β-Glucosidase from the cellulolytic system of Alternaria alternata autolyzed cultures

Abstract A β-glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G-200, CM-Biogel A and DEAE-Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a M _r of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β-glucosidase is inhibited by gluconolactone but not by 10 mM glucose.     

Role of uronic acids present in phytopathogenic fungi as inducers of polygalacturonases during autolysis

The presence of uronic acids in the culture fluid and mycelium of the fungi: Alternaria alternata, Botrytis cinerea, Drechslera halodes, Fusarium culmorum, Fusarium oxysporum, Monilinia fructigena, Mucor mucedo, Rhizopus stolonifer and Trichoderma hamatum was detected and quantified. In these fungi the concentration of uronic acids increased during the growth phase and the maximal concentrations were found at the end of the growth phase or onset of autolysis both in the mycelium as well as in the culture fluid. The uronic acids were metabolized during the first days of autolysis decreasing to constant levels until the end of the autolytic period studied.The variations in the activity of polygalacturonase and polymethylgalacturonase present in the culture fluid were determined at the onset and during autolysis in these fungi. These enzymic activities were found in the culture fluid of these fungi, with exception of M. rouxii, and they showed an increasing activity in the first days of autolysis and later a slight increase or decrease was observed. The presence of uronic acids in these phytopathogenic or saprophytic fungi and the low levels detected during autolysis could be related to the induction of pectic enzymes and the pathogenicity of these fungi.     

Protoplast production from filamentous fungi with their own autolytic enzymes

Abstract Production of protoplasts in different genera of filamentous fungi with their own lytic enzymes obtained from autolyzed cultures, as well as the regeneration of these protoplasts, has been studied. The results support the idea that the use of these autolytic enzymes could be a general method of production of protoplasts from filamentous fungi.     

Effect of the level of the carbon source on the activity of some lytic enzymes released during autolysis of Aspergillus niger

Changes in the activity of -N-acetylglucosaminidase, chitinase, invertase, esterases, glucanases and phosphatases liberated into the culture fluid were followed during the autolytic phase of growth of Aspergillus niger on media with various initial levels of the carbon source. The general pattern was of an accumulation of these lytic enzymes in the culture fluid during autolysis, but some enzymes reached maximum activity and then declined. The initial level of the carbon source affected the enzyme pattern during autolysis. Maximum activity for the various enzymes was always observed either for the lowest initial level of carbon or the highest (3.5 mM glucose, 111 mM glucose). The highest specific activities were those for exopolygalacturonidase (500 mU/mg at 3.45 mM glucose), and for -amylase (about 500 mU/mg at 3.45 mM glucose). Cellulase, chitinase and esterase showed the weakest activity. Acid phosphatase was most active (about 200 mU/mg) at 3.45 mM initial glucose, whereas alkaline phosphatase was most active (45 mU/mg) at 111 mM glucose, both during the autolytic phase of growth.     

Lytic enzymes in the autolysis of filamentous fungi

The degrees of autolysis attained by five different genera of filamentous fungi during an incubation period of 60 days, under the same culture conditions were: 87.3% for Penicillium oxalicum; 65.9% for Neurospora crassa; 62.7% for Polystictus versicolor; 51.7% for Aspergillus niger and 23.5% for Nectria galligena. N. crassa, A. niger and P. versicolor reached the end of the autolysis during this incubation period (60 days), whereas P. oxalicum and N. galligena did not.The excretion of the lytic enzymes -N-acetyl-glucosaminidase, -1–3 glucanase, chitinase, invertase and acid phosphatase into the culture medium during growth and autolysis was investigated. The excretion of these enzymes was consistent with the degree of autolysis reached, the maximum excretion belonging to P. oxalicum and the minimum to N. galligena. The N. crassa invertase was excreted into the culture liquid at levels very much higher than the other enzymes studied, and at levels very much higher than the invertases excreted by the other fungi.     

