Recent Trends in molecular Diagnostic towards Gray Mold control in Different Host Plants

Summary

Vegetables, fruit and flower loss due to gray mold is still a major factor in plant crop production worldwide. Botrytis cinerea is an important plant pathogen causing gray mold on many economically important crops and ornamental plants. The integrated pest management is the most efficient strategy for disease control, as the use of fungicides become increasingly unacceptable and the use of biocontrol is not always sufficient. This goal can be achieved either by applying fungicides only when needed, thus eliminating unnecessary sprays, or by integrating chemical and non-chemical measures leading to the reduction in fungicide use. Approaches based on how to reduce fungal resistance towards fungicides include the understanding of fungicide resistance mechanisms. Multiple drug resistance (MDR) is common in human pathogenic microbes but rarely described for plant pathogens. The major fungicide resistance mechanism in fungi is target site alteration, so-called multidrug resistance (MDR), can also be an important mechanism of Botrytis under field conditions (Hahn and Leroch, 2015). Different types of MDR, would be studied as they have been discovered and elucidated on the molecular level (Kretschmer et al., 2009; Hahn and Leroch, 2015). In recent years, B. cinerea populations from strawberry fields showed dramatically increased fungicide resistance frequencies and high proportions of multi-resistant strains that are resistant against several or even all of the currently registered fungicides (Hahn, 2014). Determining the resistance profile of B. cinerea on many plants in Egypt will help assess the current status of sensitivity of B. cinerea to commonly used fungicides. This will benefit growers in the long run by extending the effective lifetime of available fungicides and reducing losses due to poor disease control or money spent on fungicides that don’t work. Consequently, we could make decisions about applications of fungicides and the status of crop marketing versus export.

Moreover, recent advances in understanding the biology of pre and postharvest pathogens, and the complex interactions between host plants and pathogens, offer opportunities for novel ways to better manage these diseases. Approaches based on fungal biology include the possible use of fungal viruses (mycoviruses) as biocontrol agents, the use of reduced-pathogenicity strains of pathogens as biocontrol agents, and using fungal genomic data and molecular techniques to identify key pathogenicity factors as targets for novel control products. The current project develops, on one hand, the recent molecular diagnostic tools to detect and quantify Botrytis cinerea especially non or reduced pathogenicity isolates (hypovirulent isolates) from different host plants like grape, strawberry, onion and lettuce in order to develop new strategies for the biological control of gray mold. On the other hand, isolation and screening the existence of multi-resistant isolates, from different host plants, that are widely distributed and dominant in Egyptian fields using a rapid and reliable methods such as allele-specific (AS)-polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP), and primer-introduced restriction enzyme analysis (PIRA)-PCR. These PCR- based methods are more convenient than the traditional antifungal susceptibility test, as the sequence mutation may confer the fungicide resistance. In this study, we will develop new assay protocols for detecting mutations that cause resistance to multiple fungicides currently used. Two adjacent probes will be used to recognize changes in the fungal genomic by real-time PCR instrument. This current project would focus on the use of advanced molecular methods to detect such isolates in order to improve the future integrated management of Botrytis infections.

Botrytis cinerea is an important plant pathogen causing gray mold on many economically important crops and ornamental plants. Managing gray mold is difficult and requires applications of fungicides at timely manner, and resistance is inadequate in many crops. In fact, the growers rely on fungicides for management of Botrytis rot, caused by the fungus B. cinerea, which is a threat to crop yield and quality. The number of effective fungicides for Botrytis is relatively limited since this fungus is not sensitive to many fungicides commonly used for most other fungi. For instance, multifungicide resistance is common in Botrytis on crops. Usually, the more fungicide applications are made per season, the greater the risk of fungicide resistance development for those fungicides that are prone to it. Developing biological control would be an alternative and could be a component of an integrated management package. In fact, B. cinerea not only infects plants, it also hosts several mycoviruses itself. A range of phenotypic alterations due to the mycoviral infection have been observed from symptomless to mild impact, or more severe phenotypic changes including reduction in growth/suppression of mycelia, sporulation and sclerotia production, formation of abnormal colony sectors and virulence. Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Most mycoviruses lead 'secret lives', some reduce the ability of their fungal hosts to cause disease in plants. This property, known as hypovirulence, has attracted attention owing to the importance of fungal diseases in agriculture and the limited strategies that are available for the control of these diseases. Exploitation of alternative measures to control B. cinerea, including biological control, has attracted the interest of many researchers (Fravel, 2015). Successful control of some diseases with hypovirulent (HV) strains of the causal pathogen was achieved in Europe, and has inspired further studies on fungal hypovirulence and its potential for control of other plant pathogenic fungi (Nuss, 2005.). Studies on hypovirulence fungi showed that the mechanism involved in this biocontrol strategy is through the transmission of hypovirulent elements or factors from HV strains to virulent strains via hyphal anastomosis, thereby resulting in debilitation of pathogenicity of virulent strains. The presence of single-stranded (ss) RNA or dsRNA-mycoviruses in mycelia of B. cinerea has been reported in previous studies (Howitt et al., 2001; Howitt et al., 2005). Using one pathogen to control another is appealing, both intellectually and ecologically. However, pathogenicity tests showed that hypovirulence was not always associated with infection of B. cinerea by mycoviruses. For example, it was reported that there was no significant difference in infection of beans between dsRNA-containing and dsRNA-free strains of B. cinerea. B. cinerea is a pathogen of numerous economically important crops (Li et al., 2002). However, there are no reports on the presence of dsRNA or mycoviruses in B. cinerea in Egypt. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal–virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.

