Soil and entomopathogenic fungi with potential for biodegradation of insecticides: degradation of flubendiamide in vivo by fungi and in vitro by laccase

Purpose

Flubendiamide is a highly toxic and persistent insecticide that causes loss of insect muscle functions leading to paralysis and death. The objective was to screen for filamentous fungi in soils where insecticides had been applied, to isolate entomopathogenic fungi from insect larva (Anticarsia gemmatalis) that infest soybean crops, and to use these in biodegradation of insecticides.

Method

Filamentous fungi were isolated from soils, and growth inhibition was evaluated on solid medium containing commercial insecticides, Belt® (flubendiamide) and Actara® (thiamethoxam). A total of 133 fungi were isolated from soil and 80 entomopathogenic fungi from insect larva. Based on growth inhibition tests, ten soil fungi, 2 entomopathogenic fungi, and Botryosphaeria rhodina MAMB-05 (reference standard) were selected for growth on commercial insecticides in solid media. Fungi were grown in submerged fermentation on media containing commercial insecticides and assayed for laccase activity.

Result

Isolates JUSOLCL039 (soil), JUANT070 (insect), and MAMB-05 performed best, and were respectively inhibited by 48.41%, 75.97%, and 79.23% when cultivated on 35 g/L Actara®, and 0.0, 5.42%, and 43.39% on 39.04 g/L Belt®. JUSOLCL039 and JUANT070 were molecularly identified as Trichoderma koningiopsis and Neurospora sp., respectively. The three fungal isolates produced laccase constitutively, albeit at low activities. Fungal growth on pure flubendiamide and thiamethoxam resulted in only thiamethoxam inducing high laccase titers (10.16 U/mL) by JUANT070. Neurospora sp. and B. rhodina degraded flubendiamide by 27.4% and 9.5% in vivo, while a crude laccase from B. rhodina degraded flubendiamide by 20.2% in vitro.

Conclusion

This is the first report of fungi capable of degrading flubendiamide, which have applications in bioremediation.

     

Laccase activity and putative laccase genes in marine-derived basidiomycetes

Studies of laccases from marine-derived fungi are limited. In the present work, putative laccase genes from three marine-derived basidiomycetes and their laccase activities were evaluated. High amounts of laccase were produced by the fungal strains Marasmiellus sp. CBMAI 1062 (971.2 U L⁻¹) and Peniophora sp. CBMAI 1063 (709.03 U L⁻¹) when grown for 21 d at 28 °C in MA2ASW medium prepared with artificial seawater. Marine-derived basidiomycetes produced multiple distinct laccase sequences of about 200 bp with 73–90 % similarity to terrestrial basidiomycete laccases. Marasmiellus sp. CBMAI 1062 and Tinctoporellus sp. CBMAI 1061 showed the greatest laccase gene diversity with three and four distinct putative laccase sequences, respectively. This is the first report of laccase genes from marine-derived fungi, and our results revealed new putative laccases produced by three basidiomycetes.     

Phylogenetic relationships of ascomycetes and basidiomycetes based on comparative genomics analysis

The relationship between ascomycetes and basidiomycetes, the two main phyla of non-flagellated fungi, has rarely been investigated. In this study, we performed a comparative genomics analysis of genome sequences of 55 ascomycetes and 26 basidiomycetes species and detected 81 universal markers, 875 homologous genes and a conserved contig in the glucose-regulated protein gene. In dendrograms based on simple sequence repeat markers and homologous genes, ascomycetes and basidiomycetes formed distinct clusters, with each set of taxa having a high coefficient of relatedness. Ascomycetes and basidiomycetes also constituted distinct groups in a phylogenetic tree based on a conserved contig in the glucose-regulated protein gene. These results provide evidence that basidiomycetes may be derived from ascomycetes but are definitely genetically differentiated at the genomic level. The phylogenetic relationships of ascomycetes and basidiomycetes uncovered in this study provide new insights for future research related to fungal classification and evolution.     

