RESUMEN
Soybean is a crucial crop for the Brazilian economy, but it faces challenges from the biotrophic fungus Phakopsora pachyrhizi, which causes Asian Soybean Rust (ASR). In this study, we aimed to identify SNPs associated with resistance within the Rpp1 locus, which is effective against Brazilian ASR populations. We employed GWAS and re-sequencing analyzes to pinpoint SNP markers capable of differentiating between soybean accessions harboring the Rpp1, Rpp1-b and other alternative alleles in the Rpp1 locus and from susceptible soybean cultivars. Seven SNP markers were found to be associated with ASR resistance through GWAS, with three of them defining haplotypes that efficiently distinguished the accessions based on their ASR resistance and source of the Rpp gene. These haplotypes were subsequently validated using a bi-parental population and a diverse set of Rpp sources, demonstrating that the GWAS markers co-segregate with ASR resistance. We then examined the presence of these haplotypes in a diverse set of soybean genomes worldwide, finding a few new potential sources of Rpp1/Rpp1-b. Further genomic sequence analysis revealed nucleotide differences within the genes present in the Rpp1 locus, including the ULP1-NBS-LRR genes, which are potential R gene candidates. These results provide valuable insights into ASR resistance in soybean, thus helping the development of resistant soybean varieties through genetic breeding programs.
Asunto(s)
Alelos , Resistencia a la Enfermedad , Estudio de Asociación del Genoma Completo , Glycine max , Phakopsora pachyrhizi , Enfermedades de las Plantas , Polimorfismo de Nucleótido Simple , Glycine max/genética , Glycine max/microbiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Resistencia a la Enfermedad/genética , Phakopsora pachyrhizi/fisiología , Phakopsora pachyrhizi/genética , Haplotipos , Genes de Plantas , Basidiomycota/fisiologíaRESUMEN
Soybean rust is an economically significant disease caused by the fungus Phakopsora pachyrhizi that negatively impacts soybean (Glycine max [L.] Merr.) production throughout the world. Susceptible plants infected by P. pachyrhizi develop tan-colored lesions on the leaf surface that give rise to funnel-shaped uredinia as the disease progresses. While most soybean germplasm is susceptible, seven genetic loci (Rpp1 to Rpp7) that provide race-specific resistance to P. pachyrhizi (Rpp) have been identified. Rpp3 was first discovered and characterized in the soybean accession PI 462312 (Ankur), and it was also determined to be one of two Rpp genes present in PI 506764 (Hyuuga). Genetic crosses with PI 506764 were later used to fine-map the Rpp3 locus to a 371-kb region on chromosome 6. The corresponding region in the susceptible Williams 82 (Wm82) reference genome contains several homologous nucleotide binding site-leucine rich repeat (NBS-LRR) genes. To identify Rpp3, we designed oligonucleotide primers to amplify Rpp3 candidate (Rpp3C) NBS-LRR genes at this locus from PI 462312, PI 506764, and Wm82 using polymerase chain reaction (PCR). Five Rpp3C genes were identified in both Rpp3-resistant soybean lines, and co-silencing these genes compromised resistance to P. pachyrhizi. Gene expression analysis and sequence comparisons of the Rpp3C genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3, is responsible for Rpp3-mediated resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.
Asunto(s)
Resistencia a la Enfermedad , Glycine max , Phakopsora pachyrhizi , Enfermedades de las Plantas , Proteínas de Plantas , Glycine max/microbiología , Glycine max/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Resistencia a la Enfermedad/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Phakopsora pachyrhizi/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Mapeo CromosómicoRESUMEN
Soybean rust (SBR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is a devastating foliar disease threatening soybean production. To date, no commercial cultivars conferring durable resistance to SBR are available. The development of long-lasting SBR resistance has been hindered by the lack of understanding of this complex pathosystem, encompassing challenges posed by intricate genetic structures in both the host and pathogen, leading to a gap in the knowledge of gene-for-gene interactions between soybean and P. pachyrhizi. In this review, we focus on recent advancements and emerging technologies that can be used to improve our understanding of the P. pachyrhizi-soybean molecular interactions. We further explore approaches used to combat SBR, including conventional breeding, transgenic approaches and RNA interference, and how advances in our understanding of plant immune networks, the availability of new molecular tools, and the recent sequencing of the P. pachyrhizi genome could be used to aid in the development of better genetic resistance against SBR. Lastly, we discuss the research gaps of this pathosystem and how new technologies can be used to shed light on these questions and to develop durable next-generation SBR-resistant soybean plants.