Assessing estuarine quality under the ecosystem services scope: Ecological and socioeconomic aspects

An increasing need for integrative assessments that measure the contributions of the environment to human welfare has recently been recognised. In the present study, a preliminary assessment of the services provided by the Mondego Basin in terms of system ecological quality was carried out. The ecological, economic, and societal relations of the Mondego estuarine services were analysed. An inventory of the main ecosystem services provided by the Mondego system was performed. The conditions and trends of the main services (food production, recreation and water quality maintenance) were determined, and the scale dependence of this assessment was interpolated on three different scales: Mondego Basin, Lower Mondego, and Mondego Estuary. The interdependence among services was quantified; an ecological assessment regarding water quality and ecological conditions was performed, and a preliminary valuation of the food production, recreation and tourism in the region was undertaken. In the study system, from 1992 to 2006, there was an increase in recreation activities and water uses and a simultaneous decrease in services such as food production (i.e., strong interdependence among services). Ecological quality improvement is reflected in both local communities’ diversity and water quality. The market prices method was used to estimate the values for the three services considered; however, the Mondego catchment's full value cannot be calculated without estimating the real wetlands value because these are prone to underestimation. Uncertainties and shortcomings regarding the reliability of this kind of assessment for implementation on estuarine ecosystems are discussed.     

Increased Diversity of Arbuscular Mycorrhizal Fungi in a Long-Term Field Experiment via Application of Organic Amendments to a Semiarid Degraded Soil

In this study, we tested whether communities of arbuscular mycorrhizal (AM) fungi associated with roots of plant species forming vegetative cover as well as some soil parameters (amounts of phosphatase and glomalin-related soil protein, microbial biomass C and N concentrations, amount of P available, and aggregate stability) were affected by different amounts (control, 6.5 kg m⁻², 13.0 kg m⁻², 19.5 kg m⁻², and 26.0 kg m⁻²) of an urban refuse (UR) 19 years after its application to a highly eroded, semiarid soil. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, single-stranded conformation polymorphism analysis, sequencing, and phylogenetic analyses. One hundred sixteen SSU rRNA sequences were analyzed, and nine AM fungal types belonging to Glomus groups A and B were identified: three of them were present in all the plots that had received UR, and six appeared to be specific to certain amendment doses. The community of AM fungi was more diverse after the application of the different amounts of UR. The values of all the soil parameters analyzed increased proportionally with the dose of amendment applied. In conclusion, the application of organic wastes enhanced soil microbial activities and aggregation, and the AM fungal diversity increased, particularly when a moderate dose of UR (13.0 kg m⁻²) was applied.The semiarid Mediterranean areas of Southeastern Spain are affected by environmental degradation and erosive processes due to the fact that they are characterized by a set of climatic conditions that includes irregular and scarce rainfall and long, dry, and hot summers. Under these conditions, the soil organic matter content decreases, and the availability of nutrients and water for plants is reduced. Consequently, soil productivity decreases, levels of below-ground microbially diverse populations decline, and the water deficit limits plant growth so that the vegetation cover of natural soils cannot be sustained. Therefore, the development of revegetation techniques to reduce erosion, to remediate the effects of degradation, and, thus, to allow the restoration of biodiversity is needed. It was previously demonstrated that the application of organic amendments, such as urban refuse (UR), to soil increases the organic matter content of soil and improves the quality and productivity of degraded soils (17, 44, 57). Also, it was previously shown that the organic residues yield an improvement in levels of microbially diverse populations in the soil (43).A substantial part of the soil microbial communities belongs to the arbuscular mycorrhizal (AM) fungi, an ancient group of fungi belonging to the phylum Glomeromycota (49), which form mutualistic associations with the roots of the majority of land plants. These fungi have a variety of beneficial effects on their host plants, such as increasing the uptake of mineral nutrients, particularly phosphorus and nitrogen (41, 52); reduction of pathogen infections (7); improvement of water relations (12) and soil stability (58); and the limitation of heavy metal uptake (34). It is evident that AM fungi are an important factor contributing to the maintenance of terrestrial ecosystem functioning. Studies have shown that the diversity of AM fungal populations in the soil can affect plant diversity and productivity and ecosystem stability (62). Therefore, information on the species composition of the AM fungal community in roots is important for an understanding of mycorrhizal function as well as for the effective management and preservation of the diversity of AM fungal populations in ecological field studies.Thanks to advances in molecular techniques in recent years, it is possible to apply PCR-based molecular methods in order to analyze the diversity of AM fungi colonizing the roots of an individual plant at any given time. Traditional identification based on spore morphology is often problematic, and the abundance of spores in the soil may not accurately reflect AM fungal community composition and dynamics (8). The single-stranded conformation polymorphism (SSCP) approach is a very sensitive and reproducible technique for analyzing the sequence diversity of AM fungi within roots (30). This method is based on nucleotide differences between homologous sequence strands, which are detected by electrophoresis of single-stranded DNA under nondenaturing conditions (38).It is known that the application of organic amendments can have a positive effect on the proliferation of natural AM fungi in crop systems (20, 26). The stimulatory effects of the addition of organic matter on the development of AM fungi could be related to an improvement in the extensive network of AM fungal mycelium in the soil. In this way, the colonized plants are able to effectively exploit nutrients and water from soil (52). Moreover, AM fungi are able to exploit nutrients released by the mineralization of organic matter due to the activities of mineralizing microorganisms (28). However, there are many previous reports that showed a strong negative impact on the presence of AM fungal populations and mycorrhizal colonization when composted urban waste was added to the soil (19, 46). Also, research using trap cultures of host plants showed a decrease in the level of diversity of AM fungal species in soils amended with sewage sludge (25, 61).In a previous study carried out in 1992 at the site that is also the subject of the current work, Roldán and Albaladejo (43) found that the application of UR decreased levels of AM fungal populations in the first year after amendment; however, they observed an increase in levels of these populations 3 years after the addition. We hypothesized that after a long period of time, the application of UR could alter the diversity of AM fungal populations in a highly eroded, semiarid soil and that this effect could be influenced by the refuse application rate. In order to verify this hypothesis, we studied the diversity of the AM fungi associated with the roots of plant species forming the vegetative cover of five plots that received different amounts of UR 19 years after the amendment. Also, we determined whether there was an improvement in soil quality parameters related to soil microbial activity.     

