Aggressiveness of Small-Spored Alternaria spp. and Their Sensitivity to Succinate Dehydrogenase Inhibitor Fungicides.

Brown leaf spot of potato is caused by a number of small-spored Alternaria spp. Alternaria alternata sensu stricto, A. arborescens, and A. tenuissima have been reported with increasing frequency in commercial potato fields. Potato cultivars with resistance to small-spored Alternaria spp. have yet to be developed; therefore, the application of foliar fungicides is a primary management strategy. Greenhouse inoculation assays demonstrated that isolates of these three small-spored Alternaria spp. were pathogenic. Significant differences in aggressiveness were observed across isolates; however, there was no trend in aggressiveness based on species. Significant fungicide by isolate interactions in in vitro fungicide sensitivity and significant differences between baseline and nonbaseline isolates were observed in all three small-spored Alternaria spp. The ranges of in vitro sensitivity of A. alternata baseline isolates to boscalid (EC50 <0.010 to 0.89 µg/ml), fluopyram (<0.010 to 1.14 µg/ml) and solatenol (<0.010 to 1.14 µg/ml) were relatively wide when compared with adepidyn (<0.010 to 0.023 µg/ml). The baseline sensitivities of A. arborescens and A. tenuissima isolates to all four fungicides were <0.065 µg/ml. Between 10 and 21% of nonbaseline A. alternata isolates fell outside the baseline range established for the four succinate dehydrogenase inhibitor (SDHI) fungicides evaluated. In A. arborescens, 10 to 80% of nonbaseline isolates had higher sensitivities than the baseline. A. tenuissima isolates fell outside the baseline for boscalid (55%), fluopyram (14%), and solatenol (14%), but none fell outside the baseline range for adepidyn. Evaluations of in vivo fungicide efficacy demonstrated that most isolates were equally controlled by the four SDHI fungicides. However, reduced boscalid efficacy was observed for four isolates (two each of A. arborescens and A. tenuissima) and reduced fluopyram control was observed in one A. alternata isolate. Results of these studies demonstrate that isolates of all three species could be contributing to the brown leaf spot pathogen complex and that monitoring both species diversity and fungicide sensitivity could be advantageous for the management of brown leaf spot in potatoes with SDHI fungicides.

Phylogenetics of tobacco rattle virus isolates from potato (Solanum tuberosum L.) in the USA: a multi-gene approach to evolutionary lineage.

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed.

Impact of SDH Mutations in Alternaria solani on Recently Developed SDHI Fungicides Adepidyn and Solatenol.

Early blight, caused by Alternaria solani, is observed annually in all midwestern potato production areas. The use of foliar fungicides remains a primary management strategy. However, A. solani has developed reduced sensitivity or resistance to many single-site fungicides such as quinone outside inhibitor (QoI, FRAC group 11), succinate dehydrogenase inhibitor (SDHI, FRAC group 7), demethylation inhibitor (DMI, FRAC group 3), and anilinopyrimidine (AP, FRAC group 9) fungicides. Boscalid, fluopyram, solatenol, and adepidyn are EPA-registered SDHI fungicides used commercially on a variety of crops, including potato. Five SDH mutations have been characterized previously in A. solani that affect the efficacy of boscalid while only one of these mutations has been demonstrated to negatively affect fluopyram efficacy. Conidial germination assays were used to determine if a shift in sensitivity has occurred in these SDHI fungicides. A. solani isolates collected prior to the commercial application of SDHI fungicides (baseline) were compared with recently collected isolates (nonbaseline). Greenhouse evaluations were conducted also to evaluate the efficacy of boscalid, fluopyram, solatenol, and adepidyn on A. solani isolates possessing individual SDH mutations. Additionally, field trials were conducted to determine the effects of application of these SDHI fungicides on the frequency of SDH mutations. Fluopyram, solatenol, and adepidyn had high intrinsic activity against A. solani when compared with boscalid, based on in vitro assays. The application of adepidyn and solatenol resulted in greater early blight control than the application of boscalid and fluopyram in greenhouse experiments. Molecular characterization of A. solani isolates collected from the field trials determined that the frequency of the H134R-mutation can increase in response to more recently developed SDHI fungicides. In contrast, the H278R/Y- and H133R-mutations decreased to the point of being nearly absent in these field experiments.

Development and Application of a Real-Time Reverse-Transcription PCR and Droplet Digital PCR Assays for the Direct Detection of Potato mop top virus in Soil.

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 R2 > 0.99. Furthermore, the RT-ddPCR assay also demonstrated a high degree of linearity (R2 > 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 × 102 g-1 to 1.2 × 106 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.

Detection and Quantification of Spongospora subterranea Sporosori in Soil by Quantitative Real-Time PCR.

Powdery scab on potato tubers is caused by the obligate soilborne biotroph Spongospora subterranea and is known to cause substantial losses in potato production. The pathogen also infects roots of susceptible hosts, forming galls which can negatively affect root function. S. subterranea is also the vector of Potato mop-top virus, which causes a tuber necrosis disease that can, depending on temperature and cultivar, render potato tubers unmarketable. In this study, we adapted a published protocol to develop a sensitive and robust quantitative real-time PCR (qPCR) assay using specific primers and probes for detecting and quantifying S. subterranea sporosori in soil types that differ in physical properties, including organic matter content and soil pH. For the first time, an external control was utilized and applied directly to the soil prior to DNA extraction, which facilitated normalization of S. subterranea sporosori soil levels from sample to sample. The duplex qPCR protocol was demonstrated to be highly sensitive, capable of detecting and quantifying as few as 1 sporosorus/g of soil, with consistently high qPCR efficiency and the coefficient of determination (R2) values ranging from 94 to 99% and 0.98 to 0.99, respectively. The protocol was successfully implemented in enumerating S. subterranea sporosori in naturally infested field soil collected from several states and in artificial potting mixes with high organic matter content ranging from 64 to 71%. The qPCR method developed can be useful for potato growers to avoid agricultural soils highly infested with S. subterranea and in the development of risk assessment models in the future that incorporate cultivar susceptibility to powdery scab and soil infestation levels.

Spatial and Temporal Distribution of Mutations Conferring QoI and SDHI Resistance in Alternaria solani Across the United States.

The application of succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicide chemistries is a primary tactic in the management of early blight of potato, caused by Alternaria solani. Resistance to QoIs in A. solani has been attributed to the F129L mutation, while resistance to SDHIs is conferred by five different known point mutations on three AsSdh genes. In total, 1,323 isolates were collected from 2013 through 2015 across 11 states to determine spatial and temporal frequency distribution of these mutations. A real-time polymerase chain reaction (PCR) was used to detect the presence of the F129L mutation. Molecular detection of SDHI-resistant isolates was performed using SDH multiplex PCR specific for point mutations in AsSdhB, AsSdhC, or AsSdhD genes and mismatch amplification analysis PCR detecting the point mutations in AsSdhB. Previous work in our research group determined that substitutions of histidine for tyrosine (H278Y) or arginine (H278R) at codon 278 on the AsSdhB gene were the most prevalent mutations, detected in 46 and 21% of A. solani isolates, respectively, collected in 2011 to 2012, and uniformly distributed among six sampled states. In contrast, the substitution of histidine for arginine (H134R) at codon 134 in the AsSdhC gene was the most prevalent mutation in 2013 through 2015, identified in 36% of isolates, compared with 7.5% of isolates recovered in 2011 to 2012. Substitutions of histidine for arginine (H133R) at codon 133 and aspartic acid for glutamic acid (D123E) at codon 123 in the AsSdhD gene were detected in 16 and 12%, respectively, in the A. solani population by 2015 and were recovered across a wide range of states, compared with 15 and 1.5% of isolates collected in 2011 to 2012, respectively. Overall, SDHI- and QoI-resistant isolates were detected at high frequencies across all years, with evidence of significant spatial variability. Future research will investigate whether these results are due to differences in parasitic fitness.

A Real-Time PCR Assay for the Detection of Clavibacter michiganensis subsp. sepedonicus Based on the Cellulase A Gene Sequence.

Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot (BRR) of potato (Solanum tuberosum), is a globally important quarantine pathogen that is managed in North America using zero tolerance regulations in the certified seed industry. C. michiganensis subsp. sepedonicus is well documented to cause symptomless infections in potato, contributing to its persistence in certified seed stocks. Reliable laboratory methods to detect symptomless infections with a high degree of sensitivity could assist in the reduction of inoculum in certified seed potato stocks. A real-time polymerase chain reaction (PCR) assay was developed using the cellulase A (CelA) gene sequence as the basis for primer design. CelA primers were specific to C. michiganensis subsp. sepedonicus grown in vitro and did not detect any other coryneform bacteria or potato pathogenic bacteria but did detect 69 strains of C. michiganensis subsp. sepedonicus. The CelA real-time PCR assay was more sensitive than immunofluorescence (IFA) and Cms50/72a PCR assays in detecting C. michiganensis subsp. sepedonicus in infected potato tuber cores blended with healthy tuber cores in simulated seed lot contamination experiments. CelA primers detected nonmucoid and mucoid strains with equivalent sensitivity. In naturally infected seed lots, CelA PCR primers also were more sensitive in detecting symptomless infections of C. michiganensis subsp. sepedonicus in seed tubers prior to planting compared to Cms50/72a PCR primers, IFA, and enzyme-linked immunosorbent assay. A real-time PCR format using the newly developed CelA primers proved to be a very robust detection tool for C. michiganensis subsp. sepedonicus with the added advantage of detecting only virulent strains of the ring rot bacterium.

Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells.

Androgen receptor plays a critical role in the development and maintenance of cancers in the prostate. Earlier, we have shown that Cdc6, a regulatory protein for initiation of DNA replication, is down regulated in androgen-insensitive prostate cancer cells. In this report, we studied the involvement of androgen, mediated through androgen receptor (AR) in regulation of Cdc6 expression. Our results demonstrated that androgen treatment stimulated Cdc6 expression in xenograft tumors and androgen-sensitive prostate cancer cells. We also showed that androgen treatment stimulated Cdc6 transcription through possible interaction of AR with the ARE sequence in the Cdc6 promoter and that the stimulatory effect of androgen required intact E2F binding sites in the promoter. Androgen treatment differentially altered nuclear availability of E2F1 and E2F3, and increased the amount of hypophosphorylated retinoblastoma protein (pRb) in the nucleus in a time dependent fashion. We further showed that AR interacted with E2F transcription factors in a ligand-independent manner and that ligand-bound AR was less efficient in interacting with E2F proteins. DNA-protein interaction assays indicated that androgen treatment altered binding of E2F1 to the Cdc6 promoter in prostate cancer cells. We conclude that AR regulates Cdc6 transcription through interaction with the Cdc6 promoter, and complex formation with E2F1 and E2F3 in a differential manner.

Recognition of secretory proteins in Escherichia coli requires signals in addition to the signal sequence and slow folding.

BACKGROUND: The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway. RESULTS: In the current work, we tested this hypothesis using a mutant form of lambda repressor that folds slowly. No export of the mutant protein was observed, even in a prl strain. We then examined binding of the mutant lambda repressor to SecB. We did not observe interaction by either of two assays, indicating that slow folding is not sufficient for SecB binding and targeting to translocase. CONCLUSIONS: These results strongly suggest that to be targeted to the export pathway, secretory proteins contain signals in addition to the canonical signal sequence and the rate of folding.