High-Fat Feeding Protects Mice From Ventilator-Induced Lung Injury, Via Neutrophil-Independent Mechanisms.

OBJECTIVE: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury. DESIGN: In vivo study. SETTING: University research laboratory. SUBJECTS: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12-14 weeks, or age-matched lean controls. INTERVENTIONS: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes. MEASUREMENTS AND MAIN RESULTS: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells. CONCLUSIONS: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance.

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

Transient Microgeographic Clines during B Chromosome Invasion.

The near-neutral model of B chromosome evolution predicts that the invasion of a new population should last some tens of generations, but the details on how it proceeds in real populations are mostly unknown. Trying to fill this gap, we analyze here a natural population of the grasshopper Eyprepocnemis plorans at three time points during the last 35 years. Our results show that B chromosome frequency increased significantly during this period and that a cline observed in 1992 had disappeared in 2012 once B chromosome frequency reached an upper limit at all sites sampled. This indicates that, during B chromosome invasion, transient clines for B chromosome frequency are formed at the invasion front on a microgeographic scale. Computer simulation experiments showed that the pattern of change observed for genotypic frequencies is consistent with the existence of B chromosome drive through females and selection against individuals with a high number of B chromosomes.

Microscope studies of symptomless growth of Botrytis cinerea in Lactuca sativa and Arabidopsis thaliana.

The grey mould pathogen Botrytis cinerea forms systemic associations in some hosts, spreading into plant organs produced a considerable time after initial infection. These infections may have no macroscopic symptoms during much of the hosts' lifetime and are at least partially within the host tissue. The aim of the studies reported here was to locate and visualize these infections at a cellular level in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless but infected plants were produced by dry spore inoculation of plants growing in conditions previously shown to result in fungal spread from the initial inoculation site to newly developing plant organs. Tissue taken from inoculated plants was examined using confocal laser scanning microscopy. Two B. cinerea isolates were used: B05.10 and its GFP-labelled derivative Bcgfp1-3. Spore germination on leaf surfaces was followed by development of subcuticular inclusions and plant cell damage in single infected epidermal cells and sometimes a few nearby cells. Sparsely branched long hyphae arose and spread from the inclusions, mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells, where further inclusions formed. This was consistent with the pattern in time of recovery of B. cinerea from surface-sterilized leaf tissue. In the late symptomless phase, mycelium arising from internal fungal inclusions formed mycelial networks on the surface of leaves. Symptomless exterior mycelium grew on the roots in A. thaliana.

Emergence of a novel swine-origin influenza A (H1N1) virus in humans.

BACKGROUND: On April 15 and April 17, 2009, novel swine-origin influenza A (H1N1) virus (S-OIV) was identified in specimens obtained from two epidemiologically unlinked patients in the United States. The same strain of the virus was identified in Mexico, Canada, and elsewhere. We describe 642 confirmed cases of human S-OIV infection identified from the rapidly evolving U.S. outbreak. METHODS: Enhanced surveillance was implemented in the United States for human infection with influenza A viruses that could not be subtyped. Specimens were sent to the Centers for Disease Control and Prevention for real-time reverse-transcriptase-polymerase-chain-reaction confirmatory testing for S-OIV. RESULTS: From April 15 through May 5, a total of 642 confirmed cases of S-OIV infection were identified in 41 states. The ages of patients ranged from 3 months to 81 years; 60% of patients were 18 years of age or younger. Of patients with available data, 18% had recently traveled to Mexico, and 16% were identified from school outbreaks of S-OIV infection. The most common presenting symptoms were fever (94% of patients), cough (92%), and sore throat (66%); 25% of patients had diarrhea, and 25% had vomiting. Of the 399 patients for whom hospitalization status was known, 36 (9%) required hospitalization. Of 22 hospitalized patients with available data, 12 had characteristics that conferred an increased risk of severe seasonal influenza, 11 had pneumonia, 8 required admission to an intensive care unit, 4 had respiratory failure, and 2 died. The S-OIV was determined to have a unique genome composition that had not been identified previously. CONCLUSIONS: A novel swine-origin influenza A virus was identified as the cause of outbreaks of febrile respiratory infection ranging from self-limited to severe illness. It is likely that the number of confirmed cases underestimates the number of cases that have occurred.

