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BACKGROUND: Transgenic event DAS44406-6 (E3) makes soybeans that are herbicide [glyphosate (Gly), 2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate] and caterpillar resistant. The E3 soybean was commercially released for the 2021/2022 harvest in Brazil. We conducted this study to test whether Gly and 2,4-D applied alone and in a commercial mixture affect Asian soybean rust (ASR). Assays were conducted in detached leaves and in vivo, in a controlled environment using the herbicides Gly, 2,4-D and Gly + 2,4-D, and pathogen inoculation. Disease severity and spore production were evaluated. RESULTS: Only the herbicides Gly and Gly + 2,4-D inhibited ASR in detached leaves and in vivo. When applied preventively and curatively in vivo, these herbicides reduced the disease severity and spore production of the fungus. In vivo, inhibition of disease severity reached 87% for Gly + 2,4-D and 42% for Gly. A synergistic effect was observed with the commercial Gly + 2,4-D mixture. Application of 2,4-D alone in the in vivo assays did not reduce or increase disease severity. Gly and Gly + 2,4-D act residually in inhibiting the disease. Growing E3 soybeans may combine weed and caterpillar management benefits with ASR inhibition. CONCLUSION: Application of Gly and Gly + 2,4-D herbicides in resistant E3 soybean shows inhibitory activity for ASR. © 2023 Society of Chemical Industry.

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Colletotrichum isolates from apple leaves with symptoms of Glomerella leaf spot (GLS) can cause fruit rot and several small lesion spots, here called Colletotrichum fruit spot (CFS). This work investigated the epidemiological relevance of Colletotrichum species obtained from leaves with GLS in causing diseases in immature apple fruit by comparing different fruit sizes (phenological stages) for symptom development. In the first experiment, five Colletotrichum species were inoculated in 'Gala' (Ø = 5.5 cm) and 'Eva' (Ø = 4.8 cm) fruit in the field (2016/17 season). Subsequently, C. chrysophilum and C. nymphaeae were inoculated in fruit of different sizes (Ø = 2.4 to 6.3 cm) in the field (2017/18 and 2021/22 seasons) and in the laboratory according to the phenological stages of growing fruit. At harvest of the immature inoculated fruit in the field, only CFS symptoms were observed in both cultivars. For Gala, the CFS incidence reached 50% regardless of season, pathogen species, and fruit size. For Eva, CFS symptoms were observed after inoculation with C. melonis in the 2016/17 season and in smaller fruit inoculated with C. chrysophilum and C. nymphaeae in 2021/22. During postharvest, bitter rot symptoms developed, but did not seem to come from CFS symptoms. It can be concluded that the Gala cultivar has a high susceptibility to CFS caused by the two Colletotrichum species of the greatest epidemiological importance for GLS in Brazil in all fruit sizes tested.

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Glomerella leaf spot (GLS) and bitter rot (BR), caused by Colletotrichum spp., are major diseases on apple in southern Brazil. Among integrated pest management tools for disease management in commercial orchards, fungicides remain an important component. This study aimed to identify Colletotrichum spp. from cultivar Eva in Paraná state orchards; evaluate their in vitro sensitivity to cyprodinil, tebuconazole, iprodione, and fluazinam; and determine the baseline in vitro sensitivity of these isolates to benzovindiflupyr and natamycin. Most isolates belonged to Colletotrichum melonis and C. nymphaeae of the C. acutatum species complex. The two species varied in sensitivity to fluazinam and tebuconazole, but no variability was found for any other fungicide. The lowest 50% effective concentration (EC50) values of Colletotrichum spp. were observed for cyprodinil (mean EC50 < 0.02) and benzovindiflupyr (mean EC50 < 0.05); EC50 values were intermediate for fluazinam (mean EC50 < 0.33) and tebuconazole (mean EC50 < 0.14), and they were highest for natamycin (mean EC50 < 5.56) and iprodione (mean EC50 > 12). Cyprodinil and fluazinam are registered for use in Brazil for apple disease management but not specifically for GLS and BR. Tebuconazole is one of the few products registered for Colletotrichum spp. control in apples. In conclusion, flowers and fruitlets can serve as sources of inoculum for GLS and BR disease; C. acutatum was the predominant species complex in these tissues; cyprodinil and fluazinam applications may suppress GLS and BR; and benzovindiflupyr and natamycin warrant further investigation for GLS and BR disease control of apple due to comparably high in vitro sensitivity.

