First report of Colletotrichum chlorophyti causing soybean anthracnose in Brazil.

Soybean [Glycine max (L.) Merr.] is one of the world's five major food crops, and Brazil produces the highest share at around 42%. Anthracnose caused by Colletotrichum is an important limiting factor to soybean production. In November 2013, anthracnose symptoms, characterized by brown irregular-shaped lesions on petioles, stems, and pods were observed in soybean fields (1% of incidence) in Vera, Mato Grosso State, Brazil. From the five plants gathered in the field, three leaves along with their corresponding petioles were meticulously chosen for the removal of symptomatic tissues. Sampling of these tissues involved carefully cutting a 0.5 × 0.5 cm fragment in the lesion area. The fragments were disinfected with 70% ethanol for 1 min, followed by 1% sodium hypochlorite for 2 min. Then the fragments were rinsed three times in sterile distilled water, placed on water-agar, and incubated at 25 °C for four days, in a 12/12 h photoperiod. Hyphal tips were transferred to potato dextrose agar (PDA) plates and incubated as previously described for seven days. A Colletotrichum sp. single-spore isolate (LFN0461) was selected, grown, preserved in filter paper, and stored at -80 °C. In 2023, it was reactivated for molecular characterization. On PDA, colony showed a rough-like mycelial growth, violaceous-black (front/reverse), with curved-shaped conidia 14.7 - 28.2 × 2.1 - 8.96 µm (average 18.4 × 4.7 µm). The DNA was extracted from 10-day-old mycelium using the cetyltrimethylammonium bromide (CTAB) method. The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone (HIS3), and ß-tubulin 2 (TUB2) regions were amplified by polymerase chain reaction (PCR), using the primer pairs ITS-1F + ITS-4 (Gardes and Bruns 1993; White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), CYLH3F + CYLH3R (Crous et al. 2006), and Bt2A + Bt2B (Glass and Donaldson 1995), respectively. The sequences were deposited in the GenBank database (accession numbers: PP209207 - ITS; PP213392 - GAPDH; PP213393 - HIS3; MN688797 - TUB2). The reconstruction of the multilocus phylogenetic tree revealed that the LFN0461 isolate clustered with C. cholorophyti reference strain (IMI 103806) with 99.9% of Bayesian probability. Given the seed-borne nature of soybean anthracnose (Boufleur et al. 2021; Yang et al. 2013), pathogenicity tests were carried out by soybean seeds inoculation. Fifty seeds of NS6220 IPRO (Nidera) cultivar were inoculated by water restriction method, with LFN0461 colonies grown on PDA amended with mannitol (Machado et al. 2004), while 50 seeds were placed on PDA amended with mannitol as negative control. Soybean seeds remained in contact with the inoculum for 48 hours. Subsequently, seeds were sown in 2 L pots (n = 10) containing sterilized substrate, which were placed in a greenhouse at 25 ± 5 ºC. After 10 days, inoculated soybean seedlings exhibited characteristic necrotic lesions on cotyledons and hypocotyls, while negative control plants remained asymptomatic. Colletotrichum chlorophyti was successfully reisolated from the symptomatic tissues. Currently, C. chlorophyti has been reported to cause soybean anthracnose and infect seeds in the United States (Yang et al. 2013, 2012). Although this pathogen has not been reported since our first observation in 2013 in Brazil, many Colletotrichum isolates are misidentified due to reliance on morphology (Boufleur et al. 2021). To our knowledge, this study is the first report of C. chlorophyti causing soybean anthracnose in Brazil, joining a new group of emergent Colletotrichum spp. associated with this disease.

Survival of Colletotrichum truncatum as Microsclerotia in Soil.

Anthracnose has become one of the main threats to soybean production and is considered the most important disease in some soybean-producing areas. Colletotrichum truncatum is the species most commonly associated with anthracnose and produces microsclerotia. However, the role of microsclerotia in the epidemiology of soybean anthracnose disease has not yet been described. The aim of this study was to determine whether C. truncatum microsclerotia can survive and maintain pathogenicity for a period of up to 246 days, corresponding to the off-season period of soybean cultivation in Brazil. Therefore, microsclerotia of two pathogenic isolates of C. truncatum (CMES1059 and LFN0297) were produced and placed in polyester bags, which were kept under field conditions either on the soil surface under maize straw or buried 8-cm deep. The bags were collected monthly for a period of up to 246 days to assess the viability of microsclerotia based on their germination and typical colony growth. The logistic regression model was used for data analysis considering viable and nonviable microsclerotia. In addition, periodic sowing of soybean was done in the soil infested with LFN0297 microsclerotia to test pathogenicity up to 246 days after soil infestation. C. truncatum microsclerotia survived from 92 to 246 days in the field soil, with the highest recovery of viable microsclerotia at 153 days. C. truncatum was reisolated from soybean plants sown in infested soil at 245 days postinoculation. The isolates from the last microsclerotia sampling from the field (246 days) and those obtained from a plant at the last sowing date (245 days) had the same genotypic profile for 12 microsatellite loci as the isolates used to perform the experiments. C. truncatum microsclerotia in soil may serve as the primary inoculum for soybean anthracnose.

