Diagnosis Of Plant Virus Diseases Research Articles

Rapid detection of Potato leafroll virus and Potato virus Y by reverse transcription loop-mediated isothermal amplification method in north-east India

Potato leafroll virus (PLRV) and Potato virus Y (PVY) are two important viruses causing serious potato yield losses in the North-east region and other planting areas in India. As a consequence, it is urgent to develop an efficient and quick method for the identification and diagnosis in the field. The results presented here showed that the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was efficient and sensitive than reverse transcription-polymerase chain reaction (RT-PCR) for the detection of PLRV and PVY. The RT-LAMP primers specifically targeted PLRV and PVY (including PVYO, PVYN, and PVYNTN strains) and resulted in typical sigmoidal amplification curves. Ten-fold serial dilutions of PLRV and PVY total RNA indicated that RT-LAMP is faster and at least a hundred times more sensitive than RT-PCR in detecting both the viruses. Additionally, samples that RT-PCR could not detect at a diluted concentration of 10−3 and 10-4 ng/μl were identified by RT-LAMP. Thus, RT-LAMP offers many advantages over RT-PCR such as low cost and high accuracy, sensitivity, and specificity for the rapid diagnosis of plant virus diseases. In conclusion, the results highlighted the efficacy of the RT-LAMP method in quickly detecting PLRV and PVY in infected plants.

Reverse transcription loop-mediated isothermal amplification to rapidly detect Rice ragged stunt virus.

Rice ragged stunt virus (RRSV) is a very important virus that infects rice and causes serious yield losses in Asian countries and other major rice planting areas. Thus, it is urgent to establish an efficient and practical approach for identification and diagnosis in the field. Our results indicated that reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions are more efficient and sensitive than RT-PCR for RRSV detection. The optimal LAMP conditions were as follows: 0.4–1.2 μM internal primers, 0.2–0.25 μM external primers, 0.8 μM loop primers, and incubation at 62 °C or 63 °C for 30 min. Furthermore, the RT-LAMP primers specifically targeted RRSV virus and resulted in typical waterfall-like bands by gel electrophoresis and sigmoidal amplification curves. The primers could not be used to amplify other common plant viruses including Papaya ringspot virus (PRSV), Rice yellow stunt virus (RYSV), Sorghum mosaic virus (SrMV), Cactus virus X (CVX), Melon yellow spot virus (MYSV) and Southern rice black-streaked dwarf virus (SRBSDV). Ten-fold serial dilutions of RRSV cDNA indicated that RT-LAMP is much faster and at least ten times more sensitive than RT-PCR in detecting the virus. The waterfall-like product bands could be observed within one hour. In the field study, about 77% samples were identified as RRSV. RT-LAMP has many benefits over RT-PCR such as low cost and high accuracy, sensitivity, and specificity. This technology meets the requirements for rapid diagnosis of plant virus diseases in the field to best guide management practices for growers.

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by TEM

A rapid and unambiguous diagnosis of plant virus diseases is of great importance for agriculture. Investigations of ultrastructural changes induced by viruses within plants by using transmission electron microscopy (TEM) are often necessary to clearly identify the viral agent. Nevertheless, with conventional methods such investigations can take several days and are not suited for the rapid diagnosis of plant virus diseases [1]. Microwave assisted plant sample preparation can help to drastically reduce sample preparation time for TEM investigations with similar or even better ultrastructural results as observed after sample preparation with conventional methods [1,2]. This massive reduction of sample preparation time can be attributed to dielectric heating induced by microwave irradiation which causes a temperature rise inside the whole sample whereas conventional heating starts at the specimen surface. The increase in temperature can then enhance and accelerate the diffusion of reagents, protein crosslinking and the polymerization of the resin [3,4].

Rapid immunohistochemical diagnosis of tobacco mosaic virus disease by microwave-assisted plant sample preparation.

Immunoelectron microscopy is a powerful method to diagnose viral diseases and to study the distribution of the viral agent within plant cells and tissues. Nevertheless, current protocols for the immunological detection of viral diseases with transmission electron microscopy (TEM) in plants take between 3 and 6 days and are therefore not suited for rapid diagnosis of virus diseases in plants. In this study, we describe a method that allows rapid cytohistochemical detection of tobacco mosaic virus (TMV) in leaves of tobacco plants.With the help of microwave irradiation, sample preparation of the leaves was reduced to 90 min. After sample sectioning, virus particles were stained on the sections by immunogold labelling of the viral coat protein, which took 100 min. After investigation with the TEM, a clear visualization of TMV in tobacco cells was achieved altogether in about half a day. Comparison of gold particle density by image analysis revealed that samples prepared with the help of microwave irradiation yielded significantly higher gold particle density as samples prepared conventionally at room temperature.This study clearly demonstrates that microwave-assisted plant sample preparation in combination with cytohistochemical localization of viral coat protein is well suited for rapid diagnosis of plant virus diseases in altogether about half a day by TEM.

