Resistance to the SDHI fungicides boscalid and fluopyram in Podosphaera xanthii from commercial cucurbit fields in Spain

Powdery mildew elicited by Podosphaera xanthii is a devastating disease of cucurbits worldwide and one of the most important diseases affecting these crops in Spain. Application of fungicides is the main control practice for managing P. xanthii; however, isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish cucurbit powdery mildew population. Succinate dehydrogenase inhibitors (SDHIs) constitute a relatively novel class of fungicides registered for powdery mildew control representing new alternatives for cucurbit growers. In the present study, 30 P. xanthii isolates were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to boscalid and fluopyram. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 180 P. xanthii isolates collected from several commercial cucurbit fields in Spain during 2017-2018. Three SDHI resistance patterns were observed in our population, which include patterns I (resistance to boscalid), II (resistance to fluopyram), and III (resistance to boscalid and fluopyram). The amino acid changes associated with these resistance patterns in the Sdh protein were also examined. Based on our results, SDHI fungicides are good alternatives for cucurbit powdery mildew control, although they should be applied with caution.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Coordinated role of chitin-triggered immunity suppression mechanisms of Podosphaera xanthii.

Fungal pathogens are the main destructive microorganisms for terrestrial plants and pose increasing challenges for global agricultural production. Chitin is a vital building block for fungal cell walls and a widely effective inducer for plant immunity that, through chitin-triggered immunity, can defend against fungi attack. That is why the phytopathogenic fungi have developed different virulence factors that allow them to suppress the activation of this defensive response. In this study, the molecular mechanisms of chitin-triggered suppression, previously identified in the cucurbit powdery mildew Podosphaera xanthii, have been evaluated in detail. These mechanisms consist of the modification of chitin immunogenic oligomers (CDA), the binding to these oligomers (CHBE) and their degradation (EWCAs). For this, the RNA interference (RNAi) technology, which consists of the application of double-stranded RNA (dsRNA) designed to suppress the expression of target genes, was used. The preliminary results obtained using this strategy significantly reduces the development of the fungus and the symptoms of powdery mildew disease in melon, suggesting that chitin signaling suppression mechanisms are essential for the development of P. xanthii. This work was supported by AEI (PDC2021-121373-C21).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The combined function of Podosphaera xanthii's mechanisms to suppress immunity triggered by chitin.

Fungal pathogens are the main destructive microorganisms for terrestrial plants and pose increasing challenges for global agricultural production. Chitin is a vital building block for fungal cell walls and a widely effective inducer for plant immunity. Chitin-triggered immunity is a powerful plant defense response against fungi. Therefore, phytopathogenic fungi have developed different virulence factors that allow them to suppress the activation of this defensive response. In this study, we intend to evaluate the molecular mechanisms of suppression the chitin-triggered previously identified in Podosphaera xanthii, the main causal agent of powdery mildew in cucurbits. These mechanisms consist of the modification of chitin immunogenic oligomers (CDA), the binding to these oligomers (CHBE) and their degradation (EWCAs). For this, we used RNA interference (RNAi) technology, which consists of the application of double-stranded RNA (dsRNA) designed to suppress the expression of the PxCDA and PxEWCA genes, which would result in the reduction of the three mechanisms of suppression of chitin signaling mentioned above, since CDA and CHBE proteins are products of the same PxCDA gene. Application of dsRNA was carried out using leaf disc assays and infiltration of melon cotyledons. The preliminary results obtained indicate that the application of dsRNA significantly reduces the development of the fungus and the symptoms of powdery mildew disease in melon, suggesting that chitin signaling suppression mechanisms are essential for the development of P. xanthii.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Resistance to SDHI fungicides in Botrytis cinerea from strawberry fields in Spain

Gray mold, caused by Botrytis cinerea Pers., is one of the most economically important diseases of strawberries and its control involves the application of fungicides throughout the strawberry growing season. Succinate dehydrogenase inhibitors (SDHIs) constitute a novel class of fungicides representing new alternatives for strawberry growers. In the present study, B. cinerea isolates were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) and to obtain discriminatory doses to monitor SDHI fungicides over the course of three-year monitoring period. The overall frequencies of resistance to the SDHI fungicides boscalid, fluopyram, fluxapyroxad and penthiopyrad were 56.9, 6.9, 12.9, and 24.6%, respectively. Four SDHI resistance patterns were observed in our population. Patterns I (resistance to boscalid) and II (resistance to boscalid and penthiopyrad) were associated with the amino acid substitutions H272R/Y; pattern III (resistance to boscalid, fluxapyroxad, and penthiopyrad) was associated only with the H272Y mutation; and finally, pattern IV (resistance to boscalid, fluopyram, fluxapyroxad and penthiopyrad) was associated with the N230I allele in the SdhB subunit. For gray mold management, it is suggested that the simultaneous use of boscalid and penthiopyrad should be limited to one application per season. The use of fluxapyroxad and fluopyram could be used as valid SDHI alternatives for our strawberry growers, but they should be applied with caution.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The gene of a GPI-anchoring protein a promising new target for the control of the cucurbit powdery mildew Podosphaera xanthii.

