Biology subjects

Lopez-Ruiz, F. J.

Publications and source records attributed to Lopez-Ruiz, F. J..

13 recordsLinked to original sources

bioRxiv2025

Evidence of a distinct Blumeria graminis f. sp. tritici pathotype structure in Australian wheat powdery mildew: implications for resistance breeding.

The obligate biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) is the causal agent of wheat powdery mildew. To investigate virulence diversity, 30 Australian Bgt isolates collected between 2020 and 2024 were screened against a panel of 24 wheat lines carrying defined resistance (R) genes. Pathotyping analysis indicated that the Australian Bgt population is diverse in its virulence profile. Non-metric multidimensional scaling analysis of disease phenotyping revealed evidence of six distinct Bgt pathotype groups. In addition, we identified wheat varieties carrying defined R genes and their combinations that remain effective against the contemporary Australian Bgt. Notably, wheat cvs Khapli and Tabasco, possessing Pm4a and Pm2, respectively, in addition to unknown R gene(s), were highly resistant to all the isolates tested. Furthermore, elite European varieties that possess durable resistance, such as Alchemy and Cordiale, display complete to moderate resistance against all selected isolates representing the Australian pathotype groups.

microbiology↗

bioRxiv2025

Dissecting allele-specific fungicide resistance mechanisms by heterologous expression of the demethylase inhibitor target gene Cyp51 in a phytopathogen model

BACKGROUNDFungicide resistance is a major concern both in agriculture and clinical disease control. Whilst several mechanisms of resistance have been elucidated, assigning phenotype to genotype is often difficult and reliant on correlations. Resistance to demethylase inhibitor (DMI) fungicides was recently reported in the economically important filamentous fungal barley (Hordeum vulgare) pathogen Pyrenophora teres f. teres (Ptt) in Australia. The target of DMI fungicides is encoded by the Cyp51 gene family; single allele of Cyp51B and two copies of the Cyp51A gene1. Five Cyp51A alleles (W1-A1, 9193-A1, KO103-A1, W1-A2 and 9193-A2) were identified in Ptt with KO103-A1 containing the mutation F489L (F495L) which correlates with resistance to various DMIs.1 RESULTSWe replaced the coding region of the native Cyp51B gene of the filamentous fungal Dothideomycete wheat pathogen Parastagonospora nodorum with each of the five Ptt Cyp51A alleles to compare the phenotypic effects of each allele in isolation. The native Cyp51B of P. nodorum could be functionally replaced by Cyp51-A1 but not Cyp51-A2. Transformants carrying KO103-A1 exhibited significantly higher gene expression than 9193-A1 and W1-A1, suggesting the mechanism of gene regulation lies within the coding sequence and is conserved between Ptt and P. nodorum. The EC50 values of the KO103-A1 transformants were significantly higher than any other transformants or wild type isolates for metconazole, prochloraz and tebuconazole but lower for epoxiconazole. CONCLUSIONThis system permits the functional characterisation of fungicide target genes in an isogenic background that mimics the physiological environment of plant pathogens. We suggest the system will prove useful in dissecting the impact of genetic mutations on a spectrum of fungicides and permit the design of fungal strains for screening active ingredients that may control strains resistant to existing fungicides.

microbiology↗

bioRxiv2025

Monitoring fungicide resistance frequencies -- a case study of barley net blotch

Decreased sensitivity to fungicides impacts the effectiveness of fungicide applications for managing plant disease. Knowledge of the frequency of decreased sensitivity in field populations is critical for evaluating risks for disease control. This study has applied a droplet digital PCR detection approach to assess pathogen populations and quantify the frequencies of alleles associated with decreased sensitivity to either demethylation inhibitor or succinate dehydrogenase inhibitor fungicides in Pyrenophora teres causing net blotch on barley in Western Australia. Pyrenophora teres f. maculata was the most frequent form of the pathogen in the sampled region. Frequencies of decreased fungicide sensitivity alleles varied, being as great as 92% for the PtTi insertion in the CYP51A promoter, 88% for C-S75 and 28% for L489-2. Impacts of cultivar selection and weed hosts on the presence and survival of decreased fungicide sensitivity were observed. Determining the dynamics of alleles within different field populations of P. teres provides an important perspective on the impact of fungicides, the fitness associated with decreased fungicide sensitivity alleles and the susceptibility of barley cultivars.