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

Microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

2009년 우리나라 주요 작물 바이러스병 발생 상황

Among the plant specimens requested from agricultural actual places of farmers, Agency of agricultural extension services and so forth for the diagnosis of plant virus diseases in 2009, the rate of crop types was 87.5% for vegetables, 4.0% for upland crops and 3.5% for orchids. In vegetables, the crops damaged severely by viral diseases were red pepper and tomato by the infection rate of 51.6% and 26.5%, orderly. Virus species occurring vegetables were 19 and the economically important viruses were Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV), Pepper mild mottle virus (PMMoV) with the infection rate of 33.2%, 16.9%, 16.1% and 7.4%, respectively. Rice stripe virus (RSV) occurred at the whole areas of west coast in Korea in 2009, and its incidence was 14.2% mainly on the susceptible cultivars and yield loss was estimated up to 50%. TYLCV was spread at 34 areas of Si and/or Gun, 22 areas in 2009 and 12 in 2008. Distribution of TSWV was expanded newly in 6 areas of Si and/or Gun including Gangryung, Gangwondo in 2009, and its occurrence areas were 23 Si and/or Gun after first incidence at Anyang area in 2004. Tomato bushy stunt virus (TBSV) was incited newly at Gimcheon area in 2009 with the infection rate of 65.2%, and its soil transmission rate was 55.0% in average.

Rapid diagnosis of plant virus diseases by transmission electron microscopy

A clear and rapid diagnosis of plant virus diseases is of great importance for agriculture and scientific experiments in plant phytopathology. Even though negative staining and transmission electron microscopy (TEM) are often used for detection and identification of viral particles and provide rapid and reliable results, it is necessary to examine ultrastructural changes induced by viruses for clear identification of the disease. With conventional sample preparation for TEM it can take several days to obtain ultrastructural results and it is therefore not suitable for rapid diagnosis of virus diseases of plants. The use of microwave irradiation can reduce the time for sample preparation for TEM investigations. Two model virus–plant systems [ Nicotiana tabacum plants infected with Tobacco mosaic virus (TMV), Cucurbita pepo plants infected with Zucchini yellow mosaic virus (ZYMV)] demonstrate that it is possible to diagnose ultrastructural alterations induced by viruses in less than half a day by using microwave irradiation for preparation of samples. Negative staining of the sap of plants infected with TMV and ZYMV and the examination of ultrastructure and size were also carried out during sample preparation thus permitting diagnosis of the viral agent by TEM in a few hours. These methods will contribute towards a rapid and clear identification of virus diseases of plants and will be useful for diagnostic purposes in agriculture and in plant phytopathology.

Testing Methods For Seed‐transmitted Viruses: Principles and Protocols ‐ S. E. Albrechtsen

Testing Methods For Seed-transmitted Viruses: Principles and Protocols. S. E. Albrechtsen. 288 pp. Wallingford, UK: CABI Publishing (http://www.cabi-publishing.org), 2005. £55. ISBN 0 85 199016 9 (spiralbound softback). I was recently asked to recommend some key plant virology texts for a new diagnostic laboratory. Good reference material is easy: buy the latest edition of Matthews’ Plant Virology (Hull, 2002) and then go online for VIDE (http://image.fs.uidaho.edu/vide/refs.htm) and DPVweb (http://www.dpvweb.net). There are also some excellent crop-specific texts available, e.g. Viruses and Virus Diseases of Poaceae (Lapierre & Signoret, eds, 2004). But what about practical guides? Well, then it gets more tricky: Methods in Plant Virology (Hill, 1984) is long out of press. There is Diagnosis of Plant Virus Diseases (Matthews, 1993), which is excellent on the principles but is somewhat dated, e.g. PCR only gets one page. The more recent and soon to be revised Plant Virology Protocols (Foster & Taylor, 1998) is much better for molecular methods, but is aimed more at research and development rather than diagnostic requirements. Beyond that there is little else; that is, I believe until now, with the publication of this new book. As far as I am aware, this is the first book to be dedicated to seed virus-testing, but it goes well beyond that and actually provides one of the best single-source, practical guides to plant virus diagnostic methods available. Split into seven chapters, the main bulk of the book is dedicated to the three methods chapters: biological assays, serological tests and nucleic acid-based tests. All provide excellent coverage of their respective fields, with good, established protocols. I particularly like the fact that there are plenty of options given, e.g. a table of different recipes for one-step RT-PCR. I also appreciated that support techniques such as isolate storage are fully covered. As anyone who has run a routine ELISA laboratory will know, maintaining a supply of positive control material is a major problem. All laboratories need good advice and protocols on this, and this book provides it. And good advice on troubleshooting is another strong point, as is the fact that the latest techniques, such as real-time PCR methods, are covered. I guess one disappointment is that the author did not go as far as including any real-time protocols, in contrast to the comprehensive coverage given for conventional PCR. While real-time PCR is still a relatively new technique, it has become a routine technique in some laboratories and is rapidly establishing itself. Similarly electron microscopy does not really feature. However in fairness, EM is not a great tool for seed testing, which has very much become a niche technique used routinely by very few. The other good thing about this book is that it really does do what it says in the title; covering both principles and protocols. It is often easy for books to become either laboratory manuals with precious little background or reference texts without practical use. However, this is a book that is equally at home in the laboratory and the library. It has excellent review chapters covering the background to seedborne viruses, providing information on a range of subjects including the mechanism of seed transmission, control and economic importance – all good stuff. It also has an extremely useful section (co-written by Steve Roberts) on sampling and testing strategies, arguably the most important step in any seed test. The quick reference list of seedborne viruses is another helpful addition. Overall, this is an excellent guide to seed testing and virological diagnostic methods in general. It is a very welcome addition to the literature and a book that should have a place on the shelf of every diagnostic plant virology laboratory, whether or not they are involved in seed testing.

