New Field-Based Tool for Detection of Multiple Thrips Transmitted Plant Viruses

Image of ImmunoComb for detection of plant viruses with flowers in the background

Figure 1. The new Thrips Spot Viruses ImmunoComb® for convenient detection of INSV, TSV and TSWV.

Agdia, Inc. is happy to announce the addition of a new product to their ImmunoComb® line to help growers detect and combat plant viruses that cause similar symptoms in addition to sharing a common insect vector. The new Thrips Spot Viruses ImmunoComb® brings together three existing ImmunoStrips® in one easy-to-use format.

Impatiens necrotic spot virus (INSV), Tobacco streak virus (TSV) and Tomato spotted wilt virus (TSWV) are all commonly spread by thrips and can cause similar spot-like symptoms on many plant species. Such visual similarities in the presence of a common vector underscore the unreliability of visually identifying the causal pathogen, further emphasizing the need for reliable and easy-to-use onsite diagnostic test methods.

Impatiens necrotic spot virus (INSV, Orthotospovirus) is an enveloped and spherical-shaped virus that infects over 600 plant species. While the virus is primarily spread by western flower thrips (WFT), it can also spread effectively via cuttings (vegetative propagation). From entire fields of lettuce in the Southwest to widespread distribution of infected African Violet cuttings on the East Coast, INSV outbreaks have covered much of the United States in recent years.

Symptoms of INSV infection vary by host, but often include brown, purple or sunken spots on leaves, brown spots on stems, chlorosis, necrosis, stunting and ringspots.

Tobacco streak virus (TSV, Ilarvirus) has a wide host range consisting of nearly 200 plant species, causing significant economic losses in crops ranging from dahlias and other ornamentals to vegetable and field crops.

While some infected plants are asymptomatic, symptoms vary between hosts and include spots, mosaic patterns, necrotic streaking, stunting, apical necrosis and leaf deformation. Tobacco streak virus is spread by several thrips species but can also be spread via propagative cuttings and infected seed.

Tomato spotted wilt virus (TSWV) is another member of the Orthotospovirus genus capable of infecting many different crops of economic significance. According to the Centre for Agriculture and Biosciences International (CABI), TSWV is now considered to be among the top 10 “most economically destructive plant viruses with worldwide losses exceeding 1 billion dollars annually.” It is spread by several different species of thrips. Symptoms include (but are not limited to) small brown spots or flecks, ring spots, chlorosis, necrotic patches and more.

The Thrips Spot Viruses ImmunoComb® for INSV, TSV and TSWV allows growers to conveniently and simultaneously detect or rule out infections of each virus in a single sample. This product is sold in kits of 8 combs consisting of 3 ImmunoStrips® each, and kits include the buffer-filled extraction bags necessary to perform a test.  Agdia provides a one-year warranty on all purchased products along with an unrivaled level of customer support.

About Agdia

A leading provider of diagnostic solutions for agriculture, Agdia, Inc. has been serving plant breeders, propagators, growers, universities, and private testing laboratories since 1981. The company offers a comprehensive portfolio of validated, easy-to-use diagnostics for identifying plant pathogens, hormones, and transgenic traits.  In addition, Agdia operates an ISO accredited, in-house, testing services laboratory. Agdia’s quality management system is ISO 9001:2015 certified and their Testing Services Laboratory is ISO 17025:2017 accredited.  Visit the company’s website at www.agdia.com

ImmunoStrip® and ImmunoComb® are registered trademarks of Agdia, Inc

Acknowledgements: Thank you to Robert Wick (University of Massachusetts, Bugwood.org) for providing Figures 2 and 4, and to Howard F. Schwartz (Colorado State University, Bugwood.org) for providing Figure 3.

New Rapid Molecular Test Kit and Testing Service for Blueberry Scorch Virus (BlScV) Launched by Agdia, Inc.

Image of a table showing results comparison between new rapid molecular test and other established methods for blueberry scorch virus

Figure 1. BlScV diagnostic sensitivity and specificity method comparison

Agdia, Inc. is happy to announce the launch of a new rapid, field-deployable RNA-based assay for the detection of Blueberry scorch virus (BlScV). The new test outperforms all commercially available serological (ELISA) tests and matches the performance of the most well-established PCR methods in use today.

Blueberry scorch virus has been a significant threat to blueberry production in the Pacific Northwest of the United States for many years, spreading more recently across the country as far as the US Northeastern Seaboard.

While this single-stranded, RNA Carlavirus is known to be aphid-borne, it can also be spread through vegetative propagation from stock plants. Symptoms of infection include dieback, “scorch” symptoms on flowers, reduced fruit yield, marginal leaf chlorosis and red line patterns. Symptoms take a long time to develop (1-2 years from the point of infection, according to the North Central IPM Center).

Such a wide range of symptom onset and type highlights the necessity of testing to ensure clean nursery stock as well as regular diagnostic monitoring of production sites to cull infected bushes before aphids transmit the disease to neighboring plants.

As part of our continual product improvement efforts, Agdia has identified that most, if not all commercially available BlScV serological assays were prone to false-negative results due to either low sensitivity or lack of specificity for serologically divergent viral isolates.  Since low sensitivity and lack of specificity in serological assays cannot be addressed by even the best antibodies, we shifted our development focus to molecular identification via AmplifyRP® XRT, where primer design could be targeted to ensure detection of the infected samples/isolates.

AmplifyRP® XRT for BlScV is the rapid diagnostic output of these efforts, and we think the results speak for themselves. The new molecular assay outperforms all ELISA offerings on the market and is comparable to PCR methods used in diagnostic laboratories, without costly and labor-intensive RNA purification step(s).  Additionally, AmplifyRP® technology is generally more resistant than RT-PCR to the various inhibitors found in fruit leaves. See Figure 1 for a results comparison across test formats. A full validation report is also available for more details on test performance.

AmplifyRP® XRT for BlScV is compatible with most qPCR instruments on the market as well as with the AmpliFire® isothermal fluorometer (Figure 2) when portability and ultimate protocol simplicity are desired. This test is also being deployed by Agdia’s Testing Services division for customers who prefer to submit samples to a laboratory with an ISO 17025-accredited quality management system rather than test onsite.

About Agdia

A leading provider of diagnostic solutions for agriculture, Agdia, Inc. has been serving plant breeders, propagators, growers, universities, and private testing laboratories since 1981. The company offers a comprehensive portfolio of validated, easy-to-use diagnostics for identifying plant pathogens, hormones, and transgenic traits.  In addition, Agdia operates an ISO accredited, in-house, testing services laboratory. Agdia’s quality management system is ISO 9001:2015 certified and their Testing Services Laboratory is ISO 17025:2017 accredited.  Visit the company’s website at www.agdia.com, e-mail info@agdia.com, phone 1-574-264-2615 (toll-free 800-622-4342) or fax 1-574-264-2153.

AmplifyRP® and AmpliFire® are registered trademarks of Agdia, Inc.

Agdia Releases ImmunoStrip® for Detection of Angelonia Flower Break Virus (AnFBV)

Agdia's new ImmunoStrip for AnFBV

Figure 1. Agdia’s new Angelonia flower break virus (AnFBV) ImmunoStrip®

Agdia, Inc. of Elkhart, IN is happy to announce the commercial release of a rapid diagnostic for the detection of Angelonia flower break virus on their popular ImmunoStrip® platform.

Symptomology of Angelonia flower break virus (AnFBV, Alphacarmovirus) was first observed in 2005 on Angelonia angustifolia plants grown in Germany, Israel and the U.S.  Symptoms included leaf mottling, flower break and stunting, leading researchers to suspect viral etiology.  Subsequent sequence analysis of plant products revealed the presence of an uncharacterized virus, most closely related to Pelargonium flower break virus (PFVB) and Carnation mottle virus (CarMV), both in the genus Carmovirus.  Following the completion of Koch’s postulates, the new virus was named Angelonia flower break virus.  Initially, the German isolate was referred to as Angelonia flower mottle virus, which is now recognized as a synonym for AnFBV.  In 2015, the genus Carmovirus was divided into three discrete genera: AlphacarmovirusBetacarmovirus and Gammacarmovirus.

Since its characterization, the natural host list for AnFBV has grown to include cultivars within the genera NemesiaPhlox and Verbena, in addition to Angelonia.  Angelonia flower break virus can be spread between production facilities via the movement of infected propagative materials.  Thereafter, AnFBV is spread mechanically via the movement of viruliferous plant sap on hands and implements.  No arthropod vectors are known to transmit AnFBV, and seed transmission is believed to be inconsequential to epidemiology.  The full extent of AnFBV infections within the ornamental industry is unclear, and many believe it is more widespread than initially suspected.

Agdia states their new ImmunoStrip® for detection of AnFBV was tested against a broad exclusivity panel of potential cross-reactors, including Alfalfa mosaic virus, Alternanthera mosaic virus, Calibrachoa mottle virus, Carnation mottle virus, Impatiens necrotic spot virus, Nemesia ring necrosis virus, Pelargonium flower break virus, Tobacco mosaic virus and Tomato spotted wilt virus, with no cross-reactions observed.  Furthermore, the product will detect the original AnFBV isolates from Germany, Israel and the U.S., in addition to a discrete isolate from California.  Agdia’s new ImmunoStrip® can be used to test leaf and petiole tissue.  Please see Agdia’s product page for the AnFBV ImmunoStrip® for a comprehensive validation report.

Agdia’s ImmunoStrip® for detection of AnFBV is sold in kits of five and 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on all purchased products.  A diagnostic assay for the detection of this pathogen is also available in an enzyme linked immunosorbent assay (ELISA) format.  For more information on these products, in addition to Agdia’s full catalog of pathogen detection products, visit the company’s website at www.agdia.com.

ImmunoStrip® is a registered trademark of Agdia, Inc.

Agdia Expands Trait Catalog with Release of ImmunoStrip® for Detection of Glyphosate Tolerance in Canola

Immunostrip test for GAT4621 in canola

Fig. 1. Agdia’s new ImmunoStrip® test for Optimum® GLY GAT4621 in canola.

Agdia, Inc. (Elkhart, IN), a leading provider of agricultural diagnostic products and services, has commercialized an ImmunoStrip® assay for the detection of the GAT4621 gene product, which confers glyphosate tolerance to transgenic canola.

Agdia’s new ImmunoStrip® detects the presence of GAT4621 in transgenic canola, marketed as Optimum® GLY by Corteva Agriscience.  The GAT4621 gene produces the glyphosate N-acetyltransferase enzyme, thereby conferring tolerance to glyphosate herbicides to transformed plants.  This ImmunoStrip® was developed to be used with leaf and bulk grain samples and has a limit of detection of nine transgenic seeds in 1,000 non-transgenic seeds (0.9% LOD).  Agdia claims this ImmunoStrip® does not cross-react with other commercially available transgenic traits present in canola, including NPTII, CP4 EPSPS and PAT/bar.