From pattern to process: species and functional diversity in fungal endophytes of Abies beshanzuensis

The biodiversity-functional relationship in fungal ecology was recently developed and debated, but has rarely been addressed in endophytes. In this study, an integrative culture system was designed to capture a rich fungal consortium from the conifer Abies beshanzuensis. Results indicate an impressive diversity of fungal lineages (a total of 84 taxa classified in Dikarya) and a relatively high proportion of hitherto unknown species (27.4%). The laccase gene was used as a functional marker due to its involvement in lignocellulose degradation. Remarkable diversity of laccase genes was found across a wide range of taxa, with at least 35 and 19 distinct sequences in ascomycetes and basidiomycetes respectively, were revealed. Many groups displayed variable ability to decompose needles. Furthermore, many ascomycetes, including three volatile-producing Muscodor species (Xylariaceae), showed the ability to inhibit pathogens. Notably, most laccase-producing species showed little or no antibiosis and vice versa. Clavicipitalean and ustilaginomycetous fungi, specifically toxic to insects, were inferred from taxonomic information. Intra-specific physiological variation in Pezicula sporulosa, a second dominant species, was clearly high. We conclude that a suite of defensive characteristics in endophytes contributes to improving host fitness under various stresses and that a diversity of laccase genes confers an ecological advantage in competition for nutrients. Intra-specific diversity may be of great ecological significance for ecotypic adaptation. These findings suggest a fair degree of functional complementarity rather than redundancy among endemic symbionts of natural plant populations.     

Biosorption of lanthanum and samarium by viable and autoclaved mycelium of Botryosphaeria rhodina MAMB-05

The ascomycetous fungus, Botryosphaeria rhodina MAMB-05, secretes a (1→3)(1→6)-β-D-glucan, and the scaled-up production of this β-glucan results in large amounts of mycelial biomass being produced that represent a potentially cost-effective biosorbent for rare-earth elements. High sorption capacities for both La(III) and Sm(III) were demonstrated for viable and autoclaved lyophilized fungal mycelium. Fourier-transformed infra-red absorption spectra and the point of zero charge (PZC) were similar for the viable and inactive fungal mycelia. The rare-earth lanthanide elements (La and Sm) binding increased at initial pH values greater than 5.0, which was also observed for the PZC determination. The maximum La(III) uptake capacity was observed at lower amounts of La(III) ions in solution, decreasing from 100.0 to 25.3% when the initial lanthanide concentration increased from 15 to 100 mg/L. Lanthanide biosorption by B. rhodina MAMB-05 mycelia followed the Langmuir model, and the affinity of biosorbent functional groups was similar for La(III) and Sm(III).     

Allergenicity of airborne basidiospores and ascospores: need for further studies

Many known fungal species are grouped among basidiomycetes and ascomycetes. Active mechanisms of spore release into air currents are among the main features of these fungi. Aerobiological studies have described their presence in many regions worldwide. In some areas, fungi have been described as the predominant outdoor airborne biological particulate with much higher concentrations than pollen. Other studies have determined that among the fungal aerospora, the highest concentrations belong to basidiospores and ascospores. Nevertheless, the allergenic potential of spores from basidiomycetes and meiotic forms of ascomycetes has not been studied to the extent of mitosporic fungi and allergens from other sources. The need to further evaluate the role of basidiomycetes and meiotic ascomycetes in allergies is evidenced by the few genera with characterized allergens and limited studies that had demonstrated levels of sensitization similar or higher to that of mitosporic fungi and other allergens. In this review, based on the existing aerobiological, epidemiological, immunological, and molecular biology studies, we provide evidence that the role of basidiomycetes and ascomycetes deserves more attention with respect to their roles as potential aeroallergens.     

Are Basidiomycete Laccase Gene Abundance and Composition Related to Reduced Lignolytic Activity Under Elevated Atmospheric NO<Subscript>3</Subscript><Superscript>−</Superscript> Deposition in a Northern Hardwood Forest?

