Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil

Production of extracellular enzymes participating in the degradation of biopolymers was studied in 29 strains of nonbasidiomycetous microfungi isolated from Quercus petraea forest soil based on the frequency of occurrence. Most of the isolates were ascomycetes and belonged to the genera Acremonium, Alternaria, Cladosporium, Geomyces, Hypocrea, Myrothecium, Ochrocladosporium, and Penicillium (18 isolates), and two isolates were zygomycetes. Only six isolates showed phenol oxidation activity which was low and none of the strains were able to degrade humic acids. Approximately half of the strains were able to degrade cellulose and all but six degraded chitin. Most strains produced significant amounts of the cellulolytic enzymes cellobiohydrolase and ??-glucosidase and the chitinolytic enzymes chitinase, chitobiosidase, and N-acetylglucosaminidase. The highest cellulase activities were found in Penicillium strains, and the highest activity of chitinolytic enzymes was found in Acremonium sp. The production of the hemicellulose-degrading enzymes ??-galactosidase, ??-galactosidase, and ??-mannosidase was mostly low. The microfungal strains were able to produce significant growth on a range of 41?C87, out of 95 simple C-containing substrates tested in a Biolog? assay, monosaccharides being for all strains the most rapidly metabolized C-sources. Comparison with saprotrophic basidiomycetes from the same environment showed that microfungi have similar cellulolytic capabilities and higher chitinase activities which testifies for their active role in the decomposition of both lignocellulose and dead fungal biomass, important pools of soil carbon.     

Fungal lysis by a soil bacterium fermenting cellulose

Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.     

Fungal contributions to carbon flow and nutrient cycling during decomposition of standing Typha domingensis leaves in a subtropical freshwater marsh

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Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B

Vibrio alginolyticus, Streptomyces griseus, Arthrobacter G12, Bacillus sp. and Cytophaga sp. were grown on solid and liquid media containing soluble and insoluble carbon sources. Arthrobacter G12, Bacillus sp. and Cytophaga sp. grew well on media which contained fungal cell walls or fungal biomass as the main carbon source. All bacteria produced extracellular proteases and all bacteria except Arthrobacter G12 produced extracellular chitinases. Growth of Cytophaga sp. on colloidal chitin was paralleled by the accumulated chitinase activity in the culture filtrate, and growth of Cytophaga sp. and Arthrobacter G12 on cell walls of Geotrichum candidum and cell walls of Candida pseudotropicalis was paralleled by the accumulation of laminarinase activity in the culture filtrate, but little or no extracellular chitinase activity was observed in these cultures. Mycolases purified from the culture filtrates of Cytophaga sp. grown on colloidal chitin on cell walls of C. pseudotropicalis potentiated the antifungal activity of amphotericin B.     

Post-harvest sugarcane residue degradation by autochthonous fungi

Several fungal species were isolated from different sources: post-harvest sugarcane residue, soil, decomposing forest litter and from mycelia obtained from the inner parts of fresh fungal fruiting bodies collected in Las Yungas region (Argentina). These isolates were first screened for their ability to produce carboxymethyl cellulose (CMC) degradation and guaiacol oxidation. After primary screening, seventeen isolates were further tested for their ligninolytic ability by assessing polyphenoloxidase, laccase, manganese peroxidase and endoxylanase activities. Based on their lignocellulolytic activities, five isolates (named Bjerkandera sp. Y-HHM2, Phanerochaete sp. Y-RN1, Pleurotus sp. Y-RN3, Hypocrea nigricans SCT-4.4 and Myrothecium sp. S-3.20) were selected for liquid and solid-state fermentation assays in culture media including sugarcane debris. Lignocellulolytic enzymes production, dry mass loss and phenol concentration in the water soluble fraction were then evaluated. Results suggest that native strains with lignocellulolytic activity are suitable to increase post-harvest sugarcane residue decomposition and support the use of these strains as an alternative to pre and post-harvest burning. Biological treatments using Phanerochaete sp. Y-RN1, Pleurotus sp. Y-RN3 and Myrothecium sp. S-3.20 could be used to degrade and increase the accessibility to lignocellulose components of sugarcane residue.     

Induction and Impact of Cell Wall Degrading Enzymes in Nutrient Solution of Closed Hydroponic Systems

The potential of the microflora in nutrient solutions to produce cell wall degrading enzymes (CWDE) was investigated by adding glucose or substrates of CWDE, such as chitin, cellulose, curdlan and preparations of fungal mycelia (0, 0.01 and 0.1%, w/v). The results indicate the potential of the microflora in nutrient solutions to produce proteolytic, chitinolytic, cellulolytic as well as β‐1,3‐glucanolytic enzymes. All enzyme complexes were induced by addition of preparations of Fusarium oxysporum f. sp. cyclaminis (Focy) and Pythium ultimum, respectively. In contrast, addition of glucose to nutrient solution resulted in only slight increase of protease and chitinase. No correlation between increased activity of CWDE and survival of Focy was found.     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

Extracellular chitinase production by some members of the saprophytic Entomophthorales group