Asunto(s)
Basidiomycota , Phakopsora pachyrhizi , Phakopsora pachyrhizi/genética , Glycine max/genética , Fitomejoramiento , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most destructive foliar diseases that affect soybeans. Developing resistant cultivars is the most cost-effective, environmentally friendly, and easy strategy for controlling the disease. However, the current understanding of the mechanisms underlying soybean resistance to P. pachyrhizi remains limited, which poses a significant challenge in devising effective control strategies. In this study, comparative transcriptomic profiling using one resistant genotype and one susceptible genotype was performed under infected and control conditions to understand the regulatory network operating between soybean and P. pachyrhizi. RNA-Seq analysis identified a total of 6540 differentially expressed genes (DEGs), which were shared by all four genotypes. The DEGs are involved in defense responses, stress responses, stimulus responses, flavonoid metabolism, and biosynthesis after infection with P. pachyrhizi. A total of 25,377 genes were divided into 33 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with pathogen defense. The DEGs were mainly enriched in RNA processing, plant-type hypersensitive response, negative regulation of cell growth, and a programmed cell death process. In conclusion, these results will provide an important resource for mining resistant genes to P. pachyrhizi infection and valuable resources to potentially pyramid quantitative resistance loci for improving soybean germplasm.
Asunto(s)
Phakopsora pachyrhizi , Transcriptoma , RNA-Seq , Phakopsora pachyrhizi/genética , Glycine max/genética , Resistencia a la Enfermedad/genética , GenotipoRESUMEN
The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.
Asunto(s)
Phakopsora pachyrhizi , Phakopsora pachyrhizi/genética , Glycine max/genética , Proteínas Repetidas Ricas en Leucina , Genes de Plantas/genética , Sitios de Unión , Enfermedades de las Plantas/genéticaRESUMEN
KEY MESSAGE: Eight soybean genomic regions, including six never before reported, were found to be associated with resistance to soybean rust (Phakopsora pachyrhizi) in the southeastern USA. Soybean rust caused by Phakopsora pachyrhizi is one of the most important foliar diseases of soybean [Glycine max (L.) Merr.]. Although seven Rpp resistance gene loci have been reported, extensive pathotype variation in and among fungal populations increases the importance of identifying additional genes and loci associated with rust resistance. One hundred and ninety-one soybean plant introductions from Japan, Indonesia and Vietnam, and 65 plant introductions from other countries were screened for resistance to P. pachyrhizi under field conditions in the southeastern USA between 2008 and 2015. The results indicated that 84, 69, and 49% of the accessions from southern Japan, Vietnam or central Indonesia, respectively, had negative BLUP values, indicating less disease than the panel mean. A genome-wide association analysis using SoySNP50K Infinium BeadChip data identified eight genomic regions on seven chromosomes associated with SBR resistance, including previously unreported regions of Chromosomes 1, 4, 6, 9, 13, and 15, in addition to the locations of the Rpp3 and Rpp6 loci. The six unreported genomic regions might contain novel Rpp loci. The identification of additional sources of rust resistance and associated genomic regions will further efforts to develop soybean cultivars with broad and durable resistance to soybean rust in the southern USA.
Asunto(s)
Basidiomycota , Phakopsora pachyrhizi , Genes de Plantas , Estudio de Asociación del Genoma Completo , Genómica , Genotipo , Indonesia , Japón , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Glycine max/genética , Glycine max/microbiología , VietnamRESUMEN
To obtain direct evidence for the influence of an effector on the virulence or pathogenicity of a pathogen, it is necessary to knock out, knock down, or silence the respective gene. Since genetic transformation is not yet possible for rust fungi, silencing the gene is the only option. Posttranscriptional gene silencing uses RNAi. RNAi in plant pathogens can be accomplished by introducing dsRNA either by direct application of in vitro synthesized dsRNA or through positive-strand or double-strand RNA plant viruses. For studying effectors in Phakopsora pachyrhizi, we have implemented a host-induced silencing procedure based on virus-induced gene silencing using the bean pod mottle virus system. Here, procedures and interpretations of results are described and limitations of the system are discussed.
