Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

<p>Abstract</p> <p>Background</p> <p>A recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, <it>Peronosclerospora sorghi</it>. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.</p> <p>Results</p> <p>Among the 55 primers pairs designed from clones from pathotype 3 of <it>P. sorghi</it>, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from <it>P. sorghi</it>, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (<it>P. maydis & P. philippinensis</it>), sugar cane (<it>P. sacchari</it>), pearl millet (<it>Sclerospora graminicola</it>) and rose (<it>Peronospora sparsa</it>) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to <it>P. philippinensis </it>(one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 <it>Peronosclerospora, Peronospora and Sclerospora </it>spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of <it>Peronosclerospora sorghi </it>in cluster I, five isolates of <it>P. maydis </it>and three isolates of <it>P. sacchari </it>in cluster II and five isolates of <it>Sclerospora graminicola </it>in cluster III).</p> <p>Conclusion</p> <p>To our knowledge, this is the first attempt to extensively develop SSR markers from <it>Peronosclerospora </it>genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.</p

Isolation and characterization of the grain mold fungi \u3ci\u3eCochliobolus\u3c/i\u3e and \u3ci\u3eAlternaria\u3c/i\u3e spp. from sorghum using semiselective media and DNA sequence analyses

Mold diseases, caused by fungal complexes including Alternaria, Cochliobolus, and Fusarium species, limit sorghum grain production. Media were tested by plating Fusarium thapsinum, Alternaria sp., and Curvularia lunata, individually and competitively. Dichloran chloramphenicol rose bengal (DRBC) and modified V8 juice (ModV8) agars, found to be useful, were compared with commonly used agar media, dichloran chloramphenicol peptone (DCPA) and pentachloronitrobenzene (PCNB). Radial growth, starting with mycelia or single-conidia and hyphal tips, demonstrated an effect of media. For isolation of grain fungi, DRBC and ModV8 were similar or superior to DCPA and PCNB. When seedlings were inoculated with conidia of C. lunata, Alternaria sp., F. thapsinum, or mixtures, the percentage of root infection ranged from 28% to 77%. For mixed inoculations, shoot weights, lesion lengths, and percentage of root infections were similar to F. thapsinum inoculations; most colonies recovered from roots were F. thapsinum. For Alternaria grain isolates, 5 morphological types, including Alternaria alternata, were distinguished by colony morphologies and conidial dimensions. Sequence analysis using a portion of the endo-polygalacturonase gene was able to further distinguish isolates. Cochliobolus isolates were identified morphologically as C. lunata, Curvularia sorghina, and Bipolaris sorghicola. Multiple molecular genotypes were apparent from rRNA internal transcribed spacer region sequences from Cochliobolus grain isolates. La moisissure produite par des complexes de champignons formés des espèces Alternaria, Cochliobolus et Fusarium limite la production de sorgho. Des milieux de culture ont été étudiés en ensemençant Fusarium thapsinum, Alternaria sp. et Curvularia lunata de manière individuelle et compétitive. La gélose Dichloran Rose-bengale Chloramphénicol (DRBC) et la gélose au jus V8 modifiée (ModV8), qui s’étaient avérées utiles, ont été comparées a` d’autres géloses couramment utilisées, la gélose Dichloran Chloramphénicol Peptone (DCPA) et la gélose pentachloronitrobenzène (PCNB). L’examen de la croissance radiale, commençant par les mycéliums ou les conidies uniques et les têtes des hyphes, a démontré que les milieux exerçaient un effet. Les milieux DRBC et ModV8 étaient similaires ou supérieurs au DCPA et au PCNB quant a` l’isolement des champignons des céréales. Lorsque les semis étaient inoculés avec les conidies de C. lunata, Alternaria sp., F. thapsinum ou des mélanges de celles-ci, le pourcentage d’infection des racines allait de 28 % a` 77 %. La taille des pousses, la longueur des lésions et le pourcentage d’infection obtenus après une inoculation mixte ou une inoculation avec F. thapsinum étaient similaires ; la plupart des colonies récupérées des racines appartenaient a` F. thapsinum. En ce qui concerne les isolats de Alternaria présents sur les céréales, cinq types morphologiques dont Alternaria alternata se distinguaient par la morphologie des colonies et les dimensions des conidies. L’analyse de séquence d’une portion du gène codant l’endo-polygalacturonase a permis de distinguer davantage les isolats. Les isolats de Cochliobolus ont été identifiés morphologiquement comme étant C. lunata, Curvularia sorghina et Bipolaris sorghicola. De multiples génotypes moléculaires étaient mis en évidence par les séquences de la région transcrite de l’espaceur interne de l’ARNr des isolats de Cochliobolus provenant des céréales

Respuesta de germoplasma de sorgo de Etiopía a la antracnosis.

