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A new mechanism for reduced sensitivity to demethylation-inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis.

ABSTRACT: The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

SUBMITTER: Diaz-Trujillo C

PROVIDER: S-EPMC6637983 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Project description:Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Project description:The Sigatoka disease complex, caused by the closely-related Dothideomycete fungi Pseudocercospora musae (yellow sigatoka), Pseudocercospora eumusae (eumusae leaf spot), and Pseudocercospora fijiensis (black sigatoka), is currently the most devastating disease on banana worldwide. The three species emerged on bananas from a recent common ancestor and show clear differences in virulence, with P. eumusae and P. fijiensis considered the most aggressive. In order to understand the genomic modifications associated with shifts in the species virulence spectra after speciation, and to identify their pathogenic core that can be exploited in disease management programs, we have sequenced and analyzed the genomes of P. eumusae and P. musae and compared them with the available genome sequence of P. fijiensis. Comparative analysis of genome architectures revealed significant differences in genome size, mainly due to different rates of LTR retrotransposon proliferation. Still, gene counts remained relatively equal and in the range of other Dothideomycetes. Phylogenetic reconstruction based on a set of 46 conserved single-copy genes strongly supported an earlier evolutionary radiation of P. fijiensis from P. musae and P. eumusae. However, pairwise analyses of gene content indicated that the more virulent P. eumusae and P. fijiensis share complementary patterns of expansions and contractions in core gene families related to metabolism and enzymatic degradation of plant cell walls, suggesting that the evolution of virulence in these two pathogens has, to some extent, been facilitated by convergent changes in metabolic pathways associated with nutrient acquisition and assimilation. In spite of their common ancestry and shared host-specificity, the three species retain fairly dissimilar repertoires of effector proteins, suggesting that they likely evolved different strategies for manipulating the host immune system. Finally, 234 gene families, including seven putative effectors, were exclusively present in the three Sigatoka species, and could thus be related to adaptation to the banana host.

Project description:In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.

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A new mechanism for reduced sensitivity to demethylation-inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis.

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