Chemical Composition, Enantiomeric Analysis, AEDA Sensorial Evaluation and Antifungal Activity of the Essential Oil from the Ecuadorian Plant Lepechinia mutica Benth (Lamiaceae).

This study describes the GC-FID, GC/MS, GC-O, and enantioselective GC analysis of the essential oil hydrodistilled from leaves of Lepechinica mutica (Lamiaceae), collected in Ecuador. GC-FID and GC/MS analyses allowed the characterization and quantification of 79 components, representing 97.3% of the total sample. Sesquiterpene hydrocarbons (38.50%) and monoterpene hydrocarbons (30.59%) were found to be the most abundant volatiles, while oxygenated sesquiterpenes (16.20%) and oxygenated monoterpenes (2.10%) were the minor components. In order to better characterize the oil aroma, the most important odorants, from the sensorial point of view, were identified by Aroma Extract Dilution Analysis (AEDA) GC-O. They were α-Pinene, ß-Phellandrene, and Dauca-5,8-diene, exhibiting the characteristic woody, herbaceus, and earthy odors, respectively. Enantioselective GC analysis of L. mutica essential oil revealed the presence of twelve couples and two enantiomerically pure chiral monoterpenoids. Their enantiomeric excesses were from a few percent units to 100%. Moreover, the essential oil exhibited moderate in vitro activity against five fungal strains, being especially effective against M. canis, which is a severe zoophilic dermatophyte causal agent of pet and human infections.

Fungal biodiversity in the periglacial soil of Dosdè Glacier (Valtellina, Northern Italy).

Periglacial areas are one of the least studied habitats on Earth, especially in terms of their fungal communities. In this work, both molecular and culture-dependent methods have been used to analyse the microfungi in soils sampled on the front of the East Dosdè Glacier (Valtellina, Northern Italy). Although this survey revealed a community that was rich in fungal species, a distinct group of psychrophilic microfungi has not been detected. Most of the isolated microfungi were mesophiles, which are well adapted to the sensitive climatic changes that occur in this alpine environment. A discrepancy in the results that were obtained by means of the two diagnostic approaches suggests that the used molecular methods cannot entirely replace traditional culture-dependent methods, and vice versa.

Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides.

Two distinct extracellular lipases were obtained from Penicillium solitum 194A, isolated from domestic compost, and Cladosporium cladosporioides 194B, isolated from dairy wastewater. These alkaline enzymes had molecular masses of 42 and 30 kDa, respectively. The P. solitum 194A lipase differed in mass from previously reported enzyme, indicating that it is a novel lipase, and indicating that penicillia can secrete lipase isoenzymes. The C. cladosporioides lipase was more active on esters of medium-chain acids, whereas the P. solitum lipase was more active on longer chained substrates. The C. cladosporioides enzyme displayed higher thermal stability than the P. solitum lipase, preserving full activity up to 48 °C and showing a T50 (10 min) of 60 °C. Their different catalytic properties and good protein stability should make these enzymes suitable for biotechnological applications. Furthermore, the combined use of these two fungal strains may prove to be valuable in lipid-rich waste management.

Functional analysis of the degradation of cellulosic substrates by a Chaetomium globosum endophytic isolate.

Most photosynthetically fixed carbon is contained in cell wall polymers present in plant biomasses, the largest organic carbon source in the biosphere. The degradation of these polymers for biotechnological purposes requires the combined action of several enzymes. To identify new activities, we examined which enzymes are activated by an endophytic strain of Chaetomium globosum to degrade cellulose-containing substrates. After biochemical analyses of the secretome of the fungus grown on cellulose or woody substrates, we took advantage of the available genomic data to identify potentially involved genes. After in silico identification of putative genes encoding either proteins able to bind to cellulose or glycohydrolases (GHs) of family 7, we investigated their transcript levels by reverse transcription-quantitative PCR (RT-qPCR). Our data suggest that eight genes compose the core of the cellulose-degrading system of C. globosum. Notably, the related enzymes belong structurally to the well-described GH families 5, 6, 7, 16, and 45, which are known to be the core of the cellulose degradation systems of several ascomycetes. The high expression levels of cellobiose dehydrogenase and two GH 61 enzymes suggest the involvement of this oxidoreductive synergic system in C. globosum. Transcript analysis along with relevant coding sequence (CDS) isolation and expression of recombinant proteins proved to be a key strategy for the determination of the features of two endoglucanases used by C. globosum for the first attack of crystalline cellulose. Finally, the possible involvement of transcriptional regulators described for other ascomycetes is discussed.

Evaluation of microbiological air quality and of microclimate in university classrooms.

