Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

UNASSIGNED: Potassium and phosphorus are essential macronutrients for plant growth and development. However, most P and K exist in insoluble forms, which are difficult for plants to directly absorb and utilize, thereby resulting in growth retardation of plants under P or K deficiency stress. The Aspergillus aculeatus fungus has growth-promoting characteristics and the ability to dissolve P and K.
UNASSIGNED: Here, to investigate the physiological effects of A. aculeatus on bermudagrass under P or K deficiency, A. aculeatus and bermudagrass were used as experimental materials.
UNASSIGNED: The results showed that A. aculeatus could promote tolerance to P or K deficiency stress in bermudagrass, decrease the rate of leaf death, and increase the contents of crude fat as well as crude protein. In addition, A. aculeatus significantly enhanced the chlorophyll a+b and carotenoid contents. Moreover, under P or K deficiency stress, bermudagrass inoculated with A. aculeatus showed higher N, P, and K contents than non-inoculated plants. Furthermore, exogenous A. aculeatus markedly decreased the H2O2 level and CAT and POD activities. Based on our results, A. aculeatus could effectively improve the forage quality of bermudagrass and alleviate the negative effects of P or K deficiency stress, thereby playing a positive economic role in the forage industry.

Chemical epigenetic regulation (CER) is an effective method to activate the silent pathway of fungal secondary metabolite synthesis. However, conventional methods for CER study are laborious and time-consuming. In the meantime, the overall profile of the secondary metabolites in the fungi treated by the CER reagent is not well characterized. In this study, suberohydroxamic acid (SBHA), a histone deacetylase inhibitor, was added to a culture of Aspergillus aculeatus DL1011 and a new strategy based on LC-MS/MS analysis integrated with various metabolomic tools (MetaboAnalyst, MS-DIAL, SIRIUS and GNPS) was developed to characterize the profile of induced metabolites. As a result, 13.6%, 29.5% and 27.2% of metabolites were identified as newly biosynthesized, increasing and decreasing in abundance by CER, respectively. The structures of the 18 newly induced secondary metabolites were further identified by the new strategy to demonstrate that 72.2% of them (1 novel compound and 12 known compounds) were first discovered in A. aculeatus upon SBHA treatment. The accuracy of the new approach was confirmed by purification and NMR data analysis of major newly biosynthesized secondary metabolites. The bioassay showed that the newly biosynthesized compounds, roseopurpurin analogues, showed selective activities against DPPH scavenging, cytotoxicity and SHP1 inhibition. Our research demonstrated that CER was beneficial for changing the secondary metabolic profile of fungi and was an effective means of increasing the diversity of active metabolites. Our work also supplied a metabolomic strategy to characterize the profile changes and determine the newly induced compounds in the secondary metabolites of fungi treated with the chemical epigenetic regulator.

Blue mold caused by Penicillium italicum is a severe postharvest disease in citrus fruits. In this study, the fermentation product (FP-E) of Aspergillus aculeatus GC-09, an endophytic fungus isolated from a citrus plant, was found to exhibit antifungal activity against P. italicum with a MIC of 0.3125 mg/mL. The fungus A. aculeatus GC-09 was identified based on the studies of morphology and ITS nucleotide sequence. FP-E significantly inhibited the spore germination and mycelial growth of P. italicum. Scanning electron microscopy (SEM) results of P. italicum treated with FP-E showed shrunken, distorted and collapsed hyphae and conidiospores, indicative of the cell membrane damage, which was further confirmed by the propidium iodide (PI) fluorescent staining analysis. Consistent with the microscopy observation, FP-E led to the leakage of cellular constituents from P. italicum, which is evident from the increase in electrical conductivity and nucleic acid contents in the mycelial solution incubated with FP-E. In addition, FP-E treatment considerably increased the intracellular reactive oxygen species (ROS) content, and reduced the enzyme activities of both catalase (CAT) and peroxidase (POD) in P. italicum cells. Furthermore, orange fruits treated with FP-E showed fewer disease symptoms compared to the untreated fruits. These results suggested that the antifungal activity of FP-E might be associated with the disruption of cell membrane integrity, the accumulation of ROS level, and the reduction of the antioxidant enzymes activity of P. italicum. Therefore, A. aculeatus GC-09 might be a potential microbial resource for the biocontrol of citrus postharvest blue mold.

Three new indole diterpenoids, aculeatupenes A-C (1-3), together with four known compounds (4-7), were isolated from the mycelium of Aspergillus aculeatus KKU-CT2. Their structures were established by spectroscopic evidence and absolute configurations of 1-3 were determined by comparison of their experimental and calculated ECD spectra. Compounds 1, 2, and emindole SB (4) showed weak cytotoxicity against HelaS3, KB, HepG2, MCF-7, and A549 cancer cell lines with IC50 values in the range of 11.12-67.81 μM. Compound 3 showed weak cytotoxicity against HelaS3 cell lines with an IC50 value of 17.48 μM but non-cytotoxicity against Vero cell line. In addition, compound 1 exhibited weak antibacterial activity against Bacillus cereus.

Perennial ryegrass (Lolium perenne) is a cool-season grass whose growth and development are limited by drought and high temperature. Aspergillus aculeatus has been reported to promote plant growth and counteract the adverse effects of abiotic stresses. The objective of this study was to assess A. aculeatus-induced response mechanisms to drought and heat resistance in perennial ryegrass. We evaluated the physiological and biochemical markers of drought and heat stress based on the hormone homeostasis, photosynthesis, antioxidant enzymes activity, lipid peroxidation, and genes expression level. We found out that under drought and heat stress, A. aculeatus-inoculated leaves exhibited higher abscisic acid (ABA) and lower salicylic acid (SA) contents than non-inoculated regimes. In addition, under drought and heat stress, the fungus enhanced the photosynthetic performance, decreased the antioxidase activities, and mitigated membrane lipid peroxidation compared to non-inoculated regime. Furthermore, under drought stress, A. aculeatus induced a dramatic upregulation of sHSP17.8 and DREB1A and a downregulation of POD47, Cu/ZnSOD, and FeSOD genes. In addition, under heat stress, A. aculeatus-inoculated plants exhibited a higher expression level of HSP26.7a, sHSP17.8, and DREB1A while a lower expression level of POD47 and FeSOD than non-inoculated ones. Our results provide an evidence of the protective role of A. aculeatus in perennial ryegrass response to drought and heat stresses.