pathology↗

bioRxiv2023

Exploiting long read sequencing to detect azole fungicide resistance mutations in Pyrenophora teres using unique molecular identifiers

Resistance to fungicides is a global challenge as target proteins under selection can evolve rapidly, reducing fungicide efficacy. To manage resistance, detection technologies must be fast and flexible enough to cope with a rapidly increasing number of mutations. The most important agricultural fungicides are azoles that target the ergosterol biosynthetic enzyme sterol 14-demethylase (CYP51). Mutations associated with azole resistance in the Cyp51 promoter and coding sequence can co-occur in the same allele at different positions and codons, increasing the complexity of resistance detection. Resistance mutations arise rapidly and cannot be detected using traditional amplification-based methods if they are not known. To capture the complexity of azole resistance in two net blotch pathogens of barley we used the Oxford Nanopore MinION to sequence the promoter and coding sequence of Cyp51A. This approach detected all currently known mutations from biologically complex samples increasing the simplicity of resistance detection as multiple alleles can be profiled in a single assay. With the mobility and decreasing cost of long read sequencing, we demonstrate this approach is broadly applicable for characterizing resistance within known agrochemical target sites.

microbiology↗

bioRxiv2023

Workflows for detecting fungicide resistance in net form and spot form net blotch pathogens

Fungicide resistance in Pyrenophora teres f. maculata and P. teres f. teres has become an important disease management issue. Control of the associated barley foliar diseases, spot form and net form net blotch, respectively, relies on three major groups of fungicides, demethylation inhibitors (DMI), succinate dehydrogenase inhibitors (SDHI) and quinone outside inhibitors (QoI). However, resistance has been reported for the DMI and SDHI fungicides in Australia. To enhance detection of different resistance levels, phenotyping and genotyping workflows were designed. The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time PCR assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to a net blotch collection from 2019 consisting predominantly of P. teres f. teres from South Australia and P. teres f. maculata from Western Australia. For South Australia the Cyp51A L489-3 and SdhC-R134 alleles, associated with resistance to tebuconazole and fluxapyroxad, respectively, were the most prevalent. These alleles were frequently found in single isolates with dual resistance. This study also reports the first detection of a 134 base pair insertion located at position -66 (PtTi-6) in the Cyp51A promoter of P. teres f. maculata from South Australia. For Western Australia, the PtTi-1 insertion was the most common allele associated with resistance to tebuconazole. These workflows will be valuable for screening P. teres populations for fungicide resistance, and informing appropriate management strategies.

pathology↗

bioRxiv2023

Emergence of resistance to succinate dehydrogenase inhibitor fungicides in Pyrenophora teres f. teres and P. teres f. maculata in Australia

