An-Dong Gong

Antagonistic mechanism of iturin A and plipastatin A from Bacillus amyloliquefaciens S76-3 from wheat spikes against Fusarium graminearum.

Controlling toxigenic Fusarium graminearum (FG) is challenging. A bacterial strain (S76-3, identified as Bacillus amyloliquefaciens) that was isolated from diseased wheat spikes in the field displayed strong antifungal activity against FG. Reverse-phase high performance liquid chromatography and electrospray ionization mass spectrometry analyses revealed that S76-3 produced three classes of cyclic lipopeptides including iturin, plipastatin and surfactin. Each class consisted of several different molecules. The iturin and plipastatin fractions strongly inhibited FG; the surfactin fractions did not. The most abundant compound that had antagonistic activity from the iturin fraction was iturin A (m/z 1043.35); the most abundant active compound from the plipastatin fraction was plipastatin A (m/z 1463.90). These compounds were analyzed with collision-induced dissociation mass spectrometry. The two purified compounds displayed strong fungicidal activity, completely killing conidial spores at the minimal inhibitory concentration range of 50 µg/ml (iturin A) and 100 µg/ml (plipastatin A). Optical and fluorescence microscopy analyses revealed severe morphological changes in conidia and substantial distortions in FG hyphae treated with iturin A or plipastatin A. Iturin A caused leakage and/or inactivation of FG cellular contents and plipastatin A caused vacuolation. Time-lapse imaging of dynamic antagonistic processes illustrated that iturin A caused distortion and conglobation along hyphae and inhibited branch formation and growth, while plipastatin A caused conglobation in young hyphae and branch tips. Transmission electron microscopy analyses demonstrated that the cell walls of conidia and hyphae of iturin A and plipastatin A treated FG had large gaps and that their plasma membranes were severely damaged and separated from cell walls.

Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum

Trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) are required for trehalose biosynthesis in yeast and filamentous fungi, including Fusarium graminearum. Three null mutants Δtps1, Δtps2 and Δtps1-Δtps2, each carrying either a single deletion of TPS1 or TPS2 or a double deletion of TPS1-TPS2, were generated from a toxigenic F. graminearum strain and were not able to synthesize trehalose. In contrast to its reported function in yeasts and filamentous fungi, TPS1 appeared dispensable for development and virulence. However, deletion of TPS2 abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. The cell polarity alteration was exhibited as reduced apical growth and increased lateral growth and branching with increased hyphal and cell wall widths. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution in its conidia and vegetative hyphae. The Δtps2 mutant also had 62% lower mycelial growth on potato dextrose agar and 99% lower virulence on wheat compared with the wild-type. The Δtps1, Δtps2 and Δtps1-Δtps2 mutants synthesized over 3.08-, 7.09- and 2.47-fold less mycotoxins, respectively, on rice culture compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively, with significantly changed expression profiles compared with the wild-type. Further dissection of this pathway will provide new insights into regulation of fungal development, virulence and trichothecene biosynthesis.

Type II myosin gene in Fusarium graminearum is required for septation, development, mycotoxin biosynthesis and pathogenicity.

Type II myosin is required for cytokinesis/septation in yeast and filamentous fungi, including Fusarium graminearum, a prevalent cause of Fusarium head blight in China. A type II myosin gene from the Chinese F. graminearum strain 5035, isolated from infected wheat spikes, was identified by screening a mutant library generated by restriction enzyme-mediated integration. Disruption of the Myo2 gene reduced mycelial growth by 50% and conidiation by 76-fold, and abolished sexual reproduction on wheat kernels. The Δmyo2 mutants also had a 97% decrease in their pathogenicity on wheat, and mycotoxin production fell to just 3.4% of the normal level. The distribution of nuclei and septa was abnormal in the mutants, and the septal ultrastructure appeared disorganized. Time-lapse imaging of septation provided direct evidence that Myo2 is required for septum initiation and formation, and revealed the dynamic behavior of GFP-tagged Myo2 during hyphal and macroconidia development, particularly in the delimiting septum of phialides and macroconidial spores. Microarray analysis identified many genes with altered expression profiles in the Δmyo2 mutant, indicating that Myo2 is required for several F. graminearum developmental processes and biological activities.

The Shewanella algae strain YM8 produces volatiles with strong inhibition activity against Aspergillus pathogens and aflatoxins.