Asunto(s)
Basidiomycota , Phakopsora pachyrhizi , Basidiomycota/genética , Silenciador del Gen , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Glycine max/genéticaRESUMEN
Succinate dehydrogenase inhibitors (SDHIs) fungicides are used to control Asian soybean rust (Phakopsora pachyrhizi), and the SdhC-I86F mutation is related to pathogen resistance. The objective of this study was to determine whether fitness penalties are associated with SDHI resistance (SdhC-I86F mutation) in P. pachyrhizi populations. Moreover, the study investigated whether the SdhC-I86F mutation remained stable after the fungus propagation both in the absence and presence of fungicide. The populations used in this study presented mutations for all genes analyzed (Cyp51, Cytb, and SdhC), except for a wild-type population (WTSdhC) found with no SdhC-I86F mutation. The frequencies of the SdhC-I86F mutant populations were stable after 36 generations in the absence of fungicide. However, in the case of the WTSdhC population, the SdhC-I86F mutation was further detected after one generation of the fungus in the presence of the SDHI fungicide, according to the results of a detached leaf assay. Three tests were performed to evaluate fitness components and sensitivity to fungicides (half maximal effective concentration). SdhC-I86F mutant populations were more sensitive to osmotic and oxidative stress than the WTSdhC population; however, the sensitivity to ultraviolet radiation was similar for both populations. All mutated populations were less sensitive than the WTSdhC when using SDHI (azoxystrobin + benzovindiflupyr), but more sensitive to mancozeb. The presence of fitness penalties, the mutation stability, and the sensitivity to mancozeb presented by the SdhC-I86F mutant populations can be relevant to the management of the disease in the field.
Asunto(s)
Fungicidas Industriales , Phakopsora pachyrhizi , Farmacorresistencia Fúngica/genética , Fungicidas Industriales/farmacología , Mutación , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas/microbiología , Rayos UltravioletaRESUMEN
BACKGROUND: Fungicides of the succinate dehydrogenase inhibitors (SDHIs) group have been used in soybean to control Asian soybean rust (ASR) caused by Phakopsora pachyrhizi. Fungal populations with less sensitivity to SDHI fungicides have been reported since 2015. RESULTS: In this study, fungal sensitivity to benzovindiflupyr (BZV) and fluxapyroxad (FXD) was assessed using a total of 770 P. pachyrhizi populations sampled over four soybean growing seasons. Cross-resistance, intrinsic activity, and frequency of SDHC-I86F mutation were also analyzed. The average effective concentration to inhibit 50% (EC50 ) and SDHC-I86F frequency increased over the 2015/2016, 2016/2017, 2017/2018 and 2018/2019 soybean-seasons. Fourteen P. pachyrhizi populations had the EC50 value above 10 mg L-1 for both carboxamides. No difference was found in intrinsic active to BZV and FXD fungicides for sensitive P. pachyrhizi populations. For P. pachyrhizi classified as less sensitive BZV showed the highest fungitoxicity effect. High frequency of the C-I86F mutation was observed in samples collected in volunteer soybean plants. The maximum frequency of SDHC-I86F mutation in the population was 50% and resulting in ASR populations with low sensitivity to SDHIs. A low correlation between bioassay and SDHC-I86F mutation was observed possible due to the dikaryotic nature of rust fungi or other mutations in the other succinate dehydrogenase subunits. CONCLUSION: The present work provides an overview of a large sampling size of P. pachyrhizi populations and their performance over the four crop seasons. The high frequency of SDHC-I86F mutation and low sensitivity to SDHIs are widely distributed in the main soybean growing regions in Brazil and present in volunteer plants in the soybean-free period. Further detailed studies are needed to identify novel point mutations affecting the effectiveness of SDHIs. © 2021 Society of Chemical Industry.
Asunto(s)
Fungicidas Industriales , Phakopsora pachyrhizi , Succinato Deshidrogenasa/genética , Amidas , Brasil , Fungicidas Industriales/farmacología , Tasa de Mutación , Norbornanos , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas , PirazolesRESUMEN
BACKGROUND: Phakopsora pachyrhizi is a biotrophic fungal pathogen responsible for the Asian soybean rust disease causing important yield losses in tropical and subtropical soybean-producing countries. P. pachyrhizi triggers important transcriptional changes in soybean plants during infection, with several hundreds of genes being either up- or downregulated. RESULTS: Based on published transcriptomic data, we identified a predicted chitinase gene, referred to as GmCHIT1, that was upregulated in the first hours of infection. We first confirmed this early induction and showed that this gene was expressed as early as 8 h after P. pachyrhizi inoculation. To investigate the promoter of GmCHIT1, transgenic soybean plants expressing the green fluorescence protein (GFP) under the control of the GmCHIT1 promoter were generated. Following inoculation of these transgenic plants with P. pachyrhizi, GFP fluorescence was detected in a limited area located around appressoria, the fungal penetration structures. Fluorescence was also observed after mechanical wounding whereas no variation in fluorescence of pGmCHIT1:GFP transgenic plants was detected after a treatment with an ethylene precursor or a methyl jasmonate analogue. CONCLUSION: We identified a soybean chitinase promoter exhibiting an early induction by P. pachyrhizi located in the first infected soybean leaf cells. Our results on the induction of GmCHIT1 promoter by P. pachyrhizi contribute to the identification of a new pathogen inducible promoter in soybean and beyond to the development of a strategy for the Asian soybean rust disease control using biotechnological approaches.