This introduction is the 24th ImmunoStrip® product in Agdia’s comprehensive catalog of transgenic trait identification assays.  The GAT4621 ImmunoStrip® assay can be performed in the field or the lab and does not require special equipment or prior diagnostic experience.  Test protocol is straightforward, and results can be visualized in ten minutes as a single control line or a control and test line for negative or positive results, respectively.

The GAT4621 ImmunoStrip® is available in kits containing 50 strips. Agdia provides a one-year warranty and full technical support on purchased kits.  For more information on Agdia’s full catalog of trait and pathogen detection products, please visit the company website at www.agdia.com, email info@agdia.com, or phone 1-574-264-2615 (toll-free 800-622-4342)

Optimum® GLY is a trademark of Corteva Agriscience and its licensed affiliates.

ImmunoStrip® is a trademark of Agdia, Inc.

Agdia Launches Rapid Molecular Test Kit for Phytophthora ramorum (Sudden Oak Death)

Agdia, Inc. (Elkhart, IN) is proud to announce the commercialization of an isothermal molecular assay for the detection and identification of Phytophthora ramorum on their AmplifyRP® XRT platform.

Since its characterization in the early 2000’s, Phytophthora ramorum has killed tens of millions of native tanoaks and true oak species across western hardwood forests in the U.S., thereby altering natural ecosystems and carbon sequestration while increasing wildfire risk (Figure 1.)  Furthermore, this pathogen has moved throughout Europe and the U.S. and into Canada on infected nursery stock, such as woody ornamental shrubs and understory trees.  The lethality of sudden oak death on native species combined with the mobility of ramorum shoot and leaf blight on propagated hosts have caused significant ecological change and turmoil among foresters, ecologists, nursery associations and Federal and state regulatory agencies.

Symptoms of what we now designate as sudden oak death were first observed in the mid-1990’s in Marin County, California.  Rapid onset of bleeding stem and trunk cankers, overall decline and death were observed in large numbers of coast live oak (Quercus agrifolia) and tanoak (Lithocarpus densiflorus), two species native to California and Oregon coastal ranges.  At that time, etiology was undetermined, but drought, insect pests and opportunistic fungal pathogens were investigated as potential suspects, independently and in ensemble.

In 2000, a seemingly unknown and non-native species of the oomycete pathogen Phytophthora was isolated from canker material and identified as the causal agent of the symptomology observed in California.  Shortly thereafter, it was determined this same organism was causing foliar blight, tip blight and stem cankers on nursery-grown rhododendrons and viburnums in Europe, where this discrete, non-lethal symptomology was first observed in the early 1990’s.  It is believed that P. ramorum was introduced to Europe and the U.S. in two separate events, apparently on nursery stock.  Current research suggests P. ramorum is native to Southeast Asia, but the extent of its endemic range is unknown.  The species was named P. ramorum in 2001, due to the branch infection paradigm observed across all symptomologies; ramus is Latin for branch, and ramorum is the genitive plural of ramus.  Therefore, Phytophthora ramorum translates to “plant destroyer of branches.”

It is known that P. ramorum infects at least 150 species of herbaceous perennials, cultivated woody ornamentals and native canopy and understory trees and shrubs.  For a full list of proven and associated plant hosts, please visit the USDA APHIS website.  Symptoms vary by host and exist on a spectrum from lethal to little more than associative sporulation.  Furthermore, not all hosts exhibit naturally occurring infections and only through artificial inoculation do symptoms present.  Nevertheless, two distinct natural infection strategies have been identified in North America with P. ramorum, and symptomologies are known discretely as sudden oak death and ramorum leaf and shoot blight.  In 2009, P. ramorum was identified causing lethal symptoms on Japanese larch (Larix kaempferi) in timber plantations in the UK, and Ireland shortly thereafter.  This is known as sudden larch death and exhibits symptoms ostensibly similar to those of the sudden oak death paradigm.

Sudden oak death symptoms manifest as bleeding cankers on the trunks of several members of Fagaceae, including tanoak, coast live oak, and additional species within the Quercus genus.  Phytophthora ramorum infects through the outer bark and colonizes the inner bark and phloem of trunk hosts, thereby restricting the movement of photosynthates from source to sink and compromising physical plant defense components.  Internal colonization of wood causes resinous, reddish-brown exudate to “bleed” from intact and cracked outer bark, resulting in characteristic external symptomology (Figure 2.).  Trunk cankers can enlarge to three meters in length and girdle infected trees, resulting in crown wilting, limb dieback and host mortality, typically within five years of initial infection (Figures 3. and 4.).

Furthermore, infected trees become hosts for opportunistic species of wood-boring beetles and decay fungi, such as Annulohypoxylon thouarsianum.  Presently, the sudden oak death pathosystem has been confined to coastal forests in California and Oregon, where USDA quarantines are in effect to limit human-assisted distribution.

Ramorum blight is polycyclic and can affect leaves and stems of multiple species of woody ornamentals and native trees and shrubs, other than those subject to sudden oak death.  These are typically referred to as foliar hosts and include species popular in the nursery trade, such as all species and cultivars of Camellia, Kalmia, Pieris, Rhododendron and Viburnum.

Symptoms of ramorum blight include necrotic lesions on leaf surfaces, twig dieback and defoliation on some hosts.  Leaf infections can occur through petioles as stem infections move downward from terminal buds, killing woody tissue.  Furthermore, foliar infections can appear anywhere on leaf surfaces and are indistinguishable from those caused by additional Phytophthora species and fungal pathogens.  This symptomology is not typically lethal on mature hosts.  Nevertheless, symptomatic nursery plants such as Rhododendron function as vehicles of pathogen dissemination and potential sources of primary inoculum in areas beyond current sudden oak death epidemics, e.g., Midwest and eastern U.S. hardwood forests (Figure 5.).

Natural dispersal of P. ramorum inoculum in forests occurs via splashing water and wind currents, moving from sporulating foliar hosts to trunk hosts.  In the U.S., the primary foliar host in western forests is the California bay laurel (Umbellularia californica) (Figure 6.).  Most trunk hosts are considered dead ends, as sporulation does not occur, and infections are lethal.  Nevertheless, foliar sporulation is known to occur on tanoak and Japanese larch.  Human-assisted dispersal of inoculum is via movement of infested soil on equipment, vehicle tires and shoes and infested firewood, mulch and timber products outside of regions under quarantine.  Interstate and international movement of infected nursery stock is the primary means of long-distance dispersal of P. ramorum.  It is believed this is the dispersal paradigm responsible for introduction to both the U.S. and UK in separate events.  Phytophthora ramorum is considered a high-risk quarantine pathogen of A2 status in the European and Mediterranean Plant Protection Organization (EPPO).  In the U.S., USDA APHIS regulates the movement of nursery stock and restricts movement of forest stock and wood products from areas within quarantine.        

Management of P. ramorum and its infection cycles is multi-faceted and requires the coordinated efforts of private industry and government regulatory agencies, local, state and federal.  Management protocol includes a combination of material quarantines, eradication of infected hosts and exclusion from areas free of P. ramorum (Figure 7.).  The cornerstone of this protocol is successful diagnosis of infections caused by P. ramorum specifically; symptomology on foliar hosts is indistinguishable from that caused by abiotic conditions and foliar pathogens exempt from quarantine status.  Therefore, species level diagnosis at intercept locations is indispensable to preventing outbreaks while allowing for the unimpeded movement of nursery products originating within regions of quarantine.

Current identification protocol for P. ramorum can include 1.) genus-level detection in the field on regulated plant tissue with a lateral flow device, 2.) genus-level confirmation in regional laboratories on plant tissue with an ELISA product, and 3.) species identification via approved qPCR technology.  Agdia’s new AmplifyRP® XRT assay for identification of Phytophthora ramorum was developed to function as a frontline diagnostic for all links in the regulatory chain.  It will provide a new tool for use in the ongoing effort to minimize the human-assisted spread of P. ramorum while simplifying the identification process for regulators and all those who rely on the timely delivery of regulated items as means of income.

AmplifyRP® XRT technology promotes the rapid amplification and detection of nucleic acid targets, DNA or RNA, while maintaining a single operating temperature of 42oC.  The AmplifyRP® XRT products achieve target sensitivity and specificity comparable to qPCR 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, 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 (Figure 8.).

Agdia’s new assay is specific for P. ramorum and will detect both European lineages and both North American lineages.  This product was validated against a comprehensive panel of potential cross reactors, including numerous species of PhytophthoraPhytopythium and Pythium, and no cross reactions were observed.  This product was developed to be used with leaf tissue.

About Agdia

A leading provider of diagnostic solutions for agriculture, Agdia, Inc. has been serving plant breeders, propagators, growers, universities, regulatory organizations and private testing laboratories since 1981.  The company offers a comprehensive portfolio of validated, easy-to-use diagnostics for identifying plant pathogens, plant hormones, and transgenic traits.  Furthermore, Agdia operates an ISO accredited, in-house, testing services laboratory.  For more information on Agdia’s full line of products, visit the company’s website at www.agdia.com

AmplifyRP® and AmpliFire® are registered trademarks of Agdia, Inc.

Agdia Commercializes ImmunoStrip® Product for Detection of Corn Belt Pathogen Causing Stewart’s Wilt

An image of Agdia's new ImmunoStrip lateral flow device for Pantoea stewartii subsp. stewartii

Figure 1. Positive result observed using new ImmunoStrip® for Pantoea stewartii subsp. stewartii (PStew)

Agdia, Inc. (Elkhart, IN) is happy to announce the commercialization of an ImmunoStrip® assay for the detection of Pantoea stewartii subsp. stewartii, the causal agent of Stewart’s wilt on corn.

Pantoea stewartii subsp. stewartii (Syn. Erwinia stewartii), previously Pseudomonas stewartii, is the species of bacterium responsible for Stewart’s wilt on field or dent corn (Zea mays), sweetcorn (Zea mays subsp. mays) and popcorn (Zea mays subsp. everta).  Disease symptomology was first observed in the late 19th century; however, definitive claim to the initial discovery is subject to debate.  In the late 1880’s, T.J. Burrill described a new bacterial disease of field corn in southern Illinois.  Despite isolating bacteria from symptomatic plant tissue, Burrill was unable to inoculate field corn plants for further study.  Thereafter, symptomology was attributed to drought, high pressure from chinch bugs and the unknown bacteria.  Many believe Burrill’s was the first description of what we now know to be Stewart’s wilt.

