Category Archives: Wiley: Plant Pathology

Genome‐wide mapping in an international isolate collection identifies a transcontinental erg11/CYP51 promoter insertion associated with fungicide resistance in Leptosphaeria maculans

Posted on 27 March 2024 by nJack L. Scanlan, nAlexander Idnurm, nAngela P. Van de Wouwn

Insertion of a remnant transposable element in the promoter region of the erg11/CYP51 demethylase inhibitor (DMI) target gene confers resistance to DMI fungicides worldwide.

Abstract

Fungicide resistance is often conferred through the mutation of genes encoding fungicide targets or proteins that remove fungicides from cells, but mechanisms can vary widely between taxa. Discovering the specific resistance alleles present in pathogen populations is essential for monitoring the evolution and movement of resistant genotypes. In this study, we explored the genomic basis of demethylase inhibitor (DMI) resistance in Leptosphaeria maculans, the main pathogen of the canola crop Brassica napus. Using an international collection of over 200 genome-sequenced isolates, we assayed in vitro sensitivity to the DMI tebuconazole and conducted a genome-wide association study on a variant set including single-nucleotide polymorphisms (SNPs), small indels and structural variants. The main resistance allele identified was a 237 bp remnant transposable element insertion in the promoter of the erg11/CYP51 DMI target gene in a large proportion of isolates from Europe, an allele known to confer DMI resistance in Australia. Several associated loci were identified, none of which are commonly linked to DMI resistance in other phytopathogens. We also found little to no relationship between DMI tolerance and baseline growth rate, suggesting minimal fitness effects of fungicide resistance in these isolates. This study indicates common DMI resistance alleles in L. maculans are shared across continents and erg11/CYP51 coding mutations, which are near-ubiquitous in other fungal pathogens, may not underpin DMI resistance in this species. Furthermore, that resistance occurs frequently in numerous canola-growing regions suggests management is essential for growers.

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

Posted on 26 March 2024 by

Plenodomus lingam (Phoma leaf spot/stem canker) populations in western Europe (Germany, Ireland and the United Kingdom) predominantly contain CYP51 promoter inserts leading to deceased azole (DMI) fungicide sensitivity.

Abstract

Plenodomus lingam (Leptosphaeria maculans) and P. biglobosus (L. biglobosa) are fungi causing Phoma leaf spot/stem canker, an international 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 and is mediated by CYP51 promoter inserts resulting in target site overexpression. In this study using in vitro sensitivity testing, we report decreased DMI (prothioconazole-desthio, mefentrifluconazole) sensitivity in modern western European P. lingam isolates (collected 2022–2023) compared to older baseline (1992–2005) isolates. Around 85% of 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, most commonly a 237 bp fragment of the Sahana transposable element. Inserts were associated with an approximately 3- to 10-fold decrease in sensitivity to the DMIs tested. In contrast to P. lingam, PCR screening revealed CYP51 promoter inserts were absent in modern western European P. biglobosus isolates (2021–2023). 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 evidence for substantive sensitivity shifts to the QoI (pyraclostrobin) or SDHI (boscalid) fungicides tested was obtained for either Plenodomus species.

Botryosphaeria dothidea causes stem canker of Idesia polycarpa in China

Posted on 23 March 2024 by

This is the first report of Botryosphaeria dothidea as the pathogen of stem canker of Idesia polycarpa based on morphological and molecular characterization and pathogenicity identification.

Abstract

Idesia polycarpa is a woody oil plant with great development and application prospects. However, stem canker disease of I. polycarpa was frequently observed in Henan and Hubei provinces of China in 2020–2022. The seriousness of the disease caused a large number of trees deaths, which affected seedling production and fruit yield, restricting the development of the I. polycarpa industry. We performed isolation, purification and pathogenicity analysis of canker samples. Pathogenicity tests reproduced typical canker disease symptoms on detached branches of I. polycarpa. Based on morphological observations, conidial morphology and phylogenetic analysis of isolates with high similarity to the rDNA internal transcribed spacer (ITS), EF-1α and TUB2 sequences screened in GenBank by BLAST, the pathogen of canker disease on I. polycarpa was identified as Botryosphaeria dothidea. This is the first report of B. dothidea as the pathogen of stem canker of I. polycarpa.