Asunto(s)
Quitinasas/genética , Glycine max/enzimología , Glycine max/genética , Phakopsora pachyrhizi/fisiología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Quitinasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/microbiologíaRESUMEN
Phakopsora pachyrhizi, the causal agent of soybean rust (SBR), is a global threat to soybean production. Since the discovery of SBR in the continental United States, quantitative polymerase chain reaction assays based on the internal transcribed spacer (ITS) ribosomal DNA locus were established for its rapid detection. However, insufficient data were initially available to test assays against factors that could give rise to misidentification. This study aimed to reevaluate current assays for (i) the potential for false-positive detection caused by nontarget Phakopsora species and (ii) the potential for false-negative detection caused by intraspecific variation within the ITS locus of P. pachyrhizi. A large amount of intraspecific and intragenomic variation in ITS was detected, including the presence of polymorphic ITS copies within single leaf samples and within single rust sori. The diagnostic assays were not affected by polymorphisms in the ITS region; however, current assays are at risk of false positives when screened against other species of Phakopsora. This study raises caveats to the use of multicopy genes (e.g., ITS) in single-gene detection assays and discusses the pitfalls of inferences concerning the aerobiological pathways of disease spread made in the absence of an evaluation of intragenomic ITS heterogeneity.
Asunto(s)
Variación Genética , Phakopsora pachyrhizi , Enfermedades de las Plantas , ADN Espaciador Ribosómico/genética , Técnicas de Diagnóstico Molecular/normas , Patología Molecular , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas/microbiología , Glycine max/microbiología , Estados UnidosRESUMEN
Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.
Asunto(s)
Basidiomycota/efectos de los fármacos , Basidiomycota/genética , Panicum/microbiología , Fosfitos/farmacología , Enfermedades de las Plantas/microbiología , Basidiomycota/patogenicidad , Resistencia a la Enfermedad , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Panicum/efectos de los fármacos , Panicum/metabolismo , Phakopsora pachyrhizi/efectos de los fármacos , Phakopsora pachyrhizi/genética , Phakopsora pachyrhizi/patogenicidad , Hojas de la Planta/microbiología , Especies Reactivas de Oxígeno/metabolismo , Glycine max/efectos de los fármacos , Glycine max/metabolismo , Glycine max/microbiologíaRESUMEN
Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.
Asunto(s)
Proteínas Fúngicas/metabolismo , Metaboloma , Nicotiana/microbiología , Phakopsora pachyrhizi/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Análisis por Conglomerados , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Ontología de Genes , Metaboloma/genética , Familia de Multigenes , Phakopsora pachyrhizi/genética , Filogenia , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Hojas de la Planta/microbiología , Glycine max/microbiología , Nicotiana/inmunología , Transcriptoma/genéticaRESUMEN
Soybean rust (Phakopsora pachyrhizi) in Brazil is mainly controlled with applications of fungicides, including demethylation inhibitors (DMI) and quinone outside inhibitors (QoI). Isolates with less sensitivity to DMI and QoI have been reported, and these have been found to have mutations in the CYP51 and CYTB genes, respectively. There have been no reports of fitness costs in isolates with mutations in CYP51 and CYTB, and the aim of this work was to compare the competitive ability of isolates with lower DMI or QoI sensitivities with that of sensitive (wild-type) isolates. Urediniospores of sensitive wild-type isolates and isolates with different CYP51 or CYTB alleles were mixed and inoculated on detached soybean leaves. After 3 weeks, urediniospores were harvested and used as inoculum for the next disease cycle. Frequencies of relevant target site mutations were monitored using the pyrosequencing method over four disease cycles. Isolates with lower DMI sensitivity and different CYP51 alleles had competitive disadvantages compared with a DMI-sensitive, wild-type CYP51 isolate. In contrast, the isolate with the F129L mutation in the CYTB gene competed equally well with a QoI-sensitive, wild-type CYTB isolate under the conditions of this experiment. The CYP51 and CYTB alleles were stable in all isolates over four disease cycles when cultivated alone.
