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Potato mop top virus (PMTV) is a continuing threat to potato production throughout the world. It has the potential to persist in the soil for long periods in the sporosori of its vector Spongospora subterranea f. sp. subterranea , which is as an important source for PMTV infection and dissemination. In this study, we used real-time quantitative reverse-transcription PCR (qRT-PCR) and reverse-transcription droplet digital PCR (RT-ddPCR) assays of the total RNA extracted directly from the soil to develop a simple, fast, and sensitive method to detect PMTV in soil samples using a specific primer with high efficiency despite a minimal amount of viral RNA. The designed primers are resilient in the presence of various PCR inhibitors in the soil when RNA is extracted. Both assays detected PMTV in all soil types used and supported the detection of <10 PMTV copies µl -1 in the RNA sample. With qRT-PCR, detection was linear, with amplification efficiencies ranging from 93.3 to 105.3% for silt loam, loamy sand, sand, and sandy loam in various experiments with R 2 > 0.99. Furthermore, the RT-ddPCR assay also demonstrated a high degree of linearity ( R 2 > 0.99 and P < 0.0001) with the RNA extracted from the soil samples representing different textures and physiochemical characteristics that were artificially spiked with infested S. subterranea f. sp. subterranea sporosori. Additionally, both assays successfully detected PMTV in different types of naturally infested soil with PMTV carrying S. subterranea f. sp. subterranea sporosori levels ranging from 6.2 × 10 2 g -1 to 1.2 × 10 6 g -1 in soils with pH ranging from 4.9 to 7.5 and organic matter ranging from 0.9 to 5.1%, demonstrating the potential to detect PMTV in a wide variety of soils. To our knowledge, this is the first report of the development of real-time PCR and ddPCR methods for the direct detection of a soilborne virus in soil.

'Candidatus Liberibacter solanacearum' contains two solanaceous crop-infecting haplotypes, A and B. Two haplotype A draft genomes were assembled and compared with ZC1 (haplotype B), revealing inversion and relocation genomic rearrangements, numerous single-nucleotide polymorphisms, and differences in phage-related regions. Differences in prophage location and sequence were seen both within and between haplotype comparisons. OrthoMCL and BLAST analyses identified 46 putative coding sequences present in haplotype A that were not present in haplotype B. Thirty-eight of these loci were not found in sequences from other Liberibacter spp. Quantitative polymerase chain reaction (qPCR) assays designed to amplify sequences from 15 of these loci were screened against a panel of 'Ca. L. solanacearum'-positive samples to investigate genetic diversity. Seven of the assays demonstrated within-haplotype diversity; five failed to amplify loci in at least one haplotype A sample while three assays produced amplicons from some haplotype B samples. Eight of the loci assays showed consistent A-B differentiation. Differences in genome arrangements, prophage, and qPCR results suggesting locus diversity within the haplotypes provide more evidence for genetic complexity in this emerging bacterial species.

An overview is provided for the aspects of history, biology, genomics, genetics, and epidemiology of zebra chip (ZC), a destructive disease of potato (Solanum tuberosum) that represents a major threat to the potato industries in the United States as well as other potato-production regions in the world. The disease is associated with a gram-negative, phloem-limited, insect-vectored, unculturable prokaryote, 'Candidatus Liberibacter solanacearum', that belongs to the Rhizobiaceae family of α-Proteobacteria. The closest cultivated relatives of 'Ca. L. solanacearum' are members of the group of bacteria known as the α-2 subgroup. In spite of the fact that Koch's postulates sensu stricto have not been fulfilled, a great deal of progress has been made in understanding the ZC disease complex since discovery of the disease. Nevertheless, more research is needed to better understand vector biology, disease mechanisms, host response, and epidemiology in the context of vector-pathogen-plant interactions. Current ZC management strategies focus primarily on psyllid control. The ultimate control of ZC likely relies on host resistance. Unfortunately, all commercial potato cultivars are susceptible to ZC. Elucidation of the 'Ca. L. solanacearum' genome sequence has provided insights into the genetic basis of virulence and physiological and metabolic capability of this organism. Finally, the most effective, sustainable management of ZC is likely to be based on integrated strategies, including removal or reduction of vectors or inocula, improvement of host resistance to the presumptive pathogen and psyllid vectors, and novel gene-based therapeutic treatment.

ABSTRACT Isolates of Helminthosporium solani, the causal agent of silver scurf of potato, collected from multiple locations consistently show white sectoring and rings, differential coloration, and reduced sporulation in culture. It has been accepted that this growth pattern is normal for H. solani cultures. Scanning electron microscopy confirmed the presence of a contaminating fungus in close association with cultures of H. solani. Repeated hyphal tip isolation techniques were used to separate H. solani from the fungal contaminant. Resultant pure cultures of H. solani were uniformly black in color, without white sectors or rings. The contaminating fungus was identified as Acremonium strictum. The purpose of this study was to elucidate the relationship between A. strictum and H. solani, and evaluate the impact of the fungicolous A. strictum on the growth and biology of H. solani. In vitro studies demonstrated that A. strictum significantly reduced sporulation of H. solani isolates from 65 to 35%, spore germination from 53 to 43%, and mycelial growth from 40 to 32% compared with noncontaminated cultures of H. solani. These data indicate that A. strictum is antagonistic to H. solani, and can be considered a mycoparasite. A. strictum reduced H. solani conidia production on minitubers, thereby reducing inoculum for infection. However, treatment with A. strictum does not reduce silver scurf of previously infected tubers. Further studies are warranted to determine the full potential of A. strictum as a biological control agent of H. solanii-incited silver scurf of stored potato tubers and the most effective manner of use.

ABSTRACT Amplified fragment length polymorphism (AFLP) using three primer sets was used to characterize 211 Colletotrichum coccodes isolates from North America, 112 of which were assigned to six vegetative compatibility groups (VCGs) using nitrate nonutilizing (nit) mutants. These isolates clustered into five corresponding groups by unweighted pairgroup method with arithmetic means-based cluster analysis of AFLP banding patterns. Isolates of C. coccodes belonging to NA-VCG1 and NA-VCG3 were closely related, as were isolates belonging to NA-VCG2 and NA-VCG5. Based on bootstrap analysis of AFLP data, the two isolates originally assigned to NA-VCG4 clustered with isolates belonging to NA-VCG2 and NA-VCG5. C. coccodes isolates that clustered with two isolates belonging to NA-VCG6 were the most diverged from other groups, including seven isolates collected from hosts other than potato. As opposed to the bootstrap analysis, a quadratic discriminant analysis (QDA) of AFLP data correctly categorized the two isolates of NA-VCG4. Furthermore, in isolates where VCG determinations had been made, this model correctly classified isolates of all VCGs. QDA classifications were identical to those made by the bootstrap analysis, with the exception of VCG4. Overall, classifications made by the QDA model were strongly correlated (r = 0.970, P < 0.001) to the VCGs assigned by traditional methods. All 99 C. coccodes isolates evaluated only by AFLP also were subjected to QDA, leading to the assignment of a presumptive VCG for each isolate. No isolates of VCG4 or VCG6 were identified by QDA within this population. Symptoms of black dot developed in plants inoculated with isolates collected from both potato and non-potato hosts. However, total yield was not significantly reduced by infection with non-potato isolates. The lack of any additional groups identified by AFLP analysis may be an indicator of a limited level of genetic variation among North American C. coccodes isolates. AFLP is a much more efficient technique for subspecific characterization in C. coccodes than VCG analysis utilizing nit mutants and will provide an effective means by which the population biology of this pathogen can be further investigated worldwide.