Differential Effects of Pseudomonas mendocina and Glomus intraradices on Lettuce Plants Physiological Response and Aquaporin PIP2 Gene Expression Under Elevated Atmospheric CO2 and Drought

Arbuscular mycorrhizal (AM) symbiosis and plant-growth-promoting rhizobacterium (PGPR) can alleviate the effects of water stress in plants, but it is unknown whether these benefits can be maintained at elevated CO₂. Therefore, we carried out a study where seedlings of Lactuca sativa were inoculated with the AM fungus (AMF) Glomus intraradices N.C. Schenk & G.S. Sm. or the PGPR Pseudomonas mendocina Palleroni and subjected to two levels of watering and two levels of atmospheric CO₂ to ascertain their effects on plant physiological parameters and gene expression of one PIP aquaporin in roots. The inoculation with PGPR produced the greatest growth in lettuce plants under all assayed treatments as well as the highest foliar potassium concentration and leaf relative water content under elevated [CO₂] and drought. However, under such conditions, the PIP2 gene expression remained almost unchanged. G. intraradices increased significantly the AMF colonization, foliar phosphorus concentration and leaf relative water content in plants grown under drought and elevated [CO₂]. Under drought and elevated [CO₂], the plants inoculated with G. intraradices showed enhanced expression of the PIP2 gene as compared to P. mendocina or control plants. Our results suggest that both microbial inoculation treatments could help to alleviate drought at elevated [CO₂]. However, the PIP2 gene expression was increased only by the AMF but not by the PGPR under these conditions.     

Estimation by PLFA of Microbial Community Structure Associated with the Rhizosphere of Lygeum spartum and Piptatherum miliaceum Growing in Semiarid Mine Tailings

The objective of this study was to compare the microbial community composition and biomass associated with the rhizosphere of a perennial gramineous species (Lygeum spartum L.) with that of an annual (Piptatherum miliaceum L.), both growing in semiarid mine tailings. We also established their relationship with the contents of potentially toxic metals as well as with indicators of soil quality. The total phospholipid fatty acid (PLFA) amount was significantly higher in the rhizosphere soil of the annual species than in the rhizosphere soil of the perennial species. The fungal/bacterial PLFA ratio was significantly greater in the perennial species compared to the annual species. The fatty acid 16:1ω5c, the fungal/bacterial PLFA ratio and monounsaturated/saturated PLFA ratio were correlated negatively with the soluble contents of toxic metals. The cyc/prec (cy17:0 + cy19:0/16:1ω7 + 18:1ω7) ratio was correlated positively with the soluble contents of Pb, Zn, Al, Ni, Cd, and Cu. The results of the PLFA analysis for profiling microbial communities and their stress status of both the plant species indicate that perennial and annual gramineous species appear equally suitable for use in programmes of revegetation of semiarid mine tailings.