FgFAD12 Regulates Vegetative Growth, Pathogenicity and Linoleic Acid Biosynthesis in Fusarium graminearum.

Polyunsaturated fatty acids (PUFAs), as important components of lipids, play indispensable roles in the development of all organisms. ∆12 fatty acid desaturase (FAD12) is a speed-determining step in the biosynthesis of PUFAs. Here, we report the characterization of FAD12 in Fusarium graminearum, which is the prevalent agent of Fusarium head blight, a destructive plant disease worldwide. The results demonstrated that deletion of the FgFAD12 gene resulted in defects in vegetative growth, conidial germination and plant pathogenesis but not sexual reproduction. A fatty acid analysis further proved that the deletion of FgFAD12 restrained the reaction of oleic acid to linoleic acid, and a large amount of oleic acid was detected in the cells. Moreover, the ∆Fgfad12 mutant showed increased resistance to osmotic stress and reduced tolerance to oxidative stress. The expression of FgFAD12 did show a temperature-dependent manner, which was not affected at a low temperature of 10 °C when compared to 25 °C. RNA-seq analysis further demonstrated that most genes enriched in fatty acid metabolism, the biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, fatty acid degradation, steroid biosynthesis and fatty acid elongation pathways were significantly up-regulated in the ∆Fgfad12 mutants. Overall, our results indicate that FgFAD12 is essential for linoleic acid biosynthesis and plays an important role in the infection process of F. graminearum.

Burkholderia ambifaria H8 as an effective biocontrol strain against maize stalk rot via producing volatile dimethyl disulfide.

BACKGROUND: Maize stalk rot (MSR) caused by Fusarium graminearum is the primary factor contributing to the reduction in maize yield and quality. However, this soil-borne disease presents a significant challenge for sustainable control through field management and chemical agents. The screening of novel biocontrol agents can aid in developing innovative and successful strategies for MSR control. RESULTS: A total of 407 strains of bacteria were isolated from the rhizosphere soil of a resistant maize inbred line. One strain exhibited significant antagonistic activity in plate and pot experiments, and was identified as Burkholderia ambifaria H8. The strain could significantly inhibit the mycelial growth and spore germination of F. graminearum, induce resistance to stalk rot, and promote plant growth. The volatile compounds produced by strain H8 and its secondary metabolites in the sterile fermentation broth exhibited antagonistic activity. The primary volatile compound produced by strain H8 was identified as dimethyl disulfide (DMDS) using gas chromatography tandem mass spectrometry. Through in vitro antagonistic activity assays and microscopic observation, it was confirmed that DMDS was capable of inhibiting mycelial growth and disrupting the mycelial structure of F. graminearum, suggesting it may be the major active compound for strain H8. The transcriptome data of F. graminearum further indicated that strain H8 and its volatile compounds could alter pathogenic fungi metabolism, influence the related metabolic pathways, and potentially induce cell apoptosis within F. graminearum. CONCLUSION: Our results showed that B. ambifaria H8 was capable of producing the volatile substance dimethyl disulfide, which influenced the synthesis and permeability of cell membranes in pathogens. Thus, B. ambifaria H8 was found to be a promising biological control agent against MSR. © 2024 Society of Chemical Industry.

Transcriptome-based variations effectively untangling the intraspecific relationships and selection signals in Xinyang Maojian tea population.

As one of the world's top three popular non-alcoholic beverages, tea is economically and culturally valuable. Xinyang Maojian, this elegant green tea, is one of the top ten famous tea in China and has gained prominence for thousands of years. However, the cultivation history of Xinyang Maojian tea population and selection signals of differentiation from the other major variety Camellia sinensis var. assamica (CSA) remain unclear. We newly generated 94 Camellia sinensis (C. sinensis) transcriptomes including 59 samples in the Xinyang area and 35 samples collected from 13 other major tea planting provinces in China. Comparing the very low resolution of phylogeny inferred from 1785 low-copy nuclear genes with 94 C. sinensis samples, we successfully resolved the phylogeny of C. sinensis samples by 99,115 high-quality SNPs from the coding region. The sources of tea planted in the Xinyang area were extensive and complex. Specifically, Shihe District and Gushi County were the two earliest tea planting areas in Xinyang, reflecting a long history of tea planting. Furthermore, we identified numerous selection sweeps during the differentiation of CSA and CSS and these positive selection genes are involved in many aspects such as regulation of secondary metabolites synthesis, amino acid metabolism, photosynthesis, etc. Numerous specific selective sweeps of modern cultivars were annotated with functions in various different aspects, indicating the CSS and CSA populations possibly underwent independent specific domestication processes. Our study indicated that transcriptome-based SNP-calling is an efficient and cost-effective method in untangling intraspecific phylogenetic relationships. This study provides a significant understanding of the cultivation history of the famous Chinese tea Xinyang Maojian and unravels the genetic basis of physiological and ecological differences between the two major tea subspecies.

