Laetisaria Arvalis Research Articles

Comparison of Fungi with Sclerotia as Sustainable Materials for Product Design

Due to the increasing problems related to environmental pollution, sustainable and biodegradable materials gained importance. Fungi are among the most promising sources as biomaterials and have recently been used in product design. Sclerotia are compact hyphal masses produced by fungi under certain conditions, especially for survival. In this study four sclerotia forming fungi; Botrytis cinerea, Laetisaria arvalis, Macrophomina phaseolina and Rhizoctonia solani were compared in terms of features of sclerotia and possible usage as sustainable materials. Fungi were grown on a solid medium (potato dextrose agar) for two weeks and sclerotia formations were compared by daily observations. L. arvalis and M. phaseolina had smaller sclerotia homogeneously scattered all over the medium surface, while B. cinerea and R. solani formed sparse and bigger sclerotia. Shapes, colors and hardness of the sclerotia were also different from each other. Like fungal mycelia, sclerotia with more resistant structures may also be used as biomaterials in product design.

Characterization of extracellular secondary metabolites in Oudemansiella canarii BRM-044600 displaying antifungal activity against the phytopathogen Sclerotinia sclerotiorum.

White mold disease, caused by the phytopathogen Sclerotinia sclerotiorum, provokes severe productivity losses in several economically important crops. Biocontrol agents, especially antagonist filamentous fungi, are environmentally friendly alternatives to the chemical fungicides used in white mold management. The objective of this study was to screen for basidiomycete fungi capable of inhibiting S. sclerotiorum and investigate their bioactive metabolites responsible for antifungal activities. Two out of 17 tested basidiomycete isolates inhibited the mycelial growth of S. sclerotiorum in pair culture experiments on agar plates, namely Oudemansiella canarii BRM-044600 and Laetisaria arvalis ATCC52088. O. canarii BRM-044600 liquid culture filtrate exhibited the greatest antifungal activity and was selected for further investigation. UHPLC-MS analysis suggests that six putative strobilurins, including strobilurin A and/or stereoisomers of this compound (m/z 259.1299, [M + H]+) and three putative strobilurins with m/z 257.1184 ([M + H]+) are likely responsible for the antifungal activity observed in the culture filtrate. For the first time, this work demonstrated the potential of O. canarii for white mold biocontrol and strobilurin production.

Co-culturing of micro- and macro-fungi for producing highly active enzyme cocktail for producing biofuels

Mesocarp fiber (MF) and oil palm decanter cake (OPDC) are inexpensive substrates from oil palm refineries with potential to reduce fungal enzyme costs. This work demonstrated the producing enzymes method from mono- and co-cultures of micro- and macro-fungi cultured in MF or OPDC. Macrofungal species evaluated include Panus lecomtei, Trametes versicolor, Schizophyllum commune, Coprinus sp., Chrysosporium lucknowense, and Laetisaria arvalis. Secreted enzyme activities from individually cultured macrofungal strains were compared with Trichoderma reesei enzymes. Fungi co-culturing were evaluated in plates and submerged fermentation. Enzyme cocktail produced by co-culturing T. reesei and P. lecomtei exhibited higher laccase, peroxidases, hemicellulase and cellulase activities. Pretreated sugarcane bagasse (PSB) saccharification, with co-cultured enzyme cocktail, produced 12.3 g.L−1 of glucose, which was 44.7% higher when compared with T. reesei that produced 8.5 g.L−1. This work is the first to report co-culturing fungi to produce superior enzymes cocktail from OPDC that will benefit future lignocellulosic biorefinery.

