Using rapid diag-nostic kits fordetecting viraldiseases ing r e e n h o u s e

crops can be very beneficial.These kits produce results in ashort period of time and are reli-able, sensitive, cost effective anduser friendly. In fact, tests are

usually as sensitive as their com-parable ELISA test counterpartsthat are used in diagnostic labs.Results from rapid diagnostic kittests are ready in about 10-30minutes and each test costsbetween $3.00 and $7.00.

BEFORE STARTINGBefore using these kits, it is

important that you know yourcrops and what pathogens caninfect them. You also need to knowif the crop is a local host for thevirus, meaning the virus does notspread throughout the plant, or asystemic host, meaning the virusmoves through the entire plant.Finally, you need to know whatsymptoms the virus produces in thecrop or if it produces any symp-toms at all.

Viruses often cause diseasesthat have visual manifestations inornamental plants. However, thereare viruses that do not producesymptoms at all and often appearto have no effect on the plant.These are called latent viruses,and the most common are ringspot viruses. But a word of cau-tion, these viruses will often pro-duce strong symptoms if the plantbecomes stressed or is infectedwith another virus.

Viral infection often reduces thenumber of cuttings that a stockplant can produce and can affect theflower color and quality of theplant. Viral infected plants are oftenunmarketable because of poorflower quality and the presence ofsymptoms. All of these problemscombined lead to lost profits.

COMMON PLANT VIRUSES

The four most common plantviruses that infect ornamentalplants and other crops are cucum-ber mosaic virus (CMV), impatiensnecrotic spot virus (INSV), tobaccomosaic virus (TMV) and tomatospotted wilt virus (TSWV). The fol-lowing are some of the most com-mon virus symptoms.

Lesions — are localized spotsthat may be chlorotic (yellow) ornecrotic (brown). The virus doesnot go systemic in the plant.

Line patterns — a series ofchlorotic lines or patterns.

Mottle — a diffuse pattern of var-ious shades of green, see calibrachoamottle virus (CbMV) bottom left.

Mosaic — a definite pattern oflight green/dark green areas, seeCMV left.

Ringspots — concentric ring pat-terns that may be chlorotic or necrot-ic, see pelargonium flower breakvirus (PFBV) and tomato ringspotvirus (ToRSV) bottom right.

Calibrachoas present an interest-ing situation. Many cultivars devel-op virus-like symptoms when theplants are infected with CbMV.There are other calibrachoa cultivarsthat do not produce any virus-likesymptoms when infected withCbMV or TMV. However, whenthese plants are tested with the rapiddiagnostic kit, they do produce apositive result for CbMV or TMV. Ifyou inoculate sap from theseasymptomatic calibrachoas ontobioindicator plants, virus-like symp-toms are produced, and these plantstest positive for CbMV or TMV.

USING RAPIDDIAGNOSTIC KITS

All of the current rapid diagnos-tic kits are designed to detect thepathogen in symptomatic plant tis-sue. You might obtain a positiveresult in asymptomatic tissue, butyou cannot predict what tissue tosample to ensure a positive result.Also a negative result does notassure that the tested plant does notcontain the pathogen; it only ➧

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Grower 101:

Rapid Diagnostic Kit

Top: Anthurium showing strong mosaic symptoms of CMV; Bottom left: Calibrachoashowing mottling symptoms of CbMV; Bottom right: Geranium showing ringspotsymptoms of PFBV/ToRSV. (Photos courtesy of Agdia)

By Mike Tiffany

Using rapid diagnostic kits to detect viral diseases in the greenhouseis a good way to prevent wide-scale infestation and crop loss.

means that the plant tested negativeat that point in time. This problemis due to latent infections.

Follow the instructions. It isimportant that you follow the specif-ic instructions provided by the man-

ufacturer for the kit. Even thoughinstructions might be similarbetween kits, it has been observedthat mixing the instructions or com-ponents from multiple kits couldresult in false results.

