Unlike most pathogenic organisms, viruses require a special approach to establish their presence in a diseased plant, since they are not visible, even with a light microscope. Due to the tremendous damage of crop plants caused by viruses various methods and techniques have been developed to enable the investigation of these pathogens. A variety of protocols for determining the properties and identity of a virus or its behaviour in an infected plant are described in this manual. For teaching purposes, a number of class exercises have been added. The protocols have been selected in such a way as to be useful for plant viruses in both tropical and non-tropical parts of the world.

Klappentext

Viruses require a special approach to establish their presence in a diseased plant since they are not visible, even under a light microscope. This manual describes in detail a variety of protocols for determining the properties and identity of a virus and its behavior in infected plants. A Springer Lab Manual.

Inhalt

Preface Abbreviations Part I Virus Inoculation Introduction I Virus Inoculation Protocol 1 Mechanical inoculation of plants Protocol 2 Inoculation of protoplasts isolated from leaves 2.1 Inoculation of cowpea protoplasts with cowpea mosaic Comovirus in PEG 2.2 Inoculation of barley protoplasts with brome mosaic Bromovirus-RNA by electroporation Protocol 3 Differential temperature treatment of infected plants Exercise1 Infectivity of tobacco mosaic Tobamovirus Part II Symptomatology Introduction II Symptomatology Protocol 4 Description of external symptoms - Cross-protection (premunity, interference) Protocol 5 Determination of internal symptoms 5.1 Staining with trypan blue 5.2 Staining with phloxine-methylene blue 5.3 Staining with methyl green-pyronin 5.4 Staining with Azure A 5.5 Staining with orange-green (O-G) Exercise 2 Systemic acquired resistance (SAR) Exercise 3 Demonstration of phloem necrosis in plants affected by potato leafroll Exercise 4 Inclusions in hair cells of leaves of tobacco infected with tobacco mosaic Tobamovirus Part III Infectivity assays Protocol 6 Infectivity assay on local-lesion hosts 6.1 Infectivity assay on detached leaves 6.2 Infectivity assay on non-detached leaves Protocol 7 Infectivity assay on hosts producing starch lesions Protocol 8 Infectivity assay on hosts producing systemic symptoms only Exercise 5 Effect of temperature on the susceptibility of plants Exercise 6 Effect of abrasives on the infection Exercise 7 Effect of inhibitors on the infection Exercise 8 Effect of light on the susceptibility of plants Exercise 9 Effect of time of the day on the susceptibility of plants Part IV Infectivity of virus in crude sap Introduction IV Infectivity of virus in crude sap Protocol 9 Determination of the dilution end-point Protocol 10 Determination of the thermal inactivation point Protocol 11 Determination of the longevity in vitro Exercise 10 Determination of the dilution end-point Exercise 11 Determination of the thermal inactivation point Exercise 12 Determination of the longevity in vitro Part V Transmission of viruses Introduction V Transmission of viruses Protocol 12 Virus transmission by grafting 12.1 Bud grafting 12.2 Wedge grafting 12.3 Tuber grafting Protocol 13 Virus transmission through soil 13.1 Testing of roots for the presence of virus 13.2 Use of bait plants to reveal the presence of viruliferous vectors Protocol 14 Virus transmission by nematodes Protocol 15 Virus transmission by fungi Protocol 16 Virus transmission by aphids 16.1 Non-persistent transmission 16.2 Persistent transmission of a circulative virus 16.3 Acquisition of a circulative virus by aphids feeding on artificial diets Protocol 17 Transmission by leafhoppers (Cicadellidae) 17.1 Transmission of semi-persistent viruses 17.2 Non-transovarial transmission of a circulative-propagative virus 17.3 Transovarial transmission of a circulative-propagative virus 17.4 Glasshouse mass screening for resistance to rice tungro disease Protocol 18 Transmission by whiteflies (Adeyrodidae) Protocol 19 Transmission by thrips (Thripidae) Exercise 13 Wedge-grafting of tobacco Exercise 14 Separation of a non-persistently and a persistently transmitted virus by differential transmission Exercise 15 Visualisation of salivary sheaths formed by aphids Exercise 16 Separation of a semi-persistently transmitted and acirculative-propagative virus by differential transmission Part VI Maintenance of virus isolates Introduction VI Maintenance of virus isolates Protocol 20 Freezing Protocol 21 Dehydration Protocol 22 Freeze-drying Protocol 23 Short-term storage Part VII Production of virus-free material from infected plants Introduction VII Production of virus-free material from infected plants Protocol 24 Heat treatment Protocol 25 Meristem-tip culture Protocol 26 Heat treatment combined with meristem-tip culture Part VIII Virus isolation and purification Introduction VIII Virus isolation and purification Protocol 27 Density-gradient centrifugat