Engineering the glyoxylate cycle for chemical bioproduction.

With growing concerns about environmental issues and sustainable economy, bioproduction of chemicals utilizing microbial cell factories provides an eco-friendly alternative to current petro-based processes. Creating high-performance strains (with high titer, yield, and productivity) through metabolic engineering strategies is critical for cost-competitive production. Commonly, it is inevitable to fine-tuning or rewire the endogenous or heterologous pathways in such processes. As an important pathway involved in the synthesis of many kinds of chemicals, the potential of the glyoxylate cycle in metabolic engineering has been studied extensively these years. Here, we review the metabolic regulation of the glyoxylate cycle and summarize recent achievements in microbial production of chemicals through tuning of the glyoxylate cycle, with a focus on studies implemented in model microorganisms. Also, future prospects for bioproduction of glyoxylate cycle-related chemicals are discussed.

The Inhibitory Effect of Pseudomonas stutzeri YM6 on Aspergillus flavus Growth and Aflatoxins Production by the Production of Volatile Dimethyl Trisulfide.

Aspergillus flavus and the produced aflatoxins cause great hazards to food security and human health across all countries. The control of A. flavus and aflatoxins in grains during storage is of great significance to humans. In the current study, bacteria strain YM6 isolated from sea sediment was demonstrated effective in controlling A. flavus by the production of anti-fungal volatiles. According to morphological characteristics and phylogenetic analysis, strain YM6 was identified as Pseudomonas stutzeri. YM6 can produce abundant volatile compounds which could inhibit mycelial growth and conidial germination of A. flavus. Moreover, it greatly prevented fungal infection and aflatoxin production on maize and peanuts during storage. The inhibition rate was 100%. Scanning electron microscopy further supported that the volatiles could destroy the cell structure of A. flavus and prevent conidia germination on the grain surface. Gas chromatography/mass spectrometry revealed that dimethyl trisulfide (DMTS) with a relative abundance of 13% is the most abundant fraction in the volatiles from strain YM6. The minimal inhibitory concentration of DMTS to A. flavus conidia is 200 µL/L (compound volume/airspace volume). Thus, we concluded that Pseudomonas stutzeri YM6 and the produced DMTS showed great inhibition to A. flavus, which could be considered as effective biocontrol agents in further application.

Dual activity of Serratia marcescens Pt-3 in phosphate-solubilizing and production of antifungal volatiles.

BACKGROUND: Soil fertility decline and pathogen infection are severe issues for crop production all over the world. Microbes as inherent factors in soil were effective in alleviating fertility decrease, promoting plant growth and controlling plant pathogens et al. Thus, screening microbes with fertility improving and pathogen controlling properties is of great importance to humans. RESULTS: Bacteria Pt-3 isolated from tea rhizosphere showed multiple functions in solubilizing insoluble phosphate, promoting plant growth, producing abundant volatile organic compounds (VOCs) and inhibiting the growth of important fungal pathogens in vitro. According to the 16S rRNA phylogenetic and biochemical analysis, Pt-3 was identified to be Serratia marcescens. The solubilizing zone of Pt-3 in the medium of lecithin and Ca3(PO4)2 was 2.1 cm and 1.8 cm respectively. In liquid medium and soil, the concentration of soluble phosphorus reached 343.9 mg.L- 1, and 3.98 mg.kg- 1, and significantly promoted the growth of maize seedling, respectively. Moreover, Pt-3 produced abundant volatiles and greatly inhibited the growth of seven important phytopathogens. The inhibition rate ranged from 75.51 to 100% respectively. Solid phase micro-extraction coupled with gas chromatography tandem mass spectrometry proved that the antifungal volatile was dimethyl disulfide. Dimethyl disulfide can inhibit the germination of Aspergillus flavus, and severely destroy the cell structures under scanning electron microscopy. CONCLUSIONS: S. marcescens Pt-3 with multiple functions will provide novel agent for the production of bioactive fertilizer with P-solubilizing and fungal pathogens control activity.

Development of a defined medium for Corynebacterium glutamicum using urea as nitrogen source.