Asunto(s)
Familia 51 del Citocromo P450/genética , Citocromos b/genética , Farmacorresistencia Fúngica/genética , Glycine max/microbiología , Phakopsora pachyrhizi/fisiología , Enfermedades de las Plantas/microbiología , Alelos , Sustitución de Aminoácidos , Brasil , Proteínas Fúngicas/genética , Fungicidas Industriales/farmacología , Genotipo , Mutación , Phakopsora pachyrhizi/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Asian soybean rust, caused by Phakopsora pachyrhizi, is mostly controlled by demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides. Mutations in the cytochrome b (CYTB) gene can lead to pathogen resistance to QoIs. The occurrence of the mutations in codons 129, 137 and 143 in the CYTB gene was investigated, and a pyrosequencing assay was developed for rapid and quantitative detection of the F129L mutation. RESULTS: Molecular analysis of the CYTB gene showed the presence of the F129L mutation in field samples and monouredinial isolates, while other mutations (G143A and G137R) were not found. The pyrosequencing was an effective method for quantitative detection of the F129L mutation, and many of the P. pachyrhizi samples showed high frequency of F129L. CONCLUSION: This is the first report of the occurrence of the F129L mutation in P. pachyrhizi. The practical relevance of this mutation for field efficacy of QoIs needs further investigation. © 2015 Society of Chemical Industry.
Asunto(s)
Citocromos b/genética , Farmacorresistencia Fúngica/genética , Phakopsora pachyrhizi/genética , Sustitución de Aminoácidos/genética , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Fungicidas Industriales/farmacología , Genes Fúngicos/genética , Phakopsora pachyrhizi/efectos de los fármacos , Análisis de Secuencia de ADNRESUMEN
Background Asian soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. & Syd., is one of the main diseases affecting soybean and has been reported as one of the most economically important fungal pathogens worldwide. Knowledge of the genetic diversity of this fungus should be considered when developing resistance breeding strategies. We aimed to analyze the genetic diversity of P. pachyrhizi combining simple sampling with a powerful and reproducible molecular technique. Results We employed Amplified Fragment Length Polymorphism (AFLP) technique for the amplification of P. pachyrhizi DNA extracted from naturally SBR-infected plants from 23 production fields. From a total of 1919 markers obtained, 77% were polymorphic. The high percentage of polymorphism and the Nei's genetic diversity coefficient (0.22) indicated high pathogen diversity. Analysis of molecular variance showed higher genetic variation within countries than among them. Temporal analysis showed a higher genetic variation within a year than between years. Cluster, phylogenetic and principal co-ordinate analysis showed that samples group by year of collection and then by country sampled. Conclusions The study proposed combining a simple collection of urediniospore with a subsequent analysis by AFLP was useful to examine the molecular polymorphism of samples of P. pachyrhizi collected and might have a significant contribution to the knowledge of its genetic diversity. Also, AFLP analysis is an important and potent molecular tool for the study of genetic diversity and could be useful to carry out wider genetic diversity studies.
Asunto(s)
Enfermedades de las Plantas , Variación Genética , Marcadores Genéticos , Phakopsora pachyrhizi/genética , Glycine max , Reacción en Cadena de la Polimerasa , Análisis del Polimorfismo de Longitud de Fragmentos AmplificadosRESUMEN
Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended.
Asunto(s)
Glycine max/genética , Glycine max/microbiología , Phakopsora pachyrhizi/genética , Enfermedades de las Plantas/genética , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Esenciales , Interacciones Huésped-Patógeno , Phakopsora pachyrhizi/fisiología , Enfermedades de las Plantas/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
Soybean (Glycine max L.)-breeding programs aim to develop cultivars with high grain yields and high tolerance to Asian soybean rust (Phakopsora pachyrhizi). Considering that the traits targeted for breeding are mainly quantitative in nature, knowledge of associations between traits allows the breeder to formulate indirect selection strategies. In this study, we investigated phenotypic, genotypic, and environmental correlations between the agronomic traits of soybean plants naturally infected with P. pachyrhizi, and identified agronomic traits that would be useful in indirectly selecting soybean genotypes for high yields. The study was conducted on the Capim Branco Farm, Uberlândia, Brazil, with 15 soybean genotypes, which were cultivated in a completely randomized block design with four replications. Fourteen phenotypic traits were evaluated using the GENES software. The phenotypic and genotypic correlations were positive and of a high magnitude between the total number of pods and the number of pods with two or three grains, indicating that the total number of pods is a useful trait for the indirect selection of soybean genotypes for high grain yields. Strong environmental correlations were found between plant height at blooming and maturity and grain yield and yield components.