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Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD.

The dicarboximide fungicide iprodione has been used in Brazilian peach orchards for more than three decades to control brown rot and there are no studies on sensitivity to Monilinia fructicola in the country. In addition, isolates with reduced sensitivity to other important groups of fungicides have already been reported in Brazilian isolates. The sensitivity of M. fructicola to iprodione was evaluated using discriminatory doses and concentration resulting in 50% inhibition of mycelial growth (EC50) values. In total, 174 isolates collected from the main peach production areas were characterized for fungicide sensitivity. Disease control efficacy (DCE) was determined by comparing lesion inhibition on iprodione treated fruits with those without fungicide treatment. No resistant phenotypes were found in the M. fructicola population and 71.8% of the isolates were extremely sensitive to iprodione. The EC50 values of the isolates ranged from 0.13 to 1.01 μg/mL. DCE in wounded and non-wounded fruit was greater than 85% providing an efficient control in controlled conditions. These results suggest that iprodione could be integrated into disease management programs to prevent the establishment of resistant populations as already reported to other fungicides.

Pleoroma fotherghillae, also known as "princess flower", is an ornamental species native to Brazil and naturalized in several countries (Faravani et al. 2007). P. fotherghillae has a high economic value, with an ornamental and landscape application (Nienow et al. 2010). In September 2018, leaf spots were observed in approximately 80% of the 50 P. fotherghillae plants grown in a nursery in the municipality of Curitiba-Parana, Brazil. The spots were round-shaped, with a necrotic brown center and a reddish-brown halo, ranging from 1 to 4 mm in diameter. High leaf fall was observed among plants presenting a higher severity. Symptomatic leaves fragments were collected and disinfected as described by (Pereira et al. 2019). The fragments were transferred to a potato dextrose agar medium supplemented with streptomycin sulfate and incubated at 24 ± 1oC with a photoperiod of 12 h for 7 days. Four monosporic cultures were obtained from colonies isolated. The isolates had a grayish-white cottony aerial mycelium and reverse olive-yellow with black dots. The colonies reached approximately 60 mm in diameter, forming globular and conical pycnidia, brown to black in color with white or cream globular conidial mass. Beta conidia were hyaline, smooth, curved to the size of 19 - 25 x 1 - 1.5 μm (n = 50). No alpha nor gamma conidia were observed. The characteristics are similar to the description of Diaporthe terebinthifolli (Gomes et al. 2013). The total genomic DNA of a representative isolate, LEMIDPRPf-19-02, was extracted for amplification and sequencing of the internal transcribed spacer (ITS) region and partial of the Tubby (TUB) and thyrotroph embryonic factor (TEF) genes. The sequences of the ITS (No MN415990.1), TUB (No MW505549), and TEF (No MW505550) genes were deposited in GenBank. BLAST analysis showed similarity above 99% with D. terebinthifolli sequences (KC343219.1, KC344187.1, and KC343945.1). The multigene phylogenetic analysis, based on Bayesian Inference, grouped the isolate in a clade with other sequences of Diaporthe terebinthifolii. Four healthy plants of P. fotherghillae about 5 months old, were used for pathogenicity testing. A suspension containing 105 conidia/ml was sprayed on the surface of the leaves of four plants to the point of runoff. The plants were covered with a transparent plastic bag for 24 hours. The leaves of four other plants received sterile distilled water and served as the control treatment. The plants were kept in a greenhouse at 20±5oC. Necrotic lesions appeared 10 to 15 days after inoculation. No symptoms were observed in the control plants. The pathogen was reisolated from symptomatic leaves and had the same characteristics as the isolate LEMIDPRPf-19-02. A representative sample (MBM 331603) was deposited at the Museu do Jardim Botânico (Botanical Garden Museum) - Curitiba, Brazil. Diaporthe terebinthifolii was previously reported as endophytic in Brazil and Uruguay, isolated from Schinus terebinthifolius and Pyrus communis, respectively (Gomes et al. 2013; Sessa et al. 2017). To our knowledge, this is the first report of D. terebinthifolii causing leaf spot on P. fotherghillae in Brazil and worldwide.

The objectives of this work were to investigate the Botrytis species that cause gray mold in strawberries in Parana and to study their sensitivity to fungicides: procymidone (P), iprodione (I), boscalid (B), thiophanate-methyl (T), fluazinam (F), azoxystrobin (A) and difenoconazole (D). The identification of 150 isolates of Botrytis sp. was carried out by amplifying the DNA by PCR using specific primers. The sensitivity to fungicides was determined by in vitro assays with discriminatory doses and EC50 (effective concentration to inhibit 50% of the growth of the fungus), molecular characterization of mutations and ex vivo assays for disease control efficacy. Thirty phenotypes were selected from combinations (S-sensitive and R-resistant) for the EC50 studies. Molecular characterization was performed using specific primers for mutations in the cyt b, bos1 and β-tubulin genes to confirm the resistance genotype. Tests were carried out with fruit treated with the fungicides (P), (B), (F) and (A) x the different phenotypes. All isolates were confirmed as Botrytis cinerea. Forty-one isolates were classified as fungicide sensitive phenotypes and of these, six isolates showed resistance to all seven tested fungicides. The percentage of isolates resistant to the fungicides azoxystrobin, boscalid, difenoconazole, fluazinam, iprodione and procymidone was 90.7; 45.3; 33.3; 32.7; 44.0 and 43.3%, respectively. A total of 94.6% isolates were classified as highly resistant to thiophanate-methyl. The EC50 for the fungicides procymidone, fluazinam and boscalid ranged from less than 0.1 to greater than 100 μg/ml. For azoxystrobin, most isolates had an EC50 greater than 100 μg/ml. Isolates with more than one mutation for different fungicides were found. For the assay in fruits with infections from the field, only the fungicide fluazinam differed from the control treatment, showing lower final incidence of 33.3% and control efficiency of 55.6%. For the assay with inoculated fruits, the fungicide fluazinam best controlled the disease for the three isolates presenting an average final incidence of 33.3% and an average control efficiency of 61.9%. Therefore, disease management in the state of Parana should be reviewed in terms of registering new fungicides to gray mold and the use of integrated management measures to reduce the reliance on fungicides.

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.