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Persimmon (Diospyros kaki) anthracnose is a major threat in production areas worldwide. Most of the studies are focused on Colletotrichum horii, but other species have been reported as well. The association of distinct Colletotrichum species present in Brazilian persimmon production regions as well as their host ranges are yet elusive. The aims of this work were to identify and characterize Colletotrichum species associated with the persimmon anthracnose. In a survey performed in four production regions of Brazil, 88.7% and 11.3% out of 231 isolates were identified as members of Colletotrichum gloeosporioides species complex (Cgc) or Colletotrichum acutatum species complex (Cac), respectively. A subset of 18 isolates were identified through multilocus phylogenetic analysis, using ITS-rDNA region and two loci, namely GAPDH and TUB2. This study revealed the presence of four species: C. horii (38.8%) and Colletotrichum fructicola (27.7%) from the Cgc and Colletotrichum nymphaeae (27.7%) and Colletotrichum melonis (5.8%), from the Cac. Additionally, 13 isolates were selected for morphological, physiological, and pathogenic analyses. Contrasting characteristics were observed among species of the Cgc and Cac complexes. The optimal temperature for mycelial growth and germination were higher for Cgc species. The percentage of appressoria melanisation also varied across complexes. All the identified species were able to cause anthracnose-like symptoms on persimmon fruit, leaves, shoots, and sepals. Colletotrichum species from persimmon were also able to infect apple and pear. The findings will support decisions to manage anthracnose of persimmon under high infection risk due to multiple host susceptibility.

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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-Paraná, 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.

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Plum and peach are important crops in the southernmost regions of Brazil and in the majority, fresh fruit producers are small producers, which guarantee their family income. Tranzschelia discolor and T. pruni-spinosae are the etiological agents of rust on Prunus domestica (plum) and P. persica (peach) in Brazil (Mendes and Urben, 2020). The molecular characterization of Tranzschelia specimens revealed different clades that are not attributed to known species, showing the need for taxonomic evaluation of Tranzschelia species in the tropics (Scholler et al. 2014; 2019). As Tranzschelia species reported in Brazil were identified only by morphological characteristics, this study aimed to carry out a survey to verify the etiology of rust on plum and peach based on molecular data. In 2018, rust symptoms in peach and plum trees were observed with maximum severity of 30% and 35%, respectively, in three Brazilian states. Symptoms of plum and peach rust are yellowish-green spots visible on the adaxial side of the leaves and uredia/uredinial sori releasing the brown urediniospores on the abaxial side (Supplementary figure 1). Symptomatic leaves of plum and peach were collected at Curitiba in the states of Paraná (lat. 25°25'47" S and long. 49°16'19" W, altitude of 935 meters) in a research station, Videira in Santa Catarina (lat. 27°00'30" S and long. 51°09'06" W, altitude of 750 meters) in a research station and Paranapanema in São Paulo (lat. 23º23'19" S and long. 48º43'22" W, altitude of 610 meters) in a farmer field, and deposited in the herbarium of the Municipal Botanical Museum of Curitiba (MBM 429790 to 429795). Urediniospores collected on plum and peach leaves were all echinulate, obovoid, orange-brown, and measured 18.0 - 33.5 µm × 10.5 - 20.5 µm (n=150) and 22.5 - 40.0 µm × 11.5 - 20.5 µm (n=150), respectively. The genomic DNA of the urediniospores was extracted for amplification and sequencing of the internal transcribed spacer region (ITS) using primers ITS5-u and ITS4-u (Pfunder et al. 2001). The sequences were deposited (Accession Nos. MT786213 to MT786218) and compared to sequences in the GenBank repository using the BLASTn algorithm. The sequences of ITS showed a high percentage of identity (>99%) with sequences from T. discolor (Accession Nos. AB097449, EU014071, KU712078, KY764179, MH599069, MN545867, DQ995341, DQ354542, and KX985768). Additionally, our isolates clustered with others T. discolor in a Bayesian phylogenetic tree based on ITS sequences (study S26663 deposited in TreeBASE) (Supplementary figure 2). A pathogenicity test was carried out on plants by inoculation of a 1.5 × 105 urediniospores mL-1 suspension on the abaxial side of the leaves. Leaves sprayed with sterile water were used as controls. The plants were incubated in a growth chamber (GC) in the dark for 48 h at 23 °C and maintained with 100% RH to establish infections. The inoculated plants were afterwards kept in the GC at a photoperiod of 12 h under same conditions until 14 days when the symptoms and pathogen structures were observed to all six isolates. Control leaves remained symptomless. Tranzschelia discolor infect plants in the genus Prunus, including almond, apricot, nectarine, cherry, peach, and plum (Farr and Rossman 2021). As T. pruni-spinosae was not found, T. discolor is probably the prevalent species in the main regions of Brazil. This information reveals T. discolor as the causal agent of plum and peach rust in Brazil and helps to understand the distribution of this disease in tropics or worldwide.