Anthropogenic release of biologically available N has increased atmospheric N deposition in forest ecosystems, which may slow decomposition by reducing the lignolytic activity of white-rot fungi. We investigated the potential for atmospheric N deposition to reduce the abundance and alter the composition of lignolytic basidiomycetes in a regional network of four northern hardwood forest stands receiving experimental NO₃ ⁻ deposition (30 kg NO₃ ⁻−N ha⁻¹ year⁻¹) for a decade. To estimate the abundance of basidiomycetes with lignolytic potential, we used PCR primers targeting laccase (polyphenol oxidase) and quantitative fluorescence PCR to estimate gene copy number. Natural variation in laccase gene size permitted use of length heterogeneity PCR to profile basidiomycete community composition across two sampling dates in forest floor and mineral soil. Although past work has identified significant and consistent negative effects of NO₃ ⁻ deposition on lignolytic enzyme activity, microbial biomass, soil respiration, and decomposition rate, we found no consistent effect of NO₃ ⁻ deposition on basidiomycete laccase gene abundance or community profile. Rather, laccase abundance under NO₃ ⁻ deposition was lower (−52%), higher (+223%), or unchanged, depending on stand. Only a single stand exhibited a significant change in basidiomycete laccase gene profile. Basidiomycete laccase genes occurring in mineral soil were a subset of the genes observed in the forest floor. Moreover, significant effects on laccase abundance were confined to the forest floor, suggesting that species composition plays some role in determining how lignolytic basidiomycetes are affected by N deposition. Community profiles differed between July and October sampling dates, and basidiomycete communities sampled in October had lower laccase gene abundance in the forest floor, but higher laccase abundance in mineral soil. Although experimental N deposition significantly suppresses lignolytic activity in these forests, this change is not related to the abundance or community composition of basidiomycete fungi with laccase genes. Understanding the expression of laccases and other lignolytic enzymes by basidiomycete fungi and other lignin-decaying organisms appears to hold promise for explaining the consistent decline in lignolytic activity elicited by experimental N deposition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Screening of ecologically diverse fungi for their potential to pretreat lignocellulosic bioenergy feedstock

A widespread and hitherto by far underexploited potential among ecologically diverse fungi to pretreat wheat straw and digestate from maize silage in the future perspective of using such lignocellulosic feedstock for fermentative bioenergy production was inferred from a screening of nine freshwater ascomycetes, 76 isolates from constructed wetlands, nine peatland isolates and ten basidiomycetes. Wheat straw pretreatment was most efficient with three ascomycetes belonging to the genera Acephala (peatland isolate) and Stachybotrys (constructed wetland isolates) and two white-rot fungi (Hypholoma fasciculare and Stropharia rugosoannulata) as it increased the amounts of water-extractable total sugars by more than 50 % and sometimes up to 150 % above the untreated control. The ascomycetes delignified wheat straw at rates (lignin losses between about 31 and 40 % of the initial content) coming close to those observed with white-rot fungi (about 40 to 57 % lignin removal). Overall, fungal delignification was indicated as a major process facilitating the digestibility of wheat straw. Digestate was generally more resistant to fungal decomposition than wheat straw. Nevertheless, certain ascomycetes delignified this substrate to extents sometimes even exceeding delignification by basidiomycetes. Total sugar amounts of about 20 to 60 % above the control value were obtained with the most efficient fungi (one ascomycete of the genus Phoma, the unspecific wood-rot basidiomycete Agrocybe aegerita and one unidentified constructed wetland isolate). This was accompanied by lignin losses of about 47 to 56 % of the initial content. Overall, digestate delignification was implied to be less decisive for high yields of fermentable sugars than wheat straw delignification.     

