Touseef Hussain

A quantitative Real Time PCR based method for the detection of Phytophthora infestans causing Late blight of potato, in infested soil

A fast and simple polymerase chain reaction method has been developed for detection of Phytophthora infestans oospores, the causal agent of Late blight of Potato in soil. The method involves the disruption of oospores by grinding dry soil, using abrasive properties, in the presence of glass powder and skimmed milk powder within a short time. The latter prevents loss of DNA by adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with the DNA. After phenol/chloroform extraction; the DNA is suitable for direct PCR amplification without a precipitation step. This amplification leads to detection of pathogen in infested soils before planting of crop. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. infestans detection to confirm positive inoculum level in potato seeds and elsewhere. With increasing amounts of standard DNA templates, the respective threshold cycle (Ct) values were determined and a linear relationship was established between these Ct values and the logarithm of initial template amounts. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of P. infestans oospores on a large-scale basis.

A Quantative Real-Time PCR of Phytophthora infestans in different Indian potato cultivars

Reliable and sensitive quantification of Phytophthorainfestansin potato plant is of crucial importance in managing the multiple syndromes caused by this pathogen. A Real-Time polymerase chain reaction (PCR) assay was developed for the determination of P.infestans in host tissues. DNA levels of a highly virulent isolate were measured in different potato cultivars with varying degrees of resistance. Using SYBR Green and specific primers for P.infestans the minimal amount of pathogen DNA quantified was 0.0005ng/µl. Pathogen DNA was recorded at 24 hours postinoculation. Thereafter, the increase was rapid in susceptible cultivars and slower in resistant ones. The amount of pathogen DNA quantified in each potato cultivars correlated with susceptibility to late blight. Likewise, there was a relationship between the virulence of the pathogen and the degree of colonization. Differences also were found in pathogen amount among host tissues, with maximal pathogen biomass occurring in susceptible one. The real-time PCR technique developed in this study was sensitive and robust enough to assess both pathogen development and resistance to Late blight in different potato genotypes. Keywords: Phytophtohorainfestans, resistance, potato, cultivars, Real Time PCR

Molecular Diagnosis of Killer Pathogen of Potato: Phytophthora infestans and Its Management

Phytophthora diseases cause major losses to agricultural as well as to horticultural production in India and worldwide. Most Phytophthora diseases are soilborne except Late blight of Potato caused by Phytophthora infestans and difficult to control, making disease prevention an important component of many disease management strategies. Detection and identification of the causal agent, therefore, is an essential part of effective disease management. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. Enumerating the pathogen upon detection is crucial to estimate the potential risks with respect to diseases development and provides a useful basis for diseases management decisions. Species of the genus Phytophthora are arguably the most destructive plant pathogens causing widespread damage to many agricultural, horticultural and ornamental crops, and to native ecosystems throughout the world. Globalization has increased the volume of plants being transported over long distances and has increased the spread of Phytophthora species. The morphological characteristics of these structures and various kinds of spores produced by them have been the basis of identification up to genus/species level and classification of these pathogens into family, order and class. The Polymerase Chain Reaction (PCR) has emerged as a tool for detecting microorganisms in many diverse environments. Thus far, it is clear that DNA-based detection systems exhibit higher levels sensitivity than conventional techniques. The possibility of detecting two or more pathogens simultaneously has become bright after the development of DNA array technology. The usefulness of immunoassays for early detection and precise identification has been significantly enhanced following the development of Enzyme-Linked Immunosorbent Assay (ELISA) and monoclonal antibodies (Dipsticks) which exhibit greater sensitivity and specificity compared with isolation based methods which are laborious and time-consuming. Disease management methods based on cultural, chemical control, resistant varieties, Diseases forecasting are illustrated. New technologies promise to improve the speed and accuracy of disease diagnostics and pathogen detection. Widespread adoption of standard operating procedures and diagnostic laboratory accreditation serve to build trust and confidence among institutions. Case studies of national and international diagnostic networks are presented. This review examines the technical advances in the field and the rationale for such studies on Phytophthora.