Identification of candidate RXLR effectors from downy mildew of foxtail millet pathogen Sclerospora graminicola and functional analysis of SG_RXLR41

Posted on 20 March 2024 by

Sixty-two RXLR effectors were identified from Sclerospora graminicola. The screening revealed that SG_RXLR41 suppresses INF1-induced cell death and promotes Phytophthora capsici infection by inhibiting host immune responses.

Abstract

Downy mildew caused by Sclerospora graminicola is a systemic disease that affects the yield and quality of foxtail millet. This obligate biotrophic oomycete manipulates host physiology and immune processes through numerous effectors. A thorough comprehension of effector biology is crucial to unravel disease mechanisms and understand host plant resistance. In this study, bioinformatic analyses revealed 498 potentially secreted proteins in S. graminicola, of which 62 were identified as RXLR effectors; 46 RXLR-encoding genes exhibited upregulated expression during the early stages of infection. To elucidate the functions of these secreted proteins, a heterogeneous expression system was developed using Nicotiana benthamiana. Twenty-one RXLR effectors secreted by S. graminicola were transiently expressed in N. benthamiana, of which four could suppress INF1-triggered cell death. Various defence responses in N. benthamiana were attenuated, including inhibition of defence gene expression, reduction of reactive oxygen species (ROS) accumulation and diminished callose deposition. The expression of SG_RXLR41 also enhanced the growth of Phytophthora capsici on N. benthamiana leaves. These findings indicate that S. graminicola facilitates infection and expansion through the secretion of multiple RXLR effectors, and SG_RXLR41 is an important virulence-related effector that is involved in manipulating plant immunity by suppressing cell death.

Estimating the frequency of virulence against an Stb gene in Zymoseptoria tritici populations by bulk phenotyping on checkerboard microcanopies of wheat near‐isogenic lines

Posted on 16 March 2024 by

The BCP method leverages the relationship between the ratio of Zymoseptoria tritici lesions on wheat near-isogenic lines differing by one resistance gene (Stb16q) and the frequency of virulent strains in the inoculated population.

Abstract

Monitoring virulent strains within pathogen populations is crucial to improve host resistance deployment strategies. Such monitoring increasingly involves field pathogenomics studies of molecular polymorphisms in pathogen genomes based on high-throughput screening technologies. However, it is not always straightforward to predict virulence phenotypes from these polymorphisms, and in planta phenotyping remains necessary. We developed a method for ‘bulk phenotyping on checkerboard microcanopies of wheat near-isogenic lines’ (BPC) for estimating the frequency of virulence against a resistance gene in mixed populations of the fungal pathogen Zymoseptoria tritici, the causal agent of Septoria tritici blotch (STB) in wheat, without the need for strain-by-strain pathogen phenotyping. Our method involves the uniform inoculation of a microcanopy of two wheat lines—one with the target resistance gene and the other without it—with a multistrain mixture of isolates representative of the population to be characterized, followed by the differential quantification of infection points (lesions). Using Stb16q, a wheat resistance gene that has recently broken down in Europe, we found a robust correlation between the ratio of the mean number of lesions on each wheat line and the frequency of virulent strains in the inoculum. Using pairs of virulent and avirulent strains, as well as synthetic populations consisting of 10 virulent strains and 10 avirulent strains mixed in different proportions, we validated the principle of the method and established standard curves at virulence frequencies close to those observed in natural conditions. We discuss the potential of this method for virulence monitoring in combination with molecular methods.

