Constraints on the evolution of azole resistance in plant pathogenic fungi

The durability of azole fungicides in controlling agriculturally important pathogenic fungi is unique amongst modern single site fungicides. Today, azoles are still relied on to control pathogens of many crops including cereals, fruits and vegetables, canola and soybeans. Significantly, this widespread use continues despite many reports of azole-resistant fungal strains. In this review, recent reports of azole resistance and the mechanisms associated with resistant phenotypes are discussed. The example of the complex evolution of the azole target sterol 14-demethylase (CYP51) enzyme in modern European populations of the wheat pathogen Mycosphaerella graminicola is used to describe the quantitative and epistatic effects on fungicide sensitivity and enzyme function of target site mutations, and to explore the hypothesis that constraints on CYP51 evolution have ensured the longevity of azoles. In addition, the threats posed by alternative resistance mechanisms causing cross-resistance to all azoles or even unrelated fungicides are discussed, and postulations are made on how using new genomic technologies to gain a greater understanding of azole resistance evolution should enhance the ability to control azole-resistant strains of plant pathogenic fungi in the future

Lack of an Intron in Cytochrome b and Overexpression of Sterol 14 alpha-Demethylase Indicate a Potential Risk for QoI and DMI Resistance Development in Neophysopella spp. on Grapes

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14 alpha-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 mu g/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 mu g/ml for N. tropicalis and 0.46 mu g/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 mu g/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp

The Multi-Fungicide Resistance Status of Aspergillus fumigatus Populations in Arable Soils and the Wider European Environment

The evolution and spread of pan-azole resistance alleles in clinical and environmental isolates of Aspergillus fumigatus is a global human health concern. The identification of hotspots for azole resistance development in the wider environment can inform optimal measures to counteract further spread by minimizing exposure to azole fungicides and reducing inoculum build-up and pathogen dispersal. We investigated the fungicide sensitivity status of soil populations sampled from arable crops and the wider environment and compared these with urban airborne populations. Low levels of azole resistance were observed for isolates carrying the CYP51A variant F46Y/M172V/E427K, all belonging to a cluster of related cell surface protein (CSP) types which included t07, t08, t13, t15, t19, and t02B, a new allele. High levels of resistance were found in soil isolates carrying CYP51A variants TR34/L98H and TR46/Y121F/T289A, all belonging to CSP types t01, t02, t04B, or t11. TR46/Y121F/M172V/T289A/G448S (CSP t01) and TR46/Y121F/T289A/S363P/I364V/G448S (CSP t01), a new haplotype associated with high levels of resistance, were isolated from Dutch urban air samples, indicating azole resistance evolution is ongoing. Based on low numbers of pan-azole resistant isolates and lack of new genotypes in soils of fungicide-treated commercial and experimental wheat crops, we consider arable crop production as a coldspot for azole resistance development, in contrast to previously reported flower bulb waste heaps. This study also shows that, in addition to azole resistance, several lineages of A. fumigatus carrying TR-based CYP51A variants have also developed acquired resistance to methyl benzimidazole carbamate, quinone outside inhibitor and succinate dehydrogenase (Sdh) inhibitor fungicides through target-site alterations in the corresponding fungicide target proteins; beta-tubulin (F200Y), cytochrome b (G143A), and Sdh subunit B (H270Y and H270R), respectively. Molecular typing showed that several multi-fungicide resistant strains found in agricultural soils in this study were clonal as identical isolates have been found earlier in the environment and/or in patients. Further research on the spread of different fungicide-resistant alleles from the wider environment to patients and vice versa can inform optimal practices to tackle the further spread of antifungal resistance in A. fumigatus populations and to safeguard the efficacy of azoles for future treatment of invasive aspergillosis

A phylogenetically distinct lineage of Pyrenopeziza brassicae associated with chlorotic leaf spot of Brassicaceae in North America

Light leaf spot, caused by the ascomycete Pyrenopeziza brassicae Sutton & Rawlinson, is an established disease of Brassicaceae in the United Kingdom (UK), continental Europe, and Oceania (OC, including New Zealand and Australia). The disease was reported in North America (NA) for the first time in 2014 on Brassica spp. in the Willamette Valley of western Oregon, followed by detection in Brassica juncea cover crops and on B. rapa weeds in northwestern Washington in 2016. Preliminary DNA sequence data and field observations suggest that isolates of the pathogen present in NA might be distinct from those in the UK, continental Europe, and OC. Comparisons of isolates from these regions genetically (multilocus sequence analysis, MAT gene sequences, and rep-PCR DNA fingerprinting), pathogenically (B. rapa inoculation studies), biologically (sexual compatibility), and morphologically (colony and conidial morphology) demonstrated two genetically distinct evolutionary lineages. Lineage 1 comprised isolates from the UK, continental Europe, and OC, and included the P. brassicae type specimen. Lineage 2 contained the NA isolates associated with recent disease outbreaks in the Pacific Northwest region of the USA. Symptoms caused by isolates of the two lineages on B. rapa and B. juncea differed, so ‘chlorotic leaf spot’ is proposed for the disease caused by lineage 2 isolates of P. brassicae. Isolates of the two lineages differed in genetic diversity as well as sensitivity to the fungicides carbendazim and prothioconazole