Cross-resistance between myclobutanil and tebuconazole and the genetic basis of tebuconazole resistance in Venturia inaequalis.

BACKGROUND: Myclobutanil is one of the most widely used demethylation inhibitor (DMI) fungicides for the management of apple scab, caused by Venturia inaequalis. Strains of V. inaequalis resistant to myclobutanil have been reported across the world. Tebuconazole, another DMI fungicide, has been proposed as an alternative to myclobutanil, and the extent of cross-resistance with myclobutanil therefore needs to be evaluated. The sensitivity to tebuconazole and myclobutanil of a total of 40 isolates was determined. Half the isolates came from an isolated orchard which had never been sprayed with fungicides and half from orchards sprayed regularly with myclobutanil, but still with disease control problems. The progeny of a tebuconazole resistant (R) × sensitive (S) V. inaequalis cross were analyzed in order to improve understanding of the genetic control of tebuconazole sensitivity. RESULTS: There is cross-resistance between myclobutanil and tebuconazole (r = 0.91; P < 0.001). Sensitivity to tebuconazole of the progeny of a R × S cross varied quantitatively in a pattern which implied at least two gene loci differing between the parental strains. In addition, the asymmetric distribution of the sensitivity in the progeny implied possible epistatic effects. CONCLUSION: Resistance to myclobutanil and tebuconazole is strongly correlated. At least two genes are involved in the control of tebuconazole resistance in V. inaequalis.

The effect of competition on the control of invading plant pathogens.

New invading pathogen strains must compete with endemic pathogen strains to emerge and spread. As disease control measures are often non-specific, that is, they do not distinguish between strains, applying control not only affects the invading pathogen strain but the endemic as well. We hypothesize that the control of the invasive strain could be compromised due to the non-specific nature of the control.A spatially explicit model, describing the East African cassava mosaic virus-Uganda strain (EACMV-UG) outbreak, is used to evaluate methods of controlling both disease incidence and spread of invading pathogen strains in pathosystems with and without an endemic pathogen strain present.We find that while many newly introduced or intensified control measures (such as resistant cultivars or roguing) decrease the expected incidence, they have the unintended consequence of increasing, or at least not reducing, the speed with which the invasive pathogen spreads geographically. We identify the controls that cause this effect and methods in which these controls may be applied to prevent it.We found that the spatial spread of the invading strain is chiefly governed by the incidence at the wave front. Control can therefore be applied, or intensified, once the wave front has passed without increasing the pathogen's rate of spread.When trade of planting material occurs, it is possible that the planting material is already infected. The only forms of control in this study that reduces the speed of geographic spread, regardless of the presence of an endemic strain, are those that reduce the amount of trade and the distance over which trade takes place. Synthesis and applications. The best control strategy depends on the presence of competing endemic strains. Applying or intensifying the control can slow the rate of spread when absent but increase it if present. Imposing trade restrictions before the epidemic has reached a given area and intensifying other control methods only when the wave front has passed is the most effective way of both slowing down spread and controlling incidence when a competing endemic strain is present and is the safest approach when its presence is unknown.

Lessons from the reestablishment of Public Health Laboratory activities in Puerto Rico after Hurricane Maria.

Public Health Laboratories (PHLs) in Puerto Rico did not escape the devastation caused by Hurricane Maria. We implemented a quality management system (QMS) approach to systematically reestablish laboratory testing, after evaluating structural and functional damage. PHLs were inoperable immediately after the storm. Our QMS-based approach began in October 2017, ended in May 2018, and resulted in the reestablishment of 92% of baseline laboratory testing capacity. Here, we share lessons learned from the historic recovery of the largest United States' jurisdiction to lose its PHL capacity, and provide broadly applicable tools for other jurisdictions to enhance preparedness for public health emergencies.

The population genetic structure of clonal organisms generated by exponentially bounded and fat-tailed dispersal.

