RESUMEN
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
Fusarium verticillioides, an important maize pathogen, produce fumonisins, causes stalk rot and consequentially reduce crop growth and yield. Therefore, herein we aimed to evaluate the potential use of two farmyard soil organic manures, i.e., fresh (5-6 days old) and stored (5-6 months old) organic manure, to manage F. verticillioides infections as well as borer incidence and lodging in maize plants. After 30, 60, and 90 days of sowing, samples of soil, roots, and stems were collected to isolate F. verticillioides. Moreover, we estimated ear and kernel rot induced by F. verticillioides at the final harvest. Fresh organic manure treatment increased infection rates of F. verticillioides in soil, roots, stem and kernels compared to the control treatment. In contrast, stored organic manure plots treatments decrease F. verticillioides frequency. At 90 days after sowing, stored organic manure suppressed the survival of F. verticillioides, which reduced the F. verticillioides incidence percent. These results were similar to the effect of herbicides-and insecticide-treated plots demonstrated, which show a significant decrease in F. verticillioides incidence rates. Mycological analysis on symptomless kernels revealed a higher % of pathogen infection in opened husks variety (Balady) than closed husks variety (SC10). Compared with stored organic manure, the stem borer incidence and lodging percentage were the highest in fresh organic manure plots. Finally, these results demonstrated that storing organic manure within five to six months as farmyard manure led to high-temperature centigrade within organic manure, thereby destroying spores of F. verticillioides, whereas fresh organic manure did not.
RESUMEN
O uso sucessivo de híbridos de milho com resistência variada aos fungos causadores de podridão de grãos tem causado aumento na importância dessas doenças a cada safra. Conduziu-se este trabalho com o objetivo de avaliar pelo teste de sanidade (blotter test) a severidade do fungo Fusarium verticilioides e a incidência dos fungos Stenocarpella maydis e Stenocarpella macrospora em grãos de milho, oriundos de híbridos comerciais, com e sem a inoculação artificial a campo, em dois sistemas de cultivo, convencional e sistema de semeadura direta e em dois anos agrícolas. O experimento foi conduzido a campo, em blocos casualizados, com três repetições. Em laboratório, foram avaliadas, pelo método de "blotter test", a severidade do fungo F. verticilioides e a incidência dos fungos S. maydis e S. macrospora causadores de podridões de espiga. A avaliação pelo teste de sanidade (blotter test) evidenciou a diferença entre os híbridos quanto a reação aos fungos F.verticilioides, S. maydis e S. macrospora. Houve influência do híbrido, da safra agrícola e do sistema de cultivo sobre a infecção dos fungos causadores de podridões de espigas, sendo que o sistema plantio direto favorece o aumento dos fungos responsáveis pelos grãos ardidos em milho. A utilização da inoculação artificial, em campo, visando a selecionar genótipos resistentes aos fungos causadores do "complexo de grãos ardidos" é eficiente.
The continued use of corn hybrids with varied resistance to grain rot-causing fungal agents increases the importance of these diseases every year. The objective of this work was to evaluate, by a sanitary test (blotter test), the severity of the fungus Fusarium verticilioides and the incidence of Stenocarpella maydis and Stenocarpella macrospora in corn grain from commercial hybrids, with and without in-field artificial inoculation, under two cultivation systems (conventional and direct sowing), over two crop seasons. The experiment was carried out under field conditions, using an experimental randomized blocks design, with three replications. In the laboratory, the severity of F. verticilioides and the incidence of S. maydis and S. macrospora ,the causative agents of ear rot in corn, were evaluated by the "blotter test" method. The blotter test evaluation allowed us to detect differences between the hybrids in their reactions to the fungi F. verticilioides, S. maydis and S. macrospora. The crop season and cultivation system used had the most influence on fungal infection in the hybrids. The direct sowing system showed an increase in the fungal infection responsible for causing grain rot. The use of artificial inoculation, in-field, to select genotypes resistant to the fungal agents causing the "grain rot complex" is efficient.