Detecting & Quantifying Viruliferous vs. Non-Viruliferous Polymyxa betae | InformativeBD

Fatemeh Hassanzadeh Davarani, Saeed Rezaee,  Seyed Bagher Mahmoudi, Peyman Norouzi, and Mohammad Reza Safarnejad, from the institute of Iran. wrote a Research article about, Detecting & Quantifying Viruliferous vs. Non-Viruliferous Polymyxa betae. Entitled, Identification and quantification of viruliferous and non- viruliferous Polymyxa betae. This research paper published by the International Journal of Biosciences | IJB. an open access scholarly research journal Biosciences. under the affiliation of the International Network For Natural Sciences| INNSpub. an open access multidisciplinary research journal publisher.

Abstract

Rhizomania, caused by Beet Necrotic Yellow Vein Virus (BNYVV) is transmitted by plasmodiophorid Polymyxa betae. To investigate quantification of virulifeous and non- viruliferous P. betae isolates, different techniques including serological method (DAS- ELISA), PCR- based method and nanobiocensor method have been used. For this purpose, sugar beet susceptible cultivar (Regina) was cultivated in soils of different regions in greenhouse conditions. Six weeks after planting, lateral roots of beets from each soil were visually tested through microscopy and the of P. betae cystosori was seen and the lateral root sap was prepared. Then DAS- ELISA with polyclonal antibody against recombinant expressed fungal glutathione-s- transferase isolates of Shiraz was optimized. Optical density of different samples were calculated for both the vector and the virus using ELISA method. Simultaneously, confirmation of quantitative estimation P. betae in lateral root was conducted by nanobiosensor against vector. Nanobiosensor method was performed based on Florescent Resonance Transfer Energy (FRET) using antibody attached quantom dots and GST conjugated rhodamine. Microscopic results show presence of vector in all soils. BNYVV was found in soils Fars, Khorasan, Hamadan and Kermanshah. In soils of Azarbayjan, Gorgan, Dezfool, Kerman, Karaj and Arak were found no virus. Values of optical density of P. betae in soils with and without virus have no significantly difference. Because of high speed and sensitivity of nanobiosensor, its use for quantitative estimation of P. betae has been advised.

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Introduction

The protist Polymyxa betae Keskin is an obligate parasite of sugarbeet roots and the plasmodiophorid vector of Beet Necrotic Yellow Vein Virus (BNYVV), which causes rhizomania disease. P. betae is found in almost all soils where sugarbeet is grown, spreading from plant to plant by means of motile zoospores and survive in the soil for many years in the form of thicked-wall resting spores or cystosori (Rush, 2003). Despite its ubiquitous distribution and parasitic habitat, P. betae is generally considered to cause relatively little damage in temperate climates, although it may be pathogenic in areas of the world where sugarbeet is grown in warm soils (Blunt et al., 1991). In contrast, rhizomania disease causes severe economic losses in many countries and is spreading into new regions (McGrann et al, 2009). In Iran, it was reported from the Fars province in 1996 and is now found in nearly all sugarbeet-growing areas of the country (Izadpanah et al., 1996; Sohi and Maleki 2004). P. betae, the sole vector of BNYVV, has attracted increasing attention in recent years in Iran, because its distribution and behavior determine the incidence and severity of the disease. However, because it is an obligate parasite, epidemiological studies, and the search for potential sources of host resistance to P. betae, have required bioassays procedures, the evaluation of which can only be achieved by lengthy and laborious microscopic examination of roots (Mutassa-Gottgens et al., 2000 ).

Traditional methods to detect and quantify vector and virus in soil are based on bait plant bioassays using soil dilutions to estimate the most probable numbers (MPN) of infective propagules (Tuitert, 1990). These methods are expensive and time-consuming, taking more than 8 weeks to complete for a single soil sample. There was a need to develop a rapid, accurate and specific detection and quantification method for the P. betae in roots. DNA-based tests were developed which were able to identify the presence or absence of P. betae within the plan, but unable to quantify the relative amounts of the pathogen. Another limitation of DNA-based tests is that they cannot determine if the parasite is alive or dead (Kingsnorth et al., 2000). Serological tests that recognize proteins, which can be less stable than DNA, may also be able to distinguish between viable and nonviable cells. Using ELISA as a detection method has the main advantage that amounts of protein can be quantified. Also, it is relatively quick and easy, without the need for expensive laboratory equipment, and it can be automated for rapid on-line testing. Polyclonal antibodies have been used in ELISA tests for Spongospora subterranea (Merz et al., 2005), P. betae (Mutassa-Gottgens et al., 2000 and Kingsnorth et al., 2003a), Polymyxa. graminis (Delfosse et al., 2000) and Plasmodiophora brassica (Wakeham and White 1996). All authors reported a (semi-) quantitative detection of resting spores in plant material and soil samples.

Glutathione-S-transferase (GST), a specific immunogenic protein, is a critical enzyme expressed in P. betae`s zoospores, sporangia and resting spores and could be regarded as a good candidate for the development of the biobase of antibody and nanobiosensor. In fact, the pathogen expresses GST at high levels to overcome host defense mechanisms (Mutasa et al., 2000). Antibody to P. betae has been developed in Iran recently (Safarpour et al., 2012a) and is widely available for quantitative detection of it. One of the most important nanomaterials is fluorescent semiconductor nanocrystals, also known as quantum dots (QDs) which have been widely used for disease diagnosis (Frasco and Chaniotakis, 2009). QDs have a number of unique optical properties that are advantageous in the development of bio-analyses based on fluorescence resonance energy transfer (FRET) (Algar and Krull, 2007). QDs have been reportedly used as biosensors by coating them with specific antibodies against various pathogenic agents such as E. coli O157:H7 (Hahn et al., 2008). Moreover, a quantum dots FRET-based nanobiosensor for efficient detection of P. betae was developed in Iran (Safarpour et al., 2012b). The purpose of this study was to identify and quantify viruliferous and nonviruliferous P. betae isolates in different sugarbeet cultivation of Iran firstly using serological and nanobiosensore methods that recently were developed in Iran and PCR- based method.

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Article source : Identification and quantification of viruliferous and non- viruliferous Polymyxa betae