Long-distance dispersal (LDD) plays an important role in many population processes like colonization, range expansion, and epidemics. LDD of small particles like fungal spores is often a result of turbulent wind dispersal and is best described by functions with power-law behavior in the tails ("fat tailed"). The influence of fat-tailed LDD on population genetic structure is reported in this article. In computer simulations, the population structure generated by power-law dispersal with exponents in the range of -2 to -1, in distinct contrast to that generated by exponential dispersal, has a fractal structure. As the power-law exponent becomes smaller, the distribution of individual genotypes becomes more self-similar at different scales. Common statistics like GST are not well suited to summarizing differences between the population genetic structures. Instead, fractal and self-similarity statistics demonstrated differences in structure arising from fat-tailed and exponential dispersal. When dispersal is fat tailed, a log-log plot of the Simpson index against distance between subpopulations has an approximately constant gradient over a large range of spatial scales. The fractal dimension D2 is linearly inversely related to the power-law exponent, with a slope of approximately -2. In a large simulation arena, fat-tailed LDD allows colonization of the entire space by all genotypes whereas exponentially bounded dispersal eventually confines all descendants of a single clonal lineage to a relatively small area.

Asymptomatic Host Plant Infection by the Widespread Pathogen Botrytis cinerea Alters the Life Histories, Behaviors, and Interactions of an Aphid and Its Natural Enemies.

Plant pathogens can profoundly affect host plant quality as perceived by their insect herbivores, with potentially far-reaching implications for the ecology and structure of insect communities. Changes in host plants may have direct effects on the life-histories of their insect herbivores, which can then influence their value as prey to their natural enemies. While there have been many studies that have explored the effects of infection when plants show symptoms of disease, little is understood about how unexpressed infection may affect interactions at higher trophic levels. We examined how systemic, asymptomatic, and seed-borne infection by the ubiquitous plant pathogen Botrytis cinerea, infecting two varieties of the lettuce Lactuca sativa, affected aphids (the green peach aphid, Myzus persicae) and two widely used biocontrol agents (the parasitoid Aphidius colemani and the ladybird predator Adalia bipunctata). Lettuce varieties differed in host plant quality. Asymptomatic infection reduced chlorophyll content and dry weight of host plants, irrespective of plant variety. Aphids reared on asymptomatic plants were smaller, had reduced off-plant survival time and were less fecund than aphids reared on uninfected plants. Parasitoids showed reduced attack rates on asymptomatically infected plants, and wasps emerging from hosts reared on such plants were smaller and showed reduced starvation resistance. When given a choice in an olfactometer, aphids preferentially chose uninfected plants of one variety (Tom Thumb) but showed no preference with the second (Little Gem) variety. Parasitoids preferentially chose aphids on uninfected plants, irrespective of host plant variety, but ladybirds did not show any such preference. These results suggest that the reduced quality of plants asymptomatically infected by Botrytis cinerea negatively affects the life history of aphids and their parasitoids, and alters the behaviors of aphids and parasitoids, but not of ladybirds. Fungal pathogens are ubiquitous in nature, and this work shows that even when host plants are yet to show symptoms, pathogens can affect interactions between insect herbivores and their natural enemies. This is likely to have important implications for the success of biological control programs.

Characterization of the epidemic influenza B viruses isolated during 2004-2005 season in Taiwan.

To characterize the antigenic and genetic relationships of influenza B viruses isolated during the 2004-2005 season, a total of 11,707 clinical respiratory samples were tested of which 1572 (13.5%) were positive for influenza (463 type A and 1109 type B influenza). Of the type B viruses, 348 isolates collected in different parts of Taiwan were further analyzed. Viruses belonging to both influenza B lineages, B/Yamagata/16/88 (B/Yam) and B/Victoris/2/87 (B/Vic) were detected, although an increasing number of B/Vic lineage isolates was obtained as the season progressed. Recent B/Vic-lineage isolates were found to have additional amino acid substitutions compared to isolates from previous seasons, indicating that viruses of this lineage continue to evolve significantly and may have the capacity to become the dominant influenza B viruses worldwide. Results presented in this report demonstrate that antigenically and genetically distinct viruses within both B/Vic and B/Yam lineages co-circulate and that reassortment among these two lineages occurs frequently contributing to the genetic diversity of the circulating strains.

Dose-dependent selection drives lineage replacement during the experimental evolution of SDHI fungicide resistance in Zymoseptoria tritici.