A new species and a new combination in Waitea (Corticiales, Basidiomycota) and the phylogenetic affinity of Disporotrichum

The new corticioid species Waitea guianensis was described and illustrated from French Guiana, based on morphological and molecular phylogenetic evidence. It was characterized by thin, resupinate basidiomata on wood, isodiametric subhymenial hyphae, lack of clamps, tetrasporic clavate basidia with median constriction, and ellipsoid basidiospores. Ex-holotype DNA sequences from the nuclear ribosomal regions ITS, nLSU, and nSSU were obtained. Phylogenetic analyses confirmed a close relationship of the new species to Waitea circinata. In addition, the new combination Waitea arvalis is proposed, based on Laetisaria arvalis. The monotypic genus Disporotrichum is revealed to belong in the Corticiales, Corticiaceae. A key to the accepted species in Waitea is provided.

Tracking of enzymatic biomass deconstruction by fungal secretomes highlights markers of lignocellulose recalcitrance

BackgroundLignocellulose biomass is known as a recalcitrant material towards enzymatic hydrolysis, increasing the process cost in biorefinery. In nature, filamentous fungi naturally degrade lignocellulose, using an arsenal of hydrolytic and oxidative enzymes. Assessment of enzyme hydrolysis efficiency generally relies on the yield of glucose for a given biomass. To better understand the markers governing recalcitrance to enzymatic degradation, there is a need to enlarge the set of parameters followed during deconstruction.ResultsIndustrially-pretreated biomass feedstocks from wheat straw, miscanthus and poplar were sequentially hydrolysed following two steps. First, standard secretome from Trichoderma reesei was used to maximize cellulose hydrolysis, producing three recalcitrant lignin-enriched solid substrates. Then fungal secretomes from three basidiomycete saprotrophs (Laetisaria arvalis, Artolenzites elegans and Trametes ljubarskyi) displaying various hydrolytic and oxidative enzymatic profiles were applied to these recalcitrant substrates, and compared to the T. reesei secretome. As a result, most of the glucose was released after the first hydrolysis step. After the second hydrolysis step, half of the remaining glucose amount was released. Overall, glucose yield after the two sequential hydrolyses was more dependent on the biomass source than on the fungal secretomes enzymatic profile. Solid residues obtained after the two hydrolysis steps were characterized using complementary methodologies. Correlation analysis of several physico-chemical parameters showed that released glucose yield was negatively correlated with lignin content and cellulose crystallinity while positively correlated with xylose content and water sorption. Water sorption appears as a pivotal marker of the recalcitrance as it reflects chemical and structural properties of lignocellulosic biomass.ConclusionsFungal secretomes applied to highly recalcitrant biomass samples can further extend the release of the remaining glucose. The glucose yield can be correlated to chemical and physical markers, which appear to be independent from the biomass type and secretome. Overall, correlations between these markers reveal how nano-scale properties (polymer content and organization) influence macro-scale properties (particle size and water sorption). Further systematic assessment of these markers during enzymatic degradation will foster the development of novel cocktails to unlock the degradation of lignocellulose biomass.

Evaluation of Rhizoctonia zeae as a potential biological control option for fungal root diseases of sugar beet

AbstractSeveral common root diseases routinely damage sugar beet in Nebraska and other production areas of the Central High Plains, and it is becoming more common to find fields infested simultaneously with multiple pathogens. Owing to the shortage of available fungicides for economic management of soilborne diseases, alternative techniques such as biological control are increasingly being sought for disease management. Over the last several years, unidentified, sterile fungi have been isolated in conjunction with pathogens from infected sugar beet roots and seedlings. At least two promising isolates have been identified from in vitro assays that inhibit the radial growth of multiple sugar beet root pathogens, including Rhizoctonia solani, Fusarium oxysporum f. sp. betae, Phoma betae and Pythium aphanidermatum. Based on morphological and molecular characterisation, two isolates, ‘Hall’ and ‘R47’, were putatively identified as Rhizoctonia zeae. In vitro pathogenicity testing indicated that these isolates were not pathogens of sugar beet. Both isolates were compared with the well‐established biological control fungus Laetisaria arvalis and tested as potential treatments in a field naturally infested with multiple sugar beet root diseases. Data indicated that these fungi provided some level of protection against a complex of soilborne diseases and suggest that these isolates could have a potential fit in an integrative management strategy for several sugar beet root diseases.

Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes.

BackgroundEnzymatic breakdown of lignocellulosic biomass is a known bottleneck for the production of high-value molecules and biofuels from renewable sources. Filamentous fungi are the predominant natural source of enzymes acting on lignocellulose. We describe the extraordinary cellulose-deconstructing capacity of the basidiomycete Laetisaria arvalis, a soil-inhabiting fungus.ResultsThe L. arvalis strain displayed the capacity to grow on wheat straw as the sole carbon source and to fully digest cellulose filter paper. The cellulolytic activity exhibited in the secretomes of L. arvalis was up to 7.5 times higher than that of a reference Trichoderma reesei industrial strain, resulting in a significant improvement of the glucose release from steam-exploded wheat straw. Global transcriptome and secretome analyses revealed that L. arvalis produces a unique repertoire of carbohydrate-active enzymes in the fungal taxa, including a complete set of enzymes acting on cellulose. Temporal analyses of secretomes indicated that the unusual degradation efficiency of L. arvalis relies on its early response to the carbon source, and on the finely tuned sequential secretion of several lytic polysaccharide monooxygenases and hydrolytic enzymes targeting cellulose.ConclusionsThe present study illustrates the adaptation of a litter-rot fungus to the rapid breakdown of recalcitrant plant biomass. The cellulolytic capabilities of this basidiomycete fungus result from the rapid, selective and successive secretion of oxidative and hydrolytic enzymes. These enzymes expressed at critical times during biomass degradation may inspire the design of improved enzyme cocktails for the conversion of plant cell wall resources into fermentable sugars.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0143-5) contains supplementary material, which is available to authorized users.

Evaluation of Fungal Antagonists to Control Damping-Off of Tomato ( Lycopersicon esculentum Mill.) caused by Pythium indicum

Trichoderma viride , T. harzianum , Laetisaria arvalis and other eight isolates of Trichoderma viride , isolated locally. were tested in vitro for their antagonistic activity against Pythium indicum . the causal organism of danlping-off of tomato, by dual agar technique. Among the fungal antagonists tested, Trichoderma viride was found highly inhibitory to P . indicllm. Volatile and non-volatile antibiotics of T . viride inhibited the in vitro growth of P . indicum significantly.

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C(16) and C(18) fatty acids at pH approximately 7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.

Efficacy of several potential biocontrol organisms against Rhizoctonia solani on potato

Twenty-eight potential biocontrol organisms were tested for efficacy against Rhizoctonia solani on potato in a series of greenhouse trials. Organisms tested consisted of field isolates of Paenibacillus polymyxa, Pseudomonas fluorescens, Penicillium sp., Trichoderma sp., and Rhizoctonia zeae; known biocontrol isolates including Laetisaria arvalis, Verticillium biguttatum, Cladorrhinum foecundissimum, and Stilbella aciculosa; and commercial products of Bacillus subtilis (Kodiak), Trichoderma virens (SoilGard), and T. harzianum (RootShield). Different formulations and rates of several fungal isolates and the efficacy of combinations of effective antagonists were also investigated. None of the treatments, including a chemical control (azoxystrobin), effectively controlled stem canker and black scurf in all trials. However, B. subtilis GB03, R. zeae LRNE17E, S. aciculosa 112-B, and the chemical control were most effective in reducing stem canker severity (40–49% reduction) relative to the infested controls over all trials. L. arvalis ZH-1, R. zeae LRNE17E, and the chemical control reduced black scurf severity 54–60% relative to the infested control. Other treatments also significantly reduced stem canker and black scurf, however they were slightly less effective. Biocontrol amendment rate was not correlated with disease control, although the higher rates usually provided the best control. One combination of biocontrol organisms, B. subtilis and T. virens, demonstrated somewhat better control of stem canker than each organism alone, suggesting that this approach may provide improved biocontrol efficacy.