In 1895, F.C. Stewart described a bacterial wilt disease of sweet corn in New York.  Stewart was able to isolate the bacterium and complete Koch’s postulates on sweet corn, thus allowing for his name on the pathogen and the disease.  Initially, the genera was Pseudomonas, and Stewart believed seed to be the primary means of dissemination.  It was not until 1923 that the corn flea beetle, Chaetocnema pulicaria (Coleoptera) was identified as the primary vector of the bacterium, with seed playing a lesser role in the epidemiology of Stewart’s wilt.

Since its characterization, P. stewartii subsp. stewartii has been confirmed in several countries.  Nevertheless, these occurrences are infrequent, and the pathogen has not established itself outside of North America.  The bacterium is endemic throughout the Ohio River Valley, Mid-Atlantic region and southern corn belt states, and its distribution coincides directly with that of its insect vector.  All types of corn can be infected, but sweet corn and popcorn varieties experience greater losses than hybrid field corn varieties.  This is due primarily to the durable levels of resistance that have been bred into field corn hybrids, making notable yield losses sporadic in North America.  Varying levels of resistance are available in sweet corn and popcorn; however, susceptible varieties are still widely grown due to their desirable agronomic traits.  Significant outbreaks of Stewart’s wilt are still common in sweet corn and popcorn throughout the pathogen’s range of distribution.

Pantoea stewartii subsp. stewartii overwinters in the digestive tract of adult corn flea beetles, which emerge from the soil in the Spring.  Beetles feed on corn seedlings and transmit the bacterium to feeding wounds via their fecal matter.  Subsequent generations of beetles feed throughout the season, infecting plants in multiple stages of development.  Symptomology phase and severity varies according to varietal resistance and growth stage at time of infection.  Systemic vascular wilt occurs in susceptible varieties when plants are infected as seedlings, resulting in overall plant collapse and death.  Leaf blight occurs in later stages of plant maturity and can affect varieties with variable levels of resistance.  Leaf blight symptomology includes water-soaked lesions, chlorotic and necrotic streaking, and leaf deformation.  Lesions can coalesce to encompass total leaf area in susceptible varieties, predisposing plants to opportunistic fungal pathogens.  Moreover, susceptible varieties can experience systemic wilt as late as the V9 stage, whereas infections typically remain localized on resistant varieties throughout the season.

Seed transmission of P. stewartii subsp. stewartii is possible and inversely proportional to the level of resistance present in the variety.  Many hybrid field corn varieties are highly to moderately resistant to Stewart’s wilt, making seed transmission insignificant.  In contrast, sweet corn varieties are moderately resistant to highly susceptible.  Nevertheless, seed transmission is unimportant to the epidemiology of Stewart’s wilt in areas where the bacterium and insect vector are endemic.  Several countries place some level of quarantine restriction on corn seed for P. stewartii subsp. stewartii.

While domesticated corn varieties are the most important hosts for the bacterium, it has been isolated from several species within Poaceae, including common wheat (Triticum aestivum), eastern gamagrass (Tripsacum dactyloides), rice (Oryza sativa), Sudan grass (Sorghum sudanense) sugarcane (Saccharum officinarum) and corn’s wild progenitor, teosinte (Zea mays subsp. mexicana).  It is possible many of these function as weak alternative hosts for P. stewartii subsp. stewartii.  Moreover, the bacterium is documented to cause Stewart’s wilt on lucky bamboo (Dracaena sanderiana) and bronzing disease on the dicot jackfruit (Artocarpus heterophyllus).

Agdia’s new ImmunoStrip® for detection of P. stewartii subsp. stewartii was developed to detect multiple isolates of the bacterium from a wide geographic range.  Moreover, no cross-reactivity was observed with an extensive exclusivity panel, including Clavibacter michiganensis subsp. insidiosusClavibacter michiganensis subsp. nebraskensisClavibacter michiganensis subsp. tesselariusPseudomonas syringae pv. syringaeRalstonia solanacearumStenotrophomonas maltophiliaXanthomonas albilineans and Xanthomonas campestris pv. campestris.  Cross reactions were observed with Erwinia amylovoraPantoea agglomerans and Pantoea ananatis.  This ImmunoStrip® can be used to test leaf and stem material.

The Pantoea stewartii subsp. stewartii ImmunoStrip® is sold in kits of 5 or 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on all purchased products.  A diagnostic assay for the detection of this pathogen is also available in an enzyme linked immunosorbent assay (ELISA) format.  For more information on these products, in addition to Agdia’s full catalog of pathogen detection products, visit the company’s website at www.agdia.com

Agdia Launches Rapid Molecular Test Kit for Tomato Mottle Mosaic Virus (ToMMV)

Agdia, Inc. (Elkhart, IN) is happy to announce the commercial launch of a rapid, user-friendly, RNA-based assay, on their AmplifyRP® XRT platform, for the detection of Tomato mottle mosaic virus (ToMMV)

Global tomato and pepper production has been significantly disrupted in recent years by emerging pathogens. One such pathogen, Tomato brown rugose fruit virus (ToBRFV, Tobamovirus), is thought to have caused billions of dollars in damage to the tomato industry alone over the past few years. While advancements in breeding for pathogen resistance traits over the past two decades have largely protected tomato and pepper crop production from viral threats, ToBRFV was able to bypass resistance with devastating consequences. The virus continues to cause disruption of the global seed supply chain along with affecting yield and marketability of tomato fruit when not properly excluded from production facilities.

Much like ToBRFV, Tomato mottle mosaic virus is also able to break through well-established viral resistance traits, and thus represents yet another significant threat to tomato and pepper production worldwide.

Initially found in tomato crops in Mexico in 2013, Tomato mottle mosaic virus has since been detected in the United States, Brazil, Europe, Africa, Asia and Iran. Several other Tobamovirus-infected samples collected prior to 2013 which were previously attributed to Tobacco mosaic virus (TMV) or Tomato mosaic virus (ToMV) have since been distinguished as ToMMV infections via high-specificity molecular methods which were not previously available.

Symptoms caused by ToMMV infection include mottling, necrosis, flower abortion and leaf distortion. Much like other Tobamoviruses, ToMMV is highly transmissible via mechanical means (pruning, harvesting, etc.) and may also be present in seed, although further studies are needed to demonstrate whether vertical transmission occurs at any significant level.

Agdia’s AmplifyRP® XRT for ToMMV has been validated for use with tomato and pepper seeds and leaf in addition to other secondary matrixes such as peas (Pisum sativum) and petunia. As a rapid, field-deployable molecular method requiring far less training than traditional PCR methods, this assay provides users with greater flexibility to deploy detection capabilities where they need it, when they need it. Use cases for this assay include, but are not limited to:

  • In-field monitoring at remote production sites as a stand-alone assay.
  • Screening incoming plantlets & monitoring production crops in commercial greenhouses
  • Laboratory-based molecular diagnosis with crude or purified extracts with faster time-to-result than traditional PCR or qPCR methods. This assay can be used with Agdia’s AmpliFire® isothermal fluorometer or with most real-time PCR machines.

Agdia’s AmplifyRP® XRT for ToMMV is highly specific to ToMMV and has been proven through experimentation and in-silico analysis to detect isolates from around the world. No cross-reactivity was observed with high titer samples from other Tobamoviruses, including Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Tomato brown rugose fruit virus (ToBRFV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Zucchini Green Mottle Mosaic Virus (ZGMMV) and more.

Agdia Expands ImmunoStrip® Catalog with Release of Lettuce Mosaic Virus Assay, Improves LMV ELISA Assay

Image of lettuce with lettuce mosaic virus symptoms

Figure 1. Symptoms of LMV infection on lettuce. Image courtesy of Agdia EMEA

Agdia, Inc. (Elkhart, IN), a leading provider of plant diagnostic products and services since 1981, is happy to announce the release of a new ImmunoStrip® product for detection of Lettuce mosaic virus and the improvement of their existing ELISA product.

Lettuce mosaic virus (LMV, Potyvirus) is considered one of the most significant pathogens of lettuce worldwide due to its potential to cause total crop loss.  This virus was first described in 1921 infecting field-grown lettuce in Florida.  Since then, LMV has spread to all lettuce-producing regions across the globe on every continent, excluding Antarctica.  All lettuce types are subject to LMV infection, as are several agronomically important cultivated hosts, including chickpea, common pea, endive, safflower and spinach.  Furthermore, LMV is known to infect multiple cultivated ornamental species and common weedy plants found in close proximity to lettuce fields worldwide.  In addition to widespread incidence and potential for high virulence in lettuce crops, the LMV-lettuce pathosystem is considered an important model for studying host-virus interactions.

Symptoms of LMV infection vary by host species, environmental conditions, isolate of LMV causing infection and the stage of growth the host was in when infection occurred.  Furthermore, the variety of lettuce and corresponding presence of resistance genes therein contribute greatly to symptom severity.  Typical symptoms on lettuce include chlorosis, failure to form heads, leaf distortion, mosaic, necrosis and vein clearing (Figure 1.).  Severe necrosis and plant death are commonly observed in susceptible varieties, whereas tolerant and resistant varieties can exhibit low titer infections with no symptoms to no detectable viral infection, respectively.  Current LMV resistance is conferred by the recessive genes mo11 and mo12, and both have remained relatively durable since their introduction.  Nevertheless, these genes are absent in many lettuce varieties, and LMV isolates capable of breaking resistance when present have been documented for several decades.

Lettuce mosaic virus is a member of the genus Potyvirus and vectored locally by several species of aphids (Hemiptera) in a non-persistent, non-circulative manner.  Infection is not limited to a specific plant tissue.  Like other viruses transmitted in this manner, LMV transmission efficacy is short-lived in the aphid vector, but efficient, nevertheless.  Aphid vectors acquire LMV within seconds through stylet probes of plant tissue.  Virions are localized for transmission within the aphid’s stylet via what is referred to as the “helper strategy.” In this scenario, a protein located on the viral capsid (CP, capsid protein) binds to a secondary non-structural protein known as HC-Pro (helper component proteinase), which forms a reversible bridge with an insect protein located on the cuticular lining of the stylet.  The HC-Pro molecule is encoded by the virus and must be acquired before or at the same time as the virus to be effective.  This strategy differs from other non-persistent viruses such as Cucumber mosaic virus and Alfalfa mosaic virus, whose capsid proteins bind directly to the stylet protein.

New Agdia ImmunoStrip for Lettuce mosaic virus

Following virus acquisition, aphids must emigrate from infected plants and feed on healthy plants within a few hours for horizontal LMV transmission to occur.  It is well documented that plant viruses interact with hosts and vectors on molecular levels, manipulating both to facilitate the most efficient virus acquisition and transmission.  This is true for potyviruses; however, unique host-virus-vector interactions are context-specific.  In general, infected hosts produce altered chemical compounds to increase vector immigration to and shorten feeding times on infected plants.  Furthermore, viruliferous aphids have experienced increased fitness and increased numbers of winged adults within the population.  In combination, this manipulation of the disease cycle by the causal agent facilitates the most efficient spread of non-persistent viruses.

