Fragen Plant Virology

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1. Define the features of a plant virus

• viruses are obligate biotrophic parasites

• don‘t possess energy producing metabolism.

• Only replicate in living cells

• one or several molecules of encapsidated nucleic acid (RNA or DNA)

• protecting envelope consisting of one or more proteins and in some cases a lipoprotein-coat

2. Who are the founders of plant virology?

• 1714 Lawrence first exp. transmission of a virus from jasmine by grafting,

• 1886 Mayer mosaic disease in tobacco caused by TMV and first mech. Transmission

3. Are Viruses alive?

• they are not cellular and do not metabolise;

• they rely on their host cell metabolism

BUT: they reproduce and adapt

But that is not enough to fulfill the most accepted definition of living organsim

• thus, technically they are not living organisms.

4. Explain the experimental procedure to fulfill the Koch´s postulates of a plant virus.

5. Name the structures of representative plant viruses.

• Flexous thread-like virus

• Rigid rod-shaped virus

• Short bacillus-like virus

• Isometric polyhedral virus

• Geminivirus consisting of twin particles

6. Name the primary classifiers for virus systematic classification

• Structure of genome (DNA or RNA)

• Symmetry of capsid

• Existence of envelope coat protein (lipid layer)

• Size of virion and capsid

• Replication strategy

• Organization of genes in the genome

• Host(s)

• Vector

• Structure

7. Give an overview of the viral infection-cycle and systemic movement of an RNA virus.

1. Virus taken in by wounded cell

2. Viral nucleic acid freed from coat protein

3. Replicates in cell some move do adjacent cells through plasmodesmata

4. Viral nucleic acid or virus is carried through plant in phloem

5. Viral nucleic acid or virus reaches phloem vessel

6. Multiplies in new cells and spreads in adjacent cells

7. Some of early formed nucleic acid is coated with protein and forms virus

8. Give a definition of a viroid including composition and structure.

• Viroids are pathogenic RNAs causing virus-like diseases in plants

• Viroids are small circular molecules,

o a few hundred nucleotides long,

o high degree of secondary structure

• Viroids do not code for any polypeptides and replicate independently of any associated plant

virus

• Viroids are the smallest known self-replicating genetic units

9. Name methods for analysis and detection of viral nucleic acids.

• PCR

• RT-PCR

• Nucleic Acid Sequence Based Amplification (NASBA)

• Rolling Circle Amplification (RCA)

• Padlock probe detection,

• Southern-Blot

• Northern-Blot

• Loop mediated isothermal amplification (LAMP),

10. Describe some local and some systemic symptoms induced by plant viruses

• Local symptoms:

o Local lesions:

▪ Chlorotic lesions

▪ Necrotic lesions

▪ Ring spot lesions

▪ Starch lesions

• Systemic symptoms:

o Chlorosis

o Influence on plant height/growth reduction

o Mosaic pattern

o Mottle

o Yellowing diseases

o Deformations

o Leaf rolling/ curling

o Etc.

11. Name methods for analysis of viral proteins.

• TPIA (Tissue Print Immunosorbent Assay)

• Lateral flow technique

• detection of antigen antibody combinations

• double diffusion test

• ELISA

• Serological tests

12. Name diagnostic procedures for the detection of plant viruses

• Test plants

• Detection of viral proteins

• Electron microscopy

• Detection of viral nucleic acids

13. What are typical host functions used by plant viruses to complete the infection cycle

• Components amino acids, nucleotides

• Energy Polymerisation by ATP

• Biosynthesis of proteins Cytoplasmatic Ribosomes, tRNAs, Enzymes,

• Synthesis of nucleic acids Replication complex, viral RdRp plus Host factors/

RNA-binding proteins/Elongation factors

• DNA-Viruses DdRP for transcription in the nucleus

• Membranes: ER, Chloroplastes, Peroxisomes, Nuclear

membranes

14. What are satellites and satellite viruses?

• Satellites

o subviral agents that lack the genes encoding functions that are necessary for their

replication

o depend for their multiplication on coinfection of a host cell with a helper virus

o The genomes of satellites differ substantially or totally from those of their helper

virus.