Taxonomic identity of the Sterile Red Fungus inferred using nuclear rDNA ITS 1 sequences

The taxonomic position and properties of an unidentified fungus isolated from wheat roots was investigated. The Sterile Red Fungus (SRF) is characterised by its fast growing habit, red pigmentation, non-sporing nature and mycelial form resembling some Laetisaria and Limonomyces species. rDNA variation was used to study the relationship of the SRF to Laetisaria spp. and Limonomyces spp. Nucleotide sequence obtained from eight Laetisaria and three Limonomyces species representing the ITS 1 region, were analysed by PCR and direct sequencing. Plant growth promoting properties of the five taxa were also determined. The SRF had closest identity (98 %) to British material of Limonomyces roseipellis. UPGMA analysis of ITS 1 DNA sequence support a close relationship between SRF and L. roseipellis. The relationship inferred by nucleotide sequence data was supported by phenotypic analysis; both L. roseipellis and the SRF were shown to promote the growth of wheat plants. Unexpectedly, Laetisaria arvalis and material named as Limonomyces roseipellis from New Zealand appeared to be closely related with 98 % rDNA sequence identity, suggesting the misidentification of the New Zealand collection.

Integration of Biological and Chemical Controls for Rhizoctonia Aerial Blight and Root Rot of Rosemary.

Aerial blight, caused by Rhizoctonia solani AG-4, was identified as a major disease of greenhouse mist-produced rosemary cuttings. An isolate of the biocontrol fungus Laetisaria arvalis, selected for tolerance to the experimental fungicide CGA 173506, was used as an amendment to potting soil. Combined treatment of rosemary with L. arvalis and a foliar spray of the fungicide at one-half the recommended rate reduced disease more than treatment with either the fungus or fungicide alone. Similar synergism was not observed for combinations of Trichoderma harzianum as a soil amendment and a foliar spray of the fungicide iprodione applied at the labeled rate.

Survival ofRhizoctoniaspp. and Root Diseases in a Rotation of Corn, Snap Bean, and Peanut in Microplots

The ability of several basidiomycetous fungi occurring naturally in Georgia coastal plain soils to survive and control Rhizoctonia solani anastomosis group 2 (AG-2) and AG-4 in field microplots was determined. Undisturbed profiles of soil in field microplots were fumigated with metamsodium and infested with Rhizoctonia solani AG-2-1, AG-2-2, or AG-4, or with binuclease Rhizoctonia spp. CAG-2, CAG-3, CAG-4, or CAG-5. A corn-peanut rotation was then grown for 3 yr. in a second experiment, microplots were infested with AG-4 and AG-2 alone or in combination with the potential antagonists CAG-2, CAG-4, CAG-5, Laetisaria arvalis, and an unidentified orange basidiomycetous fungus [...]

Interactions of mycophagous collembola and biological control fungi in the suppression of Rhizoctonia solani

A rhizosphere-inhabiting collembolan, Proisotoma minuta (Insecta: Isotomidae), and three known biocontrol fungi were studied in sterilized and non-sterilized soil for suppression of Rhizoctonia solani on cotton in a greenhouse environment. R. solani in dried oat culture was incorporated into soil at four inoculum densities ranging from 10 to 150mg kg −1. Trichoderma harzianum on wheat bran and Gliocladium virens as dried oatmeal culture were incorporated at 200 and 50 mg kg −1 soil, respectively, and Laetisaria arvalis dried, micromilled mycelium was applied as a seed dressing. Each fungus was applied either alone or with a population of P. minuta at 1000 kg − soil. Most effective biological control occurred in sterilized soil when the fungal biocontrol agents were integrated with the insect population; all combinations provided more effective disease suppression than the fungal agents used alone. In non-sterilized soil, having a natural competitive microflora, only P. minuta used alone and the L. arvalis + P. minuta treatment provided consistently significant disease reduction compared to R. solaniinfested soil without added agents. Moderate disease control in non-sterilized soil was obtained with T. harzianum or G. virens when combined with the insect population. Plant-growth dry weight measurements did not consistently reflect the disease control benefit. The specific mechanisms promoting increased biocontrol capacity of insect + fungus combinations, though not clearly defined here, must lie within a complex of factors including preference of R. solani as a food source for P. minuta, aversion of the insect to the two sporulating Hyphomycetes used for biocontrol, and direct parasitism of R. solani by the fungal agents.