No safe haven: Loss of avirulence in the plant pathogen Leptosphaeria maculans by DNA duplication and repeat‐induced point mutation

Posted on 16 March 2024 by nAlexander Idnurm, nAlec J. McCallum, nAngela P. Van de Wouwn

Unlike other avirulence genes in canola pathogen Leptosphaeria maculans, AvrLm2 is protected from repeat-induced point mutation (RIP): here fungal populations were identified with RIP in AvrLm2, due to a DNA duplication event.

Abstract

Microbes can overcome the ability of plant resistance genes to confer protection against disease by mutating their corresponding avirulence genes. The fungus Leptosphaeria maculans causes blackleg disease on canola crops and subverts Brassica napus resistance genes through several DNA mutation mechanisms. One of these is repeat-induced point (RIP) mutation, which can ‘leak’ into the avirulence genes from the adjacent repetitive sequences that the mutation process is targeting. Here, we identified populations of L. maculans in Australia that have extensive RIP mutations in the avirulence gene AvrLm2 and show that this has been triggered by a duplication of the gene and surrounding DNA that includes the distant (>55 kb in total) AvrLm6 gene. This finding provides another mechanism of mutation by which pathogens can overcome host resistance, and more broadly contributes to understanding the complex balance between gene duplication and genome defence.

All‐in‐one Xylella detection and identification: A nanopore sequencing‐compatible conventional PCR

Posted on 15 March 2024 by

A new diagnostic PCR assay assesed with an Interlaboratory Test Performance Study (TPS) by five plant pathology laboratories that can accurately detect and differentiate all species and subspecies in Xylella genus.

Abstract

Xylella fastidiosa is a plant-pathogenic bacterium that poses a serious threat to the production of economically important plant species including grapes, almonds, olives and a broad range of amenity plants, causing significant economic losses worldwide. While multiple molecular detection assays have been developed for X. fastidiosa, there is a lack of molecular tools available for detection and differentiation of the closely related pear pathogen, Xylella taiwanensis. In this study, we present a novel conventional PCR assay with primers that can amplify both Xylella species. The amplified product could be sequenced and used for discrimination between the two species and the subspecies within the fastidiosa species. This PCR assay was designed using a genome-informed approach to target the ComEC/Rec2 gene of both Xylella species, ensuring a higher specificity than other previously developed PCR assays. A test performance study across five national plant diagnostic laboratories in Australia and New Zealand demonstrated this assay's high sensitivity and specificity to all known species and subspecies within the Xylella genus. This PCR assay can be used for Xylella identification at the species and subspecies level and is compatible with Sanger sequencing and nanopore sequencing for rapid turnaround time. The newly developed conventional PCR assay presented here offers rapid detection and accurate identification of both Xylella species from plant, insect vector or bacterial samples, enabling timely implementation of biosecurity measures or disease management responses.

Inheritance and genetic mapping of the first CPMMV tolerance locus in common bean

Posted on 15 March 2024 by

A QTL locus for CPMMV tolerance in common bean cv. BRS Sublime was mapped to the terminal region on chromosome Pv08, linked to the SNP_Ch_8_62396711 marker. Two LRR genes and one protein kinase were located within the confidence interval.

Abstract

Although cowpea mild mottle virus (CPMMV) has been reported in Brazil since 1983, it has only become a significant concern for researchers and farmers in recent years. The objective of this work was to investigate the genetic basis of CPMMV tolerance, mapping and reporting the first loci associated with this trait in common bean (cv. BRS Sublime). Phenotypic assays were carried out on 180 individual plants (F₂ generation) and 180 F_2:3 progenies comprising 12 plants per family and their parents (BRS Sublime, tolerant parent × CNFCT 16207, susceptible parent). CPMMV was mechanically inoculated and symptoms were evaluated at 35 days after inoculation, using a 1–5 scoring scale. A linkage map was constructed using 1695 single-nucleotide polymorphism (SNP) and SilicoDArT markers that segregated in the F₂ and F_2:3 generations as expected. Markers were distributed across 11 common bean chromosomes, resulting in a total length of 2864 cM, with an average distance between markers of 1.8 cM. Phenotypic observations revealed that tolerance in cv. BRS Sublime is controlled by a single dominant gene. The main effect quantitative trait locus (QTL; CPMMV.Pv08) associated with CPMMV tolerance was identified in the terminal region on chromosome 8 (Pv08). This QTL explained approximately 77% of phenotypic variation, indicating that the inheritance of tolerance to CPMMV is monogenic, controlled by a major locus. As far as we know, this study represents the first investigation into the inheritance and genetic mapping of CPMMV tolerance in common bean, with potential for the development of elite lines with multiple virus resistance/tolerance.