Fungicide resistance is a constant threat to agricultural production worldwide. Molecular mechanisms of fungicide resistance have been studied extensively in the wheat pathogen Zymoseptoria tritici. However, less is known about the evolutionary processes driving resistance development. In vitro evolutionary studies give the opportunity to investigate this. Here, we examine the adaptation of Z. tritici to fluxapyroxad, a succinate dehydrogenase (Sdh) inhibitor. Replicate populations of Z. tritici derived from the sensitive isolate IPO323 were exposed to increasing concentrations of fluxapyroxad with or without UV mutagenesis. After ten increases in fungicide concentration, sensitivity had decreased dramatically, with replicate populations showing similar phenotypic trajectories. Sequencing the Sdh subunit B, C, and D encoding genes identified seven mutations associated with resistance to fluxapyroxad. Mutation frequency over time was measured with a pyrosequencing assay, revealing sequential lineage replacement in the UV-mutagenized populations but not in the untreated populations. Repeating selection from set time-points with different fungicide concentrations revealed that haplotype replacement of Sdh variants was driven by dose-dependent selection as fungicide concentration changed, and was not mutation-limited. These findings suggest that fungicide field applications may select for highly insensitive Sdh variants with higher resistance factors if the fungicide concentration is increased to achieve a better disease control. However, in the absence or presence of lower fungicide concentrations, the spread of these strains might be restricted if the underlying Sdh mutations carry fitness penalties.

The effect of succinate dehydrogenase inhibitor/azole mixtures on selection of Zymoseptoria tritici isolates with reduced sensitivity.

BACKGROUND: Combining fungicides with different modes of action is regarded as one of the most effective means of slowing the selection of resistance. Field trials were used to study the effects of such mixtures on selection for Zymoseptoria tritici with reduced sensitivity to the succinate dehydrogenase inhibitors (SDHIs) and azole fungicides. The SDHI isopyrazam and the azole epoxiconazole were applied individually as solo products, and together in a preformulated mixture. All fungicide treatments were included at both full and half the recommended doses. RESULTS: Compared with using epoxiconazole alone, mixing epoxiconazole with isopyrazam led to an increase in epoxiconazole-sensitive isolates. In contrast, all treatments containing isopyrazam reduced the sensitivity of Z. tritici to isopyrazam compared with those without. Reducing doses to half the recommended rate had no effect on sensitivity of isolates to either active ingredient. In a subgroup of isolates least sensitive to isopyrazam, non-synonymous mutations were found in the SdhC and SdhD subunits, but their presence was unrelated to sensitivity. CONCLUSION: Mixing an azole and SDHI was clearly beneficial for the azole, but not for the SDHI component. This dynamic might change if strains conferring reduced sensitivity to the SDHIs were to arise. © 2015 Society of Chemical Industry.

Detection of Zymoseptoria tritici SDHI-insensitive field isolates carrying the SdhC-H152R and SdhD-R47W substitutions.

BACKGROUND: Succinate dehydrogenase inhibitor (SDHI) fungicides are important in the management of Zymoseptoria tritici in wheat. New active ingredients from this group of fungicides have been introduced recently and are widely used. Because the fungicides act at a single enzyme site, resistance development in Z. tritici is classified as medium-to-high risk. RESULTS: Isolates from Irish experimental plots in 2015 were tested against the SDHI penthiopyrad during routine monitoring. The median of the population was approximately 2 times less sensitive than the median of the baseline population. Two of the 93 isolates were much less sensitive to penthiopyrad than the least sensitive of the baseline isolates. These isolates were also insensitive to most commercially available SDHIs. Analysis of the succinate dehydrogenase coding genes confirmed the presence of the substitutions SdhC-H152R and SdhD-R47W in the very insensitive isolates. CONCLUSION: This is the first report showing that the SdhC-H152R mutation detected in laboratory mutagenesis studies also exists in the field. The function and relevance of this mutation, combined with SdhD-R47W, still needs to be determined. © 2016 Society of Chemical Industry.

Analysis of Cryptic, Systemic Botrytis Infections in Symptomless Hosts.

