Cleiltan Novais da Silva

Regional and Field-Specific Factors Affect the Composition of Fusarium Head Blight Pathogens in Subtropical No-Till Wheat Agroecosystem of Brazil

A multiyear survey of >200 wheat fields in Paraná (PR) and Rio Grande do Sul (RS) states was conducted to assess the extent and distribution of Fusarium graminearum species complex (FGSC) diversity in the southern Brazilian wheat agroecosystem. Five species and three trichothecene genotypes were found among 671 FGSC isolates from Fusarium head blight (FHB)-infected wheat heads: F. graminearum (83%) of the 15-acetyldeoxynivalenol (15-ADON) genotype, F. meridionale (12.8%) and F. asiaticum (0.4%) of the nivalenol (NIV) genotype, and F. cortaderiae (2.5%) and F. austroamericanum (0.9%) with either the NIV or the 3-ADON genotype. Regional differences in FGSC composition were observed, with F. meridionale and the NIV type being significantly (P<0.001) more prevalent in PR (>28%) than in RS (≤9%). Within RS, F. graminearum was overrepresented in fields below 600 m in elevation and in fields with higher levels of FHB incidence (P<0.05). Species composition was not significantly influenced by previous crop or the stage of grain development at sampling. Habitat-specific differences in FGSC composition were evaluated in three fields by characterizing a total of 189 isolates collected from corn stubble, air above the wheat canopy, and symptomatic wheat kernels. Significant differences in FGSC composition were observed among these habitats (P<0.001). Most strikingly, F. meridionale and F. cortaderiae of the NIV genotype accounted for the vast majority (>96%) of isolates from corn stubble, whereas F. graminearum with the 15-ADON genotype was dominant (>84%) among isolates from diseased wheat kernels. Potential differences in pathogenic fitness on wheat were also suggested by a greenhouse competitiveness assay in which F. graminearum was recovered at much higher frequency (>90%) than F. meridionale from four wheat varieties inoculated with an equal mixture of F. graminearum and F. meridionale isolates. Taken together, the data presented here suggest that FGSC composition and, consequently, the trichothecene contamination in wheat grown in southern Brazil is influenced by host adaptation and pathogenic fitness. Evidence that F. meridionale and F. cortaderiae with the NIV genotype are regionally significant contributors to FHB may have significant implications for food safety and the economics of cereal production.

Fungi Isolated from Maize (Zea mays L.) Grains and Production of Associated Enzyme Activities

Filamentous fungi produce a great variety of enzymes, and research on their biotechnological potential has recently intensified. The objective of this work was to identify, at the species level, using DNA barcoding, 46 fungal isolates obtained from maize grains with rot symptoms. We also analyzed the production of extracellular amylases, cellulases, proteases and lipases of 33 of those fungal isolates. The enzymatic activities were evaluated by the formation of a clear halo or a white precipitate around the colonies in defined substrate media. The found fungi belong to the genera Talaromyces, Stenocarpella, Penicillium, Phlebiopsis, Cladosporium, Hyphopichia, Epicoccum, Trichoderma, Aspergillus, Irpex, Fusarium, Microdochium, Mucor and Sarocladium. In the genus Fusarium, the species Fusarium verticillioides was predominant and this genus presented the highest diversity, followed by the genera Aspergillus. The best genera for lipase production were Cladosporium and Penicillium; while Cladosporium, Aspergillus and Penicillium were best for cellulase activity; Hyphopichia, Aspergillus and Irpex for amylase activity; and Cladosporium and Sarocladium for proteases activity. In conclusion, a collection of fungi from maize seeds presenting rotten symptoms were obtained, among which exist important producers of hydrolases.

New PCR Assays for the Identification of Fusarium verticillioides, Fusarium subglutinans, and Other Species of the Gibberella fujikuroi Complex

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.

