Comparative fitness of Monilinia fructicola isolates with multiple fungicide-resistant phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multi-resistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 µg/ml for TF and AZO and 0.3 µg/ml for TEB. Cyt b, CYP51, and ß-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in ß-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and detached fruit assays. Phenotypes with single-resistance displayed equal fitness in in vitro and fruit assays compared to the wild-type. In contrast, the dual and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

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.

Persimmon anthracnose: a comparative study of aggressiveness on shoot and fruit among Colletotrichum horii isolates in southern Brazil / Antracnose do caquizeiro: um estudo comparativo da agressividade em ramos jovens e frutos entre isolados de Colletotrichum horii no sul do Brasil

ABSTRACT: The persimmon tree is known for its rusticity and productivity and was first introduced to Brazil in the late 19th century. However, anthracnose disease is causing immature fruit drop and severe disease symptoms in persimmon fruit, shoots, flowers, and twigs. The causal agent was first described as the fungal species, Colletotrichum horii, which was first confirmed using only the ITS region. In this study, we compared the aggressiveness of 13 isolates of Colletotrichum spp. obtained from fruit and shoots of persimmon grown in the Metropolitan Region of Curitiba, Paraná State, Brazil. A multilocus molecular analysis was carried out based on ITS, GPDH, and EF genes, and we confirmed that the isolates were confirmed as C. horii. All isolates were pathogenic for unwounded and wounded persimmon fruit but differed in aggressiveness. Only one isolate was non-pathogenic when inoculated into unwounded persimmon shoots. Most isolates caused cankers and shoot death whether shoots were wounded or unwounded. In this study, we emphasized the importance of shoots as a source of primary inoculum. In future studies, it will be critical to further elucidate the epidemiological basis of anthracnose disease by conducting field studies to establish a more effective strategy for disease control.

RESUMO: O caquizeiro foi introduzido no Brasil no final do século XIX. Esta planta é conhecida por sua rusticidade e produtividade. No entanto, a doença antracnose está causando queda e sintomas em frutos imaturos, ramos, flores e galhos no campo. O agente causal foi descrito inicialmente como Colletotrichum hori (confirmado usando apenas a região ITS) causando cancro de galho e sintomas de folhas. Neste estudo, comparamos a agressividade em caqui utilizando uma coleção de 13 isolados de Colletotrichum spp. obtidos de frutos e ramos de caqui da região metropolitana de Curitiba, Paraná, Brasil. Uma análise molecular multilocus foi realizada com base nos genes ITS, GPDH e EF, e os isolados foram confirmados como pertencentes a C. horii. Todos os isolados foram patogênicos em frutos não feridos e feridos, mas diferiram na agressividade. Apenas um isolado não foi patogênico quando inoculado sem ferimento em ramos. A maioria dos isolados foi capaz de causar cancro e morte de ramo, independentemente do ferimento. Neste estudo, enfatizamos a importância das novas brotações como fonte de inóculo primário. Para um próximo estudo, ainda é necessário ampliar as bases epidemiológicas com estudos em campo para estabelecer uma correta estratégia de controle desta doença.