The Fungicidal Activity of Tebuconazole Enantiomers against <i>Fusarium graminearum</i> and Its Selective Effect on DON Production under Different Conditions

Tebuconazole, which consists of a pair of enantiomers with different fungicidal activities, is one of the most common fungicides used in the control of <i>Fusarium graminearum</i>. In this study, the fungicidal activity of <i>rac</i>-tebuconazole and its enantiomers against <i>F. graminearum</i> was determined at 0.997, 0.975, and 0.950 <i>a</i>_w and at 20, 25, and 30 °C on wheat-based media. Then, <i>F. graminearum</i> was treated with <i>rac</i>-tebuconazole and its enantiomers at the EC₁₀, EC₅₀, and EC₉₀ levels under different culture conditions, and DON production was measured. Finally, expression of the DON biosynthetic genes (<i>TRI5</i> and <i>TRI6</i>) was quantified by real-time RT-PCR after incubation with EC₅₀ doses of <i>rac</i>-tebuconazole and its enantiomers for 4, 8, and 14 days at 30 °C and <i>a</i>_w 0.997. The results showed that the fungicidal activity of tebuconazole was strongly influenced by temperature, <i>a</i>_w, and the combined factors. (−)-Tebuconazole is higher in fungicidal activity than (+)-tebuconazole and <i>rac</i>-tebuconazole with 24–99-fold and 1.8–6.7-fold, respectively. However, (−)-tebuconazole was generally more favorable for DON production than (+)-tebuconazole under the same conditions. Additionally, (−)-tebuconazole and <i>rac</i>-tebuconazole induced significantly increased expression of the DON biosynthetic genes (<i>TRI5</i> and <i>TRI6</i>) compared to the control by the 14th day of treatment. In this research, the combination condition of 30 °C and 0.997 <i>a</i>_w is the most suitable for DON production by <i>F. graminearum.</i> The test strains of <i>F. graminearum</i> treated with the EC₁₀ dose of (−)-tebuconazole produced the greatest amounts of DON

Quantitative Real Time PCR (qRT-PCR) validation of DGE result.

a<p>means significant difference existed between the DA treated and control samples.</p>b<p>means no significant difference.</p><p>DGE data were generated from two biological repeats. qPCR data were generated by the standard deviation from three biological repeats.</p

NCBI SRA accession numbers for the treatments at each time point.

<p>NCBI SRA accession numbers for the treatments at each time point.</p

Gene Expression Profile of <i>Bombyx mori</i> Hemocyte under the Stress of Destruxin A

<div><p>Destruxin A (DA) is a cyclo-peptidic mycotoxin from the entomopathogenic fungus <i>Metarhizium anisopliae</i>. To uncover potential genes associated with its molecular mechanisms, a digital gene expression (DGE) profiling analysis was used to compare differentially expressed genes in the hemocytes of silkworm larvae treated with DA. Ten DGE libraries were constructed, sequenced, and assembled, and the unigenes with least 2.0-fold difference were further analyzed. The numbers of up-regulated genes were 10, 20, 18, 74 and 8, as well as the numbers of down-regulated genes were 0, 1, 8, 13 and 3 at 1, 4, 8, 12 and 24 h post treatment, respectively. Totally, the expression of 132 genes were significantly changed, among them, 1, 3 and 12 genes were continually up-regulated at 4, 3 and 2 different time points, respectively, while 1 gene was either up or down-regulated continually at 2 different time points. Furthermore, 68 genes were assigned to one or multiple gene ontology (GO) terms and 89 genes were assigned to specific Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology. In-depth analysis identified that these genes putatively involved in insecticide resistance, cell apoptosis, and innate immune defense. Finally, twenty differentially expressed genes were randomly chosen and validated by quantitative real-time PCR (qRT-PCR). Our studies provide insights into the toxic effect of this microbial insecticide on silkworm's hemocytes, and are helpful to better understanding of the molecular mechanisms of DA as a biological insecticide.</p></div

Summarizing of Gene Ontology functional classification of ten DGEs by comparing DA-treated and un-treated samples in differential time intervals, which described gene products in terms of their associated biological processes, cellular components and molecular functions.

<p>Summarizing of Gene Ontology functional classification of ten DGEs by comparing DA-treated and un-treated samples in differential time intervals, which described gene products in terms of their associated biological processes, cellular components and molecular functions.</p

Biodegradation of Chlorpyrifos and Its Hydrolysis Product 3,5,6-Trichloro-2-Pyridinol by a New Fungal Strain <em>Cladosporium cladosporioides</em> Hu-01

<div><p>Intensive use of chlorpyrifos has resulted in its ubiquitous presence as a contaminant in surface streams and soils. It is thus critically essential to develop bioremediation methods to degrade and eliminate this pollutant from environments. We present here that a new fungal strain Hu-01 with high chlorpyrifos-degradation activity was isolated and identified as <em>Cladosporium cladosporioides</em> based on the morphology and 5.8S rDNA gene analysis. Strain Hu-01 utilized 50 mg·L⁻¹ of chlorpyrifos as the sole carbon of source, and tolerated high concentration of chlorpyrifos up to 500 mg·L⁻¹. The optimum degradation conditions were determined to be 26.8°C and pH 6.5 based on the response surface methodology (RSM). Under these conditions, strain Hu-01 completely metabolized the supplemented chlorpyrifos (50 mg·L⁻¹) within 5 d. During the biodegradation process, transient accumulation of 3,5,6-trichloro-2-pyridinol (TCP) was observed. However, this intermediate product did not accumulate in the medium and disappeared quickly. No persistent accumulative metabolite was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis at the end of experiment. Furthermore, degradation kinetics of chlorpyrifos and TCP followed the first-order model. Compared to the non-inoculated controls, the half-lives (<em>t</em>_1/2) of chlorpyrifos and TCP significantly reduced by 688.0 and 986.9 h with the inoculum, respectively. The isolate harbors the metabolic pathway for the complete detoxification of chlorpyrifos and its hydrolysis product TCP, thus suggesting the fungus may be a promising candidate for bioremediation of chlorpyrifos-contaminated water, soil or crop.</p> </div

Quality assessment of reads in each library.

<p>Quality assessment of reads in each library.</p

Mass spectra of 3,5,6-trichloro-2-pyridinol (TCP) produced from chlorpyrifos degradation by strain Hu-01.

<p>A: sample; B: authentic standard TCP from the National Institute of Standards and Technology (NIST, USA) library database.</p

Screening of more than 2-fold differentially expressed genes in <i>B.mori</i> hemocytes after the treatment of DA in differential time intervals.

<p>Screening of more than 2-fold differentially expressed genes in <i>B.mori</i> hemocytes after the treatment of DA in differential time intervals.</p

Degradation kinetics of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) during biodegradation studies.

<p>□, non-inoculated control (chlorpyrifos); △, non-inoculated control (TCP); ▪, introduction with strain Hu-01 (chlorpyrifos); ▴, introduction with strain Hu-01(TCP). Error bars represent the standard deviation of three replicates.</p