumigatus. Unique and common antigens were demonstrable amongst the mucoraceous species although Mort. wolfii revealed little antigenic similarity with the others. Considerable sharing of antigens between Ab. corymbifera and M. pusillus was evident. Little or no cross reactivity was seen between extracts of C. albicans and Asp. fumigatus and the mucoraeceous antisera. R. oryzae and R. arrhizus, now regarded as synonymous, revealed close antigenic similarity. On the other hand, the distinction between both M. pusillus and M. miehei--which are regarded by some as belonging to a separate genus Rhizomucor--and less thermotolerant M. racemosus was reflected in their antigenic dissimilarity. Partial separation and characterization of antigens from the crude Absidia extract was achieved by concanavalin A-Sepharose chromatography. Antigens with and without affinity for concanavalin A could be demonstrated. Cross reactivity between Absidia antigens and M. pusillus antiserum appeared to be contained predominantly in material (possibly carbohydrate) which bound to concanavalin A and could be eluted with alpha-methyl-D-mannoside.     

Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation

Aspergillus flavus, A. niger, Penicillium expansum and Rhizopus stolonifer were the most frequently isolated fungi from healthy apple fruits. Alternaria alternata was the most common organism of rotten apple fruits, followed by A. niger, A. flavus, P. expansum and R. stolonifer. The prevalent type of decay, brown rot lesion, is caused by R. stolonifer followed by A. flavus, A. niger, A. alternata and P. expansum. Sodium hypochlorite had good curative properties against fruit rots. The main natural mycotoxins produced in rotten apple were patulin and aflatoxins. The optimum temperature for patulin production by P. expansum was 15 °C after 15 days. Complete inhibition of patulin formation was attained using 0.2% lemon oil and > 90% inhibition using 0.05% lemon and 0.2% orange oils. Also significant inhibition (> 90%) of aflatoxin production was observed with 0.2% lemon oil. This revised version was published online in July 2006 with corrections to the Cover Date.