First Report of Colletotrichum graminicola Causing Maize Anthracnose in Galicia, Northwestern Spain.

Maize (Zea mays) is one of the most important crops worldwide, and fungal diseases are responsible for major losses in food production. Anthracnose caused by Colletotrichum graminicola can infect all maize tissues, although stalk rot and seedling blight cause more significant economic damage (Munkvold and White, 2016). Anthracnose stalk rot is characterized by a distinctive external blackening of the lower stalks resulting in large black streaks, while the pith turns dark brown and has a shredded appearance. Like most stalk rots, the most obvious symptom is a sudden death of plants before grain maturity, and plant lodging. Symptoms commonly appear late in the season, suspicious maize stems of cultivar Tuy exhibiting symptoms of anthracnose stalk rot were collected from a field in Pontevedra, Galicia, Spain (Geographical coordinates: 42°23'27.1" N - 8°30'46.3" W) between June and December of 2022. Stem samples, approximately 50 mm2, were dissected and surface-disinfected for 90 seconds in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one half-strength acidified potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and lactic acid 90% (1.5 mL/L) and incubated for 5 days at 25 ºC (Sukno et al. 2008). Single spores were transferred to fresh PDA plates to obtain pure culture isolates. A total of six isolates were obtained, and among them, two were selected for further characterization (SP-36820-1 and SP-36820-3). Colonies grown on PDA have dark gray aerial mycelium with orange-colored spore masses. Conidia are falcate, slightly curved, tapered toward the tips, and are produced in acervuli with setae, measuring 37.65 to 24.84 x 8.02 to 4.67 µm, respectively (n = 100). These morphological characteristics are in agreement with C. graminicola previously described by Bergstrom and Nicholson (1999). Isolates were grown in potato dextrose broth (PDB) for 3 days at 25 ºC and total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). The internal transcribed spacer region of rDNA and the manganese-type superoxide dismutase gene (SOD2) were amplified using primers ITS4/ITS5 (White et al. 1990) and SOD625/SOD507 (Fang et al. 2002) and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to strains of C. graminicola. All sequences were deposited in GenBank (see e-Xtra 1 for accession numbers). To confirm Koch's postulates, plants of a derivative of maize inbred line Mo940 (developmental stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µL) of a suspension of 3 x 105 conidia per milliliter were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23ºC. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25ºC with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After four days inoculated leaves presented brown elongated lesions with necrotic centers consistent with C. graminicola infection, whereas control plants remained asymptomatic. The strains reisolated from infected leaves were morphologically identical to the original isolates. To our knowledge, this is the first report of Colletotrichum graminicola causing maize anthracnose in Spain. Recently, maize anthracnose was also reported in Bosnia and Herzegovina and China (Duan et al. 2019; Cuevas-Fernández et al. 2019), suggesting the pathogen's geographic range is increasing, which may be a threat to maize cultivation in locations with optimal humid conditions for disease development.

Means, motive and opportunity for biological invasions: Genetic introgression in a fungal pathogen.

Invasions by fungal plant pathogens pose a significant threat to the health of agricultural ecosystems. Despite limited standing genetic variation, many invasive fungal species can adapt and spread rapidly, resulting in significant losses to crop yields. Here, we report on the population genomics of Colletotrichum truncatum, a polyphagous pathogen that can infect more than 460 plant species, and an invasive pathogen of soybean in Brazil. We study the whole-genome sequences of 18 isolates representing 10 fields from two major regions of soybean production. We show that Brazilian C. truncatum is subdivided into three phylogenetically distinct lineages that exchange genetic variation through hybridization. Introgression affects 2%-30% of the nucleotides of genomes and varies widely between the lineages. We find that introgressed regions comprise secreted protein-encoding genes, suggesting possible co-evolutionary targets for selection in those regions. We highlight the inherent vulnerability of genetically uniform crops in the agro-ecological environment, particularly when faced with pathogens that can take full advantage of the opportunities offered by an increasingly globalized world. Finally, we discuss "the means, motive and opportunity" of fungal pathogens and how they can become invasive species of crops. We call for more population genomic studies because such analyses can help identify geographical areas and pathogens that pose a risk, thereby helping to inform control strategies to better protect crops in the future.

Population Genomics Provide Insights into the Global Genetic Structure of Colletotrichum graminicola, the Causal Agent of Maize Anthracnose.

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and whole-genome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure. IMPORTANCE Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems. We employed a population genomics approach to investigate the genetic diversity and reproductive biology of the maize anthracnose pathogen (Colletotrichum graminicola) in 14 countries. We found that the populations are correlated with their geographical origin and that migration between countries is ongoing, possibly caused by the movement of infected plant material. This result has direct implications for disease management because migration can cause the movement of more virulent and/or fungicide-resistant genotypes. We conclude that genetic recombination is frequent (in contrast to the traditional view of C. graminicola being mainly asexual), which strongly impacts control measures and breeding programs aimed at controlling this disease.

Soybean anthracnose caused by Colletotrichum species: Current status and future prospects.