Epidemiological studies of pan-azole resistant Aspergillus fumigatus populations sampled during tulip cultivation show clonal expansion with acquisition of multi-fungicide resistance as potential driver

Pan-azole resistant isolates are found in clinical and environmental Aspergillus fumigatus (Af) populations. Azole resistance can evolve in both settings, with Af directly targeted by antifungals in patients and, in the environment, Af unintendedly exposed to fungicides used for material preservation and plant disease control. Resistance to non-azole fungi-cides, including methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs), have recently been reported. These fungicide groups are not used in medicine but can play an important role in further spread of pan-azole resistant genotypes. We investigated the multi-fungicide resistance status and genetic diversity of Af populations sampled from tulip field soils, tulip peel waste and flower compost heaps using fungicide sensitivity testing and a range of genotyping tools, including STRAf typing and sequencing of fungicide resistant alleles. Two major clones were present in the tulip bulb population. Comparisons with clinical isolates and literature data revealed that several common clonal lineages of TR34/L98H and TR46/Y121F/T289A strains that have expanded successfully in the environment have also acquired resistance to MBC, QoI and/or SDHI fungicides. Strains carrying multiple fungicide resistant alleles have an advantage in environments where residues of multiple fungicides belonging to different modes of action are presen

Evolutionary bi-stability in pathogen transmission mode

Many pathogens transmit to new hosts by both infection (horizontal transmission) and transfer to the infected host's offspring (vertical transmission). These two transmission modes require speci®c adap- tations of the pathogen that can be mutually exclusive, resulting in a trade-off between horizontal and vertical transmission. We show that in mathematical models such trade-offs can lead to the simultaneous existence of two evolutionary stable states (evolutionary bi-stability) of allocation of resources to the two modes of transmission. We also show that jumping between evolutionary stable states can be induced by gradual environmental changes. Using quantitative PCR-based estimates of abundance in seed and vege- tative parts, we show that the pathogen of wheat, Phaeosphaeria nodorum, has jumped between two distinct states of transmission mode twice in the past 160 years, which, based on published evidence, we interpret as adaptation to environmental change. The ®nding of evolutionary bi-stability has impli- cations for human, animal and other plant diseases. An ill-judged change in a disease control programme could cause the pathogen to evolve a new, and possibly more damaging, combination of transmission modes. Similarly, environmental changes can shift the balance between transmission modes, with adverse effects on human, animal and plant health

Evolution of decreased sensitivity to azole fungicides in western European populations of Plenodomus lingam (cause of Phoma leaf spot / stem canker on oilseed rape)

Plenodomus lingam (Leptosphaeria maculans) and P. biglobosus (L. biglobosa) are related fungal pathogens causing Phoma leaf spot and stem canker, an internationally damaging disease of oilseed rape (Brassica napus) and other brassicas. In Europe, fungicides used for disease management are mainly sterol 14α-demethylase (CYP51) inhibitors (DMIs/azoles); quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs) are also used. Decreased DMI sensitivity has emerged in Australian and eastern European P. lingam populations. Decreased sensitivity is mediated by promoter inserts in CYP51 resulting in target site overexpression. In the present study, based on in vitro sensitivity testing, we report decreased DMI (prothioconazole-desthio and mefentrifluconazole) sensitivity in modern western European isolates of P. lingam (collected 2022-23) compared to baseline historical (1992-2005) isolates. Around 85% of the modern western European P. lingam isolates collected, for which the CYP51 promoter region was sequenced, carried a promoter insert but target site alterations were not detected. Six different CYP51 promoter inserts were identified, with a 237 bp fragment of the Sahana transposable element most frequently detected. Inserts were typically associated with a 3 to 10 fold decrease in sensitivity to the DMIs tested. In contrast to P. lingam, PCR screening revealed that CYP51 promoter inserts were absent in modern western European P. biglobosus isolates (collected 2021-23). The combined data indicate P. lingam isolates lacking an insert were similarly (or slightly more) sensitive to the DMIs tested for P. biglobosus, whereas those carrying an insert were slightly less sensitive than P. biglobosus. No clear evidence for substantive sensitivity shifts to the QoI (pyraclostrobin) or SDHI (boscalid) fungicides tested was obtained for either Plenodomus species

Wheat archive links long-term fungal pathogen population dynamics to air pollution

We used the PCR to study the presence of two plant pathogens in archived wheat samples from a long-term experiment started in 1843. The data were used to construct a unique 160-yr time-series of the abundance of Phaeosphaeria nodorum and Mycosphaerella graminicola, two important pathogens of wheat. During the period since 1970, the relative abundance of DNA of these two pathogens in the samples has reflected the relative importance of the two wheat diseases they cause in U.K. disease surveys. Unexpectedly, changes in the ratio of the pathogens over the 160-yr period were very strongly correlated with changes in atmospheric pollution, as measured by SO2 emissions. This finding suggests that long-term, economically important, changes in pathogen populations can be influenced by anthropogenically induced environmental changes.&nbsp