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Spinach ( Spinacae oleracae) is a nutritious vegetable and an economically important crop worldwide. Spinach is threatened by a number of diseases, and the most significant of which is downy mildew, caused by Peronospora farinosa f. sp. spinaciae (Pfs). The spinach locus RPF1 confers resistance to 11 of the 15 known races of Pfs. To study the RPF1 locus in depth, a BAC library was constructed from DNA of a single spinach plant that was homozygous at the RPF1 locus. The BAC library contained 73,728 clones, with an average insert size of 183 kb, which represented approximately 13 spinach genome equivalents (989 Mb). The ends of 1920 BAC clones were sequenced from both directions, and 3405 BAC end sequences (BESs) ranging from 100 to 935 bp were obtained. About 38.59 % of the BESs contained repetitive sequences, including 363 copies of retrotransposable elements and 32 copies of transposable elements. Less than 3 % of the clones originated from chloroplasts or mitochondria. Various categories of protein-encoding genes, including transcription factors, stress responsive genes, and reproduction-associated genes, were identified. Putative disease resistance genes containing nucleotide binding site-leucine-rich repeat (NBS-LRR) domains were also identified, one of which was closely linked to the RPF1 locus. Simple sequence repeat (SSR) motifs were identified from 154 BESs, including various di-, tri-, tetra-, and penta-nucleotide repeats. One hundred pairs of SSR primers and 24 retrotransposon primers were designed from BESs, all of which successfully amplified spinach DNA. This spinach BAC library will provide a useful resource for spinach genetic studies and breeding efforts. [ABSTRACT FROM AUTHOR]

Receptor-like cytoplasmic kinases (RLCKs) form a large subfamily of proteins in plants. RLCKs are known to regulate plant immunity to bacterial and fungal pathogens. In this study, we analyzed the genome-wide complement of maize RLCK genes and conducted detailed studies on one maize RLCK. The maize genome encodes 192 RLCKs that largely mirror the RLCK family in other plants. Previous studies implicated Arabidopsis BOTRYTIS INDUCED KINASE1 (BIK1) and TOMATO PROTEIN KINASE 1b (TPK1b) in plant resistance to the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea. A novel maize RLCK, Zea Mays BIK1-LIKE KINASE 1 (ZmBLK1), was identified based on sequence similarity to the tomato and Arabidopsis RLCKs. We demonstrated that ZmBLK1 displays protein kinase activity in vitro and the protein localizes to the plasma membrane. Importantly, expression of ZmBLK1 partially rescued the growth and disease phenotypes of the Arabidopsis bik1 mutant plants. The expression of ZmBLK1 was induced in maize at 12 h after inoculation with Clavibacter michiganensis subsp. nebraskensis (CMN), the bacterial pathogen causing Goss's wilt. Interestingly, overexpression of ZmBLK1 in transgenic maize increased resistance to CMN but did not impact resistance to Aspergillus ear rot caused by the fungal pathogen Aspergillus flavus and the associated aflatoxin contamination. These findings support our hypothesis that ZmBLK1 contributes to plant resistance to bacterial pathogens likely by modulating events early after pathogen infection, implying that the protein may interact with other membrane proteins early in the immune response pathway. [ABSTRACT FROM AUTHOR]

Downy mildew, caused by the obligate oomycete pathogen Peronospora effusa, is the most important disease of spinach. Growing resistant cultivars is the most economical way to manage the disease, particularly for organic spinach production. Therefore, the tools that assist and expedite the development of resistant cultivars are urgently needed. Resistance to P. effusa is typically controlled by major qualitative dominant genes, and at least six resistance loci are known to exist in spinach. Three of these resistant loci, RPF1, RPF2 and RPF3 have been genetically characterized and each locus controls resistance to more than nine of the 17 described races of P. effusa. However, as downy mildew is an obligate pathogen, the bioassays used to evaluate resistance are especially challenging. Thus, having robust molecular markers closely linked to resistance loci would greatly expedite genetic selections. In this study, multiple approaches, including bulked segregant analysis (BSA) using genotyping-by-sequencing, were deployed to develop thirteen, two, and seven markers linked to the RPF1, RPF2, and RPF3 resistance loci, respectively. One marker, RPF3-3, was linked to RPF2 and RPF3, and three markers, RPF3-1, RPF3-2, and RPF3-6, were linked to all three loci, indicating that the three resistance loci are closely linked. In blind tests, the markers effectively discriminated the presence of RPF1, RPF2, and RPF3 in diverse spinach germplasm. Thus, the markers developed in this study accurately predict the presence of RPF1, RPF2, and RPF3 loci, and can accelerate the development of downy mildew resistance in spinach breeding programs. [ABSTRACT FROM AUTHOR]

Fusarium verticillioides, a kernel-rotting pathogen of maize, produces fumonisin mycotoxins that are detrimental to both human and animal health. Current knowledge about the environmental regulation of fumonisin biosynthesis is centered on the influence of pH and carbohydrate availability. In this study, we report metabolic changes in F. verticillioides associated with conditions that are conducive (pH 3) or repressive (pH 8) for fumonisin biosynthesis, as well as changes associated with targeted disruption of PAC1, a pH-responsive transcription factor. To this end, metabolic fingerprints were generated from F. verticillioides with gas chromatography-mass spectrometry. A total of 46 metabolites were detected, of which approximately one third matched reference spectra of various carbohydrates and fatty acids. Analysis of wild type and Δpac1 fingerprints by principal component analysis revealed that the biosynthesis of arabitol, mannitol, and trehalose was significantly affected by pH and disruption of PAC1. Consistent with this finding, the expression of genes involved in trehalose biosynthesis was significantly reduced in the Δpac1 strain. This study is the first report linking PAC1 to polyol biosynthesis in F. verticillioides, and could indicate a broadly conserved function for PAC1 orthologs among filamentous fungi. Additionally, by presenting a metabolic fingerprinting technique for F. verticillioides, this study provides a new resource to understand pathogenesis and mycotoxigenesis through functional genomics. [ABSTRACT FROM AUTHOR]

Taproot decline (TRD) is a disease of soybean that has been reported recently from the southern United States (U.S.). Symptoms of TRD include foliar interveinal chlorosis followed by necrosis. Darkened, charcoal-colored areas of thin stromatic tissue are evident on the taproot and lateral roots along with areas of necrosis within the root and white mycelia within the pith. Upright stromata typical of Xylaria can be observed on crop debris and emerging from infested roots in fields where taproot decline is present, but these have not been determined to contain fertile perithecia. Symptomatic plant material was collected across the known range of the disease in the southern U.S., and the causal agent was isolated from roots. Four loci, ⍺-actin (ACT), β-tubulin (TUB2), the nuclear rDNA internal transcribed spacers (nrITS), and the RNA polymerase subunit II (RPB2), were sequenced from representative isolates. Both maximum likelihood and Bayesian phylogenetic analyses showed consistent clustering of representative TRD isolates in a highly supported clade within the Xylaria arbuscula species complex in the "HY" clade of the family Xylariaceae, distinct from any previously described taxa. In order to understand the origin of this pathogen, we sequenced herbarium specimens previously determined to be "Xylaria arbuscula" based on morphology and xylariaceous endophytes collected in the southern U.S. Some historical specimens from U.S. herbaria collected in the southern region as saprophytes as well as a single specimen from Martinique clustered within the "TRD" clade in phylogenetic analyses, suggesting a possible shift in lifestyle. The remaining specimens that clustered within the family Xylariaceae, but outside of the "TRD" clade, are reported. Both morphological evidence and molecular evidence indicate that the TRD pathogen is a novel species, which is described as Xylaria necrophora. [ABSTRACT FROM AUTHOR]

Charcoal rot, caused by Macrophomina phaseolina in soybean [ Glycine max (L).Merr.] is a disease of economic significance in the United States. Chemical and cultural control are not effective and host resistance could be a potential method to control the disease. In this study we report the use of bulked-segregant analysis (BSA) to map the genetic basis of resistance to charcoal rot in soybean PI 567562A, using a QTL-seq approach. Resistant and susceptible bulks from progenies of a biparental population (PI 567562A and PI 567437) were sequenced, and reads were aligned to the PI 567562A genome and used to calculate the SNP-index at each SNP position for the resistant and susceptible bulks. The average distributions of the SNP-index and Δ SNP-index across the genome were estimated for genomic intervals using a sliding window analysis with 2-Mb window size and a 10-kb step. Three genomic regions on chromosomes 5, 8 and 14 were identified with positive values in Δ SNP-index plots that potentially correspond to QTL governing the difference between the R-bulk and S-bulk sub-populations. The current study is the first attempt employing QTL-seq to identify genomic regions that may contain QTL controlling charcoal rot disease resistance in soybean. • Genetic basis of resistance to charcoal rot in soybean. • QTL-seq approach with next-generation sequencing (NGS). • Bulked-segregant analysis (BSA). [ABSTRACT FROM AUTHOR]

Charcoal rot of soybean [Glycine max (L.) Merr.], caused by the soilborne fungus Macrophomina phaseolina (Tassi) Goid., has ranked among the most important soybean diseases in the United States. Disease management is typically done in a multifaceted approach through crop rotation, tillage, irrigation, and seed treatments aimed at minimizing damage caused by the pathogen. Development of genetic resistance to charcoal rot appears to be the most efficient strategy to control the disease; however, there are no reports of genetic regions associated with resistance to the disease. The objective of this study was to identify quantitative trait loci (QTLs) governing resistance to charcoal rot in soybean using a biparental population of PI 567562A (resistant) × PI 567437 (susceptible). A total of 140 F2–derived lines were genotyped with 5403 single nucleotide polymorphism (SNP) markers covering 20 chromosomes, of which 2283 were polymorphic. Resistance to charcoal rot was evaluated in the F2:3 lines using the cut‐stem inoculation technique under greenhouse conditions. The QTL mapping analysis indicated one QTL on chromosome 15 explaining 29.4% of phenotypic variation, and two QTLs on chromosome 16 explaining 25.4 and 8.4% of phenotypic variation for resistance to M. phaseliona. To our knowledge, this is the first report of genomic regions harboring resistance to charcoal rot in soybean, and it may facilitate breeding and molecular engineering progress to combat charcoal rot disease in the future. [ABSTRACT FROM AUTHOR]

Peronospora effusa is an obligate pathogen that causes downy mildew on spinach and is considered the most economically important disease of spinach. The objective of the current research was to assess genetic diversity of known historical races and isolates collected in 2014 from production fields in Yuma, Arizona and Salinas Valley, California. Candidate neutral single nucleotide polymorphisms (SNPs) were identified by comparing sequence data from reference isolates of known races of the pathogen collected in 2009 and 2010. Genotypes were assessed using targeted sequencing on genomic DNA extracted directly from infected plant tissue. Genotyping 26 historical and 167 contemporary samples at 46 SNP loci revealed 82 unique multi-locus genotypes. The unique genotypes clustered into five groups and the majority of isolates collected in 2014 were genetically closely related, regardless of source location. The historical samples, representing several races, showed greater genetic differentiation. Overall, the SNP data indicate much of the genotypic variation found within fields was produced during asexual development, whereas overall genetic diversity may be influenced by sexual recombination on broader geographical and temporal scales. [ABSTRACT FROM AUTHOR]

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors. [ABSTRACT FROM AUTHOR]

Background: Transposable elements (TEs) can be key drivers of evolution, but the mechanisms and scope of how they impact gene and genome function are largely unknown. Previous analyses revealed that TE-mediated gene amplifications can have variable effects on fungal genomes, from inactivation of function to production of multiple active copies. For example, a DNA methyltransferase gene in the wheat pathogen Zymoseptoria tritici (synonym Mycosphaerella graminicola) was amplified to tens of copies, all of which were inactivated by Repeat-Induced Point mutation (RIP) including the original, resulting in loss of cytosine methylation. In another wheat pathogen, Pyrenophora tritici-repentis, a histone H3 gene was amplified to tens of copies with little evidence of RIP, leading to many potentially active copies. To further test the effects of transposon-aided gene amplifications on genome evolution and architecture, the repetitive fraction of the significantly expanded genome of the banana pathogen, Pseudocercospora fijiensis, was analyzed in greater detail. Results: These analyses identified a housekeeping gene, histone H3, which was captured and amplified to hundreds of copies by a hAT DNA transposon, all of which were inactivated by RIP, except for the original. In P. fijiensis the original H3 gene probably was not protected from RIP, but most likely was maintained intact due to strong purifying selection. Comparative analyses revealed that a similar event occurred in five additional genomes representing the fungal genera Cercospora, Pseudocercospora and Sphaerulina. Conclusions: These results indicate that the interplay of TEs and RIP can result in different and unpredictable fates of amplified genes, with variable effects on gene and genome evolution. [ABSTRACT FROM AUTHOR]