26 Citrograph July/August 2012

Each issue of Citrograph features reports by the principal investigators of CRB-funded research projects. You certainly have wondered, what happens to the products

of this research? This article describes how some of these proj-ects move forward and their products reach you, the grower.

In the March/April 2012 issue, Cristina Davis’ group at the University of California, Davis reported on their prog-ress in an article titled, “Reagentless detection of citrus pathogens using differential mobility spectrometry (DMS)” (pages 54-56).

This technology uses volatile organic compounds (VOCs) as a means to detect the presence of pathogens within a plant. The hope is that these pathogens can be detected before symptoms appear that are visible to the human eye.

This research group has identified unique chemical signatures for huanglongbing (HLB) and tristeza that are detected by a relatively small piece of equipment called a differential mobility spectrometer (DMS) (Figure 1). Ted Batkin also addressed this technology in his editorial in the May/June 2012 issue (page 4).

Life after the CRB-funded research project

John Morgan, CRB laboratory technician, operating the lyophilizer equipment that will freeze/dry plant tissue samples This method preserves the tissue so that it can be sent to other laboratories and analyzed at a later time.

staff, Alexander Aksenov, a development engineer in the Davis lab, took the equipment to the street.

Ideal location for field-testing As you know, HLB-associated bacteria were detected in

a tree in Hacienda Heights in Los Angeles County. CDFA has been visually inspecting citrus trees and collecting psyl-lids and plant tissue from trees adjacent to that detection site -- (the positive tree has been removed) -- and luckily, no psyllids or plants have tested positive since. What a perfect location to test this device!

In addition, another researcher from the University of California, Davis, Carolyn Slupsky, was funded last October for a project titled, “Biomarkers for detection of Liberib-acter infection in Valencia orange trees through H-NMR-based metabolomics of leaves and woody tissues”. Her re-port will be published in a future issue of Citrograph, but she believes she has identified metabolites that are unique to trees infected with HLB-associated bacteria.

So, the plan was for CRB’s Brian Taylor to coordinate with Tina Galindo of CDFA to select appropriate residential sites for Alex Aksenov, UCD, to test the DMS equipment. At the same time, plant tissue would be collected and brought back to the CRB Jerry Dimitman Laboratory for two pur-poses: one, the tissue would be tested by lab personnel by the standard Polymerase Chain Reaction (PCR) method for HLB-associated Liberibacter. The second purpose would be to process the tissue by lyophilization (freeze-drying) and send it to the Slupsky lab in Davis.

I am happy to report that the PCR results from the Jerry Dimitman lab were negative. At the time this issue went to press, we had not yet received the data analysis from the Da-vis and Slupsky laboratories.

CRB technology transfer Many growers see the faces of CRB staff at various

meetings but may not realize the roles and responsibilities of staff behind the scenes. The Citrus Research Board doesn’t just fund research projects; often, staff members actively take part in the technology transfer of project products.

This means staff participates in making research re-sults usable, including the field validation of technology and methods and adjusting an assay method so it is usable by laboratories.

As the Asian citrus psyllid continues to move through-out Southern California, we hope that technology such as the advances mentioned in this article will detect infected trees long before HLB symptoms appear. In this way, only tens of trees will require removal instead of hundreds.

As it stands today, the Davis lab’s work with VOCs is still experimental. Although the goal of the research is the early detection of HLB, if a tree were to come back suspect using

MaryLou Polek and Brian Taylor

Readers must be cautioned that although this is promising technology, it is still in the validation phase. Should a sam-ple be found suspect using this testing method, repeated testing must be performed in order to confirm the result. Furthermore, before commercializing this methodology, it must be thoroughly vetted by the scientific community.

This is fine in a controlled laboratory situation, but what about in the real world, in your grove? Does this technology really work? Well, with the assistance of CRB and CDFA

July/August 2012 Citrograph 27

Kavitha Madishetty, CRB laboratory biologist, preparing a plate of samples for Polymerase Chain Reaction (PCR) analysis. The process of loading the PCR plate must be done under a laminar flow hood. This hood is designed so that air within the hood has been purified, thereby preventing contamination of the samples.

this method, no regulatory action could result without the substantiation by the currently approved method. Detection of HLB-associated bacteria that would result in mandated tree removal is PCR followed by sequencing the PCR prod-ucts. Then, confirmation must be made by the USDA Diag-nostic Laboratory in Beltsville, Maryland.