One of the main limitations of the cucurbit crops production is the powdery mildew disease, caused by the biotrophic fungus Podosphaera xanthii. An integrated management, using several strategies, is carried to control the disease but the application of fungicides is the most effective one. The problem is that P. xanthii has been classified by the Fungicide Resistance Action Committee (FRAC) as a pathogen with a high risk of resistance developing, in addition of the strong restrictions on the use of phytosanitary products at a European level. For this reason, new phytosanitary tools are necessary to allow a sustainable control of this devastating disease such as the use of the RNA interference (RNAi) technology. In this work, dsRNA targeting a P. xanthii gene, which encodes a protein that appears to be essential for the correct assembly of the fungal cell wall, was evaluated. Preliminary gene silencing results have shown a significant reduction of fungal development on melon plants suggesting that this gene may be a promising target for the control of powdery mildew of cucurbits. This work has been funded by AEI (PID2019-107464RB-C21).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

New promising “biofungicides” targeting the fungal cell wall to control the cucurbit powdery mildew Podosphaera xanthii.

One of the main limitations of cucurbit production is the powdery mildew disease, caused by Podosphaera xanthii. Although different management strategies are used to control it, the application of fungicides is the most effective. However, there are two major problems: the rapid emergence of resistance to fungicides by the pathogen and the strong restrictions on the use and diversity of phytosanitary products imposed at the European level. For this reason, novel targets and strategies are needed to develop new “biofungicides” for sustainable disease control. The fungal cell wall (CW) is a unique and essential structure, not present in human or animal cells, which makes it an ideal target for the development of new phytosanitary products. In this study, three P. xanthii cell wall-related genes (coding a GPI-anchored membrane protein and two proteins involved in the synthesis of rhamnose) were studied. To understand their impact on the development of P. xanthii, RNA interference (RNAi) technology was used. For this purpose, dsRNA molecules were synthesized and several assays were performed. The infiltration and leaf disc assays on melon and zucchini cotyledons, respectively, showed an effective gene silencing effect, resulting in a significant reduction of gene expression, fungal biomass and area covered by powdery mildew disease. Microscopy analysis confirmed the impact on fungal penetration points. In addition, the application of dsRNAs, through spray-induced gene silencing (SIGS) assays, resulted in a significant decrease in disease development on melon plants. According to the results obtained, these genes could serve as potential targets for the development of new generations of antifungal agents. In addition, SIGS technology could be a promising strategy to incorporate into integrated management programs for this important disease.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The potential of the emerging RNAi strategy, called SIGS, in the control of Botrytis cinerea in horticultural crops

Botrytis cinerea, the causal agent of the gray mold disease, is one of the main limiting factors of horticultural crops production worldwide, consuming up to 40% of fungicides in its control. However, this fungus has been categorized by FRAC (Fungicide Resistance Action Committee) as a phytopathogen with a high risk for fungicide resistance development, a fact that has been demonstrated in our country. In addition, and according to the "farm to fork" strategy of the recent European Green Deal, the diversity of fungicides available to growers will be reduced by 50% in 2030. For this reason, alternative control tools and molecules with fungicide activity are, more than ever, needed to control this important disease. In this study, we intend to check if the efficacy of the emerging RNA interference (RNAi) strategy, called "spray-induced gene silencing" (SIGS), could be a valid sustainable solution and an alternative to the use of conventional fungicides for the control of B. cinerea. For this purpose, several double-stranded RNA (dsRNA) has been designed against targets genes involved in the virulence/pathogenicity of the fungus. Preliminary results, obtained in in vivo assays, indicated that the application of dsRNAs significantly reduces the development of the fungus, demonstrating the potential of the SIGS technique for the control of B. cinerea. On the other hand, and to improve the application of these oligonucleotides in the field, their encapsulation to create nanoparticles will be carried out. If we succeed, new molecules with fungicidal action, could be included into the several strategies carried out to obtain a sustainable plant protection control programs in the fieldUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The potencial of the RNAi technology, via SIGS, in the control of Botrytis cinerea in horticultural crops

