Micro-organisms and dust exposure in an Italian grain mill

AIMS: In order to assess possible occupational risk for workers in a grain mill, we evaluated aerial microbiological contamination in different areas of the mill and at different points of the production line. We also measured the concentration of aerodispersed dust particles. METHODS AND RESULTS: An assessment of microbiological contamination levels based on a Global Index of Microbial Contamination per cubic metre (GIMC per m3), an Index of Mesophilic Bacterial Contamination, and an Amplification Index is proposed. The indices were obtained from total and fungal counts. The cleaning sector is the most contaminated area of the mill: the mean GIMC per m3 was 17,213.6. In this area, the average microbial contamination was 11.41 times higher than that in the external environment. The highest concentrations of aerodispersed dust (inhalable 2.763 mg m(-3); respirable 1.400 mg m(-3)) were found in the cleaning area. CONCLUSIONS: The proposed microbiological indices and the concentrations of aerodispersed dust particles show that the most hazardous section of the mill is the cleaning area. The large variation in the data does not depend on seasonal factors, but rather on not easily identifiable conditions of the internal environment which facilitate diffusion and/or proliferation of the micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed microbiological contamination indices and the evaluation of the concentration of dust particles allow the identification of critical positions during the production cycle so that suitable measures to prevent the aerial contamination can be taken.     

Airborne fungi monitoring in Santiago, Chile

Fungal airborne spores were studied from September 1996throughout August 1997 in Santiago, Chile. Total concentrationsfluctuated between 308 and 10,334 spores/m³/day withan annual mean of 2,154 per m³, the highest dispersion beingduring April and May. Forty-five percent of total fungal content wasfound in autumn. Thirteen genera and 3 other spore types wereidentified. Cladosporium, the most abundant genera in ouratmosphere, contributed with 70.9% of the total fungi counts andreached an annual mean of 1,527 spores/m³/day, itshighest frequency being in autumn. Alternaria appeared as thesecond most frequent genera, with an annual mean of 40spores/m³/day, representing a 1.9% of theannual fungal catch. Altogether, Stemphylium, Torula, Epicoccum,Ganoderma, Helminthosporium, Chaetomiun, Pleospora and othersreached relative frequencies of 0.5% or less. It is concludedthat fungi are present in Santiago's atmosphere all year round, some ofthem with a clear seasonality.     

The biodiversity of air spora in an Italian vineyard

Biodiversity indices are frequently used to provide a numerical value of the diversity of species within an ecological community. To study fungal biodiversity in the air, a 7-day recording Hirst-type spore trap was used. The daily concentration of 124 taxa was recorded in an Italian vineyard. The predominant fungi were: Cladosporium spp., unknown 3-septate fusiform spore, and Alternaria spp. Shannon’s and Simpson’s biodiversity indices and evenness were calculated first for air spora. Meteorological circumstances affected the biodiversity; positive correlations were found between Simpson’s biodiversity index and precipitation, but no significant correlations were found with Shannon’s diversity index and evenness. To predict Simpson’s index for airborne fungi, regression analysis was performed. It was shown that the best estimator, sun hours, negatively affected the index. The biodiversity of the dominant species was low on dry days, since Cladosporium alone had much higher abundance on such days than other species.     

Airborne fungal load in agricultural environment during threshing operations

The effect of threshing operations on the fungal spora of farm air, has been studied with the culture plate method. Air samples were taken before, during and after the threshing of paddy and wheat grains, at the end of the respective crop seasons. An immediate local increase in the airborne fungal load was observed during threshing operations. In case of wheat the high rise was due toAlternaria tenuissima, followed byDrechslera sp. andCladosporium herbarum. In Boro variety of paddy, the high count was contributed byAlternaria humicola andA. tenuissima, while in Aman variety,Helminthosporium oryzae was the dominant type followed byC. herbarum and unidentified yeasts.Aspergillus spp. were observed in significant numbers before the operation started; their occurrence was negligible during threshing. A considerable number of yeast was observed, particularly during threshing and after threshing operations.     

An evaluation of two methods used for microscopic analysis of airborne fungal spore concentrations from the Burkard Spore Trap

The Burkard Volumetric Spore Trap is a common and efficient instrument used to collect outdoor air samples. In North America, two slide counting methods have been widely used by aerobiologists: the single longitudinal traverse method and the twelve transverse traverse method. The purpose of this study was to compare the two counting methods by assessing fungal spore concentrations of ascospores, basidiospores, smut teliospores, Cladosporium, Alternaria, Epicoccum, Curvularia, Drechslera, Pithomyces, other spores, and total spores at two metropolitan Tulsa, Oklahoma sites (Tulsa and Hectorville) during September 1996. Results showed that both methods were sensing parallel fluctuations in average daily spore concentration, although the twelve transverse traverse method usually resulted in higher concentrations. At the Tulsa site, the twelve transverse traverse method gave statistically higher concentrations than the single longitudinal traverse method except for Epicoccum, Pithomyces, smut teliospores, and other spores. At the Hectorville site, however, only Cladosporium and basidiospores showed that the twelve transverse traverse method was statistically higher than the single longitudinal traverse method. Comparison with concentrations obtained by counting the total slide surface of two slides indicated that neither method was equivalent to the total slide spore count, although the twelve transverse traverse method gave a lower absolute percent difference from the total slide surface concentration. While the twelve transverse traverse method gave slightly better approximations of the spore concentration, the increase in accuracy may not justify the extra effort required to analyze with this method.     

Presence in the atmosphere of vine and tomato pathogens

Summary In nature fungi, together with bacteria, play an important role in the degradation of organic substances. A small group of fungi is responsible for mycosis in man and animals. A larger group made up of different fungal taxa are particularly harmful to crops. The study of agricultural fungal phytopathologies is more difficult in that many taxa are characterized by a high degree of nutritional specialization. The identification in the field of the diseases caused by these taxa takes place often too late and not always are they controllable at a specific stage of their evolution. Since the majority of fungal spores is airborne and consequently present in various concentrations at different altitudes in the atmosphere, it is possible to monitor them and sometimes identify the fungus by sampling the air with a volumetric spore trap. Its diagnostic use has allowed to samplePlasmopara sporangia and obtain their concentration/m³ in the air of a vineyard. Similar results have been obtained in tomato fields, with the identification ofPhytophthora sporangia, the presence of which has been correlated to the meteorological data of the trial period. Field monitoring also offers the possibility of evaluating the presence of other fungi such as the generaBotrytis, Oidium, Alternaria, Pyricularia, Fusarium andStemphylium which damage crops.     

Chironomids of Italian rice fields

Studies on chironomids of Italian rice fields began in 1914, but a comprehensive faunistic picture of Italian rice field chironomids was provided only by COCCHI in 1966. In 1988 and 1989 field research south of Verona (northern Italy) permitted us to confirm the results of COCCHI regardingC. sylvestris andChironomus spp., and to increase the number of known species of Italian rice field chironomids. The list of species which are injurious to rice is similar to that of other European countries, with the exception ofHarnischia falcata andTanytarsus sp. The type and period of damage is different for certain species (e.g. Cricotopus sylvestris) in Italy in comparison to other countries. A few agrotechnical steps are recommended to control pest species. The finding of the parthenogenetic speciesParatanytarsus grimmii in lab. aquaria used for rearing purposes is documented.     

Airborne fungi in the hospitals of metropolitan Delhi

Summary The fungal airspora of a large hospital in Delhi Metropolis was studied from May 1989 – April 1991, using Andersen Six Stage Volumetric Sampler and Burkard Personal Slide Sampler. Simultaneously, samples were also collected from outside the hospital to act as a control. Samplers were operated for 10 min. each time, at 10 - day intervals. Additional samples were also collected from different sections of 3 other hospitals. Some of the dominant forms encountered wereCladosporium spp.,Aspergillus flavus, Smut,Fusarium spp.,Aspergillus niger, Alternaria spp.,Penicillium citrinum, Aspergillus versicolor, andPenicillium oxalicum. Aspergillus flavus showed significantly high concentration inside hospital (n=66, x=53 CFU m^–3, p<0.05) as compared to outside air. The peak period for fungi was observed to be from June – September. The spore concentration was much lower in hospital units receiving filtered air as compared to control environment, but in naturally ventilated hospitals the concentration was similar to that of outside air.     

Isolation and selection of cellulolytic fungi from palm oil mill effluent

Cellulolytic fungi, 34 strains, were isolated from samples taken from palm oil mill residues and effluent, and high cellulase producers selected in comparison with nine known reference strains. Although 13 isolates showed good filter paper distintegration within 14 days, only eight isolates exhibited clearing zones around their colonies on carboxymethylcellulose (CMC) agar medium. Quantitative cellulase activity measurements, using CMC as carbon source, selected three of the eight isolates as potential cellulase producers. Using dried palm oil mill condensate as carbon source, only one of the isolates (F 11) showed similar results on both carbon sources. During media optimization for CMCase production, a four-fold increase from 0.058 to 0.275 U/ml was obtained using a medium, containing 0.1% (v/v) Tween 80 0.02% (w/v) NH₄NO₃, 0.025% (w/v) proteose-peptone and 0.1% (w/v) CMC dissolved in undiluted condensate from the sterilization of oil palm bunches, with an initial pH of 5.5.     

Solvent-tolerance of fungi located on an interface between an agar plate and an organic solvent

While 6 by 20 of type culture fungi could grow on an interface between organic solvent (log P, 4.12) and agar plate, 13 by 20 of strains could form a large colony after the removal of more toxic solvent, such as styrene (log P, 2.95) and tert-butyl acetate (log P, 1.76) because of viability of spores on the interface.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Natural occurrence of toxigenic fungi and mycotoxins in rice bran

Thirty four samples of rice bran, of which 9 were from raw (untreated) rice (RR) and 25 from parboiled rice (PbR) were collected from commercial rice mills in and around Madras and analysed for storage mycoflora and mycotoxins. Fungi of the Aspergillus flavus group were present in 29 of the 34 samples (8 from RR and 21 from PbR) in quantities ranging from <1–432 thousand propagules/g, though not always as the dominant mycoflora. Fungal numbers were usually higher in RR than in PbR samples.Five of the 9 RR samples and 6 of the 25 PbR samples were positive for aflatoxins. Among 29 isolates of A. flavus obtained from the bran samples, 16 isolates –6 from RR bran and 10 from PbR bran — were found to be toxigenic in vitro. Some isolates of A. candidus also seemed to produce aflatoxin and other fluorescent substances.     

Identification and characterization of bacterial endophytes of rice

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.     

Microsatellite markers reveal multiple origins for Italian weedy rice

Weedy rice (Oryza sativa L.) is one of the major issues of rice cultivation worldwide. In Italy, it infests about 70% of the total rice area. Different Weedy Rice populations can be distinguished based on variable morphological and physiological traits; however, little is known about genetic differentiation and origin of Italian weedy rice populations. The objective of this study was to genetically and morphologically characterize and compare different Italian weedy rice populations selected on the basis of different phenotypes. The main Italian rice territory was divided into 10 geographical areas in which 40 weedy rice populations were collected and grouped according to the awn traits. All the individuals of the populations were morphologically characterized according to plant and seed traits. Genetic characterization was performed using 19 SSR markers on all the collected accessions, and several rice cultivars, including some very old (late 19th century), nowadays are no longer cultivated. ANOVA showed that morphological plant and seed traits were significantly affected by the collection area and awnedness group. The importance of the awn morphology was also reflected in the Bayesian clustering where, despite a relatively low genetic diversity, the clusters displayed different awn types. An UPGMA dendrogram confirmed the clusters detected in STRUCTURE analysis and also revealed a grouping of certain old cultivars with the weedy rice, suggesting a common origin.     

Plant growth hormone production from olive oil mill and alcohol factory wastewaters by white rot fungi

In this study, olive oil mill and alcohol factory wastewaters have been tested as growth media for the production of plant growth hormones. Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 have been tested. Gibberellic acid (GA₃), abscisic acid (ABA), indole acetic acid (IAA), and cytokinin were determined in the culture media of these fungi. Both organisms produced enhanced levels of all three hormones in the presence of either of the wastewaters.     

Differential responses in vitro of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea (Cooke) Sacc. 1. Oxidative burst

Suspension cultured cells of six rice cultivars differing in their sensitivity to blast were treated with mycelial wall hydrolysates prepared from seven isolates belonging to different Pyricularia grisea lineages. Soon after elicitor addition, rice cells produced significant amounts of superoxide anion, which was rapidly converted into diffusible peroxide. Maximal effects were achieved at 50 mg L-1 elicitor. In all cases, a 7 to 13-fold increase in the basal rate of reactive oxygen species production was found. Neither differential effects among strains nor clear relationships between lineage and the resulting oxidative burst were evident. Interestingly, a good correlation was found between basal (and elicited) levels of peroxide generation and the overall tolerance of rice cultivars to the pathogen. About two days after elicitation, cell death occurred proportional to the amount of hydrogen peroxide released. Peroxide was required to trigger loss of cell viability, but the latter was not due to a direct toxic effect, suggesting the induction of programmed cell death. Results represent the first data aimed to develop in vitro tests for pathogenicity prediction of Italian blast lineages toward rice cultivars.     

Rice root colonisation by mycorrhizal and endophytic fungi in aerobic soil

Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts that form intimate associations with the majority of plants growing in aerobic soil; fungal endophytes live internally, either intercellularly or intracellularly, and asymptomatically within plant tissues. Their presence is correlated with an increased response to biotic and abiotic stress. The populations of AM and of endophytic fungi were studied in the roots of different rice varieties grown in aerobic condition, in experimental fields in Vercelli, North Italy. All the rice varieties resulted colonised by AM fungi with a percentage of arbuscularisation ranging between 4% and 28%. Preliminary molecular analyses on some rice varieties showed that the AM population was composed of fungi identified as Glomus intraradices , on the basis of 18S ribosomal gene. All the varieties analysed but one resulted in colonisation by endophytic fungi. About 300 fungal isolates were obtained, belonging mainly to the genera Neotyphodium , Stagonospora and Penicillium .     

Effect of bioaugmentation of activated sludge with white-rot fungi on olive mill wastewater detoxification

AIMS: To test the potential use of Phanerochaete chrysosporium and other white-rot fungi to detoxify olive mill wastewaters (OMW) in the presence of a complex activated sludge. To combine the aerobic with anaerobic treatment to optimize the conversion of OMW in biogas. METHODS AND RESULTS: A 25-l air lift reactor was used to pretreat OMW by white-rot fungi. Detoxification of the OMW was monitored by size exclusion HPLC analysis, chemical oxygen demand (COD)/biological oxygen demand (BOD(5)) ratio evolution, and bioluminescence toxicity test. Anaerobic treatment of OMW was performed in a 12-l anaerobic filter reactor. Efficiency of the treatment was evaluated by organic matter removal, and biogas production. By comparison with the pretreatment by activated sludge only, the bioaugmentation with Phanerochaete chrysosporium or Trametes versicolor led to high removal of organic matter, decreased the COD/BOD(5) ratio and the toxicity. The subsequent anaerobic digestion of the OMW pretreated with activated sludge-white-rot fungi showed higher biomethanization yields than that pretreated with activated sludge only. Higher loading rates (7 g COD l(-1) day(-1)) were reached without any acidification or inhibition of biomethanization. CONCLUSIONS: The use of white-rot fungi, even in the presence of complex biological consortia to detoxify OMW, proved to be possible and made the anaerobic digestion of OMW for methane production feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of fungi for OMW reuse and energy production could be adapted to industrial applications.     

Phenolic removal in olive oil mill wastewater using loofah-immobilized Phanerochaete chrysosporium

Summary Olive oil mill wastewater (OMW) has a high organic load, and this is a serious concern of the olive industry. Conventional biological wastewater treatments, despite their simplicity and suitable performance are ineffective for OMW treatment since phenolics possess antimicrobial activity. In order to carry out a proper treatment of OMW, use of a microorganism able to degrade the phenolics is thus necessary. In this study the ability of Phanerochaete chrysosporium to degrade the phenolic compounds of OMW and to decrease the chemical oxygen demand (COD) using cells immobilized on loofah was examined. The basal mineral salt solution along with glucose, ammonium sulfate and yeast extract was used to dilute the OMW appropriately. The fungus did not grow on the concentrated OMW. The extent of removal in this bio-treatment, of total phenols (TP) and the COD were 90 and 50%, respectively, while the color and aromaticity decreased by 60 and 95%, respectively. The kinetic behavior of the loofah-immobilized fungus was found to follow the Monod equation. The maximum growth rate μ_max was 0.045 h⁻¹ while the Monod constant based on the consumed TP and COD were (mg/l) 370 and 6900, respectively.