The proliferation of air-diffused microorganisms inside public buildings such as schools, hospitals, and universities, is often indicated as a possible health risk. In this research, we have illustrated the results of an investigation realized to determine the health of the air in some university classrooms, both from a microbiological and a microclimatic viewpoint, during the normal didactic activity of direct lessons. The results obtained have been expressed by means of contamination indices, already used in previous works. Very little contamination was recorded in the different phases of air treatment, which underlines the efficiency of the system and of the maintenance protocols. The Global Index of Microbial Contamination (GIMC per cubic meter) showed a value greater than the mean during the heating period (290), while the highest values (95th percentile 1,138.45) were recorded in the period using air conditioning. The index of mesophilic bacterial contamination, though it did not show any significant differences in the various modes of air treatment, showed a mean value (1.34) and the 95th percentile value (4.14), which was greater in the air-conditioning phase. Finally, the mean value of the amplification index underlined a decrease in the microbial contamination in comparison to the outside, while showing situations of increased microbial amplification during the period of simple ventilation (95th percentile 4.27). The 95th percentile values found for GICM in the three sampling periods, however, permitted us to identify the value of GIMC per cubic meter equal to 1,000 as a guide to provide a means of self-monitoring the quality of the air inside the classrooms. From a microclimatic viewpoint, two periods of the year manifested discomfort situations: during the heating phase (winter) and during the simple ventilation phase (spring). The results obtained indicate, therefore, a need to intervene on the environmental parameters, not being able, in this particular case, to intervene on other aspects that influence the microclimate.

Preliminary Investigation about Aspergillus spp. Spread in Umbrian Avian Farms.

Among the fungi responsible for deep mycosis, the genus Aspergillus plays a predominant role both in human and veterinary medicine. From a "One Health" perspective, infections by Aspergillus spp. often represent a public health problem linked to specific occupational categories that could have a greater risk of inhaling spores and developing any respiratory disease. This preliminary investigation allowed to acquire information about the spread of Aspergillus spp. in avian livestock of the Umbria region (Central Italy), their sensitivity to antifungals, and the presence of mycotoxins in the considered farms. Environmental, feed, animal, and human samples were collected for mycological investigations; chemical analyses were also performed in feed samples. Moreover, prevalence estimated of the fungal isolates were provided for each individual farm sampled. Direct fungal identification was possible in 298 out of the 559 total samples; 162 of the samples were positive for Aspergillus spp. Mycotoxins were detected in 5 out of the 21 feed samples collected. All the aspergilli tested for antifungal susceptibility were resistant to fluconazole. The results obtained show how much the genus Aspergillus is widespread in the investigated farms; therefore, the poultry livestock represents a favorable environment for the maintenance and spread of fungal spores and their potential transmission to animals and humans.

Fungal Contamination and Aflatoxin B1 Detected in Hay for Dairy Cows in South Italy.

The growth of filamentous fungi on fodder is recognized as responsible for fungal deterioration and mycotoxin contamination of the plant mass leads to economic losses in the dairy cow production system. Mycotoxin contamination has significant implications for human and animal health and is one of the major concerns in the food and feed chain. This research provides an insight into the variety of viable molds (i.e., filamentous microfungi) that can be isolated from hay produced in South Italy and destined to dairy cows. On different lots of hay (n = 55) collected from 20 dairy farms, a total of 33 different fungal species were identified. The most representative was Cladosporium cladosporioides (n = 46, 84%) followed by Alternaria alternata (n = 25, 45%), and Rhizopus stolonifer (n = 24, 44%). The species most closely related to aflatoxin (AF) contamination, Aspergillus flavus, was often isolated (n = 11, 20%). Regarding AF detection, all the hay samples were found to be scarcely contaminated by AFB1 and showed values from 0.0020 to 0.0077 mg/kg, below the limits established by European Union (EU legislation) (0.02 mg/kg). None of the samples were positive for Aspergillia and tested for AFB1 showed results exceeding established limits. Additionally, hay with moisture between 15.0 and 19.2% or crude ash on dry matter content ranging from 14.0 to 15.5% reported an increased presence of AFB1 (p < 0.05) compared to the other samples. All the analyzed hay samples, besides the presence of molds, can be considered safe for the presence of AFB1. Prevention of mold spoilage is mandatory to reduce the exposure of humans and animals to mycotoxins.

Assessment of airborne microorganism contamination in an industrial area characterized by an open composting facility and a wastewater treatment plant.

In order to assess the potential exposure hazard to workers and people living in the immediate surroundings of an area characterized by an open composting facility and a wastewater treatment plant, a quantitative and a qualitative analysis of airborne microorganisms were carried out. Air sampling was performed once a week for four consecutive weeks in summer and winter. Six sites were selected as air sampling sites: one was upwind at approximately 40 m from the facilities; the other five were downwind at increasing distances from the facilities, with the furthest at 100 m away. Monitoring permitted us to verify the influence that the composting activities and wastewater treatment had on the bacterial and fungal contamination of the air. The results obtained have been expressed by means of contamination indexes that have already been used in previous works: a major microbiological contamination near the plants was evidenced. Near the facilities, mesophilic bacteria, psychrophilic bacteria and microfungi showed the highest median concentrations, respectively, of 307.5, 327.5 and 257.5 CFU/m(3). Moreover, the season generally influenced the concentration of the bacteria as well as of the fungi; higher in summer than in winter. The contamination index global index of microbial contamination (GIMC/m(3)) showed mean values of 4058.9 in summer and 439.7 in winter and the contamination index-amplification index (AI) showed values of 4.5 and 1.1 in the same seasons, respectively. Controlling the seasonal effect, mesophilic bacteria, Pseudomonas spp. and Enterobacteriaceae showed a significant decline in concentration with respect to upwind air samples and with increasing distance. Both GIMC and AI showed a significant decline with respect to upwind air samples by increasing the distance from facilities after adjusting for the seasonal effect. In conclusion, even if these plants do not represent a potential risk for nearby populations, they may pose a potential health risk for workers.