Extraction method. All of thekits have a basic format for use.There is an extraction method fol-lowed by strip development andreading the results. There are twomethods of extraction: The sample

can be ground in a plastic meshbag containing the extractionbuffer or placed in a vial that con-tains the extraction buffer andsome small beads and shaken.

Strip development. There arealso two formats for strip devel-opment. The ImmunoStrip isplaced vertically within the meshbag with the tip of the stripimmersed in the extract.Development time is 30 minutes.The second method uses a cas-sette that remains in a horizontalposition. The extract is pipettedinto a well at one end of the cas-sette. Development time is 5-10minutes. Neither test should beremoved and read until the rec-ommended time is over. The rea-son for this is that during devel-opment of the sample test line theline might be difficult to seebecause the test area has a back-ground color. The test areabecomes a clear white zone afterdevelopment is complete. Thisenhances the ability to observefaint positive results that mightoccur because of low titer.

All of the rapid diagnostic kitsemploy the same principal in theirstrip development. All rely on astrip control line that appears atthe top of the test zone. Theappearance of this line ensuresthat the strip is working properly.The failure of this line to developindicates an invalid test, and theresults should be ignored. If thishappens, repeat the test and/orcall the manufacturer to help trou-ble shoot the test.

If the pathogen is present in theextract at detectable levels, a secondline will appear below the controlline. The intensity of the line willincrease as the titer of the pathogenincreases. The presence of just thecontrol line indicates a negativeresult. The presence of two lines indi-cates a positive result, see above. ➧

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Follow the manufacturer’s test instructionsto ensure correct strip development.

IN ADDITION TO TESTING

Testing is only one facet ofvirus prevention. Testing couldresult in the early detection ofpathogens within your green-

house, thus protecting otherplants from infection, but toensure a clean greenhouse, youshould also:

• Check the health of incomingplants.

• Monitor disease as part of ascouting program.

• Protect healthy plants frominfection.

• Assure plant health qualitybefore plants are shipped.

When you receive a shipmentof impatiens and you notice thatsome have necrotic ring spots,check them for the presence ofINSV. If they test positive forINSV, this will protect your green-

management

house from introducing the dis-ease into your production areas. Itwill also allow you to contact yourbroker faster and reach a favor-able solution sooner.

If when walking the bencheslooking for insects or diseaseproblems you spot a plant, such asa petunia, with mosaic symptoms,remove it from the area and checkit for the presence of TMV. Byusing a rapid diagnostic kit inthese two scenarios, you can pro-tect your healthy plants frombecoming infected.

Also, just as you checked yourincoming plant material, you canuse rapid diagnostic kits to spot-check the material that you shipto your customers. This way youcan reduce the chance of ship-ping infected plants. Remember,just because a plant tested nega-tive on receipt, does not mean itwill not test positive at somepoint in the future.

WHEN TO TEST Test anything that has symp-

toms. These symptoms shouldcorrelate to the symptoms of thepathogens for which you havetest kits available. Remember,there is no correlation betweenseverity of symptoms and viraltiter. You could have a negativetest result in the presence ofsevere symptoms.

Listen to the industry grapevine.If a particular crop is having aproblem with a specific pathogen,watch that crop carefully and testwhen symptoms first appear. Strictsanitation practices should alwaysbe used, but more so with a prob-lem crop. If the crop is one thatmight not produce any symptoms,then test on a frequent basis.

SUMMARYIn order for you to get the best

results from your rapid diagnostickit, adhere to the following steps:

• Use only symptomatic tissue.•Isolate any suspect plant

material.• Confirm the test result by

sending samples to a diagnostic lab.• If the results are confirmed, the

infected plants should be destroyed.• Disinfect and clean the infected

area before moving in clean stock.Following these steps should

help you detect viral pathogens in

your ornamental crops and allowyou to solve the problem in atimely manner. Remember, there isno one course of action to preventdisease in the greenhouse. Alwayspurchase certified stock; establish,follow and enforce strict sanitation

methods; and above all, know andwatch your plants.

Mike Tiffany is a senior plant pathol-ogist for Agdia. He can be reachedby phone at (574) 264-2014 or E-mailat miket@agdia.com.

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