Project description:A total of 226 sugarcane rhizosphere-associated bacterial strains from the six different cultivars were screened against three pathogenic strains of C. falcatum (cfNAV, cfCHA, and cf8436) for the suppression of red rot disease. On the basis of mycelial growth inhibition in dual culture assay, 26 bacteria were selected for further characterization of morphology, biochemical activity, plant-growth-promoting (PGP) activity, antifungal potential and molecular identity by 16S rRNA gene sequence. On the basis of the 16S rRNA gene sequencing, it was found that the isolates belonged to proteobacteria (13), Firmicutes (10), and Bacteroides (3). The antagonistic bacteria tested for PGP traits revealed that 10 strains were able to solubilize tricalcium phosphate, 11 strains were able to produce siderophore, and 14 strains were able to grow in the N-free medium. The quantitative estimation of indole-3-acetic acid production was ranged from 21.58 to 66.31??g/mL. On the basis of PGP and biocontrol traits, five strains Ochrobactrum intermedium (TRD14), Acinetobacter sp. (PK9), Bacillus sp. (RSC29 and KR91) and Escherichia sp. (VRE34) were further chosen for pot trial under greenhouse conditions on highly susceptible variety CoC671. The results showed that the pathogen-inoculated sugarcane plants were able to germinate but died within one month. However, the CoC671 inoculated with selected biocontrol strains found protected from disease and an increase in plant growth parameters on par with carbendazim fungicides. This study proves that the isolates identified in this study could be used as an alternative to chemical fungicides to control red rot pathogen of sugarcane plants.
Project description:We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T 0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.
Project description:Plants respond to pathogens and insect attacks by inducing and accumulating a large set of defense-related proteins. Two homologues of a barley wound-inducible protein (BARWIN) have been characterized in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Induction of SUGARWINs occurs in response to Diatraea saccharalis damage but not to pathogen infection. In addition, the protein itself does not show any effect on insect development; instead, it has antimicrobial activities toward Fusarium verticillioides, an opportunistic fungus that usually occurs after D. saccharalis borer attacks on sugarcane. In this study, we sought to evaluate the specificity of SUGARWIN2 to better understand its mechanism of action against phytopathogens and the associations between fungi and insects that affect plants. We used Colletotrichum falcatum, a fungus that causes red rot disease in sugarcane fields infested by D. saccharalis, and Ceratocystis paradoxa, which causes pineapple disease in sugarcane. We also tested whether SUGARWIN2 is able to cause cell death in Aspergillus nidulans, a fungus that does not infect sugarcane, and in the model yeast Saccharomyces cerevisiae, which is used for bioethanol production. Recombinant SUGARWIN2 altered C. falcatum morphology by increasing vacuolization, points of fractures and a leak of intracellular material, leading to germling apoptosis. In C. paradoxa, SUGARWIN2 showed increased vacuolization in hyphae but did not kill the fungi. Neither the non-pathogenic fungus A. nidulans nor the yeast S. cerevisiae was affected by recombinant SUGARWIN2, suggesting that the protein is specific to sugarcane opportunistic fungal pathogens.
Project description:Colletotrichum higginsianum is a hemibiotrophic ascomycetous fungus that causes economically important anthracnose diseases on numerous monocot and dicot crops worldwide. As a model pathosystem, the Colletotrichum⁻Arabidopsis interaction has the significant advantage that both organisms can be manipulated genetically. The goal of this review is to provide an overview of the system and to point out recent significant studies that update our understanding of the pathogenesis of C. higginsianum and resistance mechanisms of Arabidopsis against this hemibiotrophic fungus. The genome sequence of C. higginsianum has provided insights into how genome structure and pathogen genetic variability has been shaped by transposable elements, and allows systematic approaches to longstanding areas of investigation, including infection structure differentiation and fungal⁻plant interactions. The Arabidopsis-Colletotrichum pathosystem provides an integrated system, with extensive information on the host plant and availability of genomes for both partners, to illustrate many of the important concepts governing fungal⁻plant interactions, and to serve as an excellent starting point for broad perspectives into issues in plant pathology.