Evaluation of biological control agents for managing cucurbit powdery mildew on greenhouse-grown melon

An evaluation was made of the ability of two mycoparasite-based products AQ10® (Ampelomyces quisqualis) and Mycotal® (Lecanicillium lecanii), as well as three strains of Bacillus subtilis, to manage powdery mildew disease, caused by Podosphaera fusca on melon seedlings maintained under different regimes of relative humidity and on plants grown under greenhouse conditions in Spain. In every case fungal and bacterial biocontrol agents (BCAs) performed better under conditions of high relative humidity (90–95% RH). In greenhouse experiments, the effectiveness of the mycoparasites to manage powdery mildew was absolutely dependent on mineral oil. The strains of B. subtilis provided disease control similar to that achieved with the mycoparasites or the fungicide azoxystrobin. Microscopic analysis showed the ability of these bacterial strains to efficiently colonize leaf surfaces and revealed the occurrence of antagonistic interactions between biological agents and P. fusca structures. These results confirmed the usefulness of these BCAs for managing powdery mildew on greenhouse-grown cucurbits either as single products or as a component of integrated control programmes.Estación Experimental ‘La Mayora’ (CSIC), Algarrobo-Costa, 29750 Málaga, Spain Grupo de Microbiología y Patología Vegetal-Unidad Asociada a CSIC, Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 MálagaPeer reviewe

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 epiphytic transcriptome of Podoshaera fusca, the causal agent of cucurbit powdery mildew

3rd International Powdery Mildew Workshop, Copenhagen, Denmar

Mechanisms of resistance to QoI fungicides in phytopathogenic fungi

The major threat to crops posed by fungal diseases results in the use by growers of enormous amounts of chemicals. Of these, quinol oxydation inhibitors (QoIs) are probably the most successful class of agricultural fungicides. QoIs inhibit mitochondrial respiration in fungi by binding to the Qo site of the cytochrome bc1 complex, blocking electron transfer and halting ATP synthesis. Unfortunately, the rapid development of resistance to these fungicides and consequent control failure has become increasingly problematic. The main mechanism conferring resistance to QoIs is target site modification, involving mutations in the cytochrome b gene CYTB, such as the substitution of glycine by alanine at position 143 (G143A) that occurs in several phytopathogenic fungi. The impact of other mechanisms, including alternative respiration and efflux transporters, on resistance seems to be limited. Interestingly, in some species QoI resistance is not supported by mutations in CYTB, while in others the structure of the gene is such that it is unlikely to undergo G143A mutations. Better understanding of the biological basis of QoI resistance in a single pathogen species will facilitate the development of resistance diagnostic tools as well as proper anti-resistance strategies aimed at maintaining the high efficacy of these fungicides. [Int Microbiol 2008; 11(1):1-9

Multirresistencia a fungicidas en oídio de cucurbitáceas (Podosphaera fusca)