Evaluation ofLaetisaria arvalis as a biological control agent ofRhizoctonia solani on white potato

Laetisaria arvalis, a soil-inhabiting basidiomycete, reducedRhizoctonia solani infection of white potato. A greenhouse study that monitored population levels of both fungi showed that soil infested withL. arvalis suppressed the growth ofR. solani after eight wks. Populations ofL. arvalis andR. solani increased after 11 wks. when both fungi were infested simultaneously and whenL. arvalis was introduced to soil previously infested withR. solani. Five years of field studies showed that variousL. arvalis seed treatments and soil amendments reducedR. solani infection of stems and stolons and sclerotial formation on tubers equal to the standard chemical treatment of pentachloronitrobenzene (PCNB) + thiabendazole. No relationship was shown between populations ofL. arvalis andR. solani in field plot soils, but native or wild strains ofL. arvalis were shown to be present at detectable levels in field soils.

Biosynthesis of 8 R-hydroperoxylinoleic acid by the fungus Laetisaria arvalis

8-Hydroxylinoleic acid is known to be a fungicidal metabolite formed by the fungus Laetisaria arvalis (Bowers, W.S. et al. (1986) Science 232, 105–106). In the present report, the mechanism of formation of 8-hydroxylinoleic acid was investigated. L. arvalis metabolized [ 14C]linoleic acid to 8-hydroperoxylinoleic acid and 8-hydroxylinoleic acid as major metabolites. The identification is based on the reduction of the hydroperoxide to an alcohol with stannous chloride and gas chromatography-mass spectrometry. The absolute configuration of the hydroxyl was determined to be R by ozonolysis of the (−)-menthoxycarbonyl derivative of 8-hydroxylinoleic acid. Linoleic acid 8 R-dioxygenase activity was present in the 100 000 × g supernatant of the cell lysate. In summary, the 8 R-linoleic acid dioxygenase of L. arvalis shows many similarities with the 8 R-dioxygenase recently described in the fungus Gaeumannomyces graminis.

EFFECTS OF RHIZOCTONIA SOLAM DENSITY IN POTTING SOIL ON EFFICACY OF BIOLOGICAL AND CHEMICAL TREATMENTS IN ROSEMARY PROPAGATION

Aerial blight of rosemary (Rosemarinus officinalis) caused by Rhizoctonia solani (AG-4) is a problem in production of rooted cuttings. Two separate studies were conducted on rosemary cuttings during propagation. Four levels of R. solani were mixed into potting medium at the rates of 0, 0.01, 0.1 and 1.0 percent (w/w). Seven treatments were evaluated: Trichoderma harzianum alone, Laetisaria arvalis alone, iprodione (single application, full rate), CGA 173506 (single application, full rate), T. harzianum + iprodione (single application, 1/2x rate), L. arvalis + CGA 173506 (single application, 1/2x rate), and a control. Biocontrol agents were mixed into medium at a rate of 5g/kg medium. Mycelial growth began by day four on the medium surface in the 0.1 and 1.0 R. solani levels. By day six, cuttings showed signs of infection. Disease incidence increased with higher levels of R. solani inoculum. At levels 0.01, 0.1 and 1.0, the L. arvalis plus 1/2x rate one time application CGA 173506 and iprodione alone (full rate one time application) gave the best control of aerial blight in both experiments. In the first experiment, iprodione alone and T. harzianum plus 1/2x rate iprodione gave the most root growth at the 0.01, 0.1 and 1.0 R. solani levels. In the second experiment, L. arvalis plus 1/2x rate CGA 173506 gave best root growth. At level 0, treatments were not significantly different in either experiment.