Four marine-derived fungi for bioremediation of raw textile mill effluents

Textile dye effluents pose environmental hazards because of color and toxicity. Bioremediation of these has been widely attempted. However, their widely differing characteristics and high salt contents have required application of different microorganisms and high dilutions. We report here decolorization and detoxification of two raw textile effluents, with extreme variations in their pH and dye composition, used at 20–90% concentrations by each of the four marine-derived fungi. Textile effluent A (TEA) contained an azo dye and had a pH of 8.9 and textile effluent B (TEB) with a pH of 2.5 contained a mixture of eight reactive dyes. The fungi isolated from mangroves and identified by 18S and ITS sequencing corresponded to two ascomycetes and two basidiomycetes. Each of these fungi decolorized TEA by 30–60% and TEB by 33–80% used at 20–90% concentrations and salinity of 15 ppt within 6 days. This was accompanied by two to threefold reduction in toxicity as measured by LC₅₀ values against Artemia larvae and 70–80% reduction in chemical oxygen demand and total phenolics. Mass spectrometric scan of effluents after fungal treatment revealed degradation of most of the components. The ascomycetes appeared to remove color primarily by adsorption, whereas laccase played a major role in decolorization by basidiomycetes. A process consisting of a combination of sorption by fungal biomass of an ascomycete and biodegradation by laccase from a basidiomycete was used in two separate steps or simultaneously for bioremediation of these two effluents.     

Cuticle laccase of the silkworm,Bombyx mori: Purification,gene identification and presence of its inactive precursor in the cuticle.

Laccase is a multi-copper enzyme found in variety of organisms including plants, fungi and bacteria. In insects, laccase is thought to play an important role in cuticle sclerotization with its ability to catalyze the oxidation of phenolic compounds to their corresponding quinones. From the newly ecdysed pupae of the silkworm, Bombyx mori, we purified a dimer form of cuticular laccase with 70-kDa polypeptides. Mass spectrometric analysis of the tryptic fragments and cDNA sequence analysis revealed that the gene for the purified laccase (BmLaccase2) is an ortholog of laccase2, one of the multiple laccase genes found in insect genomes. BmLaccase2 is highly expressed in the epidermis prior to ecdysis, suggesting that the BmLaccase2 protein accumulates before ecdysis. However, the cuticle of newly ecdysed pupa does not have laccase activity, and the activity only becomes detectable several hours after ecdysis. These data suggest that cuticle laccase is synthesized as an inactive precursor, which is later activated after ecdysis. We also found that urea-solubilized cuticle protein extract contains an inactive form of laccase that can be activated by trypsin treatment.     

Comparative Studies of Extracellular Fungal Laccases

Various basidiomycetes, ascomycetes, and deuteromycetes, grown in a sugar-rich liquid medium, were compared for laccase-producing ability and for the inducing effect of 2,5-xylidine on laccase production. Clear stimulation of the extracellular enzyme formation by xylidine was obtained in the cultures of Fomes annosus, Pholiota mutabilis, Pleurotus ostreatus, and Trametes versicolor, whereas Rhizoctonia praticola and Botrytis cinerea were not affected by the xylidine, and in the case of Podospora anserina a decrease in laccase activity was observed. The laccases were purified, and electrophoresis on polyacrylamide gels indicated a particular pattern for each laccase. The bands of the induced forms appeared only with basidiomycetes. The optimal pH of R. praticola laccase was in the neutral region, whereas the optima of all the other exolaccases were significantly lower (between pH 3.0 and 5.7). All laccases oxidized the methoxyphenolic acids under investigation, but there existed quantitative differences in oxidation efficiencies which depended on pH and on the nature (noninduced or induced) of the enzyme. The sensitivity of all enzymes to inhibitors did not differ considerably.     

Enzyme activities of fungi associated with Picea abies needles

Decomposition of Picea abies needles and production of extracellular enzymes involved in decomposition of lignin, cellulose, hemicelluloses and other organic compounds were studied in fungal strains of interior needle colonizers isolated from needles in different stages of decomposition (attached to trees, and early and late decomposition stages in the litter horizon). In total, 12 strains of ascomycetes (members of Helotiales, Hypocreales, Dothideales, Diaporthales and Eurotiales) and four basidiomycetes (Polyporales, Agaricales and Russulales) were tested. Significant decomposition of needles was recorded for all fungal isolates. All isolates produced cellobiohydrolase, β-glucosidase, β-xylosidase, N-acetylglucosaminidase, α-glucosidase, phosphatase and arylsulfatase and most fungi also produced endocellulase, endoxylanase and laccase in needle litter. In addition, other hemicellulases were produced by all strains. Mn-peroxidase was only produced by two basidiomycetes. Although enzyme activities varied, fungi associated with needles on fallen trees exhibited enzyme production comparable with later litter colonizers, and there was no significant difference in enzyme production between ascomycete and basidiomycete strains.     