El oídio causado por Podosphaera fusca es un factor limitante en la producción de cucurbitáceas en España y su control depende en gran medida de la aplicación de productos fitosanitarios. La resistencia a fungicidas depende de dos factores, uno inherente al propio fungicida, de manera que hay mecanismos de acción a los cuales casi todos los organismos desarrollan resistencia en un plazo más o menos largo y un segundo factor correspondiente al propio hongo que puede de desarrollar resistencias a numerosas materias activas. Los oídios se encuentran entre los organismos que desarrollan resistencia a fungicidas con gran facilidad, por lo que las aplicaciones de fitosanitarios deben efectuarse con precaución, para evitar en lo posible la aparición de estas resistencias. Durante los últimos quince años, nuestro grupo de investigación ha estudiado la resistencia de poblaciones de P. fusca en cultivos del centro y sur de España a fungicidas de diferentes mecanismos de acción: DMI (triademol, fenarimol y miclobutanilo), QoI (kresoxim-metil, azoxistrobin y trifloxistrobin), MBC (metil-tiofanato), aza-naftalenos (quinoxifén) e hidroxi (2 amino) pirimidinas (bupirimato). Las concentraciones discriminatorias se establecieron sobre la dosis máxima autorizada en campo. El hecho más interesante es la presencia de cepas multirresistentes; en nuestro laboratorio se ha determinado la resistencia a estos nueve fungicidas en 67 cepas. Todas fueron sensibles a quinoxifén y miclobutanilo, no se ha observado resistencia a ellos en España, aunque sí hemos notado cierta disminución de la sensibilidad. Para los otros cuatro (puesto que consideramos los tres QoI como un único fungicida), treinta y tres cepas eran resistentes al menos a uno de ellos, veintidós a dos, once a tres y una única cepa a cuatro. Estos datos confirman el riesgo de aparición de cepas multirresistentes en P. fusca, por lo que deben extremarse las precauciones a la hora de efectuar tratamientos fitosanitarios.Este trabajo ha sido financiado por fondos de los proyectos del Plan Nacional AGL2001-1837-CO2-02, AGL2004-06056-CO2-02, AGL2007-65340-CO2-02 y AGL2010-21848-C02-01 y una ayuda del Plan Propio de Investigación de la Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A Method for Estimation of Population-Densities of Ice Nucleating Active Pseudomonas-Syringae in Buds and Leaves of Mango

Active ice nucleation strains of Pseudomonas syringae pv. syringae have been associated with a necrotic disease in mango trees growing in Málaga (southern Spain). In this paper a simple multiple-tube test is described to estimate the number of active ice nucleation bacteria associated with plant tissues and, also from suspensions of isolated bacterial strains.Peer reviewe

La heteroplasmia mitocondrial explica la resistencia a fungicidas QoI en Podosphaera xanthii, agente causal de oídio de cucurbitáceas

En España, el control del oídio de las cucurbitáceas (Podosphaera xanthii) depende principalmente del empleo de fungicidas como los inhibidores de la quinona externa (QoI). En un trabajo anterior, se observó una resistencia generalizada a los fungicidas QoI en poblaciones de P. xanthii en el centro-sur de España, pero no se terminaron de aclarar los mecanismos moleculares implicados. En este trabajo hemos estudiado el papel de la proteína de Rieske-FeS (risp) y de las mutaciones en el gen del citocromo b (cytb) en la resistencia a fungicidas QoI en P. xanthii. No se detectaron mutaciones en el gen risp en los aislados resistentes analizados. Para cytb, el análisis de las secuencias reveló la presencia de la sustitución G143A, previamente descrita y asociada a la resistencia a QoI en varias especies. Esta mutación siempre se detectaba en aislados resistentes a QoI; sin embargo, también se detectaba en aislados sensibles aunque con menor frecuencia. Para comprobar si la heteroplasmia del gen cytb estaba implicada en la resistencia a los fungicidas QoI en P. xanthii, se desarrolló una reacción en cadena de la polimerasa específica de alelo y cuantitativa, para determinar la abundancia relativa de los alelos G143 (sensible) y A143 (resistente). En los aislados resistentes a los fungicidas QoI se observó una abundancia relativa de los alelos A143 del 70% y en los aislados sensibles estas frecuencias variaban entre el 10% y el 60%. Nuestros datos sugieren que la mutación G143A en el gen cytb es el principal factor implicado en la resistencia a QoI en P. xanthii, pero la proporción de los alelos G143 y A143 en cada aislado puede determinar su nivel de resistencia.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Guía de gestión integrada de plagas: mango

Coordinadores: Ángel Martín Gil y Gregoria Aranda Aranda

Transient transformation of Podosphaera xanthii by electroporation of conidia

[Background] Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.[Results] To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.[Conclusion] This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.This study was supported by grants from the former “Ministerio de Ciencia e Innovación (MICINN)” (AGL2010-21848-CO2-01/AGR) and current “Ministerio de Economía y Competitividad (MINECO)” (AGL2013-41939-R), both co-financed by FEDER funds (European Union). Vela-Corcía D was supported by a PhD fellowship from the FPI programme of MICINN.Peer reviewe