Achievements

List of Publications from the Project

Abdel Wahab, H. and Younis, R.A. (2012) Early Detection of Gray Mold in Grape Using Conventional and Molecular Methods. African Journal of Biotechnology, 11, 15251-15257.

Abdel Wahab, H. and Helal, N.S. (2013) Evaluation of Pre-Harvest Bioagent Applications for both Production and Biological Control of Onion and Strawberry Plants under Natural Botrytis Infections. African Journal of Plant Science and Biotechnology, 7, 64-69.

Abdel Wahab, H. (2015) Characterization of Egyptian Botrytis cinerea Isolates from Different Host Plants.

Advances in Microbiology, 5, 177-189.

Aboelghar, M. and Abdel Wahab, H. (2013) Spectral footprint of Botrytis cinerea, a novel way for fungal characterization. Advances in Bioscience and Biotechnology, 4, 374-382.

Castro, M., Kramer, K., Valdivia, L., Ortiz, S. and Castillo, A. (2003) A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea. FEMS Microbiol. Lett, 228, 87-91.

Cortesi, P., McCulloch, C. E., Song, H.-Y., Lin, H.-Q. and Milgroom, M. G. (2001) Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica. Genetics, 159, 107-118.

Dufresne, M., Hua-Van, A., Abdel Wahab, H., Ben M’Barek, S., Vasnier, C., Teysset, L., Kema, G.H.J. and Daboussi, M.-J. (2007) Transposition of a Fungal Miniature Inverted-Repeat Transposable Element through the Action of a Tc1-Like Transposase. Genetics, 175, 441-452.

Elad, Y., Williamson, B., Tudzynski, P. and Delen, N. (2004) Botrytis spp. and diseases they cause in agricultural systems-an introduction. Pages 1-8 in: Botrytis: Biology, Pathology and Control. Y. Elad, B. Williamson, P. Tudzynski, and N. Delen, eds. Kluwer Academic Publishers, Dordrecht, the Netherlands.

Fravel, D. R. (2005) Commercialization and implementation of biocontrol. Annu. Rev. Phytopathol., 43, 337-359.

Hahn, M. (2014) The rising threat of fungicide resistance in plant pathogenic fungi:Botrytis as a case study. J. Chem. Biol., DOI 10.1007/s12154-014-0113-1.

Hahn, M., Leroch, M. (2015) The role of multidrug efflux transporters in fungicide resistance of plant pathogenic fungi. In: Fungicide Resistance in Plant Pathogens: Principles and a Guide to Practical Management (Ishii H, Hollomon D., eds.), 2015, Springer Japan KK, Tokyo.

Howitt, R. L. J., Beever, R. E., Pearson, M. N. and Forster, R. L. S. (2001) Genome characterization of Botrytis virus F, a flexuous rod-shaped mycovirus resembling plant ‘potex-like’ viruses. J. Gen. Virol., 82, 67-78.

Howitt, R. L. J., Beever, R. E., Pearson, M. N. and Forster, R. L. S. (2005) Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant ‘potex-like’ viruses. Arch. Virol., 151, 563-579.

Kretschmer, M., Leroch, M., Mosbach, A., Walker, A-S., Fillinger, S., Mernke, D., Schoonbeek, H-J., Pradier, J- M., Leroux, P., De Waard, M.A. and Hahn, M. (2009) Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea. PLoS Pathog., 5, e1000696.

Li, M.-Y., Li, X.-H., Yan, H. and Zhang, T.-T. (2002) Occurrence and control of grey mould diseases on vegetables in China.

Plant Protection Technology and Extension, 221, 31-33. (in Chinese with an English abstract)

Li, G. Q., Huang, H. C., Laroche, A. and Achaya, S. N. (2003) Occurrence and characterization of the hypovirulence in the tan-sclerotial isolate of Sclerotinia sclerotiorum. Mycol. Res., 107, 1350-1360.

López-Berges, M.S., Di Pietro, A., Daboussi, M.-J., Abdel Wahab, H., Vasnier, C., Roncero, G., Dufresne, M. and Hera, C. (2009) Identification of Virulence Genes in Fusarium oxysporum f. sp. lycopersici by Large-Scale Transposon Tagging. Molecular Plant Pathology, 10, 95-107.

Nuss, D. L. (2005) Hypovirulence: Mycoviruses at the fungal-plant interface. Nature Rev. Microbiol., 3, 632-642. van Kan, J. A. L. (2006) Licensed to kill: The lifestyle of a necrotrophic plantpathogen. Trends Plant Sci., 11, 247-253.

Partners

Project Members

Project Leaders

• Prof. Dr. Hala Abdel Wahab