Soybean (Glycine max) is one of the most important cultivated plants worldwide as a source of protein-rich foods and animal feeds. Anthracnose, caused by different lineages of the hemibiotrophic fungus Colletotrichum, is one of the main limiting factors to soybean production. Losses due to anthracnose have been neglected, but their impact may threaten up to 50% of the grain production. TAXONOMY: While C. truncatum is considered the main species associated with soybean anthracnose, recently other species have been reported as pathogenic on this host. Until now, it has not been clear whether the association of new Colletotrichum species with the disease is related to emerging species or whether it is due to the undergoing changes in the taxonomy of the genus. DISEASE SYMPTOMS: Typical anthracnose symptoms are pre- and postemergence damping-off; dark, depressed, and irregular spots on cotyledons, stems, petioles, and pods; and necrotic laminar veins on leaves that can result in premature defoliation. Symptoms may evolve to pod rot, immature opening of pods, and premature germination of grains. CHALLENGES: As accurate species identification of the causal agent is decisive for disease control and prevention, in this work we review the taxonomic designation of Colletotrichum isolated from soybean to understand which lineages are pathogenic on this host. We also present a comprehensive literature review of soybean anthracnose, focusing on distribution, symptomatology, epidemiology, disease management, identification, and diagnosis. We consider the knowledge emerging from population studies and comparative genomics of Colletotrichum spp. associated with soybean providing future perspectives in the identification of molecular factors involved in the pathogenicity process. USEFUL WEBSITE: Updates on Colletotrichum can be found at http://www.colletotrichum.org/. All available Colletotrichum genomes on GenBank can be viewed at http://www.colletotrichum.org/genomics/.

Genome Sequence Resources of Colletotrichum truncatum, C. plurivorum, C. musicola, and C. sojae: Four Species Pathogenic to Soybean (Glycine max).

Colletotrichum is a large genus of plant pathogenic fungi comprising more than 200 species. In this work, we present the genome sequences of four Colletotrichum species pathogenic to soybean: C. truncatum, C. plurivorum, C. musicola, and C. sojae. While C. truncatum is globally considered the most important pathogen, the other three species have been described and associated with soybean only recently. The genome sequences will provide insights into factors that contribute to pathogenicity toward soybean and will be useful for further research into the evolution of Colletotrichum.

Multiple Introductions Without Admixture of Colletotrichum truncatum Associated with Soybean Anthracnose in Brazil.

Knowledge of the population structure, genetic diversity, and reproductive mode of plant pathogens can help to implement effective disease management strategies. Anthracnose is one of the most prominent diseases in soybean and is mainly associated with the species Colletotrichum truncatum. However, the genetic structure of C. truncatum populations associated with soybean remains unknown. We collected C. truncatum isolates from 10 sites representing two Brazilian states (Mato Grosso and Goiás) and used 13 highly polymorphic microsatellite markers to investigate the population genetic structure of the pathogen. Analyses revealed high gene and haplotypic diversity within populations, as well low genetic differentiation and sharing of multilocus haplotypes among populations and regions. Bayesian and multivariate analysis revealed the presence of three distinct genetic clusters with at least two coexisting in all locations, and all of them coexisting in eight locations. We found limited evidence for admixture between clusters, with only two isolates showing nonzero membership with a second cluster. Analyses of linkage disequilibrium rejected the hypothesis of random mating in all clusters, but values of the index of association were low and not consistent with long-term lack of sexual reproduction. Our findings suggest that Brazilian C. truncatum populations resulted from at least three founder events that led to three genetic clusters that spread throughout the country, raising questions with respect to the factors allowing their maintenance in syntopy without evidence of admixture between them.

Efeito da adubação potássica na cultura do crambe / Effect of potassium fertilization in crambe culture

O crambe é uma planta brassica de inverno, com alto teor de óleo que apresenta grande potencial para a produção de matéria prima para biodiesel. É uma cultura de inverno que possui ciclo curto de desenvolvimento que varia de 90 a 100 dias, sendo utilizada como alternativa para a entressafra. Este trabalho foi realizado no município de Umuarama ­ PR, em Latossolo Vermelho distrófico típico, o delineamento experimental foi de blocos casualizados e teve como objetivo avaliar o efeito da aplicação de doses de potássio (0, 15, 30, 60 e 90 kg ha-1) na cultura do crambe. A aplicação de doses de potássio resultou em aumento significativo da produtividade da cultura, no entanto não influenciou na massa de 100 grãos, massa seca e teor de óleo.

Crambe abyssinica is a winter brassica, with high oil content showing great potential for source of raw material for biodiesel production. It's a winter crop with short development cycle, ranging from 90 to 100 days, being used as an option for off season. This work was conducted in Umuarama County, Parana State, Latossoil Red dystrophic typical, the experimental design was a randomized complete block and aimed to evaluate the effect of potassium growing application (0, 15, 30, 60 and 90 kg ha-1) in the crambe crop. It was possible to observe that the application of different potassium doses resulted in significant increasing in yield crop, however didn't influence mass of 100 grains, dry mass and oil content.

Efeito de doses de fósforo no desenvolvimento da cultura do crambe / Effect of phosphorus doses in the development of crambe culture

O crambe é uma brássica de inverno, com alto teor de óleo que serve como fonte de matéria prima para a produção de biodiesel. Vem chamando a atenção dos produtores por ter ciclo curto, que varia de 90 a 100 dias. O experimento teve como objetivo avaliar o efeito de doses de fósforo - P2O5 (0, 15, 30, 60 e 90 kg ha-1) na adubação de base da cultura. O experimento foi realizado no município de Umuarama­ PR, em Latossolo Vermelho distrófico típico, empregando-se o delineamento experimental em blocos casualizados, com quatro repetições. A adubação fosfatada contribuiu para o aumento da produtividade e da massa seca. Para massa de 100 grãos e teor de óleo, não houve diferença significativa entre os tratamentos.

Crambe abyssinica is a winter crucifer, with high oil content being used as a source of raw material for biodiesel production. It's getting the attention of farmers because of its short cycle, which ranges from 90 to 100 days. The experiment objectified to evaluate the effect of phosphorus doses- P2O5 (0, 15, 30, 60 and 90 kg ha-1) in basic fertilization of the crop. The experiment was conducted in Umuarama County, Parana State, in Latossoil Red dystrophic typical, by using a randomized complete blocks design with four replications. Phosphate fertilization contributed to increase yield and dry mass. For the characteristics mass of 100 grains and oil content, there was no significant difference between treatments.