Book Reviews

Books reviewed in this article: Thorpe, T. A. (ed.): In Vitro Embryogenesis in Plants. Matthews, R. E. F. (ed.): Diagnosis of Plant Virus Diseases. Van Oijen, J. W. and J. Jansen (eds): Biometrics in Plant Breeding: Application of Molecular Markers. Grout, B. (ed.): Genetic Preservation of Plant Cells in Vitro.

Search mode for rapid detection of virus particles

Electron microscopy is frequently used in preliminary diagnosis of plant virus diseases by surveying negatively stained preparations of crude extracts of leaf samples. A major limitation of this method is the time required to survey grids when the concentration of virus particles (VPs) is low. A rapid survey of grids for VPs is reported here; the method employs a low magnification, out-of-focus Search Mode similar to that used for low dose electron microscopy of radiation sensitive specimens. A higher magnification, in-focus Confirm Mode is used to photograph or confirm the detection of VPs. Setting up the Search Mode by obtaining an out-of-focus image of the specimen in diffraction (K. H. Downing and W. Chiu, private communications) and pre-aligning the image in Search Mode with the image in Confirm Mode facilitates rapid switching between Modes.

Accessions of Australian Nicotiana species suitable as indicator hosts in the diagnosis of plant virus diseases

When screening the genusNicotiana for sensitive and differential hosts for a group of mechanically transmissible plant viruses with narrow host ranges, development of systemic symptoms was alsmost exclusively observed in species of three closely related sections of the subgenusPetunioides. These species wereN. miersii (sectionAcuminatae),N. bigelovii andN. clevelandii (Bigelovianae) andN. benthamiana, N. cavicola, N. ingulba, N. occidentalis, N. rosulata andN. rotundifolia (Suaveolentes). Except forN. benthamiana andN. clevelandii, which are already known for their large virus ranges, they are new experimental hosts that appeared very useful for detection of viruses and for differentiation of viruses that closely resemble each other in host range. Accessions of the same species often varied largely in local and systemic viral response. EspeciallyN. benthamiana-9,N. miersii-33 andN. occidentalis-37B (code numbers given by Tobacco Research Laboratory, Oxford, N.C., USA) are recommended for routine inoculation tests. The sensitiveSuaveolentes species mentioned are native to the arid parts of Australia. Collections of these species deserve attention in studies on virus diseases of unknown etiology where experimental hosts are lacking.

Problems and Prospects in Plant Virus Identification1

AbstractReliable identification is a prerequisite to the study and control of plant viruses and virus diseases. Viruses are now increasingly studied for their intrinsic properties, in addition to their variable biological properties. This has caused some degree of order to emerge from the intricacy of plant viruses. Ten « commandments » are listed which permit reasonably reliable diagnosis of plant virus diseases in the scientific laboratory. New viruses are still often discovered.Routine tests for virus infection, carried out by plant protection, quarantine and especially certification services, never guarantee absolute freedom from viruses. A simple, but the least reliable of indexing methods, is that of visual observation. Infectivity tests are meant to trace the infectious agents with indicator plants. Using serology, one can detect certain viruses more directly and specifically. Electron microscopy allows the observation of the virus particles themselves.The choice of test is always a compromise between efficiency and reliability. Through scientific research both are constantly being improved. With the increasing amount of information on plant viruses, diagnosis is tending to shift from mere visual observation to the more reliable study of aetiology.

Chemical tests in the diagnosis of plant virus diseases

Viruses occupy a unique position in the biological spectrum, for they are biological entities only when combined with a susceptible host. Purified virus preparations are claimed by the chemist. All plant viruses that have so far been purified have been found to be nucleoproteins, and are indistinguishable in gross chemical characteristics from naturally occurring plant nucleoproteins. From the chemical viewpoint, therefore, viruses are just one of a group of nucleoproteins which are found in all living organisms. However, virus particles are replicated only in susceptible hosts; therefore, from the biological viewpoint, viruses are obligate pathogens with one dimension less than 200 mt ( 11). Viruses are recognized because they produce reproducible syndromes in susceptible hosts, and it is the host-virus interaction that is recognized. Some plants are symptomless carriers of certain viruses, which fact