Trichoderma as a powerful fungal disease control agent for a more sustainable and healthy agriculture: recent studies and molecular insights.

MAIN CONCLUSION: Molecular studies have elucidated Trichoderma's biocontrol mechanisms. Since fungicides have limited use, Trichoderma could control disease by new metabolic routes and epigenetic alterations. Due to environmental and health hazards, agrochemicals have been a concern since they were introduced in agriculture. Trichoderma, a well-known fungal genus with different mechanisms of action, is an alternative to pesticides and a great tool to help minimize disease incidence. Trichoderma-treated plants mainly benefit from disease control and growth promotion through priming, and these fungi can modulate plants' gene expression by boosting their immune system, accelerating their response to threats, and building stress tolerance. The latest studies suggest that epigenetics is required for plant priming and could be essential for growth promotion, expanding the possibilities for producing new resistant plant varieties. Trichoderma's propagules can be mass produced and formulated depending on the delivery method. Microsclerotia-based bioproducts could be a promising way of increasing the reliability and durability of marketed products in the field, as well as help guarantee longer shelf life. Developing novel formulations and selecting efficient Trichoderma strains can be tiresome, but patent search indicates an increase in the industrialization and commercialization of technologies and an expansion of companies' involvement in research and development in this field. Although Trichoderma is considered a well-known fungal genus, it still attracts the attention of large companies, universities, and research institutes around the world.

Colletotrichum acutatum and C. gloeosporioides Species Complexes Associated with Apple in Brazil.

Glomerella leaf spot (GLS) is an apple disease that concerns growers due to the increases in severity over the years and the difficulties in control. Species within the Colletotrichum acutatum and C. gloeosporioides species complexes cause GLS, but the proportion of species within each complex in Brazilian apple orchards is not known. The objectives of this study were to identify isolates of Colletotrichum causing GLS on apple orchards in the main Brazilian producing regions to the species level. Two hundred and seven isolates were obtained in orchards in São Paulo (SP), Parana (PR), Santa Catarina (SC), and Rio Grande do Sul (RS) states. Genomic DNA was extracted, and the ITS, GAPDH, CHS-1, and TUB2 genes were amplified and sequenced. The phylogenetic trees were generated using a concatenated alignment. One hundred and fourteen isolates were identified as belonging to the C. acutatum species complex (Cac) and 93 to the C. gloeosporioides species complex (Cgc). Five phylogenetic species were identified: C. melonis (1.9%), C. nymphaeae (47.4%), C. paranaense (2.4%), C. limetticola (3.4%), and C. fructicola (44.9%). In SC, Cgc predominates, but in the states of SP, PR, and RS, Cac was predominant. This is the first report of C. limetticola from apple.

The Point Mutation G461S in the MfCYP51 Gene is Associated with Tebuconazole Resistance in Monilinia fructicola Populations in Brazil.

The ascomycete Monilinia fructicola is the causal agent of brown rot of stone fruit in Brazil, causing major pre- and postharvest losses. For many years, the demethylation inhibitor (DMI) fungicide tebuconazole has been used as the most effective active ingredient for controlling brown rot and, as a result, strains of M. fructicola resistant to this ingredient have emerged in many Brazilian states producing stone fruit. The aim of this study was to investigate the mechanisms associated with the resistance of M. fructicola to DMI tebuconazole. By sequencing the M. fructicola CYP51 (MfCYP51) gene, encoding the azole target sterol 14α-demethylase, a mutation was identified at the nucleotide position 1,492, causing the amino acid substitution from glycine to serine at the codon position 461, associated with reduced tebuconazole sensitivity. In addition, it was observed that MfCYP51 gene expression could play a secondary role in DMI fungicide resistance of M. fructicola strains in Brazil. However, for the specific isolate found to exhibit elevated expression levels of MfCYP51, no insertions that would trigger gene expression were found. Based on the point mutation associated with tebuconazole resistance, an allele-specific polymerase chain reaction method was developed to quickly identify resistant genotypes within the Brazilian population. This is the first report determining molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil. This information provides an important advancement for risk assessment of DMI fungicides used to manage brown rot of stone fruit.

