Rust fungi on Panicum.

Rusts are economically important diseases of switchgrass (Panicum virgatum) and other Paniceae grasses. Phylogenetic analyses based on sequences of the nuc rDNA 5.8S internal transcribed spacer 2 region (ITS2), partial 28S region, and intergenic spacer region (IGS) of nuc rDNA showed that species of rust fungi infecting switchgrass are closely related within Puccinia. Variation among rbcLa sequences for the associated hosts sampled concurred with the original identifications. Five species infecting switchgrass were recognized: Puccinia graminicola (≡ Uromyces graminicola), P. pammelii (= P. panici), and the proposed new species P. amari, P. novopanici, and P. pascua. These species were distinct from P. emaculata, the species previously considered the principal rust pathogen infecting switchgrass but that was found exclusively on witchgrass (Panicum capillare) in this study. Rust fungi on switchgrass previously identified as P. emaculata were identified as the morphologically similar species P. amari, P. novopanici, and P. pammelii. The morphological species Puccinia graminicola was found to comprise three species, P. graminicola and the proposed new species P. pascua on switchgrass and P. cumminsii on Panicum sp.

Evolution of Nine Microsatellite Loci in the Fungus Fusarium oxysporum.

The evolution of nine microsatellites and one minisatellite was investigated in the fungus Fusarium oxysporum and sister taxa Fusarium redolens and Fusarium verticillioides. Compared to other organisms, fungi have been reported to contain fewer and less polymorphic microsatellites. Mutational patterns over evolutionary time were studied for these ten loci by mapping changes in core repeat numbers onto a phylogeny based on the sequence of the conserved translation elongation factor 1-α gene. The patterns of microsatellite formation, expansion, and interruption by base substitutions were followed across the phylogeny, showing that these loci are evolving in a manner similar to that of microsatellites in other eukaryotes. Most mutations could be fit to a stepwise mutation model, but a few appear to have involved multiple repeat units. No evidence of gene conversion was seen at the minisatellite locus, which may also be mutating by replication slippage. Some homoplastic numbers of repeat units were observed for these loci, and polymorphisms in the regions flanking the microsatellites may provide better genetic markers for population genetics studies of these species.

Sarcococca Blight: Use of Whole-Genome Sequencing for Fungal Plant Disease Diagnosis.

Early and accurate diagnosis of new plant pathogens is vital for the rapid implementation of effective mitigation strategies and appropriate regulatory responses. Most commonly, pathogen identification relies on morphology and DNA marker analysis. However, for new diseases, these approaches may not be sufficient for precise diagnosis. In this study, we used whole-genome sequencing (WGS) to identify the causal agent of a new disease affecting Sarcococca hookeriana (sarcococca). Blight symptoms were observed on sarcococca and adjacent Buxus sempervirens (boxwood) plants in Maryland during 2014. Symptoms on sarcococca were novel, and included twig dieback and dark lesions on leaves and stems. A Calonectria sp. was isolated from both hosts and used to fulfill Koch's postulates but morphology and marker sequence data precluded species-level identification. A 51.4-Mb WGS was generated for the two isolates and identified both as Calonectria pseudonaviculata. A single-nucleotide polymorphism at a noncoding site differentiated between the two host isolates. These results indicate that the same C. pseudonaviculata genotype has the ability to induce disease on both plant species. This study marks the first application of WGS for fungal plant pathogen diagnosis and demonstrates the power of this approach to rapidly identify causal agents of new diseases.

Highly diverse endophytic and soil Fusarium oxysporum populations associated with field-grown tomato plants.

The diversity and genetic differentiation of populations of Fusarium oxysporum associated with tomato fields, both endophytes obtained from tomato plants and isolates obtained from soil surrounding the sampled plants, were investigated. A total of 609 isolates of F. oxysporum were obtained, 295 isolates from a total of 32 asymptomatic tomato plants in two fields and 314 isolates from eight soil cores sampled from the area surrounding the plants. Included in this total were 112 isolates from the stems of all 32 plants, a niche that has not been previously included in F. oxysporum population genetics studies. Isolates were characterized using the DNA sequence of the translation elongation factor 1α gene. A diverse population of 26 sequence types was found, although two sequence types represented nearly two-thirds of the isolates studied. The sequence types were placed in different phylogenetic clades within F. oxysporum, and endophytic isolates were not monophyletic. Multiple sequence types were found in all plants, with an average of 4.2 per plant. The population compositions differed between the two fields but not between soil samples within each field. A certain degree of differentiation was observed between populations associated with different tomato cultivars, suggesting that the host genotype may affect the composition of plant-associated F. oxysporum populations. No clear patterns of genetic differentiation were observed between endophyte populations and soil populations, suggesting a lack of specialization of endophytic isolates.

A Multiplex Real-Time PCR Assay for the Detection of Puccinia horiana and P. chrysanthemi on Chrysanthemum.

Puccinia horiana, the cause of chrysanthemum white rust, is a regulated fungal plant pathogen in the United States, while P. chrysanthemi, the cause of chrysanthemum brown rust, is a widespread but less destructive pathogen. Accurate identification is essential to enforce quarantine measures, but the two species cannot be differentiated visually in the absence of mature spores or symptoms. A multiplex real-time PCR assay was developed to detect and discriminate between P. chrysanthemi and P. horiana. Species-specific hydrolysis probes labeled with different fluorescent dyes were designed based on the rDNA internal transcribed spacer region. Seven fresh samples and 270 herbarium specimens of chrysanthemum rust were tested with the assay with results confirmed using spore morphology. P. horiana and P. chrysanthemi were accurately detected from all fresh samples, and as little as 1 pg of template DNA was reproducibly detected. Of the herbarium specimens, 99% were positive for at least one species using the multiplex assay with 7% positive for both species. This multiplex assay can discriminate between P. chrysanthemi and P. horiana and provides an additional tool for identification of P. horiana to ensure appropriate application of quarantine measures.

LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach.

Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.

Microcyclic rusts of hollyhock ( Alcea rosea ).

Rust fungi infecting hollyhock and other plants in Malveae are frequently intercepted at ports of entry to the USA, particularly Puccinia malvacearum and P. heterogenea. These two species can be difficult to distinguish and can be further confused with other, less common species of microcyclic rust fungi infecting hollyhock: P. heterospora, P. lobata, P. platyspora, and P. sherardiana. Molecular phylogenetic analysis revealed that P. malvacearum and P. heterogenea are closely related, along with P. sherardiana and P. platyspora. A key to the six microcyclic Puccinia species infecting hollyhock is presented.

IMA Genome-F 3: Draft genomes of Amanita jacksonii, Ceratocystis albifundus, Fusarium circinatum, Huntiella omanensis, Leptographium procerum, Rutstroemia sydowiana, and Sclerotinia echinophila.

The genomes of fungi provide an important resource to resolve issues pertaining to their taxonomy, biology, and evolution. The genomes of Amanita jacksonii, Ceratocystis albifundus, a Fusarium circinatum variant, Huntiella omanensis, Leptographium procerum, Sclerotinia echinophila, and Rutstroemia sydowiana are presented in this genome announcement. These seven genomes are from a number of fungal pathogens and economically important species. The genome sizes range from 27 Mb in the case of Ceratocystis albifundus to 51.9 Mb for Rutstroemia sydowiana. The latter also encodes for a predicted 17 350 genes, more than double that of Ceratocystis albifundus. These genomes will add to the growing body of knowledge of these fungi and provide a value resource to researchers studying these fungi.