If your crop shows unknown growth symptoms, we can investigate whether there is a pathogen, a lack of nutrients or whether other factors on your farm are causing the damage. To make the right diagnosis, we have several analytical methods at our disposal, such as microscopy, plating methods and DNA analyses.

In order to make a correct diagnosis, a number of things are of great importance, such as the cultivation conditions, climate, origin of the plant material and the spread of symptoms in the crop. It is also important to send in the right parts of the plant for diagnosis.

Microbiological research and methods for virus analysis

In this series on microbiological research, we talked extensively in the previous Newsletter about the analyses on germinal bacteria. In the next edition, DNA analyses on plant pathogenic fungi and bacteria will be discussed. Now the focus is on analysis methods on viruses. In this article we will discuss the ToBRFV analysis.

Normec Groen Agro Control

often receives questions about viruses and how analyses are carried out. There is no universal test for testing viruses in crops. This means that a specific virus must be sought each time. Each virus requires a separate analysis to confirm or exclude the presence of this virus in the crop.

The example given here is the tomato crop, but this explanation is useful for every crop.
In general, the virus in the crop can be examined well, and is therefore viable and infectious. Also disinfected drain water, swipe samples of cleaned surfaces or washed clothes can be analysed for the presence of virus.
The concentration of the demonstrated virus may be very low or no longer viable in these types of samples, so a positive result does not say anything about the infectiousness.

To determine whether this virus is still viable (and can therefore still infect plants), an extra plant test should be carried out. In the analysis methods for virus, you only know that the demonstrated virus is still alive in living crop. In decontaminated samples, dead virus may be present which, in a virus test, gives “demonstrated” as a result.
For ToBRFV, further research is needed to test disinfectants for effectiveness of killing this persistent virus.

Tomato brown fruit virus (ToBRFV)

For some time now, Normec Groen Agro Control is able to provide a quick and reliable analysis on Tomato brown rugose fruit virus (ToBRFV). A specific real-time PCR assay has been developed that can be applied to different types of samples such as crop, water and surface swipe samples. For swipe samples, a plant test is first used, after which the PCR method is used to test whether the live virus has replicated.

Various test methods for ToBRFV in tomato are described below.

ELISA test

In an ELISA test, antibodies are used to search for a protein of a certain plant virus. If the protein is detected in the sample (plant sap), the test eventually turns yellow. Without the presence of the specific protein, the test remains colourless.

For the development of ELISA tests, we are dependent on third parties who develop and supply these tests. We still often use ELISA tests when they are available and specific (good) enough.

To test ToBRFV, use can be made of:

• ELISA-TMV that also captures the signal of ToBRFV
• ELISA for the Tobamo group, which also includes ToBRFV

ELISA sometimes has the disadvantage that quite a lot of virus (symptoms) is needed before this test indicates the presence of the virus. The detection limit is therefore quite high, which can be a disadvantage for good monitoring or for making a diagnosis. During the incubation period of ToBRFV in the plant, this test could indicate that the virus was not found, while it was present. Therefore, a PCR method is preferred in our laboratory.

Normec Groen Agro Control has the following ELISA tests available for tomato:

Required plant material is leaf

• Pepino mosaic virus
• Potato virus X
• Potato virus Y
• Tomato mosaic virus
• Cucumber mosaic virus
• Tomato spotted wilt virus
• Impatiens mottling virus
• Alfalfa mosaic virus

PCR test for virus

Normec Groen Agro Control developed a real-time PCR test for ToBRFV in late 2018 because it is a highly sensitive test and can therefore quickly detect low levels of ToBRFV. The test only reacts with ToBRFV and does not cross-react with TMV and ToMV.

The PCR test requires the virus RNA (single strand) to be ‘transcribed’ into DNA (double strand). We can analyse crop samples with this PCR test. And with additional pre-processing, also swipe samples, seeds and water samples. For these special samples, a growth test is often done first, in which test plants are infected with the sample.

As a specialist in the market, Normec Groen Agro Control often leads the way in the development of PCR tests. To help growers quickly with new diseases that are prevalent.

PCR tests for specific viruses in tomato by Normec Groen Agro Control

• Tobamovirus group
• Begomovirus group
• Tomato yellow leaf curl virus (TYLCV)
• Tomato brown rugose fruit virus (TBRFV)
• Tomato chlorosis virus (TOCV)

DNA- Sequencing

Because there can be different strains of one type of virus, which can be more or less aggressive to the plant, strain research is carried out. This is done by means of so-called sequencing, which is a way of mapping out the family tree of the species. This method is used to determine the strain of various viruses. But it is also used to isolate unknown bacteria or fungi in the plant or in the soil. Normec Groen Agro Control provides this sequencing method and has its own large database to identify species and strains of diseases.

Quick test

Growers sometimes want to be able to quickly rule out for themselves whether certain symptoms in the crop are caused by ToBRFV. So-called rapid tests are available on the market for, among other things, Tobamovirus, which includes ToBRFV. It turns out that if you do such a test as prescribed on the label, you can get unclear results. We call this false positive or false negative results. We have therefore looked at how this test can be used properly.

If the virus concentration is too high (too much leaf), the test no longer measures well. It can then wrongly indicate “not demonstrated”. It is better if you first dilute more before measuring. However, diluting too much is not good either, otherwise the virus will no longer be detected.

We have come to an improved prescription for use: “Protocol TMV test strips for ToBRFV detection“. These rapid tests are known to indicate the presence of virus only when symptoms are already visible in the crop. So there should be a reasonable amount of virus particles present in the leaf.

Photo Agdia strip (source: GAC)

This is an example of a test strip. In this case, the test strip gave a faint result.