Botrytis cinerea is one of the most important limiting factors for crop production worldwide, as it demonstrated by the enormous annual intake of fungicides used for their control to avoid crop losses. However, this fungus has been categorized by FRAC as a high-risk pathogen for fungicide resistance development. Another problem is related with the diversity of fungicides available to growers, which according with the current European legislation on pesticides and the European Green Deal, will be reduced by 50% by 2030. For all this, new low-impact sustainable solutions, obtained through new phytoprotection tools, to control B. cinerea are needed. In this study, we intend to check if some emerging strategies such as RNA interference technology (RNAi) could be valid sustainable solution and alternative to the use of conventional chemical fungicides for the control of B. cinerea in crops of relevance. To achieve this goal, the SIGS (spray-induced gene silencing) approach, which concerns the exogenous application of double-stranded RNA (dsRNA), was tested. For it, ten double-stranded RNA (dsRNAs) were designed against the fungicide target’s genes and genes encoding proteins involved in virulence/pathogenicity of this fungus. The preliminary results obtained in in vitro tests have shown that the application of the different dsRNAs, individually and in combination, have significantly reduced the development of the fungus on different culture media. In addition, this reduction was very promising on detached fruit and in planta assays, demonstrating the potential of this technique in the control of B. cinerea. On the other hand, the sustained release of the dsRNA-fungicides using nanoparticles as a carrier or stabilizer has also been analyzed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Use of oligonucleotides for the control of Botrytis cinerea in horticultural crops.

Botrytis cinerea, the causal agent of the gray mold disease, is one of the main limiting factors of horticultural crops production worldwide, consuming up to 40% of fungicides in its control. However, this fungus has been categorized by FRAC (Fungicide Resistance Action Committee) as a phytopathogen with a high risk for fungicide resistance development, a fact that has been demonstrated in our country. In addition, and according to the "farm to fork" strategy of the recent European Green Deal, the diversity of fungicides available to growers will be reduced by 50% in 2030. For this reason, alternative control tools and molecules with fungicide activity are needed to B. cinerea control. In this study, the efficacy of emerging strategies using oligonucleotides with antifungal effect has been explored. Preliminary results, obtained in in vivo assays, have shown a significantly reduction of the fungal development, demonstrating the potential of these oligonucleotides to be novel candidates to include in the different strategies of integrated control programs of the gray mold disease. This work has been funded by: AYUDAS A LA I+D+i, EN EL ÁMBITO DEL PLAN ANDALUZ DE INVESTIGACIÓN, DESARROLLO E INNOVACIÓN (PAIDI 2020). Project code: PY20_00048Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Monitorización de aislados resistentes a los fungicidas MBC en Podosphaera xanthii, oídio de cucurbitáceas, utilizando la técnica LAMP

El oídio, causado por el hongo biotrofo Podosphaera xanthii, es una de las enfermedades más importantes que afectan a los cultivos de cucurbitáceas en España. El control de esta enfermedad es principalmente químico, a través del uso continuado de fungicidas durante la campaña de cultivo; sin embargo, la rápida aparición de resistencias, por parte del patógeno, a estos compuestos es un hecho bien conocido, lo que dificulta el manejo de la enfermedad. En estudios previos, se detectaron altos niveles de resistencia a los fungicidas Metil Benzimidazol Carbamatos (MBC) en la población española de P. xanthii. La resistencia fue debida al cambio aminoacídico E198A en β-tubulina, diana de estos fungicidas. Para llevar a la práctica una agricultura más sostenible y productiva en el cultivo de cucurbitáceas, se hace necesario el desarrollo de nuevas técnicas que permitan reducir el número de aplicaciones de fungicidas y que optimicen los programas de aplicación para cada finca. En este estudio se ha usado por primera vez en el oídio de cucurbitáceas, la técnica LAMP (amplificación isotérmica mediada por bucle) para distinguir de forma rápida y fiable el genotipo mutante A198 en P. xanthii. Nuestros resultados han demostrado que la técnica LAMP es un método específico y reproducible que puede ser usado para monitorizar la resistencia a fungicidas MBC en P. xanthii en campo, proporcionando información exacta para cada finca analizada, y permitiendo a cada agricultor ajustar sus programas de aplicación de fungicidas antes de la pérdida de la cosecha.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec