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.

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.

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/.

The Climate-Driven Genetic Diversity Has a Higher Impact on the Population Structure of Plasmopara viticola Than the Production System or QoI Fungicide Sensitivity in Subtropical Brazil.

Downy mildew, caused by Plasmopara viticola, is the main disease affecting vineyards in subtropical Brazil. Here, we collected 94 P. viticola isolates from four organic and conventional vineyards in the two main grape-growing states of Brazil to evaluate the sensitivity to the quinone outside inhibitor (QoI) azoxystrobin by pheno- and genotyping assays. The impact of location, production system and sensitivity to QoI fungicides on the population genetics and structure of P. viticola was determined using 10 microsatellite markers. Cytochrome b sequencing revealed that 28 and 100% of the isolates from vineyards under organic and conventional management carried the G143A mutation, respectively. The G143A mutation was associated with high levels of azoxystrobin resistance. Three out of the 94 isolates analyzed carried the M125I alteration, not previously described in P. viticola, which was associated with a five-fold reduction in azoxystrobin sensitivity compared to wild-type isolates. Haplotype network analysis based on cytochrome b gene sequences suggested that the Brazilian populations are more closely related to the European than the North American population. A total of six haplotypes were identified, with two of them carrying the G143A mutation. Microsatellite analysis revealed high allelic and genotypic variation among the four populations. Population differentiation analyses indicated that state of origin directly influences the population biology of P. viticola, while production system and QoI sensitivity have little effect. Great genetic diversity, sexual reproduction and high levels of admixture were observed in Rio Grande do Sul State. In contrast, populations in São Paulo State were dominated by a few clonal genotypes, and no admixed genotype was detected between the two genetic pools identified in the state. This study raises the hypothesis that winter weather conditions influence the overwinter survival strategy with profound effects in the population biology of P. viticola.

Multiplex qPCR Assay for Direct Detection and Quantification of Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum in Soybean Seeds.

Precise diagnosis of plant diseases is one of the most effective tools to minimize yield losses. Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum are common soilborne pathogens that affect soybeans all over the world. We developed a multiplex quantitative real-time polymerase chain reaction (qPCR) assay to simultaneously detect and quantify the three pathogens in soybean seeds and to survey their occurrence in the main soybean production areas in Brazil. Species-specific primers and probes for C. truncatum and C. cassiicola were designed based on GAPDH and TEF1 genes, respectively, to be combined with qPCR detection of S. sclerotiorum previously reported. The multiplex qPCR assay was successful in the simultaneous detection of C. truncatum, C. cassiicola, and S. sclerotiorum, along with a host internal control. The four pathogens were detected and quantified in artificially and naturally infested soybean seeds, even in the lowest incidence level tested of 0.0625% or 1 infected seed out of 1,599 healthy ones. From 81 seed samples tested, C. truncatum was the most frequently detected pathogen and with higher incidence levels (0.25 to 0.125%), followed by S. sclerotiorum and C. cassiicola, both with lower incidence levels (0.125 to 0.0625%). Together, the results evidenced the high sensitivity of the multiplex qPCR assay, indicating its usefulness for a quick and reliable diagnosis of soybean diseases in seeds.

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.

Molecular genotyping, diversity studies and high-resolution molecular markers unveiled by microsatellites in Giardia duodenalis.

BACKGROUND: Giardia duodenalis (synonyms G. lamblia and G. intestinalis) is an enteric protozoan parasite of a wide range of mammalian hosts, including humans and various domestic and wild animals. There is considerable genetic variability in G. duodenalis and isolates of this parasite have been divided into eight genetic assemblages. Microsatellites markers can be used to discriminate isolates with a high level of sensitivity. This study was conducted to identify and characterize genomic microsatellites (simple sequence repeats-SSRs), sequences of one- to six-nucleotide motifs repeated in tandem, present in the available genomes of G. duodenalis and to develop new markers that can serve as a tool for detection and for characterizing the genetic diversity of this parasite. METHODOLOGY/ PRINCIPAL FINDINGS: For each genetic assemblage, polymorphism levels for the microsatellite markers were evaluated. After performing the analysis using the MISA and SciRoKo software, 1,853 simple sequence repeats (SSRs) were identified. In all the genomes, trinucleotide repeats were the most common class followed by tetranucleotide. Many of the SSR loci are assemblage-specific, and 36 SSR loci shared among all the genomes were identified. Together with hypothetical proteins, variant-specific surface proteins represented nearly half of the annotated SSR loci. The results regarding the most common repeat among the SSRs led us to infer that positive selection occurred to avoid frameshift mutations. Additionally, based on inter- and intra-genetic assemblages polymorphism analyses, we unveiled previously undetected genetic variation, indicating that the microsatellite markers we developed are useful molecular tools for epidemiological inferences based on population genetics patterns and processes. CONCLUSIONS: There is increasing demand for the development of new molecular markers and for the characterization of pathogens at a higher resolution level. In this study, we present 60 G. duodenalis microsatellites markers that exhibited high polymerase chain reaction (PCR) amplification efficiency among the different genetic assemblages. Twenty of these markers presented nucleotide sequence polymorphisms and may be used as a genotyping tool. The monomorphic markers can be used for the detection of the parasite at the species and genetic assemblage level. These polymorphic markers revealed a genetic diversity that was previously undetectable, thus they can be considered valuable molecular tools for high resolution markers in future studies investigating Giardia and may also be used for epidemiological inferences based on populations genetics patterns and processes.