During this next year, two important activities will occur concurrently: Drs. Davis and Aksenov will continue to refine the VOC signatures and sensing probes, and a company – Applied Nanotechnologies – will reduce the overall size of the equipment and prepare the design for manufacturing. A usable product will be in the hands of growers within the next few years.

Dr. MaryLou Polek is Vice President of Science and Technology for the Citrus Research Board which includes providing oversight for all research projects funded. Among other professional duties, she is the Chief Science Editor for Citrograph. Brian Taylor, Field Operations Director for the CRB, joined the program in 2009 with more than 30 years’ experience in the detection and eradication of exotic pests.

While in the process of moving knowledge to the field…

‘‘I was asked to help coordinate with the researchers working on the EZKnows VOC system and CDFA to test trees in the Hacienda Heights area of Los

Angeles for HLB. An EZKnows prototype had been tested on Hamlin oranges in Florida with very encouraging results, and now the researchers wanted to test the machine on California-raised varieties and under California conditions.

After numerous calls to Cristina Davis’ lab and CDFA, dates were set for the testing. I looked forward to the oppor-tunity to observe and help in finding a quicker and earlier detection technique for HLB.

“A good researcher not only needs to recognize the problem and come up with a possible solution, but they must also be able to execute that solution so that it is practical

and affordable. The use of VOCs is not new. Dogs have been used for centu-ries to detect things for humans (and yes, there are even dogs being trained to detect HLB); however, dogs have limitations that machines do not. Using VOCs at airports to test for explosives is now routine. So the Davis lab hopes that they have come up with a way to test trees for HLB by analyzing the tree’s VOCs.

“There are sometimes many steps involved in solv-ing some problems, as I appreciated once again in working with Dr. Aksenov. Equipment must be designed to collect

(Left) Dr. Alexander Aksenov working with the entire set-up of the DMS equipment. (Above) Differential mobility spectrometer (DMS) prototype equipment used to detect volatile organic compounds (VOCs) in the Los Angeles area.

Brian Taylor

28 Citrograph July/August 2012

the VOC sample and then an analysis made to deter-mine the VOC signature of a healthy tree versus an HLB-infected tree. Will the signatures be the same if the samples are brought into the laboratory or sampled in the field?

“Some of the variables that must be analyzed include not only the biological char-acteristics of the tree (will the signature of an orange be the same as a lemon?), age, pro-duction cycle; environmen-tal characteristics (climate, humidity, temperature, time of day); and also the overall health of the tree (stress, in-sect pests, etc).

“The Cristina Davis lab recognized that there was a difference in the signature of a healthy Florida orange and an orange from a known in-fected tree. The question was how to move that knowledge to the field and make it useful

“Taking a laboratory full of instrumentation and ‘com-pacting’ it was the first challenge. Davis’ lab has succeeded in reducing the equipment package to a small suitcase and a laptop computer. They hope to reduce it even further and make it truly portable.

“In Florida, the testing was done in a commercial grove and the equipment placed on the back of an ATV and run from batteries. Whereas this may be an efficient method in a commercial grove, it is not very useful in someone’s back-yard. Dr. Aksenov needed a platform for the equipment to be placed on and a dolly for the batteries. The final set-up included a small folding table and a homeowner willing to allow us to plug the equipment into their electrical outlet

Sonya Segura, left, and Lydia Rodriquez, members of CDFA’s ACP/HLB survey crew, assisted with contacting homeowners and arranging for sites to test the technology.

and run extension cords. “On the agreed-upon day, I met up with Dr. Aksenov

and CDFA Inspectors Lydia Rodriquez and Sonia Segura. Lydia and Sonia had already made contact with homeown-ers in the area and located Valencia orange trees for Dr. Aksenov to test. While Alex and I were busy setting up the equipment, Lydia and Sonia conducted a visual survey for ACP and HLB symptoms. No ACP were found, as the area had recently been treated as part of the HLB eradication program. Plant samples were taken from each tree and sent to the CRB Jerry Dimitman Laboratory in Riverside for PCR analysis.” — Brian Taylor. ●

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