The net blotches are among the most economically significant diseases of barley worldwide. There are two forms of the disease: net-form net-blotch (NFNB, causal agent Pyrenophora teres f. sp. teres [Ptt]) and spot-form net blotch (SFNB, causal agent Pyrenophora teres f. sp. maculata [Ptm]). Alongside varietal choice and cultural practices, fungicides form an important part of the regime for net blotch control. The succinate dehydrogenase inhibitors (SDHIs) are a key class of fungicides used in net blotch management. However, resistance to this group of compounds has emerged in the net blotches in recent years. Here, we describe the first cases of resistance to SDHIs in Australian populations of net blotches. This study was prompted by reports of field failures of SDHI fungicides in controlling NFNB in South Australia and SFNB in Western Australia. Target site mutations in the Sdh complex genes, previously associated with reduced sensitivity in European net blotch populations, were found in Australian isolates, and two mutations which have not been previously observed in P. teres, are also described. The mutations found in Ptt included H134R and S135R in SdhC; and H134Y and D145G in SdhD; the SdhC-H134R mutation was the most frequently observed. In Ptm, the mutations found included H277L in SdhB; S73P, N75S, H134R and S135R in SdhC; and D145G in SdhD; the SdhC-N75S mutation was the most common. These mutations were correlated with reduced in vitro SDHI fungicide sensitivity by microtiter assay. The highest resistance factors to fluxapyroxad and bixafen, the most important SDHI fungicides for net blotch control in Australia, were associated with the SdhC-H134R and SdhC-S135R mutations in Ptt, and with the SdhB-H277L, SdhC-H134R, and SdhC-S135R mutations in Ptm. Modelling of the P. teres Sdh complex showed that the two novel mutations, H277L in SdhB and H134Y in SdhD, result in a highly altered binding mode and lower binding affinity of the SDHI compound compared to the wild-type.

microbiology↗

bioRxiv2021

Species hybridisation and clonal expansion as a new fungicide resistance evolutionary mechanism in Pyrenophora teres spp.

The barley net blotch diseases are caused by two fungal species of the Pyrenophora genus. Specifically, spot form net blotch is caused by P. teres f. sp. maculata (Ptm) whereas net form net blotch is caused by P. teres f. sp. teres (Ptt). Ptt and Ptm show high genetic diversity in the field due to intraspecific sexual recombination and hybridisation of the two species although the latter is considered rare. Here we present occurrence of a natural Ptt/Ptm hybrid with azole fungicides resistance and its implication to barley disease management in Australia. We collected and sequenced a hybrid, 3 Ptm and 10 Ptt isolates and performed recombination analyses in the intergenic and whole genome level. Eleven out of 12 chromosomes showed significant (P < 0.05) recombination events in the intergenic regions while variable recombination rate showed significant recombination across all the chromosomes. Locus specific analyses of Cyp51A1 gene showed at least four recombination breakpoints including a point mutation that alter target protein function. This point mutation did not found in Ptt and Ptm collected prior to 2013 and 2017, respectively. Further genotyping of fourteen Ptt, 48 HR Ptm, fifteen Ptm and two P. teres isolates from barley grass using Diversity Arrays Technology markers showed that all HR Ptm isolates were clonal and not clustered with Ptt or Ptm. The result confirms occurrence of natural recombination between Ptt and Ptm in Western Australia and the HR Ptm is likely acquired azole fungicide resistance through recombination and underwent recent rapid selective sweep likely within the last decade. The use of available fungicide resistance management tactics are essential to minimise and restrict further dissemination of these adaptive HR Ptm isolates.

evolutionary biology↗

bioRxiv2021

Detection of Ramularia collo-cygni from barley (Hordeum vulgare) in Australia using triplex quantitative and digital PCR

Ramularia leaf spot (RLS), caused by Ramularia collo-cygni, is an emerging threat to barley (Hordeum vulgare) production. RLS has been reported in Australia; however only minimal information is available regarding its detection and distribution. Due to initial asymptomatic growth in planta, slow growth in vitro and symptomatic similarities to net blotch and physiological leaf spots, detection of this pathogen can be challenging. Quantitative PCR-based methods for R. collo-cygni-specific identification and detection have been described, however these assays (based upon the internal transcribed spacer [ITS] region) have been demonstrated to lack specificity. False-positive detections may have serious implications, thus we aimed to design a robust R. collo-cygni-specific PCR method. Using the phylogenetically informative RNA polymerase II second largest subunit (rpb2) and translation elongation factor 1- (tef1-) genes, along with the tef1- gene of H. vulgare, a triplex assay was developed for both quantitative and digital PCR. The triplex assay was used to assess DNA of barley leaves from New South Wales, South Australia, Tasmania, Victoria and Western Australia, along with DNA of seeds from Western Australia. Detection of R. collo-cygni DNA was confirmed for leaf samples from New South Wales, South Australia, Tasmania, Victoria and Western Australia, indicating a distribution ranging across the southern barley growing regions of Australia. No R. collo-cygni DNA was detected in seed from Western Australia. The R. collo-cygni-specific assay will be a valuable tool to assist with monitoring the distribution of R. collo-cygni in Australia and other regions.

plant biology↗

bioRxiv2021

Fungicide resistance characterised across seven chemical classes in a Botrytis cinerea population isolated from Australian vineyards

BACKGROUNDBotrytis bunch rot, caused by Botrytis cinerea, is an economically important disease of grapes in Australia and across grape growing regions worldwide. Control of this disease relies on canopy management and the application of fungicides. Fungicide application can lead to the selection of resistant B. cinerea populations, which has an adverse effect on management of the disease. Characterising the distribution and severity of resistant B. cinerea populations is needed to inform resistance management strategies. RESULTSIn this study, 725 isolates were sampled from 75 Australian vineyards during 2013 - 2016 and were screened against seven fungicides with different modes of action (MOAs). The resistance frequencies for azoxystrobin, boscalid, fenhexamid, fludioxonil, iprodione, pyrimethanil and tebuconazole were 5, 2.8, 2.1, 6.2, 11.6, 7.7 and 2.9% respectively. Nearly half of the resistant isolates (43.8%) were resistant to more than one of the fungicides tested. The frequency of vineyards with at least one isolate simultaneously resistant to 1, 2, 3, 4 or 5 fungicides was 19.5, 7.8, 6.5, 10.4 and 2.6%. Resistance was associated with previously published genotypes in CytB (G143A), SdhB (H272R/Y), Erg27 (F412S), Mrr1 (D354Y), Bos1 (I365S, N373S + Q369P, I365S + D757N) and Pos5 (V273I, P319A, L412F/V). Novel genotypes were also described in Mrr1 (S611N, D616G) Pos5 (V273L) and Cyp51 (P347S). Expression analysis was used to characterise fludioxonil resistant isolates exhibiting overexpression (6.3-9.6-fold) of the ABC transporter gene AtrB (MDR1 phenotype). CONCLUSIONResistance frequencies were lower when compared to most previously published surveys of B. cinerea resistance in grape and other crops. Nevertheless, continued monitoring of critical MOAs used in Australian vineyards is recommended.

molecular biology↗

bioRxiv2020

When time really is money: in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici

BackgroundThere has been an inexorable increase in the incidence of fungicide resistance in plant pathogens in recent years. Control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele specific PCR (ASqPCR) is an ideal technique for the analysis of fungicide resistance in the field as it can both detect and quantify the frequency of mutations associated with fungicide resistance. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), an obligate biotrophic fungus that causes wheat powdery mildew and is responsible for up to 25% yield loss annually. In Australia, strobilurin resistant Bgt was first discovered in samples from Tasmania and Victoria in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome bc1 enzyme (cytb) complex, resulting in a substitution of alanine for glycine at position 143 (G143A) in Cytb. ResultsWe have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures within 90 min of sample collection. The in situ analysis of field samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. We validated the analysis with a newly developed laboratory based digital PCR assay and found no significant differences between the two methods. ConclusionWe have successfully developed an in-field quantification method, for a QoI resistant allele, by pairing an ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of this type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.

molecular biology↗

bioRxiv2019

Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata

The demethylase inhibitor (DMI) or group 3 fungicides are the most important class of compounds for the control both of plant and human fungal pathogens. The necrotrophic fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot form of net blotch (SFNB), is currently the most significant disease of barley in Australia, and a disease of increasing concern worldwide. The main basis for management of SFNB is by fungicide application, and in Australia the DMIs predominate. Although reduced sensitivity to DMI fungicides has recently been described in the closely related pathogen P. teres f. sp. teres (Ptt), the mechanisms of DMI resistance have not thus far been described for Ptm. In this study, several different levels of sensitivity to DMI fungicides were identified in Western Australian strains of Ptm from 2016 onwards, and reduced sensitivity phenotypes were correlated with a number of distinct mutations in both the promoter region and coding sequence of the DMI target gene encoding cytochrome P450 sterol 14-demethylase (Cyp51A). Five insertions elements of 134-base pairs in length were found at different positions within the upstream regulatory region of Cyp51A in both highly DMI-resistant (HR) and select moderately DMI-resistant (MR1) Ptm isolates. The five insertion elements had at least 95% sequence identity and were determined to be Solo-LTR (Long Terminal Repeat) elements, all deriving from Ty1/Copia-family LTR Retrotransposons. The 134-bp elements contained a predicted promoter sequence and several predicted transcription factor binding sites, and the presence of an insertion element was correlated with constitutive overexpression of Cyp51A. The substitution of phenylalanine by leucine at position 489 of the predicted amino acid sequence of CYP51A was found in both HR and select moderately DMI-resistant (MR2) Ptm isolates. The same F489L amino acid substitution has been previously reported in Western Australian strains of Ptt, where it has also been associated with reduced sensitivity to DMI fungicides. In Ptm, the F489L amino acid change was associated with either of three different single nucleotide polymorphisms in codon 489. This suggests that, in contrast to Ptt, in Ptm the F489L mutation has emerged as a result of three distinct mutational events. Moderately DMI-resistant isolates had one or the other of the F489L substitution or a promoter insertion mutation, whereas highly DMI-resistant isolates were found to have combinations of both mechanisms together. Therefore, multiple mechanisms acting both alone and in concert were found to contribute to the observed phenomena of DMI fungicide resistance in Ptm. Moreover, these mutations have apparently emerged repeatedly and independently in Western Australian Ptm populations, by a process of convergent or parallel evolution.

plant biology↗

bioRxiv2018

Sampling for disease absence-deriving informed monitoring from epidemic traits

Monitoring for disease requires subsets of the host population to be sampled and tested for the pathogen. If all the samples return healthy, what are the chances the disease was present but missed? In this paper, we developed a statistical approach to solve this problem considering the fundamental property of infectious diseases: their growing incidence in the host population. The model gives an estimate of the incidence probability density as a function of the sampling effort, and can be reversed to derive adequate monitoring patterns ensuring a given maximum incidence in the population. We then present an approximation of this model, providing a simple rule of thumb for practitioners. The approximation is shown to be accurate for a sample size larger than 20, and we demonstrate its use by applying it to three plant pathogens: citrus canker, bacterial blight and grey mould.

epidemiology↗

bioRxiv2017

Using epidemiological principles to explain fungicide resistance management strategies: why do mixtures outperform alternations?

O_LIWhether fungicide resistance management is optimised by spraying chemicals with different modes of action as a mixture (i.e. simultaneously) or in alternation (i.e. sequentially) has been studied by experimenters and modellers for decades, largely inconclusively.\nC_LIO_LIWe use previously-parameterised and validated mathematical models of wheat septoria leaf blotch and grapevine powdery mildew to test which strategy provides better resistance management, using the total yield before fungicide-resistance causes disease control to become economically-ineffective (\"lifetime yield\") to measure effectiveness.\nC_LIO_LILifetime yield is optimised by spraying as much low-risk fungicide as is permitted, combined with slightly more high-risk fungicide than needed for acceptable initial disease control, applying these fungicides as a mixture. This is invariant to model parameterisation and structure, as well as the pathosystem in question. However if comparison focuses on other metrics, for example lifetime yield at full label dose, either mixtures or alternation can be optimal.\nC_LIO_LIOur work shows how epidemiological principles can explain the evolution of fungicide resistance, and highlights a theoretical framework to address the question of whether mixtures or alternation provide better resistance management. Our work also demonstrates that precisely how spray strategies are compared must be given extremely careful consideration.\nC_LI

epidemiology↗