Thirteen strains were isolated from different habitats, belonging to two genera, namely Conidiobolus and Basidiobolus, related to saprophytic Entomophthorales. Chitin flake colonization and agar-well diffusion tests were used to screen potential extracellular chitinase-producing strains in plate assays. Preliminary screening resulted in five chitinase producers that were further studied quantitatively. Results indicated that studied isolates of this group produced chitinase at different levels in chitin-containing as well as non-chitin-containing medium. Conidiobolus coronatus was found to be the most significant chitinase producer, giving 0.261 U/ml using colloidal chitin as a carbon source, among the isolates under study. This communication also reports the chitinolytic activity of Basidiobolus haptoporus, the effect of environmental and nutritional parameters on chitinase production, and utilization of fungal biomass as a carbon source, which hitherto had not been elaborated from this genus.     

Aquatic Actinomycete-Fungal Interactions and Their Effects on Organic Matter Decomposition: A Microcosm Study

The role of fungi in the decomposition of organic matter in streams has been well examined, although the role of bacterial antagonists in such processes has gained little attention. To examine bacterial-fungal interactions, experiments involving pairwise combinations of four actinomycete isolates (A1+ and A2+ could remove chitin from chitin-containing media, and A1? and A2? could not) and two fungal isolates (F+ a true fungus, F? an oomycote) were conducted. For each bacterial-fungal combination, 250-ml microcosms were sampled at 8 day intervals for 32 days. Microbial biomass and organic matter, as well as the activities of five extracellular enzymes, were measured. Each experiment consisted of a control group and four treatment groups. Controls comprised sterilized stream water and macrophytes. The first treatment was inoculated with only actinomycetes (~103 cells ml-1), the second treatment was inoculated with only fungi (~102 cells ml-1), the third group was inoculated simultaneously with actinomycetes and fungi, and the fourth group was inoculated with actinomycetes 2 days after fungal establishment. For all combinations, the lowest rates of organic matter decomposition were expected in the controls, as a result of only physical degradation. In contrast, the greatest rates of organic matter decomposition were predicted in treatments inoculated with F+ 2 days prior to A1? or A2?. Greater than 50% of the organic matter was decomposed in each of the fungal treatments. Fungal-actinomycete interactions resulted in reduced fungal biomass relative to the fungal-only treatments. However, when inoculated 2 days apart, combinations of F? and actinomycetes resulted in enhanced rates of organic matter decomposition, as well as greater levels of extracellular enzyme activities. These results demonstrate that actinomycete-fungal interactions and their colonization dynamics affect the accumulation of biomass, extracellular enzyme activities, and rates of organic matter decomposition.     

Diversity and ubiquity of xylariaceous endophytes in live and dead leaves of temperate forest trees

To test the hypothesis that xylariaceous endophytes were ubiquitous on live and dead leaves of various tree species in the field, xylariaceous fungi were isolated from live leaves and bleached and nonbleached portions of dead leaves of a total of 94 tree species in a cool temperate forest in Japan. The biodiversity of xylariaceous endophytes was evaluated as the richness of operational taxonomic units (OTUs) determined by phylogenetic analysis of the nucleotide sequence of the D1/D2 region of the LSU rDNA of fungal isolates. A total of 326 isolates of xylariaceous fungi were isolated from live and dead leaves and classified into 15 OTUs. The three major OTUs, Xylaria sp.1, Nemania sp., and Biscogniauxia sp., accounted for 94% (308 isolates) of the total number of isolates, and were isolated from various live and dead leaves. Xylaria sp.1 was frequently encountered on bleached portions (which were produced due to the selective decomposition of lignin) of dead leaves of broad-leaved deciduous tree species. The results suggest that xylariaceous endophytes did not show host specificity and had a saprobic phase on dead leaves in their life cycles and that Xylaria sp.1 was capable of decomposing lignin in the field conditions.     

Diversity,role in decomposition,and succession of zoosporic fungi and straminipiles on submerged decaying leaves in a woodland stream

Leaf litter is a very important primary source of energy in woodland streams. Decomposition of leaf litter is a process mediated by many groups of microorganisms which release extracellular enzymes for the degradation of complex macromolecules. In this process, true fungi and straminipiles are considered to be among the most active groups, more active than the bacteria, at least during the early stages of the process. Colonization increases the quality of the leaves as a food resource for detritivores. In this way, matter and energy enter detritus-based food chains. Previously, aquatic hyphomycetes were considered to be the major fungal group responsible for leaf litter decomposition. Although zoosporic fungi and straminipiles are known to colonize and decompose plant tissues in various environments, there is scant information on their roles in leaf decomposition. This study focuses on the communities of zoosporic fungi and straminipiles in a stream which are involved in the decomposition of leaves of two plant species, Ligustrum lucidum and Pouteria salicifolia, in the presence of other groups of fungi. A characteristic community dominated by Nowakowskiella elegans, Phytophthora sp., and Pythium sp. was found. Changes in the fungal community structure over time (succession) was observed: terrestrial mitosporic fungi appeared during the early stages, zoosporic fungi, straminipiles, and aquatic Hyphomycetes in early-to-intermediate stages, while representatives of the phylum Zygomycota were found at early and latest stages of the decomposition. These observations highlight the importance of zoosporic fungi and straminipiles in aquatic ecosystems.     