Species Identification, Genetic Diversity and Phenotypic Variation Studies on the Fusarium graminearum Complex Populations from Brazil

Fusarium graminearum sensu lato, the cause of Fusarium head blight (FHB), has been reported for more than 50 years in Brazil. However, it was only during the mid-1980s to late 1990s that FHB resurged as a primary concern of wheat production. More than a dozen studies in Brazil or elsewhere have been conducted since the year 2000 employing novel and accurate methods to explore genetic variability and identify the strains to phylogenetic species. Thus far, six species of the F. graminearum species complex (FGSC) have been reported in association with wheat, barley, oat, soybean, maize, rice and ryegrass in Brazil, and their prevalence rates appears to be dependent on the region and host surveyed. Although F. graminearum sensu stricto (s. str.) is dominant in wheat, F. meridionale is the most frequently found pathogen in maize, and F. asiaticum is the only species found in rice. Most of them preferentially exhibit specific trichothecene genotypes: F. graminearum s. str. is of the 15-acetyl-deoxynivalenol (DON) genotype, F. meridionale and F. asiaticum are of the nivalenol (NIV) genotype, and F. austroamericanum is of the 3-ADON genotype; an exception is that F. cortaderiae exhibits either the 3-ADON or NIV genotype. They have also exhibited differences in relation to their reproductive fitness, fungicide sensitivity and pathogenicity toward wheat and maize. Continuous monitoring of the genotypic and phenotypic traits of the FGSC populations will help to improve local disease management and contribute to the global knowledge of the biology of the pathogen.

Cercospora zeina is the main species causing gray leaf spot in southern and central Brazilian maize regions

Maize (Zea mays) fields from a tropical central (Goiás state) and a subtropical south region (Paraná state) of Brazil were surveyed for Cercospora species causing gray leaf spot (GLS), a foliar fungal disease of increasing concern to growers of the country. Single spore isolates were obtained from maize leaves showing GLS symptoms and species-specific primers were used for species identification. In addition, a phylogeny of isolates representative of each species was conducted based on partial DNA sequencing of the EF1-α, CAL, and HIS genes, and ITS-5.8S rDNA region. In the regional collections, Cercospora zeina was the dominant species in Goiás (68/75 isolates) and Paraná (73/75 isolates), followed by C. zeae-maydis and C. sorghi var. maydis, respectively. In three field collections of both states, C. zeina was the sole species found. The phylogenetic characterization of isolates representing C. zeina (n = 18), C. zeae-maydis (n = 6), and C. sorghi var. maydis (n = 2) confirmed the identity of the species and grouped them in well-supported clades.

Aplicação de suspensões de Agaricus blazei, Lentinula edodes e de acibenzolar-S-metil na redução da antracnose em frutos de maracujá-azedo

Anthracnose caused by Colletotrichum gloeosporioides is one of the most important diseases of passion fruit. This study evaluated the potential of Agaricus blazei and Lentinula edodes at concentrations of 20, 40 and 60% (v/v) and acibenzolar-S-methyl (ASM) at 75 and 150 mg/L, applied alone or in combination, for protecting passion fruit against anthracnose. The fruits were treated (immersion, sprayings or in coverage of cassava starch) and after 36h or 72h were inoculated with the fungus. Disease assessment was done by measuring the necrotic area in the fruits at 4, 8 and 12 dai (days after inoculation) in assays I and II and 3, 6 and 9 dai in assays III, IV and V. At three days after inoculation, the treatment with A. blazei associated with cassava starch 3% showed a significant reduction of necrotic area compared to the control (p = 0.035). The L. edodes and ASM treatments did not reduce the disease in any concentration and/or periods of the evaluations. When the compounds were applied in combination and the fruits were not washed before inoculation, there was a significant reduction of necrotic area six days after inoculation (p = 0.03). However, when fruits were treated, washed and inoculated with the fungus there was no reduction of disease in any of the periods evaluated, showing that the mode of action of the compounds used possibly occurs by a protective/residual effect coupled with an inhibitory effect on C. gloeosporioides.