RESUMO
The elicitor AsES (Acremonium strictum elicitor subtilisin) is a 34-kDa subtilisin-like protein secreted by the opportunistic fungus Acremonium strictum. AsES activates innate immunity and confers resistance against anthracnose and gray mold diseases in strawberry plants (Fragaria × ananassa Duch.) and the last disease also in Arabidopsis. In the present work, we show that, upon AsES recognition, a cascade of defense responses is activated, including: calcium influx, biphasic oxidative burst (O2â - and H2O2), hypersensitive cell-death response (HR), accumulation of autofluorescent compounds, cell-wall reinforcement with callose and lignin deposition, salicylic acid accumulation, and expression of defense-related genes, such as FaPR1, FaPG1, FaMYB30, FaRBOH-D, FaRBOH-F, FaCHI23, and FaFLS. All these responses occurred following a spatial and temporal program, first induced in infiltrated leaflets (local acquired resistance), spreading out to untreated lateral leaflets, and later, to distal leaves (systemic acquired resistance). After AsES treatment, macro-HR and macro-oxidative bursts were localized in infiltrated leaflets, while micro-HRs and microbursts occurred later in untreated leaves, being confined to a single cell or a cluster of a few epidermal cells that differentiated from the surrounding ones. The differentiated cells initiated a time-dependent series of physiological and anatomical changes, evolving to idioblasts accumulating H2O2 and autofluorescent compounds that blast, delivering its content into surrounding cells. This kind of systemic cell-death process in plants is described for the first time in response to a single elicitor. All data presented in this study suggest that AsES has the potential to activate a wide spectrum of biochemical and molecular defense responses in F. ananassa that may explain the induced protection toward pathogens of opposite lifestyle, like hemibiotrophic and necrotrophic fungi.
Assuntos
Acremonium/fisiologia , Resistência à Doença , Fragaria/imunologia , Fragaria/microbiologia , Proteínas Fúngicas/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Explosão Respiratória , Subtilisina/metabolismo , Morte Celular/genética , Parede Celular/metabolismo , Fluorescência , Fragaria/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Lignina/metabolismo , Necrose , Moléculas com Motivos Associados a Patógenos/metabolismo , Doenças das Plantas/genética , Folhas de Planta/microbiologia , Ácido Salicílico/metabolismoRESUMO
BACKGROUND: Visceral toxocariasis is a parasitic zoonosis caused by Toxocara canis. The prevalence of this parasite in dogs, soil contamination and the resistance of eggs increase human exposure to the disease. Moreover, the difficulties of the control measures justify the need for alternative ones. AIMS: The objective of this study was to evaluate the in vitro ovicidal activity of fungi isolated from soils from public places in the city of Pelotas, Rio Grande do Sul, Brazil, on Toxocara canis. METHODS: Samples of soil from ten localities were inoculated onto Petri dishes with 2% water-agar (WA) that contained antibiotics, and incubated at 25°C/21 days. Isolated fungi were tested in vitro for ovicidal activity, with five replicates. One mL of an embryonated Toxocara canis egg suspension (10(3) eggs) was poured over the fungal cultures after 10 days of growth. At intervals of 7, 14 and 21 days, 100 eggs were removed from each plaque and evaluated by optical microscopy. RESULTS: Acremonium, Aspergillus, Bipolaris, Fusarium, Gliocladium, Mucor and Trichoderma were isolated from the soil. A significant ovicidal type 3 effect was observed in Trichoderma, Fusarium solani complex and Acremonium. Those isolates from the genus Trichoderma showed their ovicidal effect on the 14th day of fungus-egg interaction. The other fungal genera tested showed a type 2 effect. CONCLUSIONS: These results suggest that the use of Trichoderma and Fusarium solani complex in biological control of T. canis is promising; however, further studies should be performed.
Assuntos
Acremonium/fisiologia , Fusarium/fisiologia , Óvulo/microbiologia , Microbiologia do Solo , Solo/parasitologia , Toxocara canis/microbiologia , Trichoderma/fisiologia , Acremonium/isolamento & purificação , Animais , Agentes de Controle Biológico , Brasil , Reservatórios de Doenças , Doenças do Cão/parasitologia , Doenças do Cão/prevenção & controle , Cães , Feminino , Fungos/isolamento & purificação , Fusarium/isolamento & purificação , Humanos , Especificidade da Espécie , Toxocara canis/fisiologia , Toxocaríase/prevenção & controle , Trichoderma/isolamento & purificação , Zoonoses/prevenção & controleRESUMO
Acremonium implicatum is a seed-transmitted endophytic fungus that forms symbiotic associations with the economically significant tropical forage grasses, Brachiaria species. To take advantage of the endophyte's plant protective properties, we developed an efficient Agrobacterium-mediated transformation system for Acremonium implicatum, using green fluorescent protein (GFP) expression and vector pSK1019 (trpC promoter) or pCAMBIA1300 (CaMV35S promoter). We found that transformation efficiency doubled for both mycelial and conidial transformation as the co-cultivation period for Agrobacterium tumefaciens and Acremonium implicatum was increased from 48 to 72h. Significantly, optimal results were obtained for either mycelial or conidial transformation with Agrobacterium tumefaciens strain AGL-1 and vector pSK1019 under the control of the trpC promoter. However, mycelial transformation consistently generated a significantly higher number of transformants than did conidial transformation. The mitotic stability of the transferred DNA was confirmed by growing ten transformants in liquid and agar media for six generations. In all cases, resistance to the selection pressure (hygromycin B) was maintained. Fluorescence emission was retained by the transformants and also expressed in Brachiaria tissues from plants inoculated with GFP-transformed A. implicatum. This technology will help in the transfer and expression of agronomically important genes in host plants.
Assuntos
Acremonium/genética , Agrobacterium tumefaciens/genética , Simbiose , Transformação Genética , Acremonium/fisiologia , Agrobacterium tumefaciens/metabolismo , Brachiaria/microbiologia , Brachiaria/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Micélio/genética , Micélio/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.