Landscape‐scale patterns and predictors of potato viruses in Scotland

Posted on 15 March 2024 by nPeter Skelseyn

ArcGIS and machine learning are used to provide a comprehensive overview of potato viruses in Scotland, a deeper understanding of landscape epidemiology, and a model that could serve as the basis of a decision support tool.

Abstract

Virus diseases represent important economic threats to seed potato production worldwide, yet relatively little is known of their epidemiology at the landscape-scale. In this study, data was compiled from the Scottish national seed potato classification scheme on the incidence of 10 different potato viruses for the years 2009–2022. A co-occurrence analysis identified that 12 virus species pairs occurred together more often than expected by chance, and potato blackleg was positively associated with eight potato viruses. ArcGIS was used to investigate spatial and spatiotemporal variation in incidence rates of the three most prevalent viruses (potato virus Y, potato leaf roll virus and potato virus A), and this revealed prominent geographic differences in long-term disease outcomes. Focusing on potato virus Y as the most commonly occurring single infection, interpretable machine-learning techniques were used to investigate the influence of key crop, management and environmental factors on patterns of incidence in space and time. The results showed that health characteristics of seed stocks were among the most important predictors of incidence, along with blackleg infection, several management features, cultivar resistance, distance to the nearest seed and ware crop, temperature variables and several soil features. This approach provides a comprehensive overview of potato viruses in Scotland, a deeper understanding of epidemiological risk factors at the landscape-scale and a forecast model that could serve as the basis of a decision support tool for improved management of potato virus Y.

Ceratocystis fimbriata alters root system architecture and causes symptom development only in detached storage roots in Bayou Belle and Beauregard sweet potato

Posted on 13 March 2024 by nArthur Q. Villordon, nCatherine D. DeRobertis, nChristopher A. Clarkn

Ceratocystis fimbriata affected root system architecture of sweet potato even though it did not cause symptoms and was not isolated from roots until swollen storage roots were detached from the plant.

Abstract

Little is known about how the sweet potato (Ipomoea batatas) root system responds to Ceratocystis fimbriata in the growth substrate. Understanding whether the fungus affects the root system before harvest of storage roots (SR) could guide timing of management efforts. Cuttings of cultivars Bayou Belle and Beauregard, both previously considered susceptible to black rot, were in unamended or infested sand. At the onset of SR formation, effects on first- and second-order lateral root (LR) length and number among and within treatments, cultivars and replicate experiments were recorded. First-order LR length in inoculated Bayou Belle plants was 31% greater than in inoculated plants of cultivar Beauregard in Expt 1, but there were no differences in Expt 2. Second-order LR length varied among, and within, cultivars, inoculum treatments and experiments in Expts 1 and 2. At 49 days after planting in Expt 1, only inoculated plants had necrotic lesions on stems in both cultivars, with more than twice as many recorded on Bayou Belle than Beauregard. SR had no lesions at harvest. Detached SR originating from inoculated treatments and stored in sampling bags for 25 days developed black rot lesions at either end of the SR (45% and 31% of total in Expt 1 and Expt 2, respectively) or elsewhere, including those centred on LR emergence sites or lenticels (55% and 69%, respectively). Exposing developing plants to inoculation may reveal differences in SR susceptibility not found when harvested SR are wounded and inoculated.