Lettuce mosaic virus is spread long distances via the movement of infected seed and transplants, to a lesser extent.  Up to 10% of market seed produced by infected mother plants will be infected, with detectable virions in the pericarp, endosperm and developing cotyledons.

Field-grown lettuce is typically sown directly into prepared seed beds using pelleted seed in specialized planters.  Infected seeds germinate and serve as primary inoculum for probing aphids.  In areas of high aphid populations, relatively low plant infection rates can facilitate catastrophic epidemics within and between neighboring fields.  Furthermore, the persistence of LMV in weedy hosts in the absence of cultivated hosts makes management of LMV a multi-faceted exercise, including planting of certified virus-free seed and transplants, use of resistant varieties, fallow periods, vector management and removal of alternative hosts.

Figure 2. Interpretation of ImmunoStrip® results

Agdia states their new ImmunoStrip® for detection of LMV was screened against a diverse inclusivity panel of LMV isolates from multiple geographic regions, including a resistance-breaking isolate.  The strip detected all true positives.  Agdia states no cross-reactivity was observed for several potential cross-reactors, including Beet western yellows virus, Broad bean wilt virus 1 and 2, Cauliflower Mosaic Virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tomato bushy stunt virus, Tomato spotted wilt virus and Turnip mosaic virus.  A slight cross-reaction was observed with Bidens mottle virus.  This product was developed to be used with leaf and seed matrices and will include separate applicable protocols.Positive vs negative results interpretation of ImmunoStrips

Agdia’s ImmunoStrip® platform provides end-users with unrivaled utility; samples can be tested in the field or lab by those having no previous diagnostic experience, and results are visualized within 30 minutes.  Furthermore, this product includes everything necessary to perform a test and does not require special equipment.  Test protocol is simple and includes 1) Sample collection and extraction in Agdia buffer bags, 2) Exposing the ImmunoStrip® to the sample extract, and 3) Allowing results to develop.  Test results are visualized as a single control line or a control and test line for negative or positive results, respectively (Figure 3.).  The introduction of Agdia’s LMV ImmunoStrip® expands their catalog to 49 plant pathogen products on this platform.  In addition to the new ImmunoStrip®, Agdia is concurrently releasing an improved version of their LMV ELISA assay with greater analytical sensitivity than the original version.  Furthermore, the new ELISA will detect a Greek isolate of LMV missed by multiple other commercially available LMV ELISA tests.

The Lettuce mosaic virus ImmunoStrip® is sold in kits of 5 or 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on all purchased products.  For more information on ImmunoStrip® and ELISA products, please see Agdia’s full catalog at the company’s website www.agdia.com

Agdia Releases ImmunoStrip® for Detection of Alfalfa mosaic virus (AMV)

Symptoms of AMV infection on eggplant

Figure 1. Symptoms of AMV infection on eggplant Image courtesy of Whitney Cranshaw, Colorado State University, Bugwood.org

Agdia, Inc. (Elkhart, IN), a leading provider of plant pathogen diagnostic products and services, is happy to announce the commercialization of a rapid, user-friendly product for detection of Alfalfa mosaic virus on their popular ImmunoStrip® platform.

Alfalfa mosaic virus (AMV) is the type member and singular species within the Alfamovirus genus, which is included in the Bromoviridae family with Bromovirus, Cucumovirus, Ilarvirus and Oleavirus genera.  Symptomology was first described in 1931 on alfalfa plants (Medicago sativa) in California, and AMV is now confirmed to infect more than 600 species of plants, causing significant crop losses in many.  Hosts include many economically important species within Amaranthaceae, Asteraceae, Fabaceae, Lamiaceae and Solanaceae, in addition to several weedy species common in regions of intensive agriculture and horticulture.  This virus is now widespread throughout Africa, Asia, Europe, North America, Oceania and South America.

Symptoms vary by host, environment, plant age and strain of AMV.  Susceptible varieties of alfalfa, also known as lucerne, can express symptoms of light green or yellow mottling and mosaic, localized and systemic necrosis, leaf and petiole distortion, and stunting.  Symptoms can be more pronounced during Spring and Fall, becoming inconspicuous in Summer months.  Furthermore, AMV infections can weaken plants and predispose stands to drought stress and cold damage.  Solanaceous plants will typically express foliar symptoms of yellow to white mosaic and deformation (Figure 1. and Figure 2.).  In addition, tomatoes can develop fruit discoloration and necrosis, and lethal vascular necrosis.  Peppers can exhibit stunted fruit, while potatoes can develop plant stunting, tuber deformation and necrosis when infected, rendering market products unsalable.

Alfalfa mosaic virus can cause similar foliar and stunting symptoms in annual bedding plants, herbaceous perennials and woody ornamentals, including Dianthus, Hibiscus, Hydrangea, Impatiens, Lavandula, Peonia, Petunia and Phlox.  Furthermore, AMV is known to infect industrial hemp (Cannabis sativa), causing many in the broader cannabis production markets to screen mother plants, propagative materials and mature plant inventories for this pathogen.

Several species of aphids (Hemiptera) are known to vector AMV in a nonpersistent, noncirculative manner.  Virus acquisition time from infected plants is brief, taking seconds to minutes.  The subsequent relationship between the aphid vector and AMV is transient in nature; transmissible virus particles do not enter vector cells, nor do they circulate within the vector.  Virions are retained at surface sites within the stylet through direct protein interactions with the virus capsid.  Thereafter, aphids remain viruliferous for short periods of time and inoculation efficacy of healthy plants is short-lived.  Nevertheless, due to their polyphagous nature, high fecundity, winged forms capable of dispersion and highly efficient feeding apparatuses, aphids are the most important method of local AMV transmission in many crops.

In addition to aphid vectors, AMV can be spread via mechanical transmission, dispersion of infected pollen and movement of infected seed.  Nevertheless, AMV is not seed-transmitted in all hosts, and localized outbreaks are typically the result of a combination of dispersal paradigms.  Therefore, these epidemics are difficult to predict, and management is multi-faceted.  Long-distance dispersal of AMV occurs via the interstate movement of infected propagative materials.  Diagnostic screening of all propagative materials, including seeds, cuttings and tissue culture products, is the most effective method for limiting the movement of AMV over long distances.

Agdia states their new ImmunoStrip® product for detection of Alfalfa mosaic virus was screened against a diverse group of isolates in Subgroup I from several geographic locations.  Furthermore, no cross reactivity was observed when tested against a broad specificity panel, including Apple mosaic virus, Arabis mosaic virus, Bean common mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Soybean mosaic virus, Tobacco mosaic virus, Tomato mosaic virus, Tobacco ringspot virus, Tobacco streak virus and Tomato spotted wilt virus.  This assay was developed to be used with leaf tissue for all hosts and was validated for use on alfalfa, pepper, soybean and tomato seeds.

Agdia’s ImmunoStrip® platform provides users with a powerful diagnostic tool, as it can be used as an effective pathogen screen in the field and lab, requiring no special equipment or diagnostic experience.  Results are obtained in 30 minutes or less and interpretation is simple.  Results appear as a control and test line, indicating a positive, or a control line only, indicating a negative (Figure 3.).

The Alfalfa mosaic virus ImmunoStrip® is sold in kits of 5 or 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on purchased kits.  A diagnostic assay for the detection of AMV is also available in an enzyme linked immunosorbent assay (ELISA) format.  For more information on these products, in addition to Agdia’s full catalog of pathogen detection products, visit the company’s website at www.agdia.com

Agdia Releases Rapid Molecular Diagnostic for Detection of Emerging Viral Pathogen in Cannabis

The legal production and marketing of the cannabis plant (Cannabis sativa and C. indica) and its derivatives in the U.S. have increased dramatically over the previous decade due to ratification of medical and recreational licensing within States.  Furthermore, industrial hemp cultivation has experienced a synchronous resurgence, owing to Federal recognition of its status as a legal agricultural crop.  Increased production and sales have provided economic benefits to many, including entrepreneurs, engineers, farmers, plant scientists and general labor seeking positions in an industry once maligned.  In addition to the fiscal benefits of market growth, the medicinal value of Cannabis has been realized by many seeking alternatives to prescription pharmaceuticals for treatment of assorted medical conditions.

As the volume of indoor and field plant inventories has increased, disease management science in Cannabis has struggled to keep pace.  Research on Cannabis as a pathosystem is relatively new, and plant disease symptoms hitherto unnoticed have become limiting factors to production.  Nevertheless, legalization of this crop has incentivized private companies and public research institutions to provide market stakeholders with practical disease diagnostic products and services.  Furthermore, peer-reviewed publications examining the epidemiology, symptomology and management of pathogens affecting Cannabis are more widespread than ever before.  Consequently, the suite of identified pathogens affecting cannabis production is expanding, along with our understanding of best management practices.

Lettuce chlorosis virus (LCV) is a member of the Crinivirus genus and known to cause disease on several economically important plant species.  This virus was first characterized in the early 1990’s, infecting sugar beets and lettuce (Figure 1) in southern California, where it is considered endemic.  Since then, LCV has been identified infecting additional hosts, including common bean in Spain, papaya in Texas, ornamental periwinkle in China, Israel and Brazil; tomato in China and several weedy species worldwide.  Furthermore, LCV was recently identified as the causal agent of severe disease symptomology on glasshouse-grown medicinal Cannabis at an authorized farm in Israel.  Symptoms of infection on Cannabis include interveinal and full-leaf chlorosis, leaf distortion, necrosis, purple leaf discoloration and stunting (Figures 2 and 3).  Collectively, the symptomology decreases quantity and quality of flower yield and processed derivatives.  These symptoms are indistinguishable from those caused by abiotic physiological disorders and nutrient imbalances.

Figure 2: Images showing symptoms from the Cannabis isolate of Lettuce chlorosis virus (LCV-can) transmitted via shoots. (a) Stunted growth of infected plant (left) compared to the growth of healthy plant (right). (b) Chlorosis of infected leaves (c) Healthy leaves. (e) Symptoms of propagated infected plants. (f) A symptomatic flower of LCV infected Cannabis plant dissected to four samples (1–4). Image provided courtesy of Dr. Aviv Dombrovsky, The Volcani Center.

Lettuce chlorosis virus is spread locally via the feeding activities of Bemisia tabaci, the silverleaf whitefly (Hemiptera), in a semi-persistent manner.  This is a phloem-limited virus and is transmitted efficiently within 24 hours of vector acquisition.  Since the late 20th century, the expansion of whitefly populations in sub-tropical and temperate climates has facilitated the synchronous expansion of their vectored viruses and plant host ranges.  Furthermore, whiteflies can exhibit prohibitive levels of resistance to many traditional insecticides.  The range and diversity of host-virus-vector interactions combined with pesticide resistance can potentially precipitate epidemics previously not witnessed in regions of whitefly expansion.  These include areas of cannabis and industrial hemp production in the U.S.

Cannabis plants produced as marijuana are propagated continually via vegetative cuttings taken from “mother plants.”  Mother plants infected with LCV can remain asymptomatic indefinitely, passing the latent virus to cuttings.  The long-distance movement of infected cuttings is the primary means of LCV dissemination to areas beyond whitefly expansion in regions where cultivated hosts are readily available.  The cultivation of industrial hemp differs from that of marijuana.  Hemp crops are often seeded directly to field soils, and plant products typically include fiber, oil, seed and processed cannabidiol (CBD).  Nevertheless, hemp plants are subject to LCV infection and the accompanying decrease in yield.  It is unclear if LCV can be transmitted efficiently on tools or in seed, and further investigation of its epidemiology is ongoing.

Figure 3: Symptoms of LCV on old leaves of vegetative-stage cannabis plants. (a) Yellowing leaves with necrosis. (b) Purple leaves. (c) Chlorotic leaves. (d) Interveinal yellowing leaves with necrosis, (e) Interveinal yellowing leaves. (f) Healthy cannabis leaves. Image provided courtesy of Dr. Aviv Dombrovsky, The Volcani Center.

No therapeutics exist for viral infections in mature plants.  Therefore, exclusion is the most important virus management strategy.  Diagnostic testing is the cornerstone of an effective management program, as it is the primary means of identifying viruses and excluding infected plants before introduction into plant inventories and shipment of propagative materials.  The burgeoning industry of cannabis production, marijuana and industrial hemp, has elucidated the necessity of reliable and robust diagnostic solutions for detection of LCV in plant inventories.  Furthermore, regulations regarding the interstate movement of Cannabis have constrained many growers to identify infections at farm level, making the field-based usability of a diagnostic product indispensable.

Agdia’s new AmplifyRP® XRT assay for detection of Lettuce chlorosis virus is based on recombinase polymerase amplification (RPA).  This isothermal technology promotes the rapid amplification and detection of nucleic acid targets, DNA or RNA, at a single operating temperature of 42o C.  When paired with Agdia’s AmpliFire® isothermal fluorometer, 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, while providing target sensitivity and specificity comparable to those of PCR.

Agdia states their assay was screened against a diverse specificity panel of confirmed isolates of LCV, including those from Brazil, China, Israel, Mexico, Spain and the U.S.  A 100% detection rate of true positives was reported.  Furthermore, no cross reactivity was observed with an extensive panel of viral and viroid pathogens, including Alfalfa mosaic virus, Arabis mosaic virus, Beet curly top virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Hop latent viroid, Hop stunt viroid, Tobacco mosaic virus, Tobacco ringspot virus, Tobacco streak virus, Tomato mosaic virus, Tomato ringspot virus and Tomato spotted wilt virus.  This assay contains an endogenous RNA control and was developed to be used on leaf, petiole, seed and stem tissue.

The introduction of Agdia’s LCV AmplifyRP® XRT assay expands Agdia’s catalog to include 22 products on this platform.  In addition to LCV, Agdia now offers AmplifyRP® XRT products for detection of Beet curly top virusFusarium oxysporum, Hop latent viroid and Hop stunt viroid, for a total of five of the most widespread and emerging pathogens in the cannabis industry.  “Market demand for highly sensitive and specific products that can be used across multiple locations by personnel with diverse backgrounds in diagnostic testing has fueled development.  Furthermore, as new pathogens emerge, we will keep pace with industry needs and concerns and encourage feedback from industry stakeholders to inform innovation on product offerings,” said Robert Emmitt, Domestic Account Manager, Agdia, Inc.  For more information on Agdia’s complete line of AmplifyRP® assays, please visit Agdia’s website www.agdia.com.

Agdia Commercializes ImmunoStrip® for Detection of Nemesia ring necrosis virus (NeRNV)

Agdia, Inc. (Elkhart, IN) is happy to announce the introduction of a rapid, user-friendly diagnostic product on their ImmunoStrip® platform for the detection of Nemesia ring necrosis virus.

Nemesia ring necrosis virus (NeRNV) is a member of the Tymovirus genus.  This virus has a relatively narrow confirmed host range, infecting ornamental varieties of Alonsoa, Diascia, Lobelia, Nemesia, Sutera and Verbena.  Infection symptomology was reported initially in Germany in 2000.  Since then, NeRNV infections have been confirmed in New Zealand, the United Kingdom and the United States, while the virus is thought to be widespread throughout Europe and North America.  Symptomology varies by host and ranges from mild foliar mosaic and chlorotic flecks to severe leaf necrosis, rendering plants unmarketable.  Furthermore, latent infections are commonplace and perpetuate the unintentional dissemination of infected propagative materials.

Nemesia ring necrosis virus can be spread locally via mechanical transmission and the feeding behavior of several species of herbivorous beetles in the families Chrysomelidae (leaf beetles) and Curculionidae (snout beetles or weevils).  Unlike many arthropod viral vectors, such as aphids, thrips and whiteflies, beetles do not transmit viruses in their saliva.  While feeding, beetles regurgitate foregut-borne viruliferous sap, bathing their mouthparts and transferring the virus in a semi-persistent manner to feeding wounds on healthy plants.  Beetles can acquire NeRNV in a single bite and transmit it in kind.  Furthermore, NeRNV is spread efficiently on contaminated tools used throughout production processes.  Consequently, vector management, greenhouse sanitation and implement sterilization are critical to managing local epidemics.

The long-distance dispersal of NeRNV is through domestic and international shipping of infected propagative materials from initial and secondary sources of infection.  The ornamental host species for NeRNV are propagated vegetatively through tissue culture and leaf and stem cuttings. These materials can be latently infected with NeRNV at the initial production facility.  Viruliferous plant parts may be shipped to secondary producers for finishing and the subsequent shipment to wholesale nurseries, garden centers and big-box retail outlets.  No therapeutics are available for plant viruses, and exclusion is the best management practice.  Diagnostic testing of plant materials is the primary means of identifying viruses at initial and secondary production steps and excluding infected plants before introduction into production inventories. Therefore, testing of all propagative materials is paramount to the management of NeRNV industry wide.

Agdia’s new ImmunoStrip® for detection Nemesia ring necrosis virus was evaluated against a diverse panel of NeRNV isolates from multiple geographic regions and detected all true positives.  Furthermore, Agdia states no cross-reactivity was observed with several potential cross-reactors widespread in ornamental production, including Alfalfa mosaic virus, Alternanthera mosaic virus, Angelonia flower break virus, Calibrachoa mottle virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Tobacco streak virus, Tomato aspermy virus, Tomato spotted wilt virus and Turnip yellow mosaic virus.  This assay does exhibit cross reactivity with Scrophularia mottle virus.  This assay was developed to be used with leaf, stem and petiole sample material.

Introduction of the NeRNV ImmunoStrip® expands Agdia’s catalog to include 47 plant pathogen products on this platform.  Agdia’s ImmunoStrip® platform provides end-users with unparalleled utility, and a high level of market demand for field-deployable, plant pathogen detection products has driven and informed this output.

The Nemesia ring necrosis virus ImmunoStrip® is sold in kits of 5 or 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on purchased kits.  A diagnostic assay for the detection of this virus is also available in an enzyme linked immunosorbent assay (ELISA) format.  For more information on these products, in addition to Agdia’s full catalog of pathogen detection products, visit the company’s website at www.agdia.com

Agdia Releases Rapid Molecular Test for Detection of Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV)

Agdia, Inc. is pleased to announce the launch of a rapid molecular diagnostic test for the detection of Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV). In addition to the do-it-yourself (DIY) test kit, detection of these pathogens is now offered as a service in Agdia’s Testing Services division for customers who wish to submit plant samples to an ISO 17025-accredited laboratory.

Dahlia mosaic virus (DMV) is the causal agent of dahlia mosaic disease and a member of the Caulimovirus genus.  In addition to DMV, a second CaulimovirusDahlia common mosaic virus (DCMV), was identified in direct association with disease symptomology.  Like DMV, DCMV is a circular, double-stranded DNA virus.  Neither virus is limited to a geographical region, and both pathogens are widespread in cultivated dahlias, with mixed infections being common.

Image of vein-clearing symptoms caused by Dahlia mosaic virus. Photo provided courtesy of Claudia Nischwitz and Melanie Stock, Utah State University

Fig. 1: Vein-clearing caused by Dahlia mosaic virus (Photo provided courtesy of Claudia Nischwitz and Melanie Stock, Utah State University)

 

Transmission of both viruses occurs via mechanical means (grafting, propagation cuttings, etc.).  Furthermore, both viruses are transmitted in a non-persistent or semi-persistent manner by aphids after feeding on infected plants.

Symptoms vary by cultivar but typically include random chlorophyll loss (chlorosis), vein clearing, necrotic lesions, leaf distortion and general stunting. Stunting of the plant often inhibits or prohibits flowering, rendering plants unmarketable.  Some cultivars can exhibit mild symptoms or remain asymptomatic indefinitely.  Early and accurate detection of DMV and DCMV is paramount to management of these viruses throughout dahlia cultivation.

Agdia’s new AmplifyRP® XRT assay for detection of DMV and DCMV 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 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.  When paired with Agdia’s AmpliFire® isothermal fluorometer, the XRT system is a user-friendly tool that can be implemented in the field or the lab by personnel with limited experience in molecular diagnostics. Total assay time is less than 30 minutes when used with the AmpliFire® as a real-time assay.

Lesions on unripe tomato fruit
Fig. 2: Symptoms caused by Dahlia mosaic virus.
Photo provided courtesy of Claudia Nischwitz and Melanie Stock, Utah State University.

Agdia states their new assay was validated against a diverse panel of DMV/DCMV isolates from multiple geographic regions, detecting all true positives.  Sensitivity for this assay is comparable to that observed with published conventional PCR, qPCR and LAMP assays to which it was compared.  Furthermore, this product exhibited a high level of exclusivity against a comprehensive panel of potential cross reactors, including Carnation etched ring virus, Cauliflower mosaic virus, Chrysanthemum stunt viroid, Cucumber mosaic virus, Dahlia latent viroid, Impatiens Necrotic Spot Virus, Potato virus Y, Rudbeckia flower distortion virus, Tobacco streak virus and Tomato Spotted Wilt Virus.  This assay does not detect the endogenous pararetroviral element (DMV-D10).  This assay includes an endogenous internal DNA control and was developed to be used with flower, leaf, petiole, root, seed, stem and tuber tissue.

About Agdia

A leading provider of diagnostic solutions for agriculture, Agdia, Inc. has been serving plant breeders, propagators, growers, universities, regulatory organizations and private testing laboratories since 1981.  The company offers a comprehensive portfolio of validated, easy-to-use diagnostics for identifying plant pathogens, plant hormones, and transgenic traits.  Furthermore, Agdia operates an ISO accredited, in-house, testing services laboratory.  Agdia’s quality management system is ISO 9001:2015 certified and their Testing Services Laboratory is ISO 17025:2017 accredited.  Visit the company’s website at www.agdia.com

Agdia Releases ELISA and ImmunoStrip® Products for the Detection of Rhizoctonia solani

Agdia, Inc. (Elkhart, IN) is happy to announce the commercialization of assays for detection of Rhizoctonia solani on ELISA and ImmunoStrip® platforms.

Rhizoctonia solani is a necrotrophic fungal pathogen known to inhabit soils throughout most of the planet’s arable regions.  It survives as a strong saprobe but can cause severe disease on numerous economically important crops, including alfalfa, canola, corn, cotton, cucumber, lettuce, potato, soybean, sugar beet, tobacco, tomato, turfgrass and several species of herbaceous and woody ornamentals.Figure 1. Damping-off symptoms on radish seedling. Image courtesy of Tom Creswell, Purdue University

Figure 1. Damping-off symptoms on radish seedling. Image courtesy of Tom Creswell, Purdue University

 

Rhizoctonia solani was first described in 1858 causing disease on potato tubers.  Since then, several species of Rhizoctonia have been identified; however, R. solani is conserved as the type species within the genus.  The genus nomenclature is derived from Greek which translates to “root murder,” while solani refers the Solanaceae family of plants to which potato belongs.  Taxonomically, R. solani exists as a species complex, and isolates are delimited into anastomosis groups (AGs) based on their ability to exchange genetic material via hyphal fusion (anastomosis).  Moreover, each AG can be subdivided according to characteristics such as infection strategy and host range.

Field-level effects of damping-off in sweet potatoes Image courtesy of Edward Sikora, Auburn University.Members of the R. solani complex utilize diverse infection strategies, causing multiple symptomologies on numerous plant species and organs.  Diseases include damping-off of seedlings (figures 1and 2.), root rots and stem lesions (figure 3.), fruit rots, crater rots and black scurf on potato tubers.  Initial infections with these diseases occur in the root zone or at the soil line.  Fruit rots, bottom rots and belly rots are prevalent on organs that remain on or near the soil, including fruits produced by cucurbits, tomatoes and brassica heads.  These diseases, depending on host, are typically catastrophic to the market product.  Symptoms of patch diseases on cool-season and warm-season turfgrasses caused by R. solani include straw-colored lesions on and dieback of individual leaves, respectively.  On close-cut turf, collective infections advance outward radially forming a characteristic patch within the stand (figure 4.).

Root and stem rot on basil Image courtesy of Mary Ann Hansen, Virginia Tech UniversityRhizoctonia solani is a soilborne pathogen that can persist as a saprobe for extended periods of time in the absence of host plants.  During unfavorable growth conditions, the fungus forms compact, hardened masses of hyphae known as sclerotia.  Sclerotia can remain inactive in soils indefinitely and function as primary inoculum when environmental conditions advantageous to growth and host infection return, e.g., elevated temperature and moisture level.  Sclerotia germinate to form infection hyphae that respond to chemical stimuli produced by host root systems and senescing plant organs.  Hyphae grow up chemical gradients, actively penetrate host tissue, kill plant cells via secreted toxins and enzymes, and colonize dead tissue.

Long-distance dispersal of R. solani takes place via the movement of sclerotia in infested soil on tools and farm equipment.  Moreover, infected propagative materials such as bulbs, corms, tubers and seed can serve as vehicles of widespread dissemination.  Localized spread of sclerotia and hyphae occurs via splashing water, flooding and wind-blown soil.  Rhizoctonia solani is not known to produce asexual spores in nature.

Large patch symptoms on zoysia grass Image courtesy of Robert Emmitt, Agdia, Inc.Management of R. solani is difficult due to its persistence in soil and expansive host range.  Fungicide pot drenches on greenhouse crops and turf applications of fungicides can be effective at limiting local epidemics.  Moreover, seed and tuber treatments combined with cultural practices such as planting time and water management are utilized with success for many field crops, including corn, potato and soybean.  Nevertheless, fungicide applications across large acreages are typically ineffective and impractical.  All fungicide treatments and applications increase production costs and must be repeated under high disease pressure to maintain plant health.  Current genetic resistance to this pathogen is scarce, and its role in management is limited overall.  Therefore, exclusion of R. solani through diagnostic testing of plant materials is paramount to managing its spread in many crops.

Agdia states their new ELISA and ImmunoStrip® products for detection of Rhizoctonia solani were screened against a diverse group of isolates representing several anastomosis groups, including AG1, AG2, AG3, AG4 and AG5, detecting all true positives.  Moreover, no cross reactivity was observed with Fusarium spp., Phytophthora spp., Pythium spp. or Thielaviopsis basicola.  Both assays were developed to be used on leaf, root, seed, stem and pure culture.

Agdia’s ImmunoStrip® platform provides end-users with unparalleled utility; samples can be tested in the field or lab by those having no previous diagnostic experience, and results are visualized within no more than 30 minutes.  The ELISA platform has been used for more than 50 years and is widespread throughout the plant diagnostic community.  It is a trusted, lab-based diagnostic that offers unparalleled cost efficiency in high-throughput applications.

Both products include a one-year warranty.  For more information on these products, in addition to Agdia’s full catalog of pathogen detection products, visit the company’s website at www.agdia.com

Agdia Commercializes ImmunoStrip® for Detection of Tomato aspermy virus (TAV)

Agdia, Inc. (Elkhart, IN), a leading provider of plant pathogen diagnostic products and services, is happy to announce the commercialization of a rapid, user-friendly product for detection of Tomato aspermy virus on their popular ImmunoStrip® platform.

Tomato aspermy virus (TAV) is a member of the Cucumovirus genus and infects a wide range of economically important hosts, including Canna, Chrysanthemum, several species of Lilium, pepper, tobacco and tomato.  This virus was first described in 1949 infecting tomato, causing severe leaf distortion and seedless fruit.  Nevertheless, TAV outbreaks are uncommon on this member of Solanaceae, and it is not considered an important virus infecting tomato.  Tomato aspermy virus is widespread in cultivated chrysanthemums, and outbreaks are common throughout regions where they are grown extensively, including Asia, Australia, Europe, New Zealand and North America.

Symptomology of TAV infection on Chrysanthemum varies by cultivar but typically includes plant stunting, chlorotic leaf mottling and severe flower break, dwarfing and distortion.  These symptoms lead to reduction in yield and render plants unmarketable for floral and landscape purposes.  Furthermore, many cultivars are asymptomatic, functioning as latent reservoirs of infected propagative materials.  And, it is through international movement of infected propagative materials, including vegetative cuttings, that long-distance dispersal of TAV is accomplished.  This dissemination paradigm is applicable to all ornamental hosts of this virus.

Tomato aspermy virus is spread locally in fields and greenhouses by several species of aphids (Order Hemiptera).  It is spread in a non-persistent manner, meaning the virus can be acquired from an infected plant via feeding within seconds of stylet insertion.  Shortly thereafter, the virus can be transmitted by the vector to healthy plants, and high populations can lead to local epidemics.  Managing vector populations is important to management of TAV in nursery and greenhouse operations.  Nevertheless, the most effective step in virus management is exclusion of viruses altogether, and introducing healthy propagative material is paramount to an effective management program.  Once infected, plants remain infected for life as no curative therapies are available for viruses.  Diagnostic testing is the primary means of identifying viruses and excluding infected plants before introduction.

Agdia states their new Tomato aspermy virus ImmunoStrip® was evaluated against a diverse panel of TAV isolates from multiple geographic regions, including Asia, Europe and North America.  The assay detected all true positives.  Furthermore, Agdia states no cross-reactivity was observed for several potential cross-reactors, including Alfalfa mosaic virusChrysanthemum virus B, Impatiens necrotic spot virus, Lily symptomless virus, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tobacco streak virus, Tomato bushy stunt virus, Tomato ringspot virus and Tomato spotted wilt virus.  This assay does exhibit mild cross reactivity with Cucumber mosaic virus and Peanut stunt virus.  This assay was developed to be used with leaf and stem material.

Agdia’s ImmunoStrip® platform provides end-users with unparalleled utility; samples can be tested in the field or lab by those having no previous diagnostic experience, and results are visualized within no more than 30 minutes.  Furthermore, these products include everything necessary to perform a test and do not require special equipment.  Test protocol is simple and includes 1) Sample collection and extraction in Agdia buffer bags, 2) Exposing the ImmunoStrip® to the sample extract, and 3) Allowing results to develop.  Test results are visualized as a single control line or a control and test line for negative or positive results, respectively.

The Tomato aspermy virus ImmunoStrip® is sold in kits of 5 or 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on purchased kits.  A diagnostic assay for the detection of TAV is also available in an enzyme linked immunosorbent assay (ELISA) format. For more information on these products, in addition to Agdia’s full catalog of pathogen detection products, visit the company’s website at www.agdia.com

Agdia Releases Rapid Isothermal Assay for Detection of Quarantine Bacterium

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 Ralstonia solanacearum race 3 biovar 2 (RsR3B2).

Ralstonia solanacearum (Rs), formerly known as Pseudomonas solanacearum, causes bacterial wilt in numerous crops of economic importance worldwide. It is the causal agent of diseases including Southern wilt of geranium, Bacterial wilt of tomato, brown rot of potato, Moko disease, Bugtok disease, and Blood Disease of banana.

Ralstonia solanacearum is listed as an A2 (high risk) quarantine pathogen in the European and Mediterranean Plant Protection Organization (EPPO). In the United States, Ralstonia solanacearum race 3 biovar 2 is a Select Agent due to the potential impact on the domestic agricultural industry.

In 2020, Ralstonia solanacearum race 3 biovar 2 was detected in a U.S. greenhouse for the first time in 16 years. The infestation was traced back to geranium cuttings from an offshore location. Extensive testing and prompt mitigation efforts were employed to successfully eliminate the pathogen.

