João Leodato Nunes Maciel

Population structure and pathotype diversity of the wheat blast pathogen Magnaporthe oryzae 25 years after its emergence in Brazil.

Since its first report in Brazil in 1985, wheat blast, caused by Magnaporthe oryzae (anamorph: Pyricularia oryzae), has become increasingly important in South America, where the disease is still spreading. We used 11 microsatellite loci to elucidate the population structure of the wheat blast pathogen in wheat fields in central-western, southeastern, and southern Brazil. No subdivision was found among the wheat-infecting populations, consistent with high levels of gene flow across a large spatial scale. Although the clonal fraction was relatively high and the two mating type idiomorphs (MAT1-1 and MAT1-2) were not at similar frequencies, the clone-corrected populations from Distrito Federal and Goiás, Minas Triangle, and São Paulo were in gametic equilibrium. Based on these findings, we propose that populations of the wheat blast pathogen exhibit a mixed reproductive system in which sexual reproduction is followed by the local dispersal of clones. Seedling virulence assays with local wheat cultivars differentiated 14 pathotypes in the current population. Detached head virulence assays differentiated eight virulence groups on the same wheat cultivars. There was no correlation between seedling and head reactions.

Resistance to QoI Fungicides Is Widespread in Brazilian Populations of the Wheat Blast Pathogen Magnaporthe oryzae

Wheat blast, caused by Magnaporthe oryzae, is an important disease across central and southern Brazil. Control has relied mainly on strobilurin fungicides (quinone-outside inhibitors [QoIs]). Here, we report the widespread distribution of QoI resistance in M. oryzae populations sampled from wheat fields and poaceous hosts across central and southern Brazil and the evolution of the cytochrome b (cyt b) gene. Sequence analysis of the cyt b gene distinguished nine haplotypes, with four haplotypes carrying the G143A mutation associated with QoI resistance and two haplotypes shared between isolates sampled from wheat and other poaceous hosts. The frequency of the G143A mutation in the wheat-infecting population increased from 36% in 2005 to 90% in 2012. The G143A mutation was found in many different nuclear genetic backgrounds of M. oryzae. Our findings indicate an urgent need to reexamine the use of strobilurins to manage fungal wheat diseases in Brazil.

Resistência parcial à brusone de genótipos de trigo comum e sintético nos estádios de planta jovem e de planta adulta

Wheat blast caused by Pyricularia grisea was first reported in Brazil in 1985 in the state of Parana. From then on, researchers have been looking for resistant genotypes to blast disease among Brazilian wheat germplasm. The objective of this work was to determine the level of partial resistance of synthetic and common wheat genotypes in young and adult plant growth stages. In young plant tests 70 wheat genotypes were challenged by 18 isolates of P. grisea. Twelve of the 70 genotypes were selected for adult plant tests. Among all genotypes evaluated BRS 229, BRS 179, CNT 8, BRS 120, and BRS Buriti showed better resistance level when tested at young plant growths stages. However, CNT 8, NE 20156-B, PF 844001, PF 964009 and PF 804002 showed less leaf and head area affected by blast in adult plant.

Pyricularia graminis-tritici, a new pyricularia species causing wheat blast

Pyricularia oryzae is a species complex that causes blast disease on more than 50 species of poaceous plants. Pyricularia oryzae has a worldwide distribution as a rice pathogen and in the last 30 years emerged as an important wheat pathogen in southern Brazil. We conducted phylogenetic analyses using 10 housekeeping loci for 128 isolates of P. oryzae sampled from sympatric populations of wheat, rice, and grasses growing in or near wheat fields. Phylogenetic analyses grouped the isolates into three major clades. Clade 1 comprised isolates associated only with rice and corresponds to the previously described rice blast pathogen P. oryzae pathotype Oryza (PoO). Clade 2 comprised isolates associated almost exclusively with wheat and corresponds to the previously described wheat blast pathogen P. oryzae pathotype Triticum (PoT). Clade 3 contained isolates obtained from wheat as well as other Poaceae hosts. We found that Clade 3 is distinct from P. oryzae and represents a new species, Pyricularia graminis-tritici (Pgt). No morphological differences were observed among these species, but a distinctive pathogenicity spectrum was observed. Pgt and PoT were pathogenic and highly aggressive on Triticum aestivum (wheat), Hordeum vulgare (barley), Urochloa brizantha (signal grass), and Avena sativa (oats). PoO was highly virulent on the original rice host (Oryza sativa), and also on wheat, barley, and oats, but not on signal grass. We conclude that blast disease on wheat and its associated Poaceae hosts in Brazil is caused by multiple Pyricularia species. Pyricularia graminis-tritici was recently found causing wheat blast in Bangladesh. This indicates that P. graminis-tritici represents a serious threat to wheat cultivation globally.

