RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi.

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed.

Suppression of Chitin-Triggered Immunity by a New Fungal Chitin-Binding Effector Resulting from Alternative Splicing of a Chitin Deacetylase Gene.

Phytopathogenic fungi have evolved mechanisms to manipulate plant defences, such as chitin-triggered immunity, a plant defensive response based on the recognition of chitin oligomers by plant-specific receptors. To cope with chitin resistance, fungal pathogens have developed different strategies to prevent chitin recognition, such as binding, breaking, or modifying immunogenic oligomers. In powdery mildew fungi, the activity of chitin deacetylase (CDA) is crucial for this purpose, since silencing of the CDA gene leads to a rapid activation of chitin signalling and the subsequent suppression of fungal growth. In this work, we have identified an unusually short CDA transcript in Podosphaera xanthii, the cucurbit powdery mildew pathogen. This transcript, designated PxCDA3, appears to encode a truncated version of CDA resulting from an alternative splicing of the PxCDA gene, which lacked most of the chitin deacetylase activity domain but retained the carbohydrate-binding module. Experiments with the recombinant protein showed its ability to bind to chitin oligomers and prevent the activation of chitin signalling. Furthermore, the use of fluorescent fusion proteins allowed its localization in plant papillae at pathogen penetration sites. Our results suggest the occurrence of a new fungal chitin-binding effector, designated CHBE, involved in the manipulation of chitin-triggered immunity in powdery mildew fungi.

Resistance to the SDHI Fungicides Boscalid and Fluopyram in Podosphaera xanthii Populations from Commercial Cucurbit Fields in Spain.

Powdery mildew is caused by Podosphaera xanthii, and is one of the most important diseases that attacks Spanish cucurbit crops. Fungicide application is the primary control tool; however, its effectiveness is hampered by the rapid development of resistance to these compounds. In this study, the EC50 values of 26 isolates were determined in response to the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid and fluopyram. From these data, the discriminatory doses were deduced and used for SDHI resistance monitoring during the 2018 and 2019 growing seasons. Of the 298 isolates analysed, 37.9% showed resistance to boscalid and 44% to fluopyram. Although different phenotypes were observed in leaf disc assays, the resistant isolates showed the same phenotype in plant assays. Compared to sensitive isolates, two amino acid changes were found in the SdhC subunit, A86V and G151R, which are associated mostly with resistance patterns to fluopyram and boscalid, respectively. Furthermore, no significant differences were observed in terms of fitness cost between the selected sensitive and resistant isolates analysed here. Lastly, a loop-mediated isothermal amplification (LAMP) assay was developed to detect A86V and G151R mutations using conidia obtained directly from infected material. Our results show that growers could continue to use boscalid and fluopyram, but resistance management practices must be implemented.

Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing.

The powdery mildew fungus Podosphaera xanthii is one of the most important limiting factors for cucurbit production worldwide. Despite the significant efforts made by breeding and chemical companies, effective control of this pathogen remains elusive to growers. In this work, we examined the suitability of RNAi technology called spray-induced gene silencing (SIGS) for controlling cucurbit powdery mildew. Using leaf disc and cotyledon infiltration assays, we tested the efficacy of dsRNA applications to induce gene silencing in P. xanthii. Furthermore, to identify new target candidate genes, we analyzed sixty conserved and non-annotated proteins (CNAPs) deduced from the P. xanthii transcriptome in silico. Six proteins presumably involved in essential functions, specifically respiration (CNAP8878, CNAP9066, CNAP10905 and CNAP30520), glycosylation (CNAP1048) and efflux transport (CNAP948), were identified. Functional analysis of these CNAP coding genes by dsRNA-induced gene silencing resulted in strong silencing phenotypes with large reductions in fungal growth and disease symptoms. Due to their important contributions to fungal development, the CNAP1048, CNAP10905 and CNAP30520 genes were selected as targets to conduct SIGS assays under plant growth chamber conditions. The spray application of these dsRNAs induced high levels of disease control, supporting that SIGS could be a sustainable approach to combat powdery mildew diseases.

Fungicide Resistance in Powdery Mildew Fungi.

Powdery mildew fungi (Erysiphales) are among the most common and important plant fungal pathogens. These fungi are obligate biotrophic parasites that attack nearly 10,000 species of angiosperms, including major crops, such as cereals and grapes. Although cultural and biological practices may reduce the risk of infection by powdery mildew, they do not provide sufficient protection. Therefore, in practice, chemical control, including the use of fungicides from multiple chemical groups, is the most effective tool for managing powdery mildew. Unfortunately, the risk of resistance development is high because typical spray programs include multiple applications per season. In addition, some of the most economically destructive species of powdery mildew fungi are considered to be high-risk pathogens and are able to develop resistance to several chemical classes within a few years. This situation has decreased the efficacy of the major fungicide classes, such as sterol demethylation inhibitors, quinone outside inhibitors and succinate dehydrogenase inhibitors, that are employed against powdery mildews. In this review, we present cases of reduction in sensitivity, development of resistance and failure of control by fungicides that have been or are being used to manage powdery mildew. In addition, the molecular mechanisms underlying resistance to fungicides are also outlined. Finally, a number of recommendations are provided to decrease the probability of resistance development when fungicides are employed.