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.

Actividad antagónica de hongos endófitos de plantas medicinales del Ecuador sobre bacterias patógenas / Antagonic activity of endophytic fungi from medicinal plants of Ecuador on pathogenic bacteria

Mediante una investigación a lo largo del sector suburbano de la ciudad de Loja-Ecuador, dedicada al cultivo de plantas medicinales, se aíslaron los hongos endófitos asociados a Baccharis latifolia (Ruiz & Pav.) Pers., Baccharis obtusifolia Kunth, Piper barbatum Kunth, Borreria laevis (Lam.) Grises, Chuquiragua jussieui J.F. Gmel y Bidens andicola Kunth. Los taxa fúngicos más frecuentes fueron: Alternaria, Aspergillus, Epicoccum, Fusarium, Nigrospora, Phoma, además de algunos representantes de los grupos: coelomycetes, sinnematosos y Zygomycota. La mayor dominancia fúngica correspondió a integrantes de los denominados Mycelia sterilia, siendo de particular interés como posibles endófitos. Se evaluó además la interacción antagónica de siete aislados fúngicos frente a bacterias: Staphylococcus aureus, Klebsiella pneumomiae, Pseudomonas aeruginosa y Escherichia coli. La mayoría de las cepas fúngicas estudiadas presentaron actividad, pero los resultados más prometedores se evidenciaron en un aislado de Mycelia sterilia.

As a result of an investigation carried out along the suburban zone of the city of Loja-Ecuador, engaged in the growing of medicinal plants, endophytic fungi associated to Baccharis latifolia (Ruiz & Pav.) Pers., Baccharis obtusifolia Kunth, Piper barbatum Kunth, Borreria laevis (Lam) Grises, Chuquiragua jussieui J.F. Gmel and Bidens andicola Kunth were isolated. Fungal taxa most frequently appearing were: Alternaria, Aspergillus,Epicoccum, Fusarium, Nigrospora, Phoma besides some representatives of the groups: coelomycetes, synnematousand Zygomycota. Major fungal dominance was found in elements of the so-called Mycelia sterilia, which hada special interest as possible endophyte. Besides the antagonic interaction of seven fungal isolations in front of bacteria was assessed: Staphylococcus aureus, Klebsiella pneumomiae, Pseudomonas aeruginosa and Escherichia coli. Most of fungal strains investigated revealed activity , however most promising results could be seen in an isolation of Mycelia sterilia.

Algunos hyphomycetes dematiaceos epifitos del norte de Italia / Some epiphytic dematiaceous hyphomycetes from northern Italy

Se comenta e ilustra el hallazgo de 5 hyphomycetes dematiáceos: morrisographium ulmi, dictyochaeta simplex, stachybotrys bisbyi, menispora anamorfo de chaetosphaeria pulviscula y hemicorynespora sp., presentes en la corteza de diversos árboles y otros sustratos vegetales en el norte de Italia

Algunas especies de bipolaris y curvularia protuberata en gramineas silvestres del norte de Italia (Lombardia) / Some species of bipolaris and curvularia protuberata in wild gramineae from norther Italy (Lombardia)

Entre mayo y noviembre de 1996, se determinó la presencia y distribución geográfica de especies fúngicas intengrantes del género curvularia y el "complex helminthosporium", en las principales gramineas silvestres presentes en cuatro zonas agrícolas de la Lombardia (Italia), adyacentes a las ciudades de Milano, Bergamo, Cremona y Voghera. En 18 de las 21 especies de gramíneas colectadas, se obtuvieron 79 aislamientos fúngicos. Más del 98 porciento de éstos, se presentaron como anamorfos de cochliobolus y los más representativos fueron: bipolaris australiensis (30,4 porciento), c.sativus (27,8 porciento) y c.cynodontis (22,8 porciento). En Voghera y Cremona se obtuvieron los más altos porcentajes de aislamientos fúngicos (33-29 porciento) y en Milano los menores (13 porciento). Las espigas y las hojas fueron las más afectadas por la colonización fungica, especialmente en verano y en el culmo en otoño. Considerando la colonización de las grámineas en el tiempo-espacio y el número de aislamientos fúngicos, se determinaron puntos temporo-espaciales, creándose tres grupos de grados de colonización fúngica (A, B, C). El grupo A soportó la menor colonización y el C la mayor. En este último, cynodon dactylon, hordeum murinum y poa pratensis, fueron las gramíneas más representativas. Se comentan sus potencialidades infestantes y como reservorio de patógenos fúngicos de interés agrícola