Laccase Production and Humic Acids Decomposition by Microscopic Soil Fungi

Laccase (para-diphenol:oxygen oxidoreductase, EC 1.10.3.2) is a phenol oxidase widespread in fungi and bacteria. In basidiomycetes, this enzyme is involved in the transformation of lignin and humic substances (HS) in soil. The role of laccases of soil ascomycetes and deuteromycetes in HS degradation is not established, and conditions of the enzyme production have been poorly studied. In the present work soil micromycetes, potential laccase producers, were isolated from typical soils of the forest, steppe, and foreststeppe zones of European Russia by plating on agar media with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid, sodium salt)) as the substrate. Their abundance, species composition, conditions of laccase production, and its relation to humic acids (HA) degradation in liquid and solid media were studied. Out of 68 strains isolated, 20 exhibited ABTS oxidation at initial plating on agar media. In pure cultures on agar media, oxidation was less pronounced, but in the presence of HA laccase production by some strains was higher than without HA. Significant and weak extracellular laccase production in liquid medium was observed for Acremonium murorum (Corda) W. Gams Z1710 and Botritis cinerea Pers. ex Fries Z1711, respectively. The level of laccase production by A. murorum was the same without inducers and in the presence of HA, while B. cinerea produced laccase only without inducers. No direct correlation was found between the presence of laccase and/or its activity and ability of the fungi to decolorize (degrade) HA. In liquid media active laccase producer A. murorum caused lower HA decolorization (43%) than B. cinerea (62%) and the fungi lacking extracellular laccase (54–81%). The role of micromycete oxidative systems in HA degradation requires further investigation.     

Fungal Pls1 tetraspanins as key factors of penetration into host plants: a role in re-establishing polarized growth in the appressorium?

The ability of plant pathogenic fungi to infect their host depends on successful penetration into plant tissues. This process often involves the differentiation of a specialized cell, the appressorium. Signalling pathways required for appressorium formation are conserved among fungi. However, the functions involved in appressorium maturation and penetration peg formation are still poorly understood. Recent studies have shown that Pls1 tetraspanins control an appressorial function required for penetration into host plants and are likely conserved among plant pathogenic fungi. Tetraspanins are small membrane proteins widely distributed among ascomycetes and basidiomycetes defining two distinct families; Pls1 tetraspanins are found in both ascomycetes and basidiomycetes and Tsp2 tetraspanins are specific to basidiomycetes. Both fungal tetraspanins families have similar secondary structures shared with animal tetraspanins. Pls1 tetraspanins are present as single genes in genomes of ascomycetes, allowing a unique opportunity to study their function in appressorium mediated penetration. Experimental evidence suggests that Pls1 tetraspanins are required for the formation of the penetration peg at the base of the appressorium, probably through re-establishing cell polarity.     

Diversity of fungi in creosote-treated crosstie wastes and their resistance to polycyclic aromatic hydrocarbons

