Agdia, Inc. (Elkhart, IN) is happy to announce the commercialization of a rapid, user-friendly, DNA-based assay, on their AmplifyRP® XRT platform, for the detection of Beet curly top virus.

Curly top disease affects numerous commercially important hosts, including common bean (Phaseolus vulgaris), cucumber (Cucumis sativus), industrial hemp (Cannabis sativa), pepper (Capsicum annuum), potato (Solanum tuberosum), spinach (Spinacia oleracea), squash and pumpkin (Cucurbita pepo), sugar and table beet (Beta vulgaris) and tomato (Solanum lycopersicum). This disease is caused by Beet curly top virus (BCTV), a Curtovirus (family Geminiviridae), existing as a complex of strains differentiated genotypically, causing symptomology on the hosts mentioned above. Additionally, strains of BCTV are known to infect more than 300 species of plants in no fewer than 44 families, many of which are asymptomatic, weedy hosts.

Symptomology of Beet curly top virus was first observed in the late 19th century in the western U.S. on sugar beets. It was, however, not recognized as being caused by a specific pathogen until 1915, when leafhopper transmission was proven, and viral etiology was proposed. Since then, BCTV has spread throughout North America where hosts are cultivated, including several states in the American West and Southwest, southwestern Canada and Mexico. Furthermore, BCTV has been identified in parts of South America and several countries in the Mediterranean basin. All strains of BCTV are considered pathogens of quarantine importance in Canada, Israel, Mexico and the European Union.

Beet curly top virus is transmitted efficiently by the beet leafhopper, Circulifer tenellus (Order Hemiptera), in a persistent circulative manner. The virus can be acquired within minutes of feeding, and insects are known to remain viruliferous for up to a month. Beet curly top virus is phloem-limited, and the leafhopper must feed on infected phloem to acquire and transmit the virus to healthy plants. Circulifer tenellus is the only known vector in North America; however, in Europe, C. opacipennis is also known to vector the virus. The robust dynamics of the host-virus-vector relationship facilitate epidemics in parts of the world where leafhopper populations are high. Furthermore, the movement of infected propagative materials can spread the virus across great distances. Mechanical transmission through infected plant sap has been accomplished under experimental conditions. Nevertheless, this scenario is not thought to contribute to the epidemiology of naturally occurring curly top infections. Seed transmission of BCTV is not known to occur in the host species listed above.

Symptoms of curly top disease vary according to host and are typically more severe when plants are infected at earlier growth stages; many plants die before reaching maturity. Symptoms of curly top include stunted and distorted plant growth; leaf curling, crumpling, yellowing, vein swelling and distortion; and necrosis and hyperplasia of the phloem (figure 1). On beets, phloem tissue becomes necrotic, and exudate appears on the leaf surface. On tomato and pepper, fruit set is greatly diminished, and fruit that does form ripens prematurely. Furthermore, veins become purple.

Industrial hemp has reemerged as an important crop within several U.S. states, due to federal legalization and the demand for fiber, seed and cannabidiol. As production has increased, the list of disease organisms infecting this crop has grown to include several fungal, bacterial, viral and viroid pathogens. Beet curly top virus has been confirmed infecting industrial hemp and appears to be widespread on this crop throughout regions where vectors are present. There is a scarcity of research on this specific pathosystem; however, the understanding of the epidemiology of BCTV on cannabis is burgeoning, along with the crop. Symptoms of infection on industrial hemp include stunting leaf deformation and chlorosis (figure 2).

Agdia’s new AmplifyRP^® XRT assay for detection BCTV is based on recombinase polymerase amplification (RPA). This technology promotes the rapid amplification and detection of nucleic acid targets, DNA or RNA, while maintaining a single operating temperature of 39 – 42 °C. The AmplifyRP^® XRT products achieve target sensitivity and specificity comparable to PCR, while having clear advantages over the lab-based technology. AmplifyRP^® XRT products do not require a nucleic acid purification step; crude sample extracts are prepared using a simple extraction buffer and tested directly. This makes the testing process simple and saves the end user valuable time. Furthermore, this facilitates the implementation of this technology at remote locations with limited resources. When paired with Agdia’s AmpliFire^® isothermal fluorometer (figure 3), the XRT system is a rapid, user-friendly tool that can be implemented in the field or the lab by personnel with limited experience in molecular diagnostics.

Agdia states their assay was screened against a diverse collection of confirmed strains, including those infecting beets, industrial hemp, peppers and tomatoes, detecting all true positives. Furthermore, no cross-reactivity was observed with an extensive panel of viral and viroid pathogens, including Alfalfa mosaic virus, Cucumber mosaic virus, Hop latent viroid, Hop stunt viroid, Tobacco mosaic virus, Tobacco ringspot virus, Tomato brown rugose fruit virus, Tomato mosaic virus, Tomato ringspot virus, Tomato spotted wilt virus and Tomato yellow leaf curl virus. Sensitivity for this assay is greater than that observed with the published RT-qPCR assay and conventional RT-PCR assay to which it was compared. This product was developed to test leaf, stem and petiole tissue.

The introduction of this product brings Agdia’s catalog to 25 assays on the AmplifyRP® platform. High levels of market demand for field-deployable, plant pathogen detection products have driven this output, and Agdia maintains they will continue to expand their product offerings.

Following decades of service to food crop and ornamental growers, Agdia’s in-house diagnostic laboratory is now offering its expansive disease testing program to the hemp market.

Deborah (Debi) Groth-Helms is the Director of Agdia’s Testing Services department. “We are excited to finally offer our comprehensive virus testing program to hemp growers and diagnosticians. For years we have worked with stock and seed producers in screening propagative tissues for the presence of key pathogens. Now we can help bolster the US hemp industry in their efforts to produce the healthiest plants possible.”

Agdia Testing Services offers multiple screening options and works with clients in customizing screens to best suit their needs. The basic Hemp Screen includes a panel of a dozen different tests for viruses, viroids, and other microorganisms.

According to Debi, “Information is being rapidly released on what causes disease in Cannabis. Having a big catalog of services already in place allows us to quickly respond to new reports. Experience working with closely related plant species helps us know what to monitor before an outbreak occurs.”

Included in the Hemp Screen are tests for Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Hop latent viroid (HLVd), and many others. The Curto-Becurtovirus Group test listed in the screen detects all of the various virus species associated with Beet curly top disease. Additional tests are also available for less common or emerging pathogens. These highly sensitive molecular assays can be used to detect diseases such as witches’ broom (Phytoplasma Group PCR) and Lettuce chlorosis virus (Closteroviridae Group PCR).

Currently the laboratory is only accepting hemp tissues from US submitters that hold an active hemp license through the USDA, their state government, or tribal entity.  Information on hemp licensing and the 2018 Farm Bill can be found on the USDA’s website. The laboratory recommends shipping plant material with the United States Postal Service (USPS) along with a completed sample submission form.

Information on how to send samples can be found on the Agdia website. Questions regarding samples, customized screens, requests for quotes, and all other inquiries can be sent to info@agdia-emea.com