First Report of Mulberry Rust Caused by Cerotelium fici on Morus nigra in Brazil.

Leaf rust caused by Cerotelium fici (Cast.) Arth. is the main disease affecting Moraceae family plants, such as Ficus and Morus species (Galleti and Rezende 2016; Srikantaswamy et al. 2006). In August 2020, rust symptoms were observed in 100% of mulberry (Morus nigra L.) trees in an experimental orchard (Piracicaba, SP, Brazil; 22°42'28"S, 47°37'42"W). Mulberry leaves with high rust severity became yellowish and fell-off prematurely. Pustules were light brown with yellowish halo and presented mean size of 0.9 mm2. Uredinial paraphyses (n = 50) measured 42.2 ± 0.67 µm long with wall uniformly ca 0.6-1.1 µm thick. Urediniospores were brownish, echinulate, globoid to broadly ellipsoid, and measured 27.1 ± 0.29 × 21.0 ± 0.27 µm with a wall thickness of 0.6 ± 0.01 µm (n = 100). The morphology of the urediniospores observed in this study was similar to that reported in the literature for C. fici on Morus alba and Ficus spp. (Gupta et al. 1994; McKenzie 1986; Hennen et al. 2005). We used a low-coverage genome-skimming approach to retrieve genetic information of the rRNA cluster and the mtDNA. Genomic DNA was extracted from 3-4 mg of stored urediniospores at -80 °C, macerated in liquid nitrogen, using a modified cetyl trimethylammonium bromide extraction procedure (Lo Piccolo et al. 2012), and sequenced with 150-bp paired-end reads on Illumina NovaSeq 6000 System. Raw data, (45,761,957 X 2 reads) were assembled with SPAdes v3.15.1 (Bankevich et al., 2012) and the output used to create a custom BLAST database. Loci used for the phylogenetic analyses were identified by BLASTn using, as a query, sequences of C. fici from Ficus sp. from Australia publicly available: Accession No. MH047210.1 for the rRNA and MW036502.1 for COX3. The retrieved sequences were deposited in GenBank under accession numbers OM296992 and OP797407 for the partial rRNA cluster and COX3, respectively. The Bayesian inference phylogenetic analysis of the three concatenate loci (18S, 28S, and COX3) revealed that the isolate obtained in this study (MN1) was clustered in a well-supported clade with C. fici type species. Pathogenicity tests were conducted using mulberry potted plants under greenhouse conditions (25 ± 5 °C). The urediniospores suspension (5 × 104 urediniospores ml-1) with 0.05% Tween 20 was sprayed with an airbrush on fully expanded leaves until run-off. As a control, mulberry plants were sprayed with distilled water and kept under the same conditions. Inoculated and mock-inoculated plants were kept in a dark moist chamber at 23 °C (± 2 °C) for 24 h. After this period, plants were moved to the greenhouse. The experimental design was completely randomized with five replicates, each replicate consisted of one potted plant and the experiment was performed twice. At 12 days post-inoculation, all inoculated plants showed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. The first symptoms were small pustules on the abaxial surface of fully expanded leaves. Small chlorotic lesions were observed on the adaxial leaf surface, which evolved into necrotic lesions. The pathogen was re-inoculated into potted plants, where it was maintained through monthly inoculations. To our knowledge, this is the first report of mulberry rust on M. nigra in Brazil. As mulberry leaves are the only natural food for silkworm (Bombyx mori L.), rust poses a significant threat to the sericulture industry because the disease can decrease production and quality of mulberry foliage.

Genome Sequence Data of the Soybean Pathogen Stagonosporopsis vannaccii: A Resource for Studies on Didymellaceae Evolution.

The genus Stagonosporopsis is classified within the Didymellaceae family and has around 40 associated species. Among them, several species are important plant pathogens responsible for significant losses in economically important crops worldwide. Stagonosporopsis vannaccii is a newly described species pathogenic to soybean. Here, we present the draft whole-genome sequence, gene prediction, and annotation of S. vannaccii isolate LFN0148 (also known as IMI 507030). To our knowledge, this is the first genome sequenced of this species and represents a new useful source for future research on fungal comparative genomics studies.