Proteomics analysis of a tobacco variety resistant to brown spot disease and functional characterization of NbMLP423 in Nicotiana benthamiana.

BACKGROUND: Tobacco brown spot disease is an important disease caused by Alternaria alternata that affects tobacco production and quality worldwide. Planting resistant varieties is the most economical and effective way to control this disease. However, the lack of understanding of the mechanism of tobacco resistance to tobacco brown spot has hindered progress in the breeding of resistant varieties. METHODS AND RESULTS: In this study, differentially expressed proteins (DEPs), including 12 up-regulated and 11 down-regulated proteins, were screened using isobaric tags for relative and absolute quantification (iTRAQ) by comparing resistant and susceptible pools and analyzing the associated functions and metabolic pathways. Significantly up-regulated expression of the major latex-like protein gene 423 (MLP 423) was detected in both the resistant parent and the population pool. Bioinformatics analysis showed that the NbMLP423 cloned in Nicotiana benthamiana had a similar structure to the NtMLP423 in Nicotiana tabacum, and that expression of both genes respond rapidly to Alternaria alternata infection. NbMLP423 was then used to study the subcellular localization and expression in different tissues, followed by both silencing and the construction of an overexpression system for NbMLP423. The silenced plants demonstrated inhibited TBS resistance, while the overexpressed plants exhibited significantly enhanced resistance. Exogenous applications of plant hormones, such as salicylic acid, had a significant inducing effect on NbMLP423 expression. CONCLUSIONS: Taken together, our results provide insights into the role of NbMLP423 in plants against tobacco brown spot infection and provide a foundation for obtaining resistant tobacco varieties through the construction of new candidate genes of the MLP subfamily.

Biochar improved the composting quality of seaweeds and cow manure mixture and altered the microbial community.

The beneficial effects of biochar addition during composting have been proved for many feedstocks, like manures and crop straws. However, the effect of biochar on the quality of composting product with seaweed as the feedstock and the bacterial response has not been investigated. In this study, the wheat straw biochar addition on the quality of the composting product and the bacterial response was explored at the rate of 0-10%. The results showed that biochar addition at the optimal rate (5%, w/w) could increase the germination index and the ratio of the optical density of humic acid at 460 nm to that at 660 nm (E4/E6) of the composting product, which indicated the decreased biotoxicity and enhanced compost maturity. The significant increase of the nitrate nitrogen (NO3 --N) content of the composting product proved the improvement of N cycling during composting process with biochar addition. The bacterial community of composting product was shifted and the relative abundance of some beneficial taxa (e.g., Muricauda and Woeseia) was significantly increased with biochar addition. Furthermore, the relative abundance of some bacterial genes related to amino acid metabolism and carbohydrate metabolism was also increased with biochar addition. The results of our study provided the positive effect of biochar addition on the composting of seaweed and could help to produce high quality seaweed fertilizer by composting with biochar addition.

Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR.

Vascular wilt, caused by Verticillium dahliae and V. longisporum, limits the quality and yield of agricultural crops. Although quantitative real-time PCR (qPCR) has greatly improved the diagnosis of these two pathogens over traditional, time-consuming isolation methods, the relatively poor detection sensitivity and high measurement bias for traceable matrix-rich samples need to be improved. Here, we thus developed a droplet digital PCR (ddPCR) assay for accurate, sensitive detection and quantification of V. dahliae and V. longisporum. We compared the analytical and diagnostic performance in detail of ddPCR and the corresponding qPCR assay against the genomic DNA (gDNA) of the two fungi from cultures and field samples. In our study, the species specificity, quantification linearity, analytical sensitivity, and measurement viability of the two methods were analyzed. The results indicated that ddPCR using field samples enhanced diagnostic sensitivity, decreased quantification bias, and indicated less susceptibility to inhibitors compared with qPCR. Although ddPCR was as sensitive as qPCR when using gDNA from cultures of V. dahliae and V. longisporum, its detection rates using field samples were much higher than those of qPCR, potentially due to the inhibition from residual matrix in the extracts. The results showed that digital PCR is more sensitive and accurate than qPCR for quantifying trace amounts of V. dahliae and V. longisporum and can facilitate management practices to limit or prevent their prevalence.

Effects of Oxathiapiprolin on the Structure, Diversity and Function of Soil Fungal Community.

Pesticides can affect non-target microorganisms in the soil and are directly related to soil microecological health and environmental safety. Oxathiapiprolin is a piperidinyl thiazole isoxazoline fungicide that shows excellent control effect against oomycete fungal diseases, including late blight, downy mildew, root rot, stem rot, and blight. Though it can exist stably in the soil for a long time, its effects on soil microbial structure and diversity are not well investigated. In the present study, the effects of oxathiapiprolin on the abundance and diversity of soil fungal communities in typical farmland were studied. The results show that the abundance and diversity of soil fungi were increased by oxathiapiprolin treatment with differences not significant on the 30th day. Oxathiapiprolin was found to change the structure of soil fungal communities, among which Ascomycota and Mortierellomycota were the most affected. Undefined saprophytic fungi increased in the treatment groups, and the colonization of saprophytic fungi can act as a major contributor to the function of soil microbial communities. This study lays a solid foundation regarding environmental behavior with the use of oxathiapiprolin in soil and details its scientific and rational use.

Toxicity of the novel fungicide oxathiapiprolin to Chlorella vulgaris: Assessments at different levels of biological organization.

Oxathiapiprolin (Otp) is the first successful oxysterol-binding protein (OSBP) inhibitor in oomycete control. It is regarded as a significant milestone in the history of fungicide discoveries and has vast application prospects. There is little available information on the ecotoxicity of Otp to aquatic organisms. In this study, we evaluated the toxic effects of Otp in the Chlorella vulgaris (C. vulgaris). The results revealed the acute toxicity of Otp to C. vulgaris, with a 96-h median effective concentration for growth inhibition of 0.74 mg/L. When algal cells were exposed to 0.5 and 1.5 mg/L Otp, their chlorophyll and carotenoid contents dropped dramatically. As suggested by the significant increase in reactive oxygen species (ROS) and malondialdehyde (MDA) levels and the remarkable changes in the activity of a series of antioxidant enzymes, Otp induces production of ROS, resulting in oxidative damage. In addition, Otp can damage cell structures and could destroy membrane integrity. Finally, the changes in endogenous substances indicated that Otp can perturb energy metabolism and photosynthesis in C. vulgaris cells. The experimental results suggest that Otp can have toxic effects on algal cells by disturbing photosynthesis and causing oxidative damage and abnormal energy metabolism in C. vulgaris cells.

Effects of biocontrol Bacillus and fermentation bacteria additions on the microbial community, functions and antibiotic resistance genes of prickly ash seed oil meal-biochar compost.

This study evaluated the effects of biocontrol Bacillus and fermenting bacteria addition on the microbial community, metabolic functions and antibiotic resistance genes (ARGs) of new prickly ash seed oil meal (PSOM)-biochar composting. The results showed that the addition of Bacillus subtilis and fermentation bacteria significantly increased the NH4+-N, bacterial abundance and fungal diversity of compost while decreasing the relative abundances (RAs) of carbon metabolism genes in mature compost. NH4+-N was significantly correlated with microbial abundance and diversity, and its increase was closely related to microbial amino acid metabolism. The addition of biocontrol and fermenting bacteria changed the RAs of ARGs, which was caused by changes in the potential hosts Proteobacteria, Bacteroidota and Firmicutes in the compost. Consequently, adding Bacillus and fermenting bacteria into PSOM to make composting was suggested as an effective method to promote nutrient transformation, regulate microbial activity and decrease RAs of tetracycline and vancomycin ARGs.

An efficient method to improve the production of methane from anaerobic digestion of waste activated sludge.

Methane production from waste activated sludge (WAS) anaerobic digestion is always low due to slow hydrolysis rate and inappropriate ratio of carbon to nitrogen (C/N). In this work, a novel approach, i.e., co-digestion of WAS and tobacco waste (TW) using ozone pretreatment, to greatly enhance the production of methane is reported. Experimental results showed the optimal C/N and ozone dosage for methane production was 24:1 and 90 mg/g suspended solids, and the corresponding methane production was 203.6 mL/g volatile suspended solids, which was 1.3-fold that in mono-WAS digestion. Further investigation showed the co-digestion of WAS and TW was beneficial to the consumptions of protein and cellulose; also, the presence of ozone enhanced the disruption of organic substrates and production of short chain fatty acids, which provided sufficient digestion substrates for methane generation. Analysis of microbial community structure suggested that members of the phyla Bacteroidetes and Firmicutes were the dominant species when ozone pretreatment was applied. The findings obtained in this work might be of great importance for the treatment of WAS and TW.