This study was conducted to generate information regarding the diversity of fungi inhabiting creosote-treated wood in a storage yard for crosstie wastes in Gwangmyeong, Korea. Additionally, the resistance to polycyclic aromatic hydrocarbons (PAHs) of indigenous fungi that mainly occupy creosote-treated wood was evaluated. We isolated fungi from the surface and inner area of crosstie wastes and identified them using a combination of traditional methods and molecular techniques. Overall, 179 isolates including 47 different species were isolated from 240 sampling sites. The identified fungal species included 23 ascomycetes, 19 basidiomycetes, and 5 zygomycetes. Three species, Alternaria alternata, Irpex lacteus, and Rhizomucor variabilis, were the most frequently isolated ascomycetes, basidiomycetes, and zygomycetes, respectively. The results of this study showed that there was a large difference in the fungal diversity between the surface and the inner area. Additionally, zygomycetes and ascomycetes were found to have a greater tolerance to PAHs than basidiomycetes. However, two basidiomycetes, Heterobasidion annosum and Schizophyllum commune, showed very high resistance to PAHs, even in response to the highest concentration (1,000 ppm), which indicates that these species may play a role in the degradation of PAHs.     

Laccase Gene Composition and Relative Abundance in Oak Forest Soil is not Affected by Short-Term Nitrogen Fertilization

Anthropogenic nitrogen (N) deposition affects a wide range of soil processes including phenol oxidase (PO) activity and soil organic matter dynamics. Depression of phenol oxidase activity in response to N saturation is believed to be mediated by the activity of white-rot basidiomycetes, whose production of extracellular oxidative enzymes can be limited by high N availability. We examined the effect of short-term N deposition on basidiomycete laccase gene diversity and relative abundance in temperate oak forest soil in which significant decreases in phenol oxidase and increased SOM have been recorded in response to experimental N deposition. UniFrac was used to compare the composition of laccase genes between three control- and three nitrogen-fertilized (80 kg⁻¹ ha⁻¹ per year) oak forest soils. The relative abundance of laccase genes was determined from qPCR analysis of laccase and basidiomycete ITS gene abundances. Our results indicate that there was no significant shift in the composition of laccase genes between control- and N-fertilized soils, nor was there a significant change in the relative abundance of laccase genes. These data suggest that N deposition effects on mineral soil PO activity do not result from changes in laccase gene diversity of white-rot basidiomycetes but are likely the result of altered microbial abundance or expression in this ecosystem type. Furthermore, laccase gene composition may be tied to factors that structure microbial communities in general, as soil laccase gene communities are more similar to other forest soils than with the corresponding litter.     

Comparison of the laccases, molecular marker proteins, and induction of pycnidia by three species of botryosphaeriaceous fungi

Three species of botryosphaeriaceous fungi,Botryosphaeria sp. isolate MAMB-5,Botryosphaeria ribis andLasiodiplodia theobromae, were compared for the production of pycnidia and laccases. Laccases were produced both intra- and extra-cellularly when the fungi were cultivated on basal medium in the presence and absence of veratryl alcohol, withBotryosphaeria sp. MAMB-5 showing the highest enzyme titres. Electrophoretic examination of intracellular marker proteins (esterases and phosphatases) and laccases indicated that the three species were genetically distinctly different, although the laccase zymograms for the three fungi showed similarity. The production of pycnidia occurred under continuous lighting at 28°C, but conditions differed among the three fungal species. Production could be induced on artificial media (potato-dextrose and oat agar) under stress-induced conditions where the mycelium was stimulated by physical abrasion, and in the case ofBotryosphaeria sp. isolate MAMB-5 on eucalypt woodchips. Evidence is presented that veratryl alcohol facillitated the secretion of intracellular-localised laccases into the extracellular medium.     

Tropical aquatic fungi

This paper reports on the distribution of fungal communities in aquatic habitats in tropical regions and highlights differences in the taxa observed in freshwater and marine habitats. Ascomycetes are dominant on substrata in marine environments, with few basidiomycetes and discomycetes. Equally, few freshwater basidiomycetes and discomycetes have been reported from the tropics. In marine habitats, Dothideomycetes dominate on mangrove substrata, and halosphaeriaceous species are most numerous on submerged woody substrata in coastal waters, while yeasts are common in seawater and estuarine habitats. In freshwater, Ingoldian anamorphic fungi are most numerous on decaying leaves, while ascomycetes (Dothideomycetes, Sordariomycetes) are prevalent on submerged/exposed woody substrata. Unique fungi are found in tropical waters and differ from those in temperate locations.