This work presents the identification and proposed biosynthetic pathway for a compound of mixed polyketide-nonribosomal peptide origin that we named acurin A. The compound was isolated from an extract of the filamentous fungus Aspergillus aculeatus, and its core structure resemble that of the mycotoxin fusarin C produced by several Fusarium species. Based on bioinformatics in combination with RT-qPCR experiments and gene-deletion analysis, we identified a biosynthetic gene cluster (BGC) in A. aculeatus responsible for the biosynthesis of acurin A. Moreover, we were able to show that a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) enzyme separately encoded by this BGC are responsible for the synthesis of the PK-NRP compound, acurin A, core structure. In comparison, the production of fusarin C is reported to be facilitated by a linked PKS-NRPS hybrid enzyme. Phylogenetic analyses suggest the PKS and NRPS in A. aculeatus resulted from a recent fission of an ancestral hybrid enzyme followed by gene duplication. In addition to the PKS- and NRPS-encoding genes of acurin A, we show that six other genes are influencing the biosynthesis including a regulatory transcription factor. Altogether, we have demonstrated the involvement of eight genes in the biosynthesis of acurin A, including an in-cluster transcription factor. This study highlights the biosynthetic capacity of A. aculeatus and serves as an example of how the CRISPR/Cas9 system can be exploited for the construction of fungal strains that can be readily engineered.

Phytases are the special class of enzymes which have excellent application potential for enhancing the quality of food by decreasing its inherent anti-nutrient components. In current study, a protease-resistant, acidic phytase from Aspergillus aculeatus APF1 was partially purified by ammonium sulfate fractionation followed by chromatography techniques. The molecular weight of partially purified phytase was in range of 25-35 kDa. The purified APF1 phytase was biochemically characterized and found catalytically active at pH 3.0 and 50 °C. The Km and Vmax values of APF1 phytase for calcium phytate were 3.21 mM and 3.78 U/mg protein, respectively. Variable activity was observed with metal ions and among inhibitors, chaotropic agents and organic solvents; phenyl glyoxal, potassium iodide, and butanol inhibited enzyme activity, respectively, while the enzyme activity was not majorly influenced by EDTA, urea, ethanol, and hexane. APF1 phytase treatment was found effective in dephytinization of flour biofortified wheat genotypes. Maximum decrease in phytic acid content was noticed in genotype MB-16-1-4 (89.98%) followed by PRH3-30-3 (82.32%) and PRH3-43-1 (81.47%). Overall, the study revealed that phytase from Aspergillus aculeatus APF1 could be effectively used in food and feed processing industry for enhancing nutritional value of food.

Effects of solid-state fermentation on rapid drying and spoilage prevention of potato pulp were evaluated. Pectin hydrolyzing and antibacterial ability of pectinase-secreting Aspergillus aculeatus and Bacillus subtilis were compared. A. aculeatus grew better in potato pulp, with highest pectinase yield of 342.71 ± 5.09 U/mL and rapid pH reduction to 3.76 ± 0.01. Next generation sequencing showed that the abundance of genera Candida and Enterobacter, which probably caused undesirable fermentation and spoilage, were significantly reduced after inoculation with A. aculeatus. In addition, fermentation with A. aculeatus significantly reduced water holding capacity from 16.63 ± 0.36 g/g to 7.78 ± 0.12 g/g, which resulted in lower viscosity and water binding capacity, and concomitantly significantly decreased moisture content from 76.05 ± 0.24% to 12.95 ± 0.19% after filtration and airflow drying. These results suggested that solid-state fermentation might be a promising technology for efficient processing and utilization of potato pulp.

Aspergillus aculeatus has been shown to stimulate plant growth, but its role in plants abiotic stress tolerance and the underlying mechanisms are not fully documented. In this study, we investigated the mechanisms of A.aculeatus-mediated drought, heat and salt stress tolerance in tall fescue. The results showed that A.aculeatus inoculation improved drought and heat stress tolerance in tall fescue as observed from its effect on turf quality (TQ) and leaf relative water content (LWC). In the same stress conditions, A.aculeatus alleviated reactive oxygen species (ROS) induced burst and cell damage, as indicated by lower H2O2, electrolyte leakage (EL) and malondialdehyde (MDA) levels. Additionally, the A.aculeatus inoculated plants exhibited higher photosynthetic efficiency than uninoculated plants under drought, heat and salt stress conditions. The fungus reduced the uptake of Na+, and inoculated plants showed lower Na+/K+, Na+/Ca2+and Na+/Mg2+ ratios compared to uninoculated ones under salt stress. Furthermore, comparative metabolomic analysis showed that A.aculeatus exclusively increased amino acid (such as proline and glycine) and sugar (such as glucose, fructose, sorbose, and talose) accumulation under drought and heat stress. However, there were no differences between inoculated and uninoculated plants except for changes in H2O2 level, Na+ in the root and photosynthetic efficiency under salt stress. Taken together, this study provides the first evidence of the protective roles of A.aculeatus in the tall fescue response to abiotic stresses, partially via protection of photosynthesis and modulation of metabolic homeostasis.