Chemical composition of litter affects the growth and enzyme production by the saprotrophic basidiomycete Hypholoma fasciculare

Chemical composition of litter has previously been reported to affect in situ decomposition. To identify its effects on a single species level, the saprotrophic basidiomycete Hypholoma fasciculare was grown on 11 types of litter with variable chemical composition (N content of 3.4–28.9 mg g⁻¹), and the mass loss of litter and lignin, production of extracellular enzymes and fungal biomass were followed. After 12 weeks, mass loss ranged from 16 % to 34 %. During early decomposition stages, litter mass loss, fungal biomass production (estimated by ergosterol content) as well as fungal substrate use efficiency all increased with increasing initial N content of the litter. The initial litter decomposition rate was significantly positively correlated with the activities of arylsulfatase, cellobiohydrolase, endoxylanase and phosphatase. Contrary to expectations, the lignin content did not affect litter mass loss, when covariation with N content was accounted for. The ratio of lignin loss to total mass loss depended on the litter type and did not reflect the activities of ligninolytic enzymes.     

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.     

Microbial activity and soil respiration under nitrogen addition in Alaskan boreal forest

Climate warming could increase rates of soil organic matter turnover and nutrient mineralization, particularly in northern high‐latitude ecosystems. However, the effects of increasing nutrient availability on microbial processes in these ecosystems are poorly understood. To determine how soil microbes respond to nutrient enrichment, we measured microbial biomass, extracellular enzyme activities, soil respiration, and the community composition of active fungi in nitrogen (N) fertilized soils of a boreal forest in central Alaska. We predicted that N addition would suppress fungal activity relative to bacteria, but stimulate carbon (C)‐degrading enzyme activities and soil respiration. Instead, we found no evidence for a suppression of fungal activity, although fungal sporocarp production declined significantly, and the relative abundance of two fungal taxa changed dramatically with N fertilization. Microbial biomass as measured by chloroform fumigation did not respond to fertilization, nor did the ratio of fungi : bacteria as measured by quantitative polymerase chain reaction. However, microbial biomass C : N ratios narrowed significantly from 16.0 ± 1.4 to 5.2 ± 0.3 with fertilization. N fertilization significantly increased the activity of a cellulose‐degrading enzyme and suppressed the activities of protein‐ and chitin‐degrading enzymes but had no effect on soil respiration rates or ¹⁴C signatures. These results indicate that N fertilization alters microbial community composition and allocation to extracellular enzyme production without affecting soil respiration. Thus, our results do not provide evidence for strong microbial feedbacks to the boreal C cycle under climate warming or N addition. However, organic N cycling may decline due to a reduction in the activity of enzymes that target nitrogenous compounds.     

Some properties of chitinase produced by a potent Aspergillus carneus strain

Summary Seven fungal isolates characterized by high chitinolytic activity were isolated from soil and identified. Aspergillus carneus in a 7-day-old shaken culture was the most promising chitinase producer. The use of chitin as a carbon source favoured production of extracellular chitinase enzymes. Maximum chitinase activity was reached at 10 g chitin/1. An initial pH value of the culture medium of 5.0 gave the highest chitinolytic activity. Some properties of the crude enzyme produced by A. carneus were studied. Maximal enzyme activity was reached at pH 4.5 and 40° C after 30 min. Thermal treatments at 70° C and pH 4.5 had the most adverse effect on enzyme activity.Offprint requests to: M. A. Abd El-Naby     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Saprotrophic basidiomycete mycelia and their interspecific interactions affect the spatial distribution of extracellular enzymes in soil

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of β-xylosidase, β-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

Filamentous fungi with high paraquat-degrading activity isolated from contaminated agricultural soils in northern Thailand

The contamination of paraquat (1,1′-dimethyl-4,4′-bipyridylium dichloride) herbicide from the farming area has become a public concern in many countries. This herbicide harms to human health and negatively effects the soil fertility. Several methods have been introduced for the remediation of paraquat. In this study, 20 isolates of the paraquat-tolerant fungi were isolated from the contaminated soil samples in northern Thailand. We found that isolate PRPY-2 and PFCM-1 exhibited the highest degradation activity of paraquat on synthetic liquid medium. About 80 and 68% of paraquat were removed by PRPY-2 and PFCM-1 respectively after 15 days of cultivation. Based on the morphological characteristic and molecular analysis, the fungal isolate PRPY-2 and PFCM-1 were identified as Aspergillus tamarii and Cunninghamella sp. respectively. The biosorption of paraquat on these fungal mycelia was also investigated. It was found that only 8–10% of paraquat could be detected on their mycelia, while 24–46% of paraquat was degraded by fungal mycelia. This is the first report on paraquat degrading ability by A. tamarii and Cunninghamella sp. It is demonstrated that these filamentous fungi are promising microorganisms available for remediation of paraquat contaminated environment.