Caracterização genética e fenotípica de isolados de Pyricularia grisea do trigo

The use of molecular tools and virulence evaluations of Pyricularia grisea, the causal agent of rice and wheat blast, has permitted identification of strains of the pathogen, especially those occurring in the rice crop. Microsatellites have already demonstrated efficiency to classify P. grisea isolates by genetic similarity groups. The objectives of this work were to characterize the genetic diversity of 18 isolates of P. griseacollected from wheat by applying 8 microsatellite primers; and to compare the molecular and virulence results of isolates, when inoculated in seedlings of 70 wheat genotypes. The most informative primer was PG 5 with 4 alleles. The primers MG 21 and PG 12 permitted separation of Py 5020 and Py 5038 in distinct groups, with less than 50% similarity in relation to others. The genetic similarity of isolates presented more than 75% similarity among them. In the virulence analysis, 15 out of 18 tested isolates showed more than 85% of similarity. The molecular pattern and virulence were similar for the isolates, except for Py 5002 isolate. The absence of resistance to all isolates confirms the need to seek new sources or better genetic combinations of wheat that are more resistant to disease.

Resistência cruzada aos fungicidas IQo azoxistrobina e piraclostrobina no patógeno da brusone do trigo Pyricularia oryzae no Brasil

As estrobilurinas estao entre os fungicidas mais comumente utilizados no controle das doencas do trigo, isoladamente ou em misturas com fungicidas inibidores da enzima succinato desidrogenase (IDHS). As estrobilurinas sao conhecidas como fungicidas inibidores da quinona oxidase ou fungicidas IQo. Por ter como alvo um unico sitio de acao nas celulas fungicas, o gene mitocondrial cyt b, o emprego continuo das estrobilurinas pode representar alto risco de emergencia de resistencia a estes fungicidas em populacoes de fitopatogenos. O presente trabalho teve como objetivo testar a hipotese de que a resistencia a azoxistrobina no fungo Pyricularia oryzae dotrigo resultou em resistencia cruzada a piraclostrobina, outro fungicida IQo. Para testar esta hipotese, foi comparado o nivel de resistencia a piraclostrobina apresentado por dois grupos de isolados do fungo P. oryzae do trigo: a) sensiveis a azoxistrobina e portadores do gene cyt b nao mutante (haplotipo H9) e b) resistentes a azoxistrobina e portadores da mutacao G143A no gene cyt b(haplotipo H1). Fungicidas pertencentes a um mesmo grupo quimico apresentam resistencia cruzada. Todos os isolados de P. oryzaedo trigo sensiveis a azoxistrobina foram tambem sensiveis a piraclostrobina. Os isolados resistentes a azoxistrobina foram, tambem, resistentes a piraclostrobina, indicando que ha resistencia cruzada para os dois fungicidas. Entre os isolados resistentes, distinguiram-se dois grupos fenotipicos: (A) altamente resistentes e (B) resistentes. Isolados de P. oryzaecom a presenca da mutacao G143A no gene cyt b foram 42 vezes mais resistentes a piraclostrobina. Esses altos niveis de resistencia a fungicidas IQo podem ser o resultado de elevada pressao de selecao exercida pelos anos consecutivos de aplicacoes de estrobilurinas para o manejo de doencas do trigo no Brasil.

Diagrammatic scale for the assessment of blast on wheat spikes

The correct quantification of blast caused by the fungus Magnaporthe oryzae on wheat (Triticum aestivum) spikes is an important component to understand the development of this disease aimed at its control. Visual quantification based on a diagrammatic scale can be a practical and efficient strategy that has already proven to be useful against several plant pathosystems, including diseases affecting wheat spikes like glume blotch and fusarium head blight. Spikes showing different disease severity values were collected from a wheat field with the aim of elaborating a diagrammatic scale to quantify blast severity on wheat spikes. The spikes were photographed and blast severity was determined by using resources of the software ImageJ. A diagrammatic scale was developed with the following disease severity values: 3.7, 7.5, 21.4, 30.5, 43.8, 57.3, 68.1, 86.0, and 100.0%. An asymptomatic spike was added to the scale. Scale validation was performed by eight people who estimated blast severity by using digitalized images of 40 wheat spikes. The precision and the accuracy of the evaluations varied according to the rater (0.82<R2<0.90, -6.12<a<2.94, 0.85<b<1.16), and systematic errors in overestimating or underestimating the disease were not found among the raters, demonstrating that the developed scale is suitable to evaluate blast on wheat spikes.

Caracterização de estirpes de Ralstonia solanacearum isoladas de plantas de batata com murcha bacteriana, por PCR-Rep e RAPD

Considered one of the most important potato (Solanum tuberosum) pathogens in tropical and subtropical regions, as well as in temperate regions with warmer climate, Ralstonia solanacearum is a species with significant genetic diversity. It has been characterized in a binary system of races and biovars based on the range of host species and on the ability to use carbon sources. The attempts to use genetic resistance as a strategy to control R. solanacearum were shown to be unstable due to climatic changes in different regions and due to the variability of the pathogenic strains. Due to different epidemiological characteristics of the biovars, strains of biovar 2 are more likely to be eradicated in an integrated control system. In a survey performed in four regions where potato is produced in Rio Grande do Sul, isolates of R. solanacearum were obtained from 25 crop fields in ten municipalities. The biochemical analyses of the isolates revealed the occurrence of biovars 1 and 2, the latter being predominant. The isolates obtained were evaluated for their genetic variability by PCR, using repetitive sequences ERIC and BOX and random primers (RAPD). The PCR-ERIC and BOX were able to clearly differentiate the biovars 1 and 2. However, neither analysis was able to demonstrate variability among isolates of biovar 2 and only PCR-BOX showed some degree of variability among isolates of biovar 1. The amplification by RAPD demonstrates the distinction between the biovars and revealed that they show characteristic profiles that are closely related to the region where they were obtained.

Gene Flow between Divergent Cereal- and Grass-Specific Lineages of the Rice Blast Fungus Magnaporthe oryzae

ABSTRACT Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae, including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species (Pyricularia graminis-tritici). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae, each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae. These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen. IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another. IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.

Wheat blast disease caused by Pyricularia graminis-tritici sp. nov.

Pyricularia oryzae is a species complex that causes blast disease on more than 50 species of poaceous plants. Pyricularia oryzae has a worldwide distribution as a rice (Oryza) pathogen and in the last century emerged as an important wheat (Triticum) pathogen in southern Brazil. Presently, P. oryzae pathotype Oryza is considered the rice blast pathogen, whereas P. oryzae pathotype Triticum is the wheat blast pathogen. In this study we investigated whether the Oryza and Triticum pathotypes of P. oryzae were distinct at the species level. We also describe a new Pyricularia species causing blast on several other poaceous hosts in Brazil, including wheat. We conducted phylogenetic analyses using 10 housekeeping loci from an extensive sample (N = 128) of sympatric populations of P. oryzae adapted to rice, wheat and other poaceous hosts found in or near wheat fields. The Bayesian phylogenetic analysis grouped the isolates into two major monophyletic clusters (I and II) with high Bayesian probabilities (P = 0.99). Cluster I contained isolates obtained from wheat as well as other Poaceae hosts (P = 0.98). Cluster II was divided into three host-associated clades (Clades 1, 2 and 3; P > 0.75). Clade 1 contained isolates obtained from wheat and other poaceous hosts, Clade 2 contained exclusively wheat-derived isolates, and Clade 3 comprised isolates associated only with rice. Our interpretation was that cluster I and cluster II correspond to two distinct species: Pyricularia graminis-tritici sp. nov. (Pgt), newly described in this study, and Pyricularia oryzae (Po). The host-associated clades found in P. oryzae Cluster II correspond to P. oryzae pathotype Triticum (PoT; Clades 1 and 2), and P. oryzae pathotype Oryza (PoO; Clade 3). No morphological or cultural differences were observed among these species, but a distinctive pathogenicity spectrum was observed. Pgt and PoT were pathogenic and highly aggressive on Triticum aestivum (wheat), Hordeum vulgare (barley), Urochloa brizantha (signal grass) and Avena sativa (oats). PoO was highly virulent on the original rice host (Oryza sativa), and also on wheat, barley, and oats, but not on signal grass. We concluded that blast disease on wheat and its associated Poaceae hosts in Brazil is caused by multiple Pyricularia species: the newly described Pyricularia graminis-tritici sp. nov., and the known P. oryzae pathotypes Triticum and Oryza. To our knowledge, P. graminis-tritici sp. nov. is still restricted to Brazil, but obviously represents a serious threat to wheat cultivation globally.Abstract Pyricularia oryzae is a species complex that causes blast disease on more than 50 species of poaceous plants. Pyricularia oryzae has a worldwide distribution as a rice (Oryza) pathogen and in the last 30 years emerged as an important wheat (Triticum) pathogen in southern Brazil. We conducted phylogenetic analyses using 10 housekeeping loci for 128 isolates of P. oryzae sampled from sympatric populations of grasses growing in or near wheat fields. Phylogenetic analyses grouped the isolates into three major clades. Clade 1 comprised isolates associated only with rice and corresponds to the previously described rice blast pathogen P. oryzae pathotype Oryza (PoO). Clade 2 comprised isolates associated almost exclusively with wheat and corresponds to the previously described wheat blast pathogen P. oryzae pathotype Triticum (PoT). Clade 3 contained isolates obtained from wheat as well as other Poaceae hosts. We found that Clade 3 is distinct from P. oryzae and represents a new species, Pyricularia graminis-tritici, (Pgt). No morphological differences were observed among these species, but a distinctive pathogenicity spectrum was observed. Pgt and PoT were pathogenic and highly aggressive on Triticum aestivum (wheat), Hordeum vulgare (barley), Urochloa brizantha (signal grass) and Avena sativa (oats). PoO was highly virulent on the original rice host (Oryza sativa), and also on wheat, barley, and oats, but not on signal grass. We conclude that blast disease on wheat and its associated Poaceae hosts in Brazil is caused by multiple Pyricularia species. Pyricularia graminis-tritici was recently found causing wheat blast in Bangladesh. This indicates that P. graminis-tritici represents a serious threat to wheat cultivation globally.