Transcriptomic analysis of Amaranthus retroflex resistant to PPO-inhibitory herbicides.

Amaranthus retroflexus L. is one of the malignant weeds which can cause a reduction in the soybean yield. We found a population of A. retroflexus (R-Q) resistant to fomesafen through the initial screening of whole-plant dose response bioassay in the research. The resistance index of the population (R-Q) was 183 times of the sensitive population (S-N). The resistant and sensitive populations were used as experimental materials in the paper. Strand-specific RNA-Seq analyses of R‒Q and S‒N populations obtained from herbicide-treated and mock-treated leaf samples after treatment were conducted to generate a full-length transcriptome database. We analyzed differentially expressed genes (DEGs) among the R-Q and S‒N A. retroflexus populations treated with recommended dose and mock-treated on the 1st (24 h) and 3rd (72 h) days to identify genes involved in fomesafen resistance. All 82,287 unigenes were annotated by Blastx search with E-value < 0.00001 from 7 databases. A total of 94,815 DEGs among the three group comparisons were identified. Two nuclear genes encoding PPO (PPX1 and PPX2) and five unigenes belonging to the AP2-EREBP, GRAS, NAC, bHLH and bZIP families exhibited different expression patterns between individuals of S‒N and R-Q populations. The A. retroflexus transcriptome and specific transcription factor families which can respond to fomesafen in resistant and susceptible genotypes were reported in this paper. The PPX1 and PPX2 genes of the target enzyme were identified. The study establishes the foundation for future research and provides opportunities to manage resistant weeds better.

The structural and functional contributions of ß-glucosidase-producing microbial communities to cellulose degradation in composting.

BACKGROUND: Compost habitats sustain a vast ensemble of microbes that engender the degradation of cellulose, which is an important part of global carbon cycle. ß-Glucosidase is the rate-limiting enzyme of degradation of cellulose. Thus, analysis of regulation of ß-glucosidase gene expression in composting is beneficial to a better understanding of cellulose degradation mechanism. Genetic diversity and expression of ß-glucosidase-producing microbial communities, and relationships of cellulose degradation, metabolic products and the relative enzyme activity during natural composting and inoculated composting were evaluated. RESULTS: Compared with natural composting, adding inoculation agent effectively improved the degradation of cellulose, and maintained high level of the carboxymethyl cellulose (CMCase) and ß-glucosidase activities in thermophilic phase. Gene expression analysis showed that glycoside hydrolase family 1 (GH1) family of ß-glucosidase genes contributed more to ß-glucosidase activity in the later thermophilic phase in inoculated compost. In the cooling phase of natural compost, glycoside hydrolase family 3 (GH3) family of ß-glucosidase genes contributed more to ß-glucosidase activity. Intracellular ß-glucosidase activity played a crucial role in the regulation of ß-glucosidase gene expression, and upregulation or downregulation was also determined by extracellular concentration of glucose. At sufficiently high glucose concentrations, the functional microbial community in compost was altered, which may contribute to maintaining ß-glucosidase activity despite the high glucose content. CONCLUSION: This research provides an ecological functional map of microorganisms involved in carbon metabolism in cattle manure-rice straw composting. The performance of the functional microbial groups in the two composting treatments is different, which is related to the cellulase activity and cellulose degradation, respectively.

The distribution of active ß-glucosidase-producing microbial communities in composting.

The composting ecosystem is a suitable source for the discovery of novel microorganisms and secondary metabolites. Cellulose degradation is an important part of the global carbon cycle, and ß-glucosidases complete the final step of cellulose hydrolysis by converting cellobiose to glucose. This work analyzes the succession of ß-glucosidase-producing microbial communities that persist throughout cattle manure - rice straw composting, and evaluates their metabolic activities and community advantage during the various phases of composting. Fungal and bacterial ß-glucosidase genes belonging to glycoside hydrolase families 1 and 3 (GH1 and GH3) amplified from DNA were classified and gene abundance levels were analyzed. The major reservoirs of ß-glucosidase genes were the fungal phylum Ascomycota and the bacterial phyla Firmicutes, Actinobacteria, Proteobacteria, and Deinococcus-Thermus. This indicates that a diverse microbial community utilizes cellobiose. The succession of dominant bacteria was also detected during composting. Firmicutes was the dominant bacteria in the thermophilic phase of composting; there was a shift to Actinomycetes in the maturing stage. Proteobacteria accounted for the highest proportions during the heating and thermophilic phases of composting. By contrast, the fungal phylum Ascomycota was a minor microbial community constituent in thermophilic phase of composting. Combined with the analysis of the temperature, cellulose degradation rate and the carboxymethyl cellulase and ß-glucosidase activities showed that the bacterial GH1 family ß-glucosidase genes make greater contribution in cellulose degradation at the later thermophilic stage of composting. In summary, even GH1 bacteria families ß-glucosidase genes showing low abundance in DNA may be functionally important in the later thermophilic phase of composting. The results indicate that a complex community of bacteria and fungi expresses ß-glucosidases in compost. Several ß-glucosidase-producing bacteria and fungi identified in this study may represent potential indicators of composting in cellulose degradation.

Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms.

The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box-Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R(2) = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 10(7) cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl-Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(µg DNA·10(7) protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.

Extraction, identification and antimicrobial activity of a new furanone, grifolaone A, from Grifola frondosa.

A furanone (1), (S)-methyl 2-(2-hydroxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl)acetate, was isolated from the edible mushroom Grifola frondosa. Mass spectrometry and NMR analyses were used to elucidate the structure of this compound, and its absolute configuration was determined using circular dichroism spectroscopy. Compound 1 exhibited specific antifungal activity against the plant pathogens, Fusarium oxysporum, Gibberella zeae and Piricularia oryzae and the opportunistic human pathogen, Pseudallescheria boydii, resulting in minimum inhibitory concentration values of 2.5, 2.5, 1.25 and 0.15 µg/mL, respectively. In contrast, the furanone showed only weak activity towards Aspergillus spp., Candida albicans and several other fungal strains tested as well as no appreciable antibacterial activity.

A new azaphilone from the entomopathogenic fungus Hypocrella sp.

This report describes the isolation of a new azaphilone, designated hypocrellone A (2), together with five known compounds (1, 3-6) from a submerged culture of the entomopathogenic fungus Hypocrella sp. (isolate WYTY-21). The absolute stereostructures of the two compounds (1 and 2) were elucidated based on 1D and 2D NMR spectroscopic and mass spectrometric data combined with the data from various chemical transformations. Hypocrellone A (2) and three (3-6) of the five known compounds were cytotoxic to hepatoma cells (cell line BEL-7404); IC50 values ranged from 6.2 to 17.4 µM. At 200 µM, none of the six compounds was toxic to normal human liver cells (cell line HL-7702) or to normal human kidney epithelial cells (cell line HEK-293T).