Agdia’s new AmplifyRP® XRT assay for detection of Ralstonia solanacearum race 3 biovar 2 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 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.  When paired with Agdia’s AmpliFire® isothermal fluorometer (figure 1), the XRT system is a user-friendly tool that can be implemented in the field or the lab by personnel with limited experience in molecular diagnostics. Total assay time is less than 30 minutes when used with the AmpliFire® as a real-time assay.

Extensive product validation was conducted to demonstrate fitness for purpose. Agdia states their new assay was screened against 62 bacterial cultures representative of other races and biovars of Rs in addition to 117 cultures of other relevant bacterial species. No cross-reactivity was observed, confirming exclusive specificity of the assay to RsR3B2. Additionally, no host reactions were observed in validation testing against geranium, tomato, potato, pepper, ginger, banana or eggplant samples. The new assay successfully detected all 19 distinct cultures of Rs race 3, biovar 2.

Note: While the new assay reliably detects Ralstonia solanacearum race 3 biovar 2, it does not change the current requirement in the United States for confirmation testing of positive R. solanacearum samples to take place in USDA-APHIS-PPQ’s Beltsville Laboratory.

Agdia Expands AmplifyRP® XRT Product Catalog with Release of Rapid Isothermal Product for Detection of Fusarium Wilt Pathogen

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 Fusarium oxysporum.

The Fusarium oxysporum species is a ubiquitous fungal inhabitant of soils throughout the world.  Members of this species complex are commonplace in the microbial communities of plant rhizospheres of cultivated crops, monocots and dicots, ranging from tropical to temperate climates.  While many strains of F. oxysporum are harmless saprobes, others are considered significant plant pathogens, even limiting factors to crop production.

The pathogens in this genus have an extensive host range at the species level.  However, individual strains of F. oxysporum, known independently as a forma specialis (f. sp.; plural: formae speciales, ff. spp.) or special form(s), exhibit highly selective host pathogenicity, typically infecting no more than a few species of plants.  Collectively, there are more than 100 recognized formae speciales of F. oxysporum, causing vascular wilt or root and crown rot on several economically important crops, including asparagus, banana, cannabis, chrysanthemum, common bean, cotton, lettuce, melon, soybean, strawberry, tomato and several cultivated members of Orchidaceae.

Amongst the symptomology induced by F. oxysporum, vascular wilts are most common.  Fungal mycelia are soil-borne and enter the plant via natural openings or damaged tissue in the root system.  Thereafter, the pathogen invades the xylem tissue and grows acropetally, clogging xylem vessels, producing microconidia and impeding the upward movement of water and nutrients.  Infected plants display severe chlorosis, unilateral wilting and overall collapse.  Plants infected with strains inducing crown and root rots display progressive necrosis of the root system and aerial basal tissue.  Mycelia do not penetrate the vascular tissue during this pathogenesis, but plant death occurs nevertheless, due to collapse of the root system.  How strains differentiate host and colonization strategy is not well characterized.  However, it is hypothesized that the fungi regulate these processes based on host plant anatomy and gene-for-gene interactions.  The research investigating this genomic basis is ongoing.

Following plant death and collapse, the fungus invades all tissues extensively until it reaches the external environment, sporulates profusely and is disseminated via wind or splashing water.  As a facultative parasite, F. oxysporum can persist in soils for decades.  Survival spores (chlamydospores) can remain dormant for several years, and mycelia can persist in minute amounts of crop debris, in the absence of cultivated hosts.  In greenhouses, infective propagules can endure on tools, work surfaces, pots and trays, and in substrate.  Furthermore, irrigation water and propagation baths can spread spores throughout facilities, fostering outbreaks.

Fusarium oxysporum is spread long distances via movement of infected plant materials such as cuttings, transplants, roots, bulbs and corms.  Furthermore, spores and mycelia can be transported in infested soil on shoes and clothing, tools, equipment and in surface water.  Its persistent nature and multiple avenues of dissemination make eradication of this pathogen impossible within affected areas.  Fungicide applications are impractical in field-grown crops, due to large acreages and the limitations of fungicide movement through soils.  Drenches are utilized in greenhouse crops with success; however, crops such as cannabis are subject to organic growing standards, precluding the use of synthetic fungicides.  Genetic resistance is effective in crops such as tomato but is not widely available amongst hosts of F. oxysporum strains.  Exclusion of the pathogen through rapid containment is the most effective management strategy.  This makes the diagnostic testing of plant materials paramount to successful disease management programs.

Agdia’s new AmplifyRP® XRT assay for detection of Fusarium oxysporum 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, 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 new assay was screened against multiple formae speciales of Fusarium oxysporum, including cannabis, chrysanthemi, fragariae, latucae, lycopersici, niveum and vasinfectum, detecting all true positives.  Furthermore, a high level of specificity was observed with an extensive exclusivity panel of multiple species of Fusarium other than F. oxysporum and additional fungal and oomycete pathogens, including Phytophthora spp, Pythium spp., Rhizoctonia solani and Verticillium dahliae.  Sensitivity for this assay is comparable to that observed with the published qPCR assay and greater than the published conventional PCR assay to which it was compared.  This product was developed to test crown, leaf, petiole, root and stem tissue.  This assay contains an endogenous internal DNA control.

Agdia now offers AmplifyRP® XRT products for detection of several important bacterial, fungal, viral and viroid pathogens across several markets, including grape, cannabis, ornamental, potato and tomato.  “We are working to develop a comprehensive portfolio of easy-to-use diagnostic products on our AmplifyRP® XRT platform.  Customer demand for assays that can be implemented across a wide spectrum of applications has driven our production.  We continue to focus on product development for established customers and burgeoning markets, such as the cannabis industry,” said Robert Emmitt, Domestic Account Manager, Agdia, Inc.  For more information on Agdia’s complete line of AmplifyRP® assays, please visit Agdia’s website.

Agdia Commercializes Rapid ImmunoStrip® Test for Detection of Five Transgenic Traits Utilized in Bollgard® 3 XtendFlex® Cotton

Agdia, Inc. (Elkhart, IN), a leading provider of agricultural diagnostic products and services, has commercialized an ImmunoStrip® assay for detection of DMO, CP4 EPSPS, Bt-Cry2Ab, Bt-Cry1Ac and Vip3A, transgenic proteins present in Bollgard® 3 XtendFlex® cotton.

Agdia’s new ImmunoStrip® detects the presence of the five transgenic proteins in Bollgard® 3 XtendFlex® cotton and was developed to be used on single-seed samples. This assay is easy to use and can be performed in the field or the lab by personnel with no previous diagnostic experience. Test protocol is straightforward, and results can be achieved in ten minutes with the visualization of five distinct test lines. Agdia claims this product does not cross-react with other commercially available transgenic traits available in cotton. This introduction is the 23rd ImmunoStrip® in Agdia’s comprehensive catalog of transgenic trait identification assays. High levels of market demand have driven this output, and Agdia maintains consumer demand is expected to continue.

This ImmunoStrip® is available in comb format, containing 12 combs of eight strips, or 50 individual strips. Agdia provides a one-year warranty and technical support on purchased kits. For more information on Agdia’s complete line of assays for detection of transgenic traits, please visit Agdia’s website at www.agdia.com.

Bollgard® and XtendFlex® are registered trademarks of Bayer
ImmunoStrip® is a registered trademark of Agdia, Inc.

Agdia Commercializes ImmunoStrip® for Detection of Xanthomonas Genus

Agdia, Inc. (Elkhart, IN) is happy to announce the commercialization of a rapid diagnostic test for detection of several phytopathogenic members of the Xanthomonas genus on their ImmunoStrip® platform.

Xanthomonas is a large genus of gram-negative bacteria containing numerous phytopathogenic species and pathovars within species.  These pathogens cause widespread outbreaks on many economically important hosts, and diseases include citrus canker, fruit and leaf spot on pepper and tomato, black rot of crucifers and blight on geranium.  Members of Xanthomonas display a great degree of host plant specificity and tissue specificity, invading either the mesophyll tissue (cankers and leaf spots) or the vascular tissue (blights and wilts).  Many of these diseases are responsible for devastating crop losses throughout the world, affecting both crop productivity and quality if unchecked.

Lesions on unripe tomato fruit
Fig. 2. Lesions on unripe tomato fruit.
Image courtesy of Robert Emmitt, Agdia, Inc.

Therefore, many species of Xanthomonas are considered organisms of quarantine status by the European and Mediterranean Plant Protection Organization (EPPO).

Outbreaks of disease caused by Xanthomonas can occur throughout the season.  Nevertheless, the proliferation of inoculum (bacterial cells) and subsequent dissemination are dependent on local climatic conditions.  Colony growth is optimal during periods of warm, wet weather.  Xanthomonas forms mucilaginous colonies, which are suspended in water and spread locally in droplets via wind, splashing rain and overhead irrigation.  Furthermore, human activities such as picking fruit and pruning can exacerbate spread within fields, greenhouses and orchards.  Long-distance dispersal is most common via movement of infected budwood, cuttings, seedlings and seed.

Tomato plant defoliation
Fig. 3: Tomato plant defoliation.
Image courtesy of Robert Emmitt, Agdia, Inc.
Leaf marginal necrosis and spotting on cabbage seedlings
Fig. 4: Leaf marginal necrosis and spotting on cabbage seedlings. Image Courtesy of Dr Francesca Rotondo, The Ohio State University

Bacterial cells enter leaves, fruit and stems through natural openings and injured tissue.  Thereafter, a wide variety of symptoms are observed, depending on the specific pathosystem.  Infected pepper and tomato plants are known to exhibit lesions on foliage, stem (figure 1.) and unripe and ripe fruit (figure 2.).  Lesions can render fruit unmarketable, while severe foliar infections can cause defoliation, leading to sunscald of fruit and overall plant deterioration (figure 3.).  Bacterial spot on peppers and tomatoes is caused by four species of XanthomonasX. euvesicatoriaX. gardneriX. perforans and X. vesicatoria.  Citrus canker, caused by X. axonopodis pv. citri, is one of the most important diseases of citrus spp. worldwide.  Symptomology is like that on pepper and tomato, including corky lesions on fruit, leaves and woody stems.  Citrus fruit exhibiting cankers are unsalable for fresh market and can drop prematurely under high disease pressure.

Xanthomonas campestris pv. campestris, the causal agent of black rot of crucifers, can cause severe disease in several species of Brassicaceae, including broccoli, Brussels sprouts, collard, cabbage, cauliflower, kohlrabi, rutabaga and turnip.  Crop losses of 50% are not uncommon under conditions conducive to rapid spread of the pathogen.  Symptoms include wilting and collapse of seedlings, necrosis at leaf margins (figure 4.) and interveinal, V-shaped lesions on mature plants (figure 5.).  Bacterial blight on geranium is caused by Xanthomonas hortorum pv. pelargonii.  This disease is specific to and one of the most important diseases of Geranium and Pelargonium.  Symptoms vary depending on species and typically include water-soaked lesions, progressing to form larger, V-shaped lesions, similar to those observed in black rot infections.  Bacteria often invade the vascular system and cause wilting and eventual plant death.

