Isolates of Alternaria solani previously collected from throughout the Midwestern United States and characterized as being azoxystrobin sensitive or reduced sensitive were tested for sensitivity to the Quinone outside inhibitor (QoI) fungicides famoxadone and fenamidone and the carboxamide fungicide boscalid. All three fungicides affect mitochondrial respiration: famoxadone and fenamidone at complex III, and boscalid at complex II. A. solani isolates possessing reducedsensitivity to azoxystrobin also were less sensitive in vitro to famoxadone and fenamidone compared with azoxystrobin-sensitive isolates, but the shift in sensitivity was of lower magnitude, approximately 2- to 3-fold versus approximately 12-fold for azoxystrobin. The in vitro EC50 values, the concentration that effectively reduces germination by 50% relative to the untreated control, for sensitive A. solani isolates were significantly lower for famoxadone and azoxystrobin than for fenamidone and boscalid; whereas, for reduced-sensitive isolates, famoxadone EC50 values were significantly lower than all other fungicides. Isolates of A. solani with reducedsensitivity to azoxystrobin were twofold more sensitive in vitro to boscalid than were azoxystrobin-sensitive wild-type isolates, displaying negative cross-sensitivity. All isolates determined to have reduced-sensitivity to azoxystrobin also were determined to possess the amino acid substitution of phenylalanine with leucine at position 129 (F129L mutation) using real-time polymerase chain reaction. In vivo studies were performed to determine the effects of in vitro sensitivity shifts on early blight disease control provided by each fungicide over a range of concentrations. Reduced-sensitivity to azoxystrobin did not significantly affect disease control provided by famoxadone, regardless of the wide range of in vitro famoxadone EC50 values. Efficacy of fenamidone was affected by some azoxystrobin reduced-sensitive A. solani isolates, but not others. Boscalid controlled azoxystrobin-sensitive and reduced-sensitive isolates with equal effectiveness. These results suggest that the F129L mutation present in A. solani does not convey cross-sensitivity in vivo among all QoI or related fungicides, and that two- to threefold shifts in in vitro sensitivity among A. solani isolates does not appreciably affect disease control.
Isolates of Alternaria solani, cause of potato early blight, collected in 1998 through 2001 from various potato growing areas across the midwestern United States, were tested for sensitivity to azoxystrobin. Isolates collected in 1998, prior to the introduction of azoxystrobin, were tested to establish the baseline sensitivity of the fungus to this fungicide. Isolates collected in subsequent years, not necessarily from the same sites as baseline isolates, were tested to determine if populations of A. solani had become less sensitive to azoxystrobin. Azoxystrobin sensitivity was determined utilizing an in vitro spore germination assay. The effective fungicide concentration that inhibited spore germination by 50% (EC50) was determined for each isolate. There was no significant difference in mean EC50 values between baseline isolates and all other isolates collected through 1999. Mean azoxystrobin EC50 values of A. solani isolates collected in 2000 and 2001 were significantly higher compared with means from previous years, and mean azoxystrobin EC50 values from 2001 were significantly higher than means from isolates collected in 2000. A subset of 54 A. solani isolates was evaluated in vitro for cross-sensitivity to pyraclostrobin and trifloxystrobin. A highly significant and strong correlation among the isolates tested for fungicide cross-sensitivity was detected between azoxystrobin and pyraclostrobin; however, the correlation between azoxystrobin and trifloxystrobin, and between trifloxystrobin and pyraclostrobin, was significant but weak. A second subset of five isolates was chosen for in vivo assessment of azoxystrobin, pyraclostrobin, and trifloxystrobin sensitivity. Disease severity on plants treated with azoxystrobin and pyraclostrobin was significantly greater with reduced-sensitive A. solani isolates compared with sensitive isolates. Disease severity was not statistically different between azoxystrobin reduced-sensitive and sensitive A. solani isolates on plants treated with trifloxystrobin. This is the first report of a shift in sensitivity to QoI fungicides in a fungus possessing only an anamorphic stage.
The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.
Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris ) is an important root rot pathogen of common bean ( Phaseolus vulgaris L.). To uncover genetic factors associated with resistance to the pathogen, the Andean (ADP; n = 273) and Middle American (MDP; n = 279) diversity panels, which represent much of the genetic diversity known in cultivated common bean, were screened in the greenhouse using R. solani anastomosis group 2-2. Repeatability of the assay was confirmed by the response of five control genotypes. The phenotypic data for both panels were normally distributed. The resistance responses of ∼10% of the ADP ( n = 28) and ∼6% of the MDP ( n = 18) genotypes were similar or higher than that of the resistant control line VAX 3. A genome-wide association study (GWAS) was performed using ∼200k single nucleotide polymorphisms to discover genomic regions associated with resistance in each panel, For GWAS, the raw phenotypic score, and polynomial and binary transformation of the scores, were individually used as the input data. A major QTL peak was observed on Pv02 in the ADP, while a major QTL was observed on Pv01 with the MDP. These regions were associated with clusters of TIR-NB_ARC-LRR (TNL) gene models encoding proteins similar to known disease resistance genes. Other QTL, unique to each panel, were mapped within or adjacent to a gene model or cluster of related genes associated with disease resistance. This is a first case study that provides evidence for major as well as minor genes involved in resistance to R. solani in common bean. This information will be useful to integrate more durable root rot resistance in common bean breeding programs and to study the genetic mechanisms associated with root diseases in this important societal legume.
Pulse crops (annual grain legumes such as field pea, lentil, dry bean, and chickpea) have become an important component of the cropping system in the northern Great Plains of North America over the last three decades. In many areas, the intensity of damping-off, seedling blight, root rot, and premature ripening of pulse crops is increasing, resulting in reduction in stand establishment and yield. This review provides a brief description of the important pathogens that make up the root rot complex and summarizes root rot management on pulses in the region. Initially, several specific Fusarium spp., a range of Pythium spp., and Rhizoctonia solani were identified as important components of the root rot disease complex. Molecular approaches have recently been used to identify the importance of Aphanomyces euteiches on pulses, and to demonstrate that year-to-year changes in precipitation and temperature have an important effect on pathogen prevalence. Progress has been made on management of root rot, but more IPM tools are required to provide effective disease management. Seed-treatment fungicides can reduce damping-off and seedling blight for many of the pathogens in this disease complex, but complex cocktails of active ingredients are required to protect seedlings from the pathogen complex present in most commercial fields. Partial resistance against many of the pathogens in the complex has been identified, but is not yet available in commercial cultivars. Cultural practices, especially diversified cropping rotations and early, shallow seeding, have been shown to have an important role in root rot management. Biocontrol agents may also have potential over the long term. Improved methods being developed to identify and quantify the pathogen inoculum in individual fields may help producers avoid high-risk fields and select IPM packages that enhance yield stability.
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