Population genetic analysis of Giardia duodenalis: genetic diversity and haplotype sharing between clinical and environmental sources.

Giardia duodenalis is a flagellated intestinal protozoan responsible for infections in various hosts including humans and several wild and domestic animals. Few studies have correlated environmental contamination and clinical infections in the same region. The aim of this study was to compare groups of Giardia duodenalis from clinical and environmental sources through population genetic analyses to verify haplotype sharing and the degree of genetic similarity among populations from clinical and environmental sources in the metropolitan region of Campinas. The results showed high diversity of haplotypes and substantial genetic similarity between clinical and environmental groups of G. duodenalis. We demonstrated sharing of Giardia genotypes among the different populations studied. The comparison between veterinary and human sequences led us to identify new zoonotic genotypes, including human isolates from genetic assemblage C. The application of a population genetic analysis in epidemiological studies allows quantification of the degree of genetic similarity among populations of Giardia duodenalis from different sources of contamination. The genetic similarity of Giardia isolates among human, veterinary, and environmental groups reinforced the correlation between clinical and environmental isolates in this region, which is of great importance for public health.

Elevation as a barrier: genetic structure for an Atlantic rain forest tree (Bathysa australis) in the Serra do Mar mountain range, SE Brazil.

Distance and discrete geographic barriers play a role in isolating populations, as seed and pollen dispersal become limited. Nearby populations without any geographic barrier between them may also suffer from ecological isolation driven by habitat heterogeneity, which may promote divergence by local adaptation and drift. Likewise, elevation gradients may influence the genetic structure and diversity of populations, particularly those marginally distributed. Bathysa australis (Rubiaceae) is a widespread tree along the elevation gradient of the Serra do Mar, SE Brazil. This self-compatible species is pollinated by bees and wasps and has autochoric seeds, suggesting restricted gene dispersal. We investigated the distribution of genetic diversity in six B. australis populations at two extreme sites along an elevation gradient: a lowland site (80-216 m) and an upland site (1010-1100 m.a.s.l.). Nine microsatellite loci were used to test for genetic structure and to verify differences in genetic diversity between sites. We found a marked genetic structure on a scale as small as 6 km (F ST = 0.21), and two distinct clusters were identified, each corresponding to a site. Although B. australis is continuously distributed along the elevation gradient, we have not observed a gene flow between the extreme populations. This might be related to B. australis biological features and creates a potential scenario for adaptation to the different conditions imposed by the elevation gradient. We failed to find an isolation-by-distance pattern; although on the fine scale, all populations showed spatial autocorrelation until ∼10-20 m. Elevation difference was a relevant factor though, but we need further sampling effort to check its correlation with genetic distance. The lowland populations had a higher allelic richness and showed higher rare allele counts than the upland ones. The upland site may be more selective, eliminating rare alleles, as we did not find any evidence for bottleneck.

Recent introduction and recombination in Colletotrichum acutatum populations associated with citrus postbloom fruit drop epidemics in São Paulo, Brazil.

Citrus crops in São Paulo State, Brazil, have been severely affected by postbloom fruit drop disease (PFD), which is caused by Colletotrichum acutatum. This disease leads to the drop of up to 100% of young fruits. Previous studies have assumed that this pathogen exhibits a clonal reproductive mode, although no population genetic studies have been conducted so far. Thus, the genetic structure of six C. acutatum populations from sweet orange orchards showing PFD symptoms was determined using nine microsatellite markers, enabling inference on predominant mode of reproduction. C. acutatum populations exhibit a nearly panmictic genetic structure and a high degree of admixture, indicating either ongoing contemporary gene flow at a regional scale or a recent introduction from a common source, since this pathogen was introduced in Brazil only very recently. Sharing haplotypes among orchards separated by 400 km suggests the natural dispersal of fungal propagules, with the possible involvement of pollinators. A significant population expansion was detected, which was consistent with an increase in host density associated with crop expansion toward new areas across the state. Findings of moderate to high levels of haplotypic diversity and gametic equilibrium suggest that recombination might play an important role in these pathogen populations, possibly via parasexual reproduction or a cryptic sexual cycle. This study provides additional tools for epidemiological studies of C. acutatum to improve prevention and management strategies for this disease.

Characterization of 10 microsatellite loci for Bathysa australis (Rubiaceae).

PREMISE OF THE STUDY: Bathysa australis is a common subcanopy tree from the Atlantic Forest that is pollinated by bees and wasps and produces autochoric seeds. This species exhibits great phenotypic plasticity along the elevational gradient of Serra do Mar in southeastern Brazil. We expect to assess the genetic diversity and gene flow between populations of this species along the elevational gradient. • METHODS AND RESULTS: We developed a microsatellite-enriched genomic library for B. australis, and 10 microsatellite loci were successfully amplified, ranging from one to 13 alleles per locus. The observed and expected heterozygosities ranged from 0.333 to 0.900 (average: 0.629) and 0.564 to 0.900 (average: 0.742), respectively. • CONCLUSIONS: These are the first microsatellite markers developed for the genus Bathysa and may be useful in other species of the Condamineeae tribe. These primers will be an important tool for studies of population ecology and conservation genetics.