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Impatiens walleriana (Balsaminaceae), popularly known as Impatiens, is an African succulent and a popular ornamental plant worldwide (GBIF, 2019). In Brazil it is broadly grown indoors and outdoors, including in public parks of Curitiba, State of Paraná (Viezzer et al. 2018). In September 2018, I. walleriana plants showing typical downy mildew symptoms were observed in wastelands and gardens in Curitiba. The symptoms included adaxial chlorotic leaf spots with abundant white sporulation on abaxial side (Supplementary figure 1). The disease led to severe defoliation of the plants and the incidence of the plant disease varied from 20 to 80% of plants in an area ranging from 400 to 40,000 m2. A representative sample was deposited in herbarium of the Museu Botânico Municipal de Curitiba (MBM 331601). The following morphology was observed: Sporangiophores (n = 30), hyaline, thin walled, emerging through stomata, 407.3 to 551.1 µm long, slightly swollen base, first branch at 165.8 to 324.7 µm from base, end branches 5.1 to 13.1 µm long, sporangia (n = 50) hyaline, thin-walled subglobose to ovoid, from 12.8 to 21.9 µm x 12.5 to 17.9 µm, slightly papillate. Due to morphological and genetic variations within the species Plasmopara obducens, Görg et al. (2017) proposed the new species P. velutina and P. destructor. The morphology of the Curitiba specimen was equivalent to that described for P. destructor (Görg et al. 2017). DNA was extracted from LEMIDPRTf-19-02 isolate and the ITS1 and cox2 regions were PCR amplified as described in Görg et al. (2017). The resulting sequences were deposited in GenBank (ITS1, MT680628; cox2, MT952335). A BLASTn analysis of the sequences revealed 100% homology with ITS (MF372742) and cox2 (MF372728) sequences of type strain of P. destructor (GLM-F107554). A Bayesian phylogenetic analysis was performed to compare the sequences from this study with reference sequences for P. obducens, P. destructor and P. velutina (Görg et al. 2017; Salgado-Salazar et al. 2018). The oomycete from Curitiba grouped in a reliable clade with P. destructor (Supplementary figure 2). Pathogenicity was carried out by ex vivo and in vivo tests. For ex vivo, stems with approximately four healthy leaves of I. walleriana (n = 10) were embedded in aluminum grid inside of gerbox with the stem bases immersed in distilled water. The inoculation of five stems was carried out by spraying a suspension with 6 x 104 sporangia mL-1 on the abaxial side of the leaves. Five stems with leaves inoculated with sterile water were used as controls. They were incubated in a growth chamber in the dark for 48 h at 20 °C and another 12 days in a 12 h light photoperiod. The confirmation of pathogenicity in plants (in vivo) was obtained with the inoculation of I. walleriana seedlings (one-month old) grown in 2 dm3 aluminum pots. The inoculation methodology and number of plants were the same as the stems test. After the inoculation, plants were incubated in a growth chamber for 48 h in the dark at 20 °C with 100% RH with nebulization, and another 10 days at a photoperiod of 12 hours of light. For both tests, abundant sporulation was observedwith morphology equivalent to Plasmopara destructor described by Görg et al. (2017). No disease developed on control plants. To our knowledge, this is the first report of P. destructor on I. walleriana in Brazil (Farr and Rossman 2019, Silva et al. 2019) representing a potential loss to flower production and a reduction in flowering period in public gardens and parks.

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Maize (Zea mays L.) is one of the most important commodities, and Brazil is the second-largest maize exporter country in the world. In April 2019, the period of the second crop maize (safrinha), it was observed black decayed lesions on roots and wilting of some maize plants, causing a "sudden death" in a commercial area in the west of Paraná state, Brazil (Figure 1A-C). Symptomatic root and stalk were collected, and tissues surface disinfected with 70% ethanol for 30 s, 1.5% NaOCl for 1 min and rinsed three times in sterile distilled water, slices of necrotic tissues were transferred to potato dextrose agar (PDA) medium and grown for 7 days at 27 ± 1ºC with a photoperiod of 12 h. Pure cultures were obtained through monosporic isolation. The fungal morphology is alike Gaeumannomyces radicicola, which is a synonym of Phialophora radicicola var. radicicola, Harpophora radicicola, P. zeicola, H. zeicola and G. graminis var. maydis (Hernández-Restrepo et al. 2016). Colonies on PDA showed flat, white to light gray at first (Fig. 1D), turning gray to black with age (Fig. 1E). Colony diameter approximately 5.2 cm on PDA in the dark after 7 days at 27ºC. Conidiophores with slightly thickened wall, mostly branched, varying in dimensions, with a range of 57.5-166.5 (avg. 128.7 µm) × 2.9-5.9 (avg. 4.2 µm) n = 25 (Fig. 1H-J). The conidia showed lunate-shaped with rounded ends, produced successively at the apex of phialide, 3.3-9.7 (avg. 6.6 µm) × 1.5-3.6 µm (avg. 2.5 µm), n = 100 (Fig. 1G-J). Morphological characteristics were comparable to the description of this specie (Cain 1952; Gams 2000; McKeen 1952). The total genomic DNA of a representative isolate, LEMIDPRZm 19-01 was extracted and the partial large subunit (28S nrDNA; LSU), internal transcribed spacer nrDNA including the intervening 5.8S nrDNA (ITS), and part of the largest subunit of the RNA polymerase II gene (RPB1) were amplified and sequenced, as following by Hernández-Restrepo et al. (2016) and Klaubauf et al. (2014). The primers to LSU - NL1 (O'Donnel, 1993) and LR5 (Vilgalys; Hester, 1990); ITS - ITS5 and ITS4 (White et al., 1990); and RPB1 - RPB1F and RPB1R (Klaubauf et al., 2014) were used in this study. The gene sequences of LSU (MT123866), ITS (MT114427), and RPB1 (MT123867) were deposited in GenBank and showed 99.67%, 99.75%, and 100% identity with type material G. radicicola CBS 296.53 (KM484962, KM484845, and KM485061). A multi-locus phylogenetic analysis based on Bayesian Inference showed the isolate LEMIDPRZm 19-01 in the G. radicicola clade (Fig. 2). To confirm pathogenicity, ex vivo assays were performed with mycelial PDA discs of 5 mm from a 7-day-old culture using detached roots (adapted method by Degani et al., 2019), on wounded and unwounded stalk and leaves, each treatment consisted of five replications. PDA discs without fungal were used in negative tissue controls. Pathogenicity tests were also conducted in vivo, two experiments performed: i) the stalk tissue was inoculated by sterilized toothpick grown on PDA with fungal mycelium and the leaves inoculated as ex vivo assay, and toothpick without fungal mycelium was used to stalk negative control, whereas PDA discs without fungal were used in the tested leaves; ii) 6 mycelial PDA discs/500 mL were placed on potato dextrose broth (PDB) media and it remained in agitation for 10 days to obtain a mycelial suspension. Subsequently, the mycelial was crushed to soil infestation, and 50 mL from this suspension were dropped in each 2 L maize pot with soil sterilization 10 days after emergence. Maize pots with soil sterilization without mycelium fungal were used as negative controls. Four replications (maize pots), for each treatment, were used in both tests. Experiments were repeated twice. In the ex vivo assay, all inoculated tissues with and without wounds showed necrotic lesions (Fig. 1K-N). In the first in vivo assay, stalk rot symptoms, including wilting of the inoculated plants causing premature plant death, were observed within 6 days (Fig. 1O-Q). In the second in vivo assay, inoculated plants had inferior growth than compared with plant control. Sixty days after inoculation, the plants were removed from the pots and it was observed a roots degeneration with symptoms of necrosis (Fig. 1R-U). No symptoms were detected in the control treatments and the pathogen was re-isolated from symptomatic tissues confirming Koch's postulate for all assays. So far, to our knowledge, the pathogen distribution was reported solely in the west area of Paraná state, but it may become a potential threat to Brazilian maize production. Further monitoring is necessary to better understand the epidemiology of this pathogen to address a strategy for disease control. The pathogen has already been detected in Canada, South Africa, and China. To our knowledge, this is the first report of G. radicicola in Brazil, as well as in South America.