▪ In satellite viruses, the satellite nucleic acid codes for its own coat protein.

15. What are phytoplasmas?

• specialised bacteria that are obligate parasites of plant phloem tissue and transmitting

insects (vectors)

• cannot be cultured in vitro in cell-free media

• lack a cell wall, a pleiomorphic or filamentous shape, normally with a diameter less than 1

μm,

• very small genomes.

• a few of them have a helical structure and are called spiroplasmas.

• Most are round to elongate but are not spiral and are called phytoplasmas.

16. Choose one Top ten plant virus from the lecture and give arguments for the assignment to this

list (biology, host range, vector transmission etc.)

TSWV (Tomato spotted wilt virus): (Optional auch hier CTV oder Plum Pox Virus die weiter unten

nochmal gefragt werden)

• More than 800 host species

• Significant crop losses resulting from infection 1 billion dollar loss/year

• Complex virion structure

• Transmitted by Thrips, transstadially

• Control of Thrips difficult

17. Describe the details of the infection and replication cycle of Tomato yellow leaf curl virus

(TYLCV)

• transmitted by whitefly (Bemesia tabaci) in a circulative manner, and transmitted by the

biotype B (or silverleaf) whitefly (Bemesia argentifolii) or grafting, not seed transmitted

• High transmission efficiency

o A single whitefly is able to inoculate a plant following a 15-min acquisition

period and a 15-min inoculation period.

• In the field, inoculation can occur immediately after transplantation.

• Influences several features of insect pathogenes:

o TYLVC is propagative (affects B. tabaci by replication in it and influences the

longevity and fertility of B. tabacci)

18. Describe the details of the infection and replication cycle of Banana bunchy top virus (BBTV)

• BBTV is the sole member of the genus Babuvirus in the family Nanoviridae

• The Banana aphid (Pentalonia nigronervosa) transmits the virus from infected to healthy

plants

• The Aphid feeds on the plant phloem tissue and acquires the virus after at least four (but

usually about 18) hours; it transmits the virus by feeding 15 min on plant tissue

• The aphid can retain the virus through its adult life 15 – 20 days

• Vector transmission is circulative and non-propagative

• Virus does not replicate within the aphids’ midgut

• Symptom appearance after about one month (30 days)

Replication:

1. penetrate host cell

2. Uncoating viral ssDNA->penetrates into nucleus

3. Converted to dsDNA

4. .production of viral mRNA and translation of viral proteins

5. Replication +strand by REP, roling cycle produces ssDNA genoms

19. Explain the control mechanism of cross protection and give an example

• Viral cross protection is known as an acquired immunity phenomen in plants

• a mild virus isolate/strain can protect plants against economic damage caused by a

severe challenge strain/isolate of the same virus.

• Example Citrus tristeza virus (CTV)

20. Name representative virus diseases that are mechanically transmitted and of economic

importance.

• CSSV Cocoa swollen shoot virus

• BSV Banana streak virus

• CaMV Cauliflower mosaic virus

• PVY Potato virus Y

• PVX Potato virus X

• PVM Potato virus M

• BtMV Beet mosaic Virus

• Etc.

21. Describe advantages and disadvantages of the test plant diagnosis

• Advantages:

1. Low investment (when greenhouse is available)

2. Techically not very demanding

3. Reliable

4. Relation of symptom expression and detection

5. Easily repeatable

• Disadvantages:

1. Results available not earlier than after one week

2. Vector must be known or mechanical inoculation possible

3. Only if symptoms are expressed, the virus can be identified

4. Space requirement in the greenhouse

5. Experience in symptom evaluation is required

6. Propagation of viruses with risk of virus spread

22. Describe the procedures during me chanical virus transmission

• Produce infected sap from young diseased leaves

• Dust abrasive powder on leaves of healthy plant

• Infected sap rubbed on healthy plant with gauze pad, hand or brush

• Inoculated plants must in some cases be rinsed of with water immediatly

• Inoculated plants kept in greenhouse or growth chamber

• Symptoms develop in 2 to 21 days

23. Explain the major steps of antibody production

1. Purified antigen (virus, bacteria, etc.) with or without adjuvant is taken up in syringe

2. Antigen is injected once or more in muscle or vein of animal

3. Lymphocytes (B-cells) produce antibodies

4. Several weeks or month later blood is obtained from ear or heart of injected animal

5. Blood is allowed to clot overnight

6. Clotted blood is centrifuged at 5000 rpm for 10 min

7. Clear antiserum (supernatant) is separated from blood cells (pellet)

8. Pellet is discarded

9. The antiserum (serum + antibodies) can be stored on ice when mixed with glycerin

24. Explain Tissue Print Immunosorbent Assays (TPIA) and name typical virus species useful to be

detected with this method

• The stem of a virus-infected plant is cut longitudinal

• The cut surface is pressed onto a specific membrane

• The plant sap leaves a tissue print on the membrane and the virus proteins contained by

plant tissue bind strongly to the membrane

• Membrane is brought to a solution with virus-specific antibodies coupled to a special enzyme

• Virus proteins on the membrane react with specific antibodies

• Membrane is transferred to a staining solution

• The special enzymes in this staining solution make the virus proteins visible

• Typical viruses are: BaYMV, BaMMV

25. Name molecules that are used for antibody coupling and their application in diagnosis

• Alkaline phosphatase

• Peroxidase

• Fluorescein

• Gold labelling

• gold labelling is used for the detection with TEM

26. Explain the principle of Transmission electron microscopy (TEM) detection of plant virus virions

• A beam of electrons is transmitted through an ultra-thin specimen

• The beam interacts with the specimen as it passes through

• An image (negative contrast) is formed from the interaction of the electrons transmitted

through the specimen

• The image is magnified and focused onto an imaging device (e.g. fluorescent screen or a

CCD camera)

27. Describe the principle of the Lateral-Flow technique

• The sample is placed on the sample pad of a diagnostic dipstick

• The sample pad contributes labelled specific antibodies

• After addition of a fluid the fluid+antibodies migrates to a conjugate pad through

capiallarity

• It gets in contact with the conjugate pad where the fluid solves the conjugate

• A reaction between the conjugate and the antigen is possible

• The fluid continuous migrating to the capture pad where molecules (one stripe) are

immobilized

• The transported molecules enrich at this stripe and can be detected by staining,

fluorescence staining or magnetic marking

• A second control line is a negative control without analyte

28. Explain Rolling circle amplification (RCA)

• RCA is an isothermal nucleic acid amplification technique where the polymerase

continuously adds single nucleotides to a primer annealed to a circular template which

results in a long concatemer ssDNA that contains tens to hundred of tandem repeats

1. Circular template ligation

2. Primer induced single-strand DNA (ssDNA) elongation

3. Amplification product detection and visualization

29. Why is dsRNA a preferred molecule for purification to detect plant viruses?

• dsRNA is stable, can be extracted easily from the majority of plant species

• no knowledge of the possible identity of the virus is required

• no antibodies, primers or probes are required

• field samples can be analyzed the day they arrive in the lab

• the number size and intensity of dsRNA segments can be determined by gel

electrophoresis within 48 hours

30. Explain briefly how Nucleic acid sequence-based amplification (NASBA) works.

1. RNA template is given to the reaction mixture, the first primer attaches to its

complementary site (3’-end of the template)

2. Reverse transcriptase synthesizes the complementary DNA strand moving upstream

along the RNA template

3. RNAse H destroys the RNA template from the DNA-RNA compound

4. The second primer attaches to the 5’-end of the DNA strand

5. Reverse transcriptase again synthesizes another DNA strand from the attached primer

resulting in double stranded DNA

6. T7 RNA polymerase binds to the promoter region on the double strand

o T7 RNA polymerase can only transcribe in the 3’ to 5’ direction the sense DNA

is transcribed and an anti-sense RNA is produced

o This is repeated, and the polymerase continuously produces complementary

RNA strands of this template which results in amplification

31. Name as many different modes/ways of plant virus plant to plant movement

• Transmission via Plant material (propagation, mechanically through sap, natural root

grafts and dodder)

• Transmission by invertebrates (insects, mites, nematodes)

• Fungal transmission of viruses

• Transmission by seed, pollen, direct contact, handling

32. What are the mechanisms behind non-persistent virus transmission?

• capsid and helper strategy

• capsid strategy: virion binding within the insect mouthparts is mediated by determinants

that are found within the coat protein (example CM)

• Helper strategy: binding is mediated by nonvirion protein that creates a “molecular

bridge” between virus and vector.