Biological Control to Reduce Inoculum of the Tan Spot PathogenPyrenophora tritici-repentisin Surface-borne Residues of Wheat Fields

Field plots of conservation-tillage wheat straw naturally infested with Pyrenophara tritici-repentis were treated with bran-based inoculum of three potential biocontrol fungi, and reduction in the number of residue-borne pseudothecia (primary inoculum of the pathogen) was determined. Limonomyces roseipellis significantly reduced residue-borne primary inoculum of P. tritici-repentis in 3 of 4 yr of experiments; and unidentified agonomycete was effective in two of three experiments, but Laetisaria arvalis was ineffective. Although L. roseipellis reduced inoculum by 60-80%, greater reductions would be necessary for acceptable control of this disease. Retention of dry bran-based inoculum on straw was enhanced by the use of alginate, but further improvements in formulation and application methods are required for effective biocontrol of pathogens in surface-borne crop residues

Screening Selected Fungi for Antagonism towardsPseudocercosporella herpotrichoides(Fron) Deighton, the Cause of Eyespot Disease of Cereals

In the laboratory, a dual culture primary screen using wheat straw agar (WSA) showed that 64 out of 152 selected fungi inhibited the in vitro growth of Pseudocercosporella herpotrichoides by 50% or more. An ‘asymmetric’ species of Penicillium isolated from wheat stubble was the most effective, inhibiting growth of the pathogen by 70%. The results were similar using both W‐type and R‐type isolates of P. herpotrichoides. Other fungi showing in vitro antagonism included: Trichoderma viride, T. koningii, T. harzianum, Chaetomium globosum, Acremonium persicum, Botryotrichum piluliferum, Sordaria alcina, Microdochium bolleyi, Rhizoctonia cerealis and Laetisaria arvalis. In a glasshouse secondary screen, the 64 fungi showing antagonism in vitro were assessed further for their ability to reduce eyespot disease symptoms of wheat seedlings using a straw collar co‐inoculation technique. Of them, 13 fungi reduced disease symptoms significantly: a Chaetomium sp. from stubble, two Fusarium spp., a F. culmorum isolate T. viride, T. koningii, T. harzianum, B. piluliferum, M. bolleyi, L. arvalis, the ‘asymmetric’ Penicillium sp. and two unidentified fungi from stubble. A F. culmorum isolate was the most effective antagonist on average, giving disease reductions of 54 and 58% for the W‐type and R‐type of P. herpotrichoides respectively. M. bolleyi, T. harzianum and the F. culmorum isolate were the most effective antagonists of the W‐type, all three reducing disease by 54%. T. viride was most effective against the R‐type, reducing disease by 64%.

Potential of Laetisaria arvalis for the biocontrol of Rhizoctonia solani

Bran preparations of the fungus Laetisaria arvalis isolates LA-1, OK-60, OK-160, OK-206 and ZH-4 reduced survival and saprophytic growth of Rhizoctonia solani (AG-4) in soil. They also prevented damping-off ( R. solani) of cotton, sugar beet, lettuce and radish in the greenhouse. The six isolates of L. arvalis grown on sterile wheat bran moistened with water and incubated for 5–15 days before addition to soil at a rate of 0.5% (w/w), were equally effective in reducing R. solani inoculum. In general, 15 day old preparations were as effective as 5 day old preparations. 7 Day old bran preparations of various L. arvalis isolates added to pathogen-infested loamy sand soil at a rate of 0.5% (w/w), prevented post-emergence damping-off of cotton, sugar beet, radish and lettuce. Several treatments resulted in seedling emergence and survival similar to those in natural soils not supplemented with R. solani. Preparations of L. arvalis also prevented damping-off of cotton in three other soils.