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Despite the resistance problems in Monilinia fructicola, demethylation inhibitor fungicides (DMIs) are still effective for the disease management of brown rot in commercial stone fruit orchards in Brazil. This study aims to investigate the sensitivity of M. fructicola isolates and efficiency of DMIs to reduce brown rot. A set of 93 isolates collected from Brazilian commercial orchards were tested for their sensitivities to tebuconazole, propiconazole, prothioconazole, and myclobutanil. The isolates were analyzed separately according to the presence or absence of the G461S mutation in MfCYP51 gene, determined by allele-specific test. The mean EC50 values for G461S mutants and wild-type isolates were respectively 8.443 and 1.13 µg/ml for myclobutanil, 0.236 and 0.026 µg/ml for propiconazole, 0.115 and 0.002 µg/ml for prothioconazole, and 1.482 and 0.096 µg/ml for tebuconazole. The density distribution curves of DMI sensitivity for both genotypes showed that myclobutanil and prothioconazole curves were mostly shifted toward resistance and sensitivity, respectively. Incomplete cross-resistance was detected among propiconazole and tebuconazole in both wild-type (r = 0.45) and G461S (r = 0.38) populations. No cross-sensitivity was observed among wild-type isolates to prothioconazole and the others DMIs tested. Fungicide treatments on detached fruit inoculated with M. fructicola genotypes showed significant DMI efficacy differences when fruit were inoculated with wild-type and G461S isolates. Protective applications with prothioconazole were more effective for control of both G461S and wild-type isolates compared with tebuconazole. Curative applications with tebuconazole were most effective in reducing the incidence and lesion size of G461S isolates. Sporulation occurred only for G461S isolates treated with tebuconazole under curative and preventative treatments. The differences found among the performance of triazoles against M. fructicola isolates will form the basis for recommendations of rational DMI usage to control brown rot in Brazil.

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Glomerella leaf spot (GLS) of apple is caused by three different Colletotrichum species complexes. This study evaluated the dispersal of Colletotrichum spores related to GLS temporal progress and defoliation. Spores were monitored by air and water runoff in different plant heights, and the temporal progress of GLS and defoliation were assessed. Spores of the pathogen were first cached in the lower part of the tree closer to the ground, confirming the importance of dead leaves on the ground as main source of primary inoculum. In plots with high primary inoculum, the disease increases exponentially during favorable weather conditions. The highest initial inoculum was found in the lower part of the tree, but the highest rate of the disease progress in the upper.

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