AZCL-Pachyman

Aspergillus hancockii sp. nov., classified in Aspergillus subgenus Circumdati section Flavi, was originally isolated from soil in peanut fields near Kumbia, in the South Burnett region of southeast Queensland, Australia, and has since been found occasionally from other substrates and locations in southeast Australia. It is phylogenetically and phenotypically related most closely to A. leporis States and M. Chr., but differs in conidial colour, other minor features and particularly in metabolite profile. When cultivated on rice as an optimal substrate, A. hancockii produced an extensive array of 69 secondary metabolites. Eleven of the 15 most abundant secondary metabolites, constituting 90% of the total area under the curve of the HPLC trace of the crude extract, were novel. The genome of A. hancockii, approximately 40 Mbp, was sequenced and mined for genes encoding carbohydrate degrading enzymes identified the presence of more than 370 genes in 114 gene clusters, demonstrating that A. hancockii has the capacity to degrade cellulose, hemicellulose, lignin, pectin, starch, chitin, cutin and fructan as nutrient sources. Like most Aspergillus species, A. hancockii exhibited a diverse secondary metabolite gene profile, encoding 26 polyketide synthase, 16 nonribosomal peptide synthase and 15 nonribosomal peptide synthase-like enzymes.

Background: Improved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry. Results: Enzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs. Conclusions: Our findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.

Field trials were conducted using 4-year-old horse chestnut (Aesculus hippocastanum L.) to assess the efficacy of potassium and silicon phosphite as plant protection agents against the bacterial pathogen Pseudomonas syringae pv. aesculi (Pae) the causal agent of Pseudomonas bleeding canker of horse chestnut. Phosphites were applied preventatively, i.e. before Pae inoculation of trees, and curatively, i.e. after Pae inoculation of trees, and, as both a foliar spray (FS) and root drench (RD). Application of both phosphite forms induced positive effects on plant vitality (increased leaf chlorophyll content, leaf chlorophyll fluorescence (F_v/F_m), enhanced defensive enzyme activity (β-1,3-glucanase, peroxidase) and reduced Pae lesion size, the main proxy of Pae success or aggressiveness. Preventative rather than curative phosphite application resulted in greater reductions in Pae severity. Little significance of mode of application (FS, RD) and phosphite anion (potassium, silicon) was demonstrated indicating both phosphites can be foliar applied or root drenched with similar degrees of resultingPae control. Significant reductions in Pae severity recorded in this study gave credence to the potential of phosphites as an alternative or compliment to conventional bactericides for Pae control.

The Central Andean Highlands are the center of origin of the potato plant (Solanum tuberosum). Ages of mutualism between potato plants and soil bacteria in this region support the hypothesis that Andean soils harbor interesting plant growth-promoting (PGP) bacteria. Therefore, the aim of this study was to isolate rhizobacteria from Andean ecosystems, and to identify those with PGP properties. A total of 585 bacterial isolates were obtained from eight potato fields in the Andes and they were screened for suppression of Phytophthora infestans and Rhizoctonia solani. Antagonistic mechanisms were determined and antagonistic isolates were further tested for phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and production of NH₃- and indole-3-acetic acid (IAA). PGP was studied in healthy and R. solani diseased plantlets under growth room conditions. Performance was compared to the commercial strain B. subtilis FZB24® WG. Isolates were dereplicated with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), and identified with 16S rRNA gene sequencing and multi locus sequence analysis (MLSA). A total of 10% of the isolates were effective antagonists, of which many were able to solubilize phosphate, and produce IAA, ACC deaminase, NH₃ and hydrogen cyanide (HCN). During growth room experiments, 23 antagonistic isolates were associated with plant growth-promotion and/or disease suppression. Ten isolates had a statistically significant impact on test parameters compared to the uninoculated control. Three isolates significantly promoted plant growth in healthy plantlets compared to the commercial strain, and seven isolates outperformed the commercial strain in in vitro R. solani diseased plantlets.

Four green-odour compounds—trans-2-hexenal, cis-3-hexenol, n-hexanal, and cis-3-hexenal—were applied (0.85 µg ml^-1 as vapour) to rice plants in laboratory conditions to observe their biological activity against the phytopathogenic fungus Maganporthe oryzae, which causes rice blast disease worldwide. Two compounds, trans-2-hexenal and cis-3-hexenal, showed remarkable disease suppression efficacy (99.7% and 100% suppression, respectively), while n-hexanal had moderate (86.5%) and cis-3-hexenol had weak (20.8%) disease-suppressing effects. Pre-application and post-application of trans-2-hexenal or cis-3-hexenal had slight effects on blast incidence, suggesting that these compounds had direct effects to suppress M. oryzae infection. In fact, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression effect on M. oryzae. Interestingly, these two compounds inhibited appressorium formation at lower concentrations than the growth suppression. Studies on the hypersensitive response (HR)-like reaction and plant β-1,3-glucanase activity in rice plant confirmed that induced resistance was not the major factor involved in the disease suppression mechanism. Results of this study conclusively showed that trans-2-hexenal and cis-3-hexenal possess potent inhibitory activities against the growth and the appressorium formation of M. oryzae and could be used as antifungal agents to significantly reduce M. oryzae infections in rice.

A rhizobacterium with high antifungal activity was isolated from a potato field at Inneruulalik, South Greenland. Phylogenetic analysis based on multi locus sequence typing showed that the bacterium was affiliated with strains of Pseudomonas fluorescens. The bacterium, denoted as Pseudomonas fluorescens In5, inhibited in vitro a broad range of phytopathogenic fungi, and the antifungal activity increased with decreasing temperature. Microcosm experiments demonstrated that P. fluorescens In5 protected tomato seedlings from Rhizoctonia solani. Transposon mutagenesis showed that the major cause for the antifungal activity of P. fluorescens In5 was a novel non-ribosomal peptide synthase (NRPS) gene. In addition, transposon mutagenesis showed that P. fluorescens In5 also contained a putative quinoprotein glucose dehydrogenase gene, which was involved in growth inhibition of phytopathogenic fungi. Although P. fluorescens In5 contained the capacity to synthesize hydrogen cyanide, β-1,3-glucanase, protease, and chitinase, these did not seem to play a role in the in vitro and microcosm antifungal assays.

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising β-1,3-glucan with β-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited β-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

The defence reactions of sorghum seedlings 7 days after inoculation with Fusarium thapsinum and F. proliferatum, and interactions with wounding and exposure to light were studied to determine whether responses to these fungi differed from those to abiotic stresses. In non-wounded plants, inoculation with both fungi increased concentrations of anthocyanins and soluble phenolics and activities of peroxidase (POX), chitinase and β-1,3-glucanase in the roots, and increased β-1,3-glucanase activity in the mesocotyls. There was no effect of inoculation on phenylalanine ammonia-lyase (PAL) activity. Wounding by itself increased anthocyanin content of mesocotyls. Wounding also had a variety of interactions with inoculation. Exposure to light had very little effect on any defence response measured. A time course experiment showed that induction of chitinase and β-1,3-glucanase occurred in less than 24 h after inoculation. POX activity increased 2 days after inoculation, followed by a transient increase in PAL activity. The content of anthocyanins and soluble phenolics in roots of inoculated seedlings increased gradually compared with controls over 6 days. The responses of sorghum seedlings to inoculation with F. thapsinum and F. proliferatum were similar to those found by other workers following challenge by necrotrophic pathogens and were different from those induced by wounding and exposure to light.

In mature kernels of oat (Avena sativa L.) and other cereals, mixed-linked (11→33; 11→34)-β-glucans and arabinoxylans are major structural polysaccharides in cell walls of the endosperm. However, (1→3)-β-glucans are deposited transiently in walls during cellularization of endosperm early in grain development [Planta 202 (1997) 414–426]. The absence of (1→3)-β-glucans in mature endosperm cell walls suggests that (1→3)-β-glucanases are active during endosperm development. To investigate the role of β-glucanases during endosperm development, a (1→3)-β-glucanase cDNA, Oglc13, was isolated from an oat (A. sativa L.) kernel cDNA library. The enzymatic activity of the protein product, OGLC13, expressed from the cDNA in an in vitro expression system, exhibited substrate specificity for (1→3)-β-glucans. Oglc13 transcripts were detected in the endosperm of portions of developing kernels with the highest steady state level of mRNA at 15 days after anthesis (DAA) and not in vegetative tissues. Antibodies raised against OGLC13 immunoprecipitated (1→3)-β-glucanase activity from endosperm extracts of 10 and 15 DAA kernels and milky endosperm extracted from 15 DAA kernels. The OGLC13 antibodies did not precipitate (1→3)-β-glucanase activity from leaf extracts. These results indicated that Oglc13 is a unique (1→3)-β-glucanase expressed early in endosperm development.