33. Compare the mechanisms of non-persistent, semi-persistent and persistent virus transmission

• Acquisition access period (AAP):The period of time given for the vector to acquire the virus

• Inoculation access period (IAP):The period of time given for the vector to transmit the virus

• Retention: The length of time after virus acquisition that the vector remains viruliferous

Nonpersistent transmission: A mode of transmission that is accomplished by acquisition

access of seconds to a few minutes and retention by the vector for no more than a few

minutes

• Semipersistent transmission: A mode of transmission requiring minutes to several hours for

acquisition access and having a retention time of several hours to a few days

34. Give an example for the helper strategy for aphid transmission.

• potyviruses

• (CaMV?)

35. Describe the routes of a plant virus during circulative insect transmission.

Circulative non-propagative:

Salivary canal food canal anterior midgut posterior midgut hindgut haemolymph

accessory salivary glands salivary pump

Circulative propagative:

Salivary canal food canal anterior midgut principal salivary glands salivary pump

36. Describe the different possible modes of fungal transmission and give one example for each.

• In-vitro transmission = transmission on surface (Olpidium brassicae)

o Infection of zoospores from soil-water and are thought to enter the zoospore

cytoplasm when the flagellum is “reeled in“.

o TNV Tobacco necrosis virus

• In-vivo transmission = transmission inside (Polymyxa betae)

o The virus is already in the zoospores, when released from veg. sporangia.

o The new host is colonized with zoospore infection

o BNYVV Beet necrotic yellow vein virus

37. Name typical control strategies to reduce losses by plant viruses and give an example for each

measurement.

• seed act (Saatgutverkehrsgesetz) (MDMV Maize dwarf mosaic virus)

• production of certified propagation material (LChV Little Cherry Virus)

• virus diagnosis

• agronomical measures and vector control (MDMV)

• breeding for resistance (MDMV)

• (Remove infected trees (LChV)) Citrus Tristeza virus (CTV)

38. Describe the virion structure and components of Tospoviruses.

• Virions of Tospoviruses like TSWV are complex compared to many plant viruses.

• There are three RNAs in the virus genome that are individually encapsidated, and are

collectively bound by a membrane envelope that is of host origin

• This complex virion structure is a characteristic that distinguishes TSWV from most other

plant viruses.

• TSWV virions are roughly spherical

• Contain glycoproteins in the envelope and several copies of the replicase protein

39. Give a short overview of the disease rice tungro.

• Tungro is the most important virus disease of rice in the Asian tropics with high economical

importance

• Composite disease caused by RTBV (Rice tungro bacilliform virus) and RTSV (Rice tungro

spherical virus)

• RTBV is a member of the genus Tungrovirus family: Caulimoviridae; has circular dsDNA

• RTSV is a member of the genus Waikavirus family Secoviridae; has ssRNA

• Symptoms when appearing together:

o Stunting

o Yellow/orange discoloration discolored leaves show irregularly shaped dark-brown

blotches

o Reduced tillering

o Younger leaves show striping or mottling and intervernal chlorosis

• Vector transmitted by leafhoppers (many different ones)

• Control: Insecticides or resistance breeding

40. Name four important viruses of sugar beet including their vectors.

• BNYVV (Beet necrotic yellow vein virus) Polymyxa betae

• BSBMV (Beet soilborne mosaic virus) Polymyxa betae

• BYV (Beet yellows Virus) Aphids

• BCTV (Beet curly top virus) Cikada

41. Describe the life cycle of the Beet necrotic yellow vein virus vector.

Life cycle of Polymyxa betae:

One has to distinguish the resting cycle from the progeny cycle

1. Resting cycle:

o beginning with the plasmodium of an infected root/ root hair which develops

cystosori (resting spores) in its surface

o The resting spores are released into the soil /soil-water where they germinate

o Primary zoospores originate and can infect new host plants and roots

2. Progeny cycle:

o Also beginning with the plasmodium. It produces a zoosporangium inside the

infected root cells

o The zoospores are released into the soil/ soil-water. They do not have to germinate

anymore

o The released zoospores are called secondary zoospores and can infect new host

plant and roots

42. Describe typical symptoms of virus diseases of fruit trees.

• Stem Grooving

• Stem pitting of rootstocks

• Basal scion swelling

• Dwarfing

• Leaves lose colour yellows, chlorosis (Rings or Spots), necrosis, Mosaic Symptoms, early

senescence

• Tatter symptoms on young leaves

• Fruits change/ lose colour, deformation, size reduction, taste might be affected

• Malformed petals

43. Describe typical symptoms of virus diseases of small grains.

• Stunting of plants and chlorosis or red/purple colour of leaves.

• reduced root mass

• loss of green color in leaves, is often more prominent on older leaves.

• Water soaking

• upright and stiff leaves and serrated leaf borders, reduced tillering and flowering, sterility

and failure to fill kernels, which results in fewer and smaller kernels and corresponding yield

losses.

• from a mild stripe mosaic to lethal necrosis;

• dwarfing, yellowing, intensive tillering, reduced winter hardiness, missing ear development,

severe yield losses

• mosaic symptoms

44. Why represents Plum pox virus one of the most important viruses in fruit trees world-wide?

Give arguments.

• Widespread in almost the whole world

• Several Strains differing in symptoms and various hosts (plum, apricot, peach, sour and sweet

cherry, some herbaceous weeds)

• Infects both leaves and fruits

• Mixed infection with other viruses

• Causes:

o Premature fruit drop and

o Bad fruit quality lead to

o Yield loss

• Transmittable by over twenty different aphid species

45. Describe the disease Citrus Tristeza in short terms and give details about the virus and its mode

of transmission.

• Common in most citrus growing areas

• Hosts: infects most varieties of citrus and relatives (wild forms)

• Is limited to scions on sour orange rootstocks, no symptoms appearing in plants on tolerant

rootstocks.

• Symptoms:

o Rapid wilting

o Root damage

o Dieback of limes

o Honeycombing

o Rapid decline causes Yellow-brown stain

o Stunting of young trees

o Stem pitting

• The virus CTV belongs to the genus Closterovirus in the family Closteroviridae

• 4 different Strains are known:

o Seedling yellow strains

o Grapefruit stem pitting strains.

o Sweet orange stem pitting strains.

o Lime dieback

• Transmission only by vectors:

o Oriental citrus aphid semi-persistent manner, no latent period;

o Greenfly loss of infectivity within 48 hours after

o Aphids acquisition

• Once introduced into a citrus-growing area, tristeza virus is spread naturally by aphids and

becomes endemic. Introduction into new areas can be prevented by quarantine measures,

budwood certification schemes, cross-protection and partly tristeza-tolerant rootstocks.

46. Name as many plant virus vectors as possible and give an example for each

47. Why have PLRV and PVX been replaced by PVY as the most important virus disease in potato?

• One of the most important virus species world-wide

• yield reduction 50-90%

• Insecticide control problematic

• PVY control in potato production requires costly certification programmes

48. What are viral quarantine diseases?

• Quarantine diseases need a strict isolation imposed to prevent the spread of disease and

economical and ecological loss.

• When quarantine diseases are detected in seeds or the field, there are specific quarantine

measures that have to be executed. Therefore, the introduction of the diseases into new

areas can eventually be prevented.

• Examples for viral quarantine diseases:

o Andean potato latent virus

o Andean potato mottle virus

o Tobacco ringspot virus

o Tomato ringspot virus

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Stefan H., Torben R., Daniel S. WiSe 2017/18