Forty-two accessions were randomly selected from the Ethiopian sorghum collection maintained by the USDA-ARS National Plant Germplasm System and were evaluated for anthracnose disease response during the 2004 dry and wet growing seasons in Isabela, Puerto Rico. A resistant response was observed for 20 accessions, with 13 accessions susceptible to the disease for the two growing seasons. This finding would suggest that Ethiopia would be an important source of anthracnose-resistant germplasm for sorghum improvement. Nine accessions showed variation for infection response within and between growing seasons. These accessions also showed low infection severity. Greater infection severity was observed during the dry growing season for the susceptible accessions; however, variation for mean infection severity for these accessions was not significantly different for the two seasons. Approximately 80% of the accessions showed a similar disease response across the two growing seasons.This percentage would suggest that large scale evaluations could be conducted throughout the year in Puerto Rico to screen the more than 7,000 Ethiopian sorghum accessions in the collection for the identification of anthracnose-resistant germplasm.Se seleccionaron al azar 42 accesiones de la colección de sorgo de Etiopía, la cual se mantiene en el Sistema Nacional de Germoplama de Plantas del Servicio de Investigación Agrícola del Departamento de Agricultura de los Estados Unidos (USDA-ARS, NGPS, por sus siglas en inglés). Se evaluó la respuesta de estas accesiones a la antracnosis durante las épocas de lluvia y de sequía en Isabela, Puerto Rico, en el 2004. Se observó resistencia a la enfermedad en 20 accesiones, mientras trece accesiones resultaron susceptibles a la enfermedad en ambas épocas. Estos resultados indican que la colección de sorgo de Etiopía es una fuente importante de germoplasma resistente a la antracnosis para utilizarse en el mejoramiento del sorgo. Nueve accesiones mostraron una variación en la respuesta a la infección dentro y fuera de las épocas de crecimiento. Estas accesiones también mostraron una baja severidad de infección. Las accesiones susceptibles a la enfermedad mostraron una infección mayor durante la época de sequía; sin embargo, la variación promedio de infección de estas accesiones no fue significativamente diferente para las dos épocas. Aproximadamente 80% de las accesiones mostraron una respuesta similar a la enfermedad a través de las dos épocas de crecimiento. Estos resultados indican que en Puerto Rico se pueden llevar a cabo evaluaciones en alta escala a través de todo el año para examinar más de 7,000 accesiones de sorgo de la colección e identificar germoplasma resistente a la antracnosis

Multi-Trait Genome-Wide Association Studies of <i>Sorghum bicolor</i> Regarding Resistance to Anthracnose, Downy Mildew, Grain Mold and Head Smut

Multivariate linear mixed models (mvLMMs) are widely applied for genome-wide association studies (GWAS) to detect genetic variants affecting multiple traits with correlations and/or different plant growth stages. Subsets of multiple sorghum populations, including the Sorghum Association Panel (SAP), the Sorghum Mini Core Collection and the Senegalese sorghum population, have been screened against various sorghum diseases such as anthracnose, downy mildew, grain mold and head smut. Still, these studies were generally performed in a univariate framework. In this study, we performed GWAS based on the principal components of defense-related multi-traits against the fungal diseases, identifying new potential SNPs (S04_51771351, S02_66200847, S09_47938177, S08_7370058, S03_72625166, S07_17951013, S04_66666642 and S08_51886715) associated with sorghum’s defense against these diseases

Response of Senegalese Sorghum Seedlings to Pathotype 5 of Sporisorium reilianum

Sporisorium reilianum causes head smut in sorghum. A total of 36 Senegalese sorghum accessions comprised of sorghum lines that have not been explored with response to pathotype 5 of S. reilianum were evaluated with 3 different treatments. First, seedling shoots were inoculated while still in soil with teliospores in agar, and then submerged under water at 4 days post inoculation. Signs of infection (noticeable spots) on the first leaf were observed up to 6 days post submergence. Second, seedlings at the same stage were inoculated by placing the teliospore impregnated agar around the stem in pots, moved to a greenhouse and grown to full panicle development stage. Third, seedings were inoculated via syringe inoculation in the greenhouse. Although soil inoculated seedlings grown in the greenhouse did not result in systemic infection as determined by lack of symptoms at panicle exsertion, 88.9% of tested cultivars showed systemic infections when syringe inoculated in the greenhouse. Inoculation of seedlings maintained under water led to broad range of noticeable spots that are assumed to be potential infection sites based on a previous study. In addition, seedling inoculation led to slightly upregulated expression of chitinase and PR10, genes that are associated with defense in aerial parts of plants

A Genome-Wide Association Study of Senegalese Sorghum Seedlings Responding to Pathotype 5 of <i>Sporisorium reilianum</i>

Sporisorium reilianum is a fungal pathogen that causes head smut in sorghum. In addition to pathotypes (P) 1-4, P5 and P6 were identified recently. In this study, seedlings of Senegalese sorghum, comprising 163 accessions, were evaluated for response to Sporisorium reilianum. Teliospores of pathotype P5 of the pathogen in dilute agar were pipetted onto seedling shoots while still in soil, and inoculated seedlings were submerged under water at 4 days post-inoculation. Signs of infection (noticeable spots) on the first leaf were checked daily up to 6 days post submergence. A genome-wide association study (GWAS) was conducted using 193,727 single-nucleotide polymorphisms (SNPs) throughout the genome based on two types of phenotypic data: whether noticeable spots were shown or not and the average time for an observation of the spots across 163 accessions. When mapped back to the reference sorghum genome, most of the top candidate SNP loci were associated with plant defense or plant stress response-related genes. The identified SNP loci were associated with spot appearance in sorghum seedlings under flooding following inoculation with P5 of Sporisorium reilianum

Genome-Wide Association Mapping of Anthracnose (Colletotrichum sublineolum) Resistance in NPGS Ethiopian Sorghum Germplasm

The National Plant Germplasm System (NPGS) Ethiopian sorghum [Sorghum bicolor (L.) Moench] collection of the United States is an important genetic resource for sorghum improvement. Anthracnose (Colletotrichum sublineolum) is one of the most harmful fungal diseases in humid sorghum production regions. Although multiple resistance sources have been identified in temperate-adapted germplasm in the Sorghum Association Panel (SAP), these resistance loci explain a limited portion of the total variation, and sources of resistance from tropical germplasm are not available for breeding programs at temperate regions. Using a core set of 335 previously genotyped NPGS Ethiopian accessions, we identified 169 accessions resistant to anthracnose. To identify resistance loci, we merged the genotypic and anthracnose response data for both NPGS Ethiopian germplasm and the SAP and performed genome-wide association scans using 219,037 single nucleotide polymorphisms and 617 accessions. The integrated data set enabled the detection of a locus on chromosome 9 present in the SAP at a low frequency. The locus explains a limited portion of the observed phenotypic variation (r2 = 0.31), suggesting the presence of other resistance loci. The locus in chromosome 9 was constituted by three R genes clustered within a 47-kb region. The presence of multiple sources of resistance in NPGS Ethiopian germplasm and SAP requires the inclusion of other resistance response evaluation that could revealed others low frequency resistance alleles in the panel

Population structure of the NPGS Senegalese sorghum collection and its evaluation to identify new disease resistant genes

<div><p>Sorghum germplasm from West and Central Africa is cultivated in rainy and high humidity regions and is an important source of resistance genes to fungal diseases. Mold and anthracnose are two important biotic constraints to sorghum production in wet areas worldwide. Here, 158 National Plant Germplasm System (NPGS) accessions from Senegal were evaluated for agronomic traits, anthracnose, and grain mold resistance at two locations, and genetically characterized according to 20 simple sequence repeat markers. A total of 221 alleles were amplified with an average of 11 alleles per locus. Each accession had a unique genetic profile (i.e., no duplicates), and the average genetic distance between accessions was 0.42. Population structure and cluster analysis separated the collection into four populations with pairwise F_ST values >0.15. Three of the populations were composed of Guinea-race sorghum germplasm, and one included multiple races. Anthracnose resistant accessions were present at high frequency and evenly distributed among the three Guinea-race populations. Fourteen accessions showed resistance to grain mold, and eight were resistant to both diseases. These results indicated that the NPGS of Senegal is a genetically diverse collection with a high frequency of disease resistant accessions. Nevertheless, its population structure suggests the presence of few sources of resistance to both grain mold and anthracnose, which are fixed in the germplasm. The phenotypic and genotypic information for these accessions provides a valuable resource for its correct use to broaden the genetic base of breeding programs.</p></div

Inoculation and Screening Methods for Major Sorghum Diseases Caused by Fungal Pathogens: <i>Claviceps africana</i>, <i>Colletotrichum sublineola</i>, <i>Sporisorium reilianum</i>, <i>Peronosclerospora sorghi</i> and <i>Macrophomina phaseolina</i>

Sorghum is the fifth most important crop globally. Researching interactions between sorghum and fungal pathogens is essential to further elucidate plant defense mechanisms to biotic stress, which allows breeders to employ genetic resistance to disease. A variety of creative and useful inoculation and screening methods have been developed by sorghum pathologists to study major fungal diseases. As inoculation and screening methods can be keys for successfully conducting experiments, it is necessary to summarize the techniques developed by this research community. Among many fungal pathogens of sorghum, here we summarize inoculation and screening methods for five important fungal pathogens of sorghum: Claviceps africana, Colletotrichum sublineola, Sporisorium reilianum, Peronosclerospora sorghi and Macrophomina phaseolina. The methods described within will be useful for researchers who are interested in exploring sorghum-fungal pathogen interactions. Finally, we discuss the latest biotechnologies and methods for studying plant-fungal pathogen interactions and their applicability to sorghum pathology

Distribution of allele sharing genetic distance among 158 NPGS Senegal sorghum accessions.

<p>Distribution of allele sharing genetic distance among 158 NPGS Senegal sorghum accessions.</p