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Development of semi-quantitative, rapid, pregnancy–like test (Lateral Flow Device) for Botrytis

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Development of semi-quantitative, rapid, pregnancy–like test (Lateral Flow Device) for Botrytis

By Dr. Molly Dewey,

Department of Viticulture and Enology,

University of California at Davis

A large percentage of the grapes coming into Californian wineries at harvest time are tested for quality by the CDFA wine grape inspection services. These tests include those for fungal rot (Bunch rot). Rot tests are done by hand-sorting samples taken from incoming trucks at the test stands. The percentage rot is calculated on a weight-for-weight basis. This hand-sorting method of determining rot levels is becoming increasingly difficult and unsatisfactory. Not only does it involve training a new work force each season, but the results no longer reflect the true levels of rot in the load. This is because most grapes are now machine harvested and rotted berries tend to burst making them difficult to identify and impossible to weigh. In the past two decades, a number of alternate methods have been explored and, although some of these methods have been shown to work, they have not proved suitable for use on the testing stands. It now seems that a 4-minute Botrytis pregnancy-like test, developed by Dr Molly Dewey and Colleagues at UC Davis may, at last, provide a suitable method to replace the hand sorting method. It is often referred to as a “Botrytis pregnancy test” because physically and technically it is very similar to the early human pregnancy test or EPT developed by Unipath. The whole test is carried out within a small, hand-held, plastic-enclosed device just like an EPT device. This device is also known as a Botrytis-Lateral Flow Device.

The test is very simple and easy to carry out and involves just three steps. First, 1 ml of grape juice is added to 19 ml of a pre-prepared buffer in a dropper bottle. The user then applies two drops of the diluted juice/buffer to the sample well of the device. Within a few seconds of applying the diluted juice, a “blue tide” can be seen moving up through the viewing window of the device. After about 1 minute, a blue line can be seen forming opposite the “c” on the plastic housing of the device. This is the control line and its appearance assures the tester that the device is working. If the juice sample came from infected grapes a second blue line will be seen forming opposite the “t” on the plastic housing. This is the test line and its appearance indicates that the test is positive.

The device is semi-quantitative. If a test band can be seen within 1 minute of applying the 2 drops (total 80µl) of diluted grape juice to the sample well, it indicates that there is more than 4% level of rot in the grapes. A test band forming within 2 minutes indicates that there is 2-3% rot. If a test band does not form within 4 minutes it indicates that there is less than 1% rot. The intensity of the test band at any set time also indicates the level of Botrytis rot in the grapes. A faint test band at 4 minutes indicates 1% rot whereas a definite band at 4 minutes indicates 2-3% rot. The absence of a test band by 4 minutes indicates that there is less than 1% rot in the grapes. The test is highly sensitive and will detect, given longer time to “develop”, Botrytis in juice from infected grapes that are otherwise symptomless.

A compact scanner, the size of a refractometer, has been developed to measure the intensity of the test band at 4 minutes. Use of this scanner has removed the need for users to make subjective judgments in visually assessing the presence or absence of test bands. The scanner was developed by KGW Enterprises Inc., Indiana, in collaboration with Dr Dewey. After the diluted juice has been added to the sample well of the Lateral Flow Device, the device is inserted into the scanner. The scanner automatically “reads” the test line after 4 minutes and ejects the device. The intensity of the test band is measured as a percentage of reflectance change and is given as a digital readout in the display window of the scanner. Wineries will be provided with a guide showing the relationship between test band intensity after 4 minutes and levels of rot.

The Botrytis Lateral Flow Devices were tested, together with the Botrytis scanner, at a number of wineries in the 2003, 2004 and 2005 harvests, by CDFA wine grape inspectors. Results were encouraging and more extensive testing is planned for future harvests with the intent of using this method of determining rot to replace the CDFA hand- sort method.

The key to the success of the Lateral Flow Device is a highly specific antibody (monoclonal antibody) raised by Dr. Dewey and Ulla Meyer at the University of Oxford, UK. This antibody binds only to molecules produced by the specific fungus (Botrytis antigens). These antigens are present in juice from infected grapes but not in juice from uninfected grapes. The antibody is used to coat small blue beads that are housed in an absorbent pad beneath the sample well. When the diluted juice sample is added, they are mobilized and move forward along the membrane beneath the absorbent pad. If the diluted juice sample comes from infected grapes, Botrytis antigens in the test sample will be captured by the Botrytis antibody coated beads. The addition of the diluted juice to the sample well mobilizes the beads in the absorbent pad and they move forward along a membrane beneath the absorbent pad. Two lines of antibodies are pre-printed onto the membrane opposite the “t” and “c” seen in the viewing window of the device. The first, or “t”-line is the test line and the second is the control or “c”-line. The test line is a line of the Botrytis antibodies and, when beads carrying the Botrytis antigens reach this line, they are arrested by this line of antibodies and gradually aggregate so that they become visible to the eye as a blue (positive) line. The control line forms when a second set of blue beads, coated with a neutral antibody, is stopped by a line of anti-control antibodies. The formation of the control line assures the tester that the device has worked. If no Botrytis antigens are present in the diluted grape juice sample, none will bind to the Botrytis-antibody-coated-beads and none of these beads will be stopped at the test line. Therefore,only the control line will be seen. In summary, at the end of the test, two bands indicate a positive result and one band indicates a negative result.

The antigen recognized by the Botrytis-specific antibody has proved to be highly stable and is not metabolized during fermentation. This means that if the antigen is present in the juice, it will also be present in the resulting wines (both table wines and “dessert” wines). A sensory group of researchers at UC Davis led by Dr Hildgarde Heymann together with Dr Molly Dewey is currently exploring the relationship between the levels of the Botrytis antigen and the characteristic aroma associated with botrytized “dessert”

wines. They are also interested in determining whether low levels of Botrytis antigens in table wines have a positive effect on aroma quality. Other fungi, such as species of Aspergillus and Penicillium, that are commonly associated with higher levels of Botrytis infections, are known to produce undesirable effects.

Development of the Lateral Flow Device was a collaborative effort by Dr Chris Danks and co- workers at the government Central Science Laboratory at York, UK and Dr Molly Dewey, funded by the CDFA. Testing was carried out, both in the lab and at the wineries, through UC Davis and the CDFA by Dr Molly Dewey and her UC Davis colleagues. The device is now available worldwide and anyone interested in helping further validate the device should contact Dr Molly Dewey by email at the following email address: fmdewey@ucdavis.edu.edu