Herbicides reduce the severity and sporulation of Phakopsora pachyrhizi in soybean with triple herbicide resistance.

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.

SdhC-I86F Mutation in Phakopsora pachyrhizi Is Stable and Can Be Related to Fitness Penalties.

Succinate dehydrogenase inhibitors (SDHIs) fungicides are used to control Asian soybean rust (Phakopsora pachyrhizi), and the SdhC-I86F mutation is related to pathogen resistance. The objective of this study was to determine whether fitness penalties are associated with SDHI resistance (SdhC-I86F mutation) in P. pachyrhizi populations. Moreover, the study investigated whether the SdhC-I86F mutation remained stable after the fungus propagation both in the absence and presence of fungicide. The populations used in this study presented mutations for all genes analyzed (Cyp51, Cytb, and SdhC), except for a wild-type population (WTSdhC) found with no SdhC-I86F mutation. The frequencies of the SdhC-I86F mutant populations were stable after 36 generations in the absence of fungicide. However, in the case of the WTSdhC population, the SdhC-I86F mutation was further detected after one generation of the fungus in the presence of the SDHI fungicide, according to the results of a detached leaf assay. Three tests were performed to evaluate fitness components and sensitivity to fungicides (half maximal effective concentration). SdhC-I86F mutant populations were more sensitive to osmotic and oxidative stress than the WTSdhC population; however, the sensitivity to ultraviolet radiation was similar for both populations. All mutated populations were less sensitive than the WTSdhC when using SDHI (azoxystrobin + benzovindiflupyr), but more sensitive to mancozeb. The presence of fitness penalties, the mutation stability, and the sensitivity to mancozeb presented by the SdhC-I86F mutant populations can be relevant to the management of the disease in the field.

First Report of Diaphorte terebinthifolii causing leaf spot on Pleoroma fotherghillae in Brazil.

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.

First Report of Gaeumannomyces radicicola causing stalk rot on maize in Brazil.

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.