Reduced Sensitivity to Azoxystrobin of Monilinia fructicola Isolates From Brazilian Stone Fruits is Not Associated With Previously Described Mutations in the Cytochrome b Gene.

Quinone-outside inhibitor (QoI) fungicides are effective tools for preharvest control of brown rot of stone fruit. These fungicides have a very specific site of action so the risk of resistance selection is high. The sensitivity of Monilinia fructicola (G. Winter) Honey isolates to azoxystrobin (QoI) was investigated in 143 isolates collected between 2002 and 2011 from four Brazilian states in orchards with different frequencies of fungicide use (0 to 6 fungicides sprays/season). Sensitivity of the isolates to azoxystrobin was determined in vitro, by inhibition of mycelial growth and spore germination on fungicide-amended media or ex vivo by pathogen inoculation in untreated or treated fruit with azoxystrobin. Potential mutations in codons 143, 137, and 129 of the cytochrome b (Cyt b) gene and the occurrence of an intron immediately after codon 143 were analyzed in a subpopulation of the isolates. The M. fructicola population of São Paulo State was less sensitive to the fungicide than the population from the states of Paraná, Santa Catarina, and Rio Grande do Sul. The low sensitivity of the isolates was confirmed also by comparing to the sensitivity of the baseline isolates. Mutations in G143A, F129L, and G137R in Cyt b gene were not found. In addition, 58 isolates tested showed an intron after codon 143 in Cyt b gene. Our results indicate that other mechanisms of selection for low sensitivity to QoI fungicides should be investigated.

Detection of the F129L mutation in the cytochrome b gene in Phakopsora pachyrhizi.

BACKGROUND: Asian soybean rust, caused by Phakopsora pachyrhizi, is mostly controlled by demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides. Mutations in the cytochrome b (CYTB) gene can lead to pathogen resistance to QoIs. The occurrence of the mutations in codons 129, 137 and 143 in the CYTB gene was investigated, and a pyrosequencing assay was developed for rapid and quantitative detection of the F129L mutation. RESULTS: Molecular analysis of the CYTB gene showed the presence of the F129L mutation in field samples and monouredinial isolates, while other mutations (G143A and G137R) were not found. The pyrosequencing was an effective method for quantitative detection of the F129L mutation, and many of the P. pachyrhizi samples showed high frequency of F129L. CONCLUSION: This is the first report of the occurrence of the F129L mutation in P. pachyrhizi. The practical relevance of this mutation for field efficacy of QoIs needs further investigation. © 2015 Society of Chemical Industry.

Sensitivity of Monilinia fructicola from Brazil to Tebuconazole, Azoxystrobin, and Thiophanate-Methyl and Implications for Disease Management.

The aim of this study was to investigate the sensitivity of Monilinia fructicola isolates to tebuconazole (demethylation inhibitor [DMI]), azoxystrobin (quinone outside inhibitor), and thiophanate-methyl (methyl benzimidazole carbamate) in 118 isolates collected from four states in Brazil from orchards with different histories of fungicide use. Sensitivity to fungicides was determined by inhibition of mycelial growth and spore germination on fungicide-amended media. Polymerase chain reaction was used to determine the frequencies of M. fructicola genotypes exhibiting high (HR) and low (LR) resistance to thiophanate-methyl among sampled populations. Resistance to tebuconazole was found in 15.8% of isolates collected from São Paulo State. The 50% effective concentration (EC50) values varied from 0.01 to greater than 100 µg/ml. The EC50 values for tebuconazole corresponded to its historic use frequency in the orchard; for instance, isolates from orchards with no DMI fungicide use had the lowest mean EC50 value (0.04 µg/ml), while those collected from orchards where more than five DMI fungicide sprays were applied per season had a mean EC50 value of 21.17 µg/ml. All isolates were sensitive to azoxystrobin but their EC50, based on tests of inhibition of conidial germination, increased from 0.05 in 2002 to 0.44 µg/ml in 2008. The EC50 values based on mycelial growth inhibition for thiophanate-methyl were >162 µg/ml, 1.99 to 12.5 µg/ml, and <1.0 µg/ml for HR, LR, and sensitive isolates, respectively.