Corynebacterium glutamicum has been widely used for bulk and fine chemicals fermentation these years. In this study, we developed a defined medium for this bacteria based on the widely used CGXII minimal medium. We evaluated the effects of different components in CGXII on cell growth of C. glutamicum ATCC 13032 and improved the medium through single-factor experiment and central composite design (CCD). Urea, K2HPO4 and MgSO4 were found to be significant factors. 7 out of the total 15 components were modified. (NH4)2SO4, KH2PO4, and protocatechuic acid were eliminated. Amounts of urea and MgSO4 were increased, and concentrations of biotin and glucose were reduced. The resulting R2 medium was proved to be more suitable for cell growth, plasmid amplification and protein production than the original recipe. Remarkably, cell biomass accumulation in R2 increased by 54.36% than CGXII. Transcriptome analysis revealed alteration of carbon metabolism, cation transport and energy synthesis, which might be beneficial for cell growth in R2. Considering the high nitrogen content and availability of urea, the new medium is simplified and cost effective, which holds attractive potential for future study. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02959-6.

Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons.

BACKGROUND: Catechins, caffeine, and theanine as three important metabolites in the tea leaves play essential roles in the formation of specific taste and shows potential health benefits to humans. However, the knowledge on the dynamic changes of these metabolites content over seasons, as well as the candidate regulatory factors, remains largely undetermined. RESULTS: An integrated transcriptomic and metabolomic approach was used to analyze the dynamic changes of three mainly metabolites including catechins, caffeine, and theanine, and to explore the potential influencing factors associated with these dynamic changes over the course of seasons. We found that the catechins abundance was higher in Summer than that in Spring and Autumn, and the theanine abundance was significantly higher in Spring than that in Summer and Autumn, whereas caffeine exhibited no significant changes over three seasons. Transcriptomics analysis suggested that genes in photosynthesis pathway were significantly down-regulated which might in linkage to the formation of different phenotypes and metabolites content in the tea leaves of varied seasons. Fifty-six copies of nine genes in catechins biosynthesis, 30 copies of 10 genes in caffeine biosynthesis, and 12 copies of six genes in theanine biosynthesis were detected. The correlative analysis further presented that eight genes can be regulated by transcription factors, and highly correlated with the changes of metabolites abundance in tea-leaves. CONCLUSION: Sunshine intensity as a key factor can affect photosynthesis of tea plants, further affect the expression of major Transcription factors (TFs) and structural genes in, and finally resulted in the various amounts of catechins, caffeine and theaine in tea-leaves over three seasons. These findings provide new insights into abundance and influencing factors of metabolites of tea in different seasons, and further our understanding in the formation of flavor, nutrition and medicinal function.

The differential expression patterns of paralogs in response to stresses indicate expression and sequence divergences.

BACKGROUND: Theoretically, paralogous genes generated through whole genome duplications should share identical expression levels due to their identical sequences and chromatin environments. However, functional divergences and expression differences have arisen due to selective pressures throughout evolution. A comprehensive investigation of the expression patterns of paralogous gene pairs in response to various stresses and a study of correlations between the expression levels and sequence divergences of the paralogs are needed. RESULTS: In this study, we analyzed the expression patterns of paralogous genes under different types of stress and investigated the correlations between the expression levels and sequence divergences of the paralogs. We analyzed the differential expression patterns of the paralogs under four different types of stress (drought, cold, infection, and herbivory) and classified them into three main types according to their expression patterns. We then further analyzed the differential expression patterns under various degrees of stress and constructed corresponding co-expression networks of differentially expressed paralogs and transcription factors. Finally, we investigated the correlations between the expression levels and sequence divergences of the paralogs and identified positive correlations between expression level and sequence divergence. With regard to sequence divergence, we identified correlations between selective pressures and phylogenetic relationships. CONCLUSIONS: These results shed light on differential expression patterns of paralogs in response to environmental stresses and are helpful for understanding the relationships between expression levels and sequences divergences.

Inhibitory Effect of Volatiles Emitted From Alcaligenes faecalis N1-4 on Aspergillus flavus and Aflatoxins in Storage.

Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 µL/L, respectively. The effective concentration for MI against A. flavus is 200 µL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.

Biodegradation of mycotoxin fumonisin B1 by a novel bacterial consortium SAAS79.

Fumonisin B1 (FB1) contamination in cereals and cereal products remains an important aspect of food safety because of its wide distribution and the potential health hazard. However, only a few microorganisms have been reported to effectively degrade FB1. In this present study, a bacterial consortium SAAS79 with highly FB1-degrading activity was isolated from the spent mushroom compost. The combination of antibiotic-driven selection and 16S rDNA sequencing identified the Pseudomonas genus as the key FB1-degrading member. The microbial consortium could degrade more than 90% of 10 µg/mL FB1 after incubation for 24 h at pH of 5-7 and temperature of 28-35 °C. The enzymes from the intracellular space were proved to be responsible for FB1 degradation, which eliminated about 90% of 10 µg/mL FB1 in 3 h. Besides, liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) analysis identified two degradation products of FB1, and their toxicity on the monkey kidney cells (MARC-145) was significantly lower (p < 0.05) compared with the parent FB1. Overall, the consortium SAAS79 and its crude enzymes may be a potential choice for the decontamination of FB1 in the feed and food industry. Also, the bacterial consortium provides a new source of genes for the development of enzymatic detoxification agent.