Botrytis species are generally considered to be aggressive, necrotrophic plant pathogens. By contrast to this general perception, however, Botrytis species could frequently be isolated from the interior of multiple tissues in apparently healthy hosts of many species. Infection frequencies reached 50% of samples or more, but were commonly less, and cryptic infections were rare or absent in some plant species. Prevalence varied substantially from year to year and from tissue to tissue, but some host species routinely had high prevalence. The same genotype was found to occur throughout a host, representing mycelial spread. Botrytis cinerea and Botrytis pseudocinerea are the species that most commonly occur as cryptic infections, but phylogenetically distant isolates of Botrytis were also detected, one of which does not correspond to previously described species. Sporulation and visible damage occurred only when infected tissues were stressed, or became mature or senescent. There was no evidence of cryptic infection having a deleterious effect on growth of the host, and prevalence was probably greater in plants grown in high light conditions. Isolates from cryptic infections were often capable of causing disease (to varying extents) when spore suspensions were inoculated onto their own host as well as on distinct host species, arguing against co-adaptation between cryptic isolates and their hosts. These data collectively suggest that several Botrytis species, including the most notorious pathogenic species, exist frequently in cryptic form to an extent that has thus far largely been neglected, and do not need to cause disease on healthy hosts in order to complete their life-cycles.

Reassortment and evolution of current human influenza A and B viruses.

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.

Has Aedes albopictus established in California?

Significant numbers of the Asian tiger mosquito, Aedes albopictus, were detected on the west coast of the USA in mid-June 2001, in containerized oceanic shipments of "lucky bamboo" (Dracaena spp.) originating from South China. Wholesale nurseries in California importing large quantities of lucky bamboo became the focal points of infestation. Greater Los Angeles County Vector Control District immediately implemented an adulticiding protocol at the Los Angeles/Long Beach Harbors, followed by larviciding soon after the shipment was delivered to the wholesale nursery. Intensive surveys are currently being conducted above ground and in the underground storm drain systems using battery-operated CDC/CO2-baited light traps and ovitraps, both enhanced with an attractant (water rinse of tiger shrimps), to determine extent of infestation and perhaps establishment of Ae. albopictus locally.

Introduction of Aedes albopictus (Skuse) in southern California and potential for its establishment.

Isolated incidences of Aedes albopictus in the continental U.S. were reported as early as 1946 and the first incidence in California was reported in 1972. These introductions were referred to as "isolated incidences" because very few immatures were observed in used tires shipped from Southeast Asia. The first major discovery of a large population and subsequent establishment of Ae. albopictus in the U.S. was reported in 1986 from Houston, TX, in a shipment of used tires from Japan. In early June 2001, infestations of this species associated with containerized oceanic shipments of "lucky bamboo" (Dracaena spp.) packaged in standing water were introduced into southern California from mainland south China. Focal points of infestation are currently at the wholesale nurseries in southern as well as northern California. A control protocol for adulticiding and larviciding has been implemented by the Greater Los Angeles County Vector Control District. Surveys are presently being conducted by local vector control agencies in southern and northern California to determine the extent of infestation. Potential forAe. albopictus establishment is discussed.

Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

Plant pathology has a long-standing tradition of classifying microbes as pathogens, endophytes or saprophytes. Lifestyles of pathogens are categorized as biotrophic, necrotrophic or hemibiotrophic. Botrytis species are considered by many to be archetypal examples of necrotrophic fungi, with B. cinerea being the most extensively studied species because of its broad host range and economic impact. In this review, we discuss recent work which illustrates that B. cinerea is capable of colonizing plants internally, presumably as an endophyte, without causing any disease or stress symptoms. The extent of the facultative endophytic behaviour of B. cinerea and its relevance in the ecology and disease epidemiology may be vastly underestimated. Moreover, we discuss the recent discovery of a novel Botrytis species, B. deweyae, which normally grows as an endophyte in ornamental daylilies (Hemerocallis), but displays facultative pathogenic behaviour, and is increasingly causing economic damage. We propose that the emergence of endophytes 'gone rogue' as novel diseases may be related to increased inbreeding of hybrid lines and reduced genetic diversity. These observations lead us to argue that the sometimes inflexible classification of pathogenic microbes by their lifestyles requires serious reconsideration. There is much more variety to the interactions of Botrytis with its hosts than the eye (or the plant pathologist) can see, and this may be true for other microbes interacting with plants.

Alterations in the predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae.

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops are increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivity of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37:erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to subinhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting that PbCYP51 evolution is ongoing and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.