Aflatoxigenic Aspergillus fungi and associated aflatoxins are ubiquitous in the production and storage of food/feed commodities. Controlling these microbes is a challenge. In this study, the Shewanella algae strain YM8 was found to produce volatiles that have strong antifungal activity against Aspergillus pathogens. Gas chromatography-mass spectrometry profiling revealed 15 volatile organic compounds (VOCs) emitted from YM8, of which dimethyl trisulfide was the most abundant. We obtained authentic reference standards for six of the VOCs; these all significantly reduced mycelial growth and conidial germination in Aspergillus; dimethyl trisulfide and 2,4-bis(1,1-dimethylethyl)-phenol showed the strongest inhibitory activity. YM8 completely inhibited Aspergillus growth and aflatoxin biosynthesis in maize and peanut samples stored at different water activity levels, and scanning electron microscopy revealed severely damaged conidia and a complete lack of mycelium development and conidiogenesis. YM8 also completely inhibited the growth of eight other agronomically important species of phytopathogenic fungi: A. parasiticus, A. niger, Alternaria alternate, Botrytis cinerea, Fusarium graminearum, Fusarium oxysporum, Monilinia fructicola, and Sclerotinia sclerotiorum. This study demonstrates the susceptibility of Aspergillus and other fungi to VOCs from marine bacteria and indicates a new strategy for effectively controlling these pathogens and the associated mycotoxin production during storage and possibly in the field.

Chicken single-chain antibody fused to alkaline phosphatase detects Aspergillus pathogens and their presence in natural samples by direct sandwich enzyme-linked immunosorbent assay.

A sensitive and specific analytical method to detect ubiquitous aflatoxigenic Aspergillus pathogens is essential for monitoring and controlling aflatoxins. Four highly reactive chicken single-chain variable fragments (scFvs) against soluble cell wall proteins (SCWPs) from Aspergillus flavus were isolated by phage display. The scFv antibody AfSA4 displayed the highest activity toward both A. flavus and A. parasiticus and specifically recognized a surface target of their cell walls as revealed by immunofluorescence localization. Molecular modeling revealed a unique compact motif on the antibody surface mainly involving L-CDR2 and H-CDR3. As measured by surface plasmon resonance, AfSA4 fused to alkaline phosphatase had a higher binding capability and 6-fold higher affinity compared with AfSA4 alone. Immunoblot analyses showed that the fusion had good binding capacity to SCWP components from the two fungal species. Direct sandwich enzyme-linked immunosorbent assays with mouse antiaspergillus monoclonal antibody mAb2A8 generated in parallel as a capture antibody revealed that the detection limit of the two fungi was as low as 10(-3) μg/mL, 1000-fold more sensitive than that reported previously (1 μg/mL). The fusion protein was able to detect fungal concentrations below 1 μg/g of maize and peanut grains in both artificially and naturally contaminated samples, with at least 10-fold more sensitivity than that reported (10 μg/g) thus far. Thus, the fusion can be applied in rapid, simple, and specific diagnosis of Aspergillus contamination in field and stored food/feed commodities.

Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5–10) and temperatures (20–37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

Enhanced overall resistance to Fusarium seedling blight and Fusarium head blight in transgenic wheat by co-expression of anti-fungal peptides

Introduction of alien genes into wheat has been proposed as a strategy to breed cultivars with improved resistance to Fusarium seedling blight (FSB) and Fusarium head blight (FHB). In this study, we co-transformed different anti-fungal peptides (AFPs) into an elite wheat cultivar Yangmai11. We identified the genetically stable transgenic wheat lines carrying single or multiple genes by PCR, qRT-PCR and Southern blot analyses. Transgenic wheat lines 451 and 513 expressing two AFPs displayed a consistent, significantly improved overall resistance to FSB and FHB, whereas only FHB resistance was observed from other lines. Furthermore, crude proteins extracted from the lines 451 and 513 showed a clear inhibitory activity against F. graminearum in vitro. Taken together, it was essential to properly combine and express AFPs in transgenic wheat in order to obtain an improved overall resistance to Fusarium pathogens.

Production of a phage-displayed mouse ScFv antibody against fumonisin B1 and molecular docking analysis of their interactions

Fumonisins produced by Fusarium pathogens are mycotoxins present in maize and other grains in the field as well as during storage worldwide and pose a serious threat to humans and domestic animals. Fumonisin B consists of different chemotypes, and fumonisin B1 (FB1) is the most predominant fumonisin found in food/feed commodities. Recombinant antibody can be deployed to analyze the fumonisin toxicological mechanism and develop a simple and cost-effective method for the detection of fumonisins, which is vitally important for monitoring and preventing fumonisins from entering food/feed chains. In this study, FB1 conjugated to keyhole limpet hemocyanin was used to immunize mice, from which RNA was isolated to construct a recombinant antibody library. Successive panning of the library by phage display was used to select monoclonal phage clones reactive to FB1 conjugated to bovine serum albumin. Subsequent phage ELISA and sequencing analyses revealed four different reactive scFv antibodies specific to FB1. Soluble expression and ELISA analysis showed that one scFv antibody, FBMA1, had the highest reactivity and could be purified from bacterial cells in large quantities. Surface plasmon resonance measurements further revealed that the FBMA1 scFv antibody had a binding kinetics of KD = 1.89 × 10–7 M. Molecular modeling and docking analyses suggested that the FBMA1 antibody shaped a proper cavity to embed the whole FB1 molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions and fumonisin toxicity, and for the development of an immunoassay for fumonisin-contaminated food/feed samples.