Genomic, molecular evolution, and expression analysis of NOX genes in soybean (Glycine max).

Reactive oxygen species (ROS) are versatile signaling molecules in sensing stresses and play critical roles in signaling and development. Plasma membrane NADPH oxidases (NOXs) are key producers of ROS, and play important roles in the regulation of plant-pathogen interactions. Here, we performed a comprehensive analysis of the NOX gene family in the soybean genome (Glycine max) and 17 NOX (GmNOX) genes were identified. Structural analysis revealed that the GmNOX proteins in soybean were as conserved as those in other plants. 8 duplicated gene pairs were formed by a Glycine-specific whole-genome duplication (WGD) event approximately 13 million years ago (Mya). The Ka/Ks ratios of GmNOX genes ranged from 0.04 to 0.28, suggesting that the GmNOX family had undergone purifying selection in soybean. Gene expression patterns showed different expression of these duplicate genes, suggesting that the GmNOXs were retained by substantial subfunctionalization during the soybean evolutionary processes. Subsequently, the expression of GmNOXs in response to drought and phytohormones were characterized via qPCR. Importantly, four GmNOXs showed strong expression in nodules, pointing to their probable involvement in nodulation. Thus, our results shed light on the evolutionary history of this family in soybean and contribute to the functional characterization of GmNOX genes in soybean.

Selective and sensitive Escherichia coli detection based on a T4 bacteriophage-immobilized multimode microfiber.

Escherichia coli bacteria have been found to be responsible for various health outbreaks caused by contaminated food and water. Accurate and rapid test of E. coli is thus crucial for protecting the public health. A fast-response, label-free bacteriophage-based detection of E. coli using multimode microfiber probe is proposed and demonstrated in this article. Due to the abrupt taper and subwavelength diameter, different modes are excited and guided in the microfiber as evanescent field that can interact with surrounding E. coli directly. The change of E. coli concentration and corresponding binding of E. coli bacteria on microfiber surface will lead to the shift of optical spectrum, which can be exploited for the application of biosensing. The proposed method is capable of reliable detection of E. coli concentration as low as 103 cfu/mL within the range of 103 to 107 cfu/mL. Owing to the advantages of high sensitivity and fast response, the microfiber probe has great potential application in the fields of environment monitoring and food safety.

Immobilized optical fiber microprobe for selective and sensitive Escherichia coli detection.

Escherichia coli (E. coli) bacteria have been found to be responsible for various health outbreaks caused by contaminated food and water. Accurate and rapid test of E. coli is thus crucial for protecting the public health. A fast-response, label-free bacteriophage-based detection of E. coli using multimode microfiber probe is proposed and demonstrated in this paper. Due to the abrupt taper and subwavelength diameter, different modes are excited and guided in the microfiber as evanescent field that can interact with surrounding E. coli directly. The change of E. coli concentration and corresponding binding of E. coli bacteria on microfiber surface will lead to the shift of optical spectrum, which can be exploited for the application of biosensing. The proposed method is capable of reliable detection of E. coli concentration as low as 103 cfu/mL within the range of 103 to 107 cfu/mL. Owing to the advantages of high sensitivity and fast response, the microfiber probe has great potential application in the fields of environment monitoring and food safety.

Proteomes and Phosphoproteomes of Anther and Pollen: Availability and Progress.

In flowering plants, anther development plays crucial role in sexual reproduction. Within the anther, microspore mother cells meiosis produces microspores, which further develop into pollen grains that play decisive role in plant reproduction. Previous studies on anther biology mainly focused on single gene functions relying on genetic and molecular methods. Recently, anther development has been expanded from multiple OMICS approaches like transcriptomics, proteomics/phosphoproteomics, and metabolomics. The development of proteomics techniques allowing increased proteome coverage and quantitative measurements of proteins which can characterize proteomes and their modulation during normal development, biotic and abiotic stresses in anther development. In this review, we summarize the achievements of proteomics and phosphoproteomics with anther and pollen organs from model plant and crop species (i.e. Arabidopsis, rice, tobacco). The increased proteomic information facilitated translation of information from the models to crops and thus aid in agricultural improvement.

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.

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.

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.

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.