Agdia’s new Xanthomonas ImmunoStrip® was screened against a diverse panel of species and pathovars within the genus, detecting 100% of true positives, including X. euvesicatoriaX. gardneriX. perforansX. vesicatoriaX. axonopodis pv. citriXanthomonas campestris pv. campestrisXanthomonas hortorum pv. pelargoniiX. oryzae pv. oryzae and X. fragrariae.

V-shaped lesions on mature cabbage leaf
Fig. 5: V-shaped lesions on mature cabbage leaf Image. Courtesy of Dr Francesca Rotondo, The Ohio State University.

Agdia states no cross-reactivity was observed for several potential cross-reactors, including Acidovorax spp., Clavibacter spp., Erwinia spp., Pseudomonas spp., and Xylella fastidiosa.  This assay was designed to be used on leaf, stem, seed, root and pure culture.

Agdia’s ImmunoStrip® platform provides end-users with unparalleled utility; samples can be tested in the field or lab by those having no previous diagnostic experience, and results are visualized within no more than 30 minutes.  Furthermore, this product includes everything necessary to perform a test and does not require special equipment.  Test protocol is simple and includes 1) Sample collection and extraction in Agdia buffer bags, 2) Exposing the ImmunoStrip® to the sample extract, and 3) Allowing results to develop.  Test results are visualized as a single control line or a control and test line for negative or positive results, respectively.  The introduction of Agdia’s Xanthomonas ImmunoStrip® expands their catalog to 44 plant pathogen products on this 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 catalog.

The XanthomonasImmunoStrip® is sold in kits of 5 or 25 strips, and kits include everything necessary to perform a test.  Agdia provides a one-year warranty on purchased kits.  A diagnostic assay for the detection of the Xanthomonas genus is also available in an enzyme linked immunosorbent assay (ELISA) format.

Agdia Releases Rapid Isothermal Assay for Detection of Emerging Pathogen in Industrial Hemp

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.

BCTV symptoms on tomato plants in outdoor cultivation
Figure 1: Beet curly top virus symptoms in outdoor tomato cultivation. Used with permission from Whitney Cranshaw, Colorado State University, Bugwood.org

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).

BCTV symptoms of leaf curling and chlorosis on cannabis plant
Figure 2: Beet curly top virus infection showing both yellowing and strong leaf curling in Cannabis spp. Used with permission from Whitney Cranshaw, Colorado State University, bugwood.org

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.

AmpliFire portable isothermal fluorometer with target and internal control curves on screen
Figure 3: AmpliFire portable isothermal fluorometer

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 virusCucumber mosaic virusHop latent viroidHop stunt viroidTobacco mosaic virusTobacco ringspot virusTomato brown rugose fruit virusTomato mosaic virusTomato ringspot virusTomato 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.

Agdia Now Accepting Hemp Samples for Pathogen Testing

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

Agdia Releases ImmunoStrip® Test for Rapid Identification of Two Invasive Ant Species and Their Hybrid.

Agdia, Inc. (Elkhart, IN) is happy to announce their commercialization of a rapid and user-friendly assay, on their ImmunoStrip® platform, for the identification of Solenopsis invicta, the red imported fire ant, Solenopsis richteri, the black imported fire ant, and the interspecific hybrid.

Imported fire ants (IFA) are highly invasive species that were introduced into the U.S. from South America in the early part of the 20th century.  It is believed that both species, Solenopsis invicta, the red imported fire ant, and Solenopsis richteri, the black imported fire ant, entered through the port of Mobile, AL in soil used as ballast in cargo ships.  Since their introduction, IFA have spread to 15 states and Puerto Rico, infesting more than 367,000,000 acres.  Solenopsis invicta is found from Virginia to Florida and west to Texas with introductions in New Mexico and California, while S. richteri is isolated to a relatively small geographic area in northeastern Mississippi, northwestern Alabama and southwestern Tennessee.  Furthermore, the interspecific hybrid, S. invicta x richteri can be found at points of contact between the two species.  In addition to the U.S., S. invicta has spread to several Caribbean islands, Australia, New Zealand and throughout Asia.  Predictive models based on average annual temperature, precipitation, geophysical characteristics and human factors revealed nearly 50% of the planet’s surface as potential habitat for S. invicta.

Both species of IFA have significant deleterious impacts on agriculture through crop injury, equipment and infrastructure damage, and livestock injury.  The USDA estimates management and repair of damage caused by IFA costs $6 billion annually, in the U.S. alone.  Furthermore, as with many invasive species, IFA poses a significant ecological threat to multiple native species, including insects, mammals, and ground-nesting birds and reptiles.  In addition to agricultural and environmental concerns, both species are potential threats to public health.  These insects are highly aggressive, attacking and stinging perceived threats collectively.  Solenopsis venom contains alkaloids known as piperidines that produce a painful burning sensation upon envenomation.  Thereafter, blisters form at the site of stings and can become infected.  Furthermore, protein components of the venom are potentially toxic, inducing anaphylaxis in susceptible individuals.

Eradication efforts for IFA were implemented on a national scale in the U.S. starting in the 1950’s.  Since then, several programs utilizing multiple residual insecticides have proven ineffective at impeding the spread of these invasive arthropods.  Many of the chemicals used were spread across wide acreages and were toxic to non-target fauna, killing indiscriminately.  The absence of natural predators and competition paved the way for re-infestations and advancement.  It is now understood that widespread eradication programs are fruitless, and the mindset has shifted to containment, assisted by a federal quarantine and localized chemical treatments.

Imported fire ants are spread by humans through the movement of infested nursey stock, baled hay and straw, grass sod, bulk soil and used soil-moving equipment.  In an effort to limit human-assisted spread of IFA, the USDA enacted a federal quarantine which mandates regular inspections of regulated items moving from quarantined areas to outside destinations.  This quarantine is enforced by the Animal and Plant Health Inspection Service (APHIS).  Rapid identification of IFA to species level is crucial to the success of the federal quarantine and the timely delivery of regulated items free of these invasive ants.  Unfortunately, visual identification of IFA is difficult, requiring samples be sent to an expert for morphological identification, thus delaying release of regulated cargo.  Due to this, a rapid and user-friendly field test for the identification of IFA is needed.

Through a collaborative effort, USDA-APHIS, USDA-ARS (Agricultural Research Service) and Agdia Inc. have developed a lateral flow assay, InvictDetect Plus™, which identifies S. invictaS. richteri and the S. invicta x richteri hybrid, discriminating them from native ant species excluded from the federal quarantine.  This product is available on Agdia’s ImmunoStrip® platform and can be implemented in the field or the lab by individuals having no expertise in ant morphology or taxonomy.  Assay protocol is relatively simple and includes 1) collection of five (5) suspect worker ants, 2) maceration of five ants in 100 µL of AEB2 extraction buffer in 1.5 mL tube, 3) placement of test strip in tube with ant extract and 4) interpretation of results.  Positive samples might show results within ten minutes; nevertheless, we recommend allowing the assay to run for 30 minutes before final determination of test result is made.

InvictDetect Plus™ provides a new tool for use in the ongoing effort to minimize the human-assisted spread of IFA.  Furthermore, Agdia endeavors to simplify the identification of IFA for regulators and all those who rely on the timely delivery of regulated items as means of income.

Agdia commercialise un kit rapide pour la détection du virus ToBRFV

Agdia, Inc. (Elkhart, IN) étoffe son offre permettant le diagnostic du virus Tomato brown rugose fruit virus (ToBRFV) avec un test bandelette rapide (ImmunoStrip®). Ce test de détection est le troisième  développé par Agdia pour la détection du ToBRFV en complément du test ELISA et du kit moléculaire d’amplification isotherme.

Image de prévisualisation YouTube

Le Tomato brown rugose fruit virus fait partie de la famille des Tobamovirus qui cause de sérieuses pertes économiques au niveau de la culture des solanacées incluant Solanum lycopersicum (tomate) et Capsicum spp. (poivron). Ce virus cause des symptômes typiques liés aux Tobamovirus comme des mosaïques et des chloroses sur feuilles, des déformations et décolorations sur fruits, les rendant non  commercialisables.

Des contrôles phytosanitaires des semences, des plants et fruits de tomates et poivrons sont aujourd’hui développés par plusieurs pays à l’international afin de réduire ou éviter l’introduction de ce virus sur leurs territoires. Le Tomato brown rugose fruit virus est classé comme organisme de quarantaine par l’OEPP (Organisation Européenne et Mediterranéenne pour la Protection des Plantes).

L’ImmunoStrip® ToBRFV développé par Agdia a été validé sur tomate, poivron, et petunia et a été testé sur différents isolats provenant de plusieurs pays.

Les tests rapides se présentent sous la forme de bandelettes et permettent une détection en quelques minutes du virus directement sur échantillon broyé dans un sachet fourni dans le kit.

Pour plus d’informations: info@agdia-emea.com

Agdia Commercializes ToBRFV ImmunoStrip® to Complete Diagnostic Suite

Agdia, Inc. (Elkhart, IN) has added an ImmunoStrip® to their Tomato brown rugose fruit virus (ToBRFV) diagnostic suite. Their ImmunoStrip® for ToBRFV is the third diagnostic assay launched by Agdia in the first half of 2021 for detection of this pathogen. Their high-specificity ELISA assay for ToBRFV was released on January 7, followed by a rapid molecular AmplifyRP® XRT for ToBRFV on January 12.

Tomato brown rugose fruit virus is a resistance-breaking Tobamovirus that causes severe economic losses in solanaceous crops, including Solanum lycopersicum (tomato) and Capsicum spp. (pepper). It causes symptoms typical of Tobamoviruses that include mosaic and chlorosis on the leaves and discoloration and deformation of the fruit.  These symptoms decrease yield and render fruit unmarketable.

Tomato and pepper seeds, transplants and fruits from certain countries are subject to a USDA-APHIS Federal Import Order in the United States. Tomato brown rugose fruit virus has also been classified as a quarantine pathogen by EPPO (European and Mediterranean Plant Protection Organization).

Agdia’s ImmunoStrip® for ToBRFV has been validated for use with tomato, pepper and petunia samples and has been tested against ToBRFV isolates from around the globe.

Cross-reactivity was observed with a Tomato mottle mosaic virus (ToMMV) sample. Very mild cross-reactivity was observed with Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) samples. No other cross-reactions were observed when testing other Tobamoviruses, including Bell pepper mosaic virus (BPeMV), Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), Pepper mