RESUMEN
Antimicrobial activity contributes to plant disease control property of composts but its source is still not clear. From composting cow manure during secondary fermentation, 50 microbial strains with antifungal activity were isolated and identified. Two bacterial strains Bacillus mojavensis B282 and Pseudomonas aeruginosa F288, antagonistic against both phytopathogenic fungi and bacteria, were respectively used as the inoculum of compost for secondary fermentation. Inoculation of B282 or F288 significantly shifted microbial community structure of compost and genera functionally linked to antagonistic activity and plant growth promotion were enriched. Notably, culturable cells of B282 increased by about 40 times during secondary fermentation. The inoculation of each strain significantly increased antifungal activity of compost extracts and enhanced disease suppressive effects of compost on wheat root rot. This study demonstrates that inoculation of compost-indigenous microorganisms could improve antimicrobial activity of compost and provides a low-cost strategy for producing bio-organic fertilizers with biocontrol function.
Asunto(s)
Compostaje , Fermentación , Antifúngicos/farmacología , Estiércol , Fertilizantes/análisis , Bacterias , Suelo/químicaRESUMEN
A novel strain AS1 with heterotrophic nitrifying-aerobic denitrifying capacity in the species of Alcaligenes aquatilis was isolated from the aerobic activated sludge. It showed a great capability of ammonia removal, and the aerobic metabolic pathways to yield gaseous-nitrogen by hydroxylamine oxidation and nitrite denitrification were proposed. AS1 could efficiently remove ammonia under a wide range of environmental conditions, including the ratio of chemical oxygen demand to total nitrogen: 15-30, pH: 6-10, NaCl: 0-60 g/L, shaking speed of 0-180 rpm, and succinate, acetate, or citrate as carbon source. In the treatment of actual piggery wastewater, 95.3%, 95.1% and 84.9% of NH4+-N was removed by AS1 when the initial ammonia concentration was 500, 1300, and 2000 mg/L, respectively, with the maximum NH4+-N removal rate of 30.5 mg/L/h and 569.7 mg/L/d. Furthermore, plate colony-counting showed that AS1 achieved an efficient proliferation. These results imply the application potential of AS1 in treating high-ammonia wastewater.
Asunto(s)
Nitrificación , Aguas Residuales , Aerobiosis , Alcaligenes , Amoníaco/metabolismo , Desnitrificación , Procesos Heterotróficos , Nitritos/metabolismo , Nitrógeno/metabolismo , Aguas Residuales/químicaRESUMEN
The emergence of antifungal resistance, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, makes fungal infections difficult to treat in clinics and agriculture. When exposed to azoles, fungi can make adaptive responses to alleviate azole toxicity and produce azole tolerance. However, except for azole efflux pumps and ergosterol biosynthesis genes, the role of most azole responsive genes in azole resistance is unknown. In this study, STK-17, whose transcription is upregulated by azoles, was characterized as a novel kinase that is required for azole resistance. Deletion or dysfunction of STK-17 led to azole hypersensitivity in Neurospora crassa and to other ergosterol biosynthesis inhibitors such as amorolfine, terbinafine, and amphotericin B, but not fatty acid and ceramide biosynthesis inhibitors. STK-17 was also required for oxidative stress resistance, but this was not connected to azole resistance. RNA-seq results showed that stk-17 deletion affected the basal expression and the response to ketoconazole of some membrane protein genes, indicating functional association of STK-17 with the membrane. Notably, deletion of stk-17 affected the normal response to azoles of erg genes, including the azole target-encoding gene erg11, and erg2, erg6, and erg24, and led to abnormal accumulation of sterols in the presence of azoles. HPLC-MS/MS analysis revealed increased intracellular azole accumulation in the stk-17 mutant, possibly due to enhanced azole influx and reduced azole efflux that was independent of the major efflux pump CDR4. Importantly, STK-17 was widely distributed and functionally conserved among fungi, thus providing a potential antifungal target. IMPORTANCE Antifungal resistance is increasing worldwide, especially to the most widely used azole class of ergosterol biosynthesis inhibitors, making control of fungal infections more challenging. A lot of effort has been expended in elucidating the mechanism of azole resistance and revealing potential antifungal targets. In this study, by analyzing azole-responsive genes in Neurospora crassa, we discovered STK-17, a novel kinase, that is required for azole resistance in several types of fungi. It has a role in regulating membrane homeostasis, responses to azole by ergosterol biosynthesis genes and azole accumulation, thus, deepening our understanding on the mechanism of azole stress response. Additionally, STK-17 is conserved among fungi and plays important roles in fungal development and stress resistance. Kinase inhibitors are broadly used for treating diseases, and our study pinpoints a potential drug target for antifungal development.
Asunto(s)
Antifúngicos/metabolismo , Azoles/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimología , Proteínas Quinasas/metabolismo , Antifúngicos/farmacología , Azoles/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/genética , Farmacorresistencia Fúngica , Ergosterol/biosíntesis , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Homeostasis , Pruebas de Sensibilidad Microbiana , Neurospora crassa/efectos de los fármacos , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas Quinasas/genéticaRESUMEN
Azoles are the most widely used antifungals for controlling fungal infections in clinic and agriculture. Fungi can adapt to azole stress by rapidly activating the transcription of a number of genes, and some of these genes can elevate resistance to azoles. We had reported the transcription factor CCG-8 as a new regulator in the adaptation to antifungal azole stress in Neurospora crassa and Fusarium verticillioides. In this study, we further investigate the mechanisms by which CCG-8 promotes fungal adaptation to azole stress using N. crassa as a model. While deletion of ccg-8 made N. crassa hypersensitive to azoles, ccg-8 overexpression strain was more resistant to azoles than wild type, which further confirmed the positive role of ccg-8 in the adaptation to antifungal azoles. Liquid chromatography-mass spectrometry analysis showed that deletion of ccg-8 resulted in decrease of ergosterol biosynthesis, and high accumulation of toxic sterol 14α-methyl-3,6-diol and ketoconazole (KTC) in the cells, whereas intracellular accumulation of ketoconazole was decreased in the ccg-8 overexpression strain as compared to wild type. For analyzing the effect of CCG-8 on azole export, we tested the contribution of predicted multidrug transporters to azole resistance and found that CDR4 is the major contributor for azole efflux in N. crassa. Interestingly, overexpression of cdr4 or erg11 in the ccg-8 deletion mutant restored its hypersensitive phenotype and overexpression of cdr4 can reduce the level of intracellular KTC. However, the double mutant of ccg-8 and cdr4 was more sensitive than each single mutant, suggesting that drug efflux pump CDR4 plays less contribution for intracellular azole accumulation in the ccg-8 deletion mutant, and CCG-8 may regulate drug uptake. Together, our results revealed that CCG-8 plays a pivotal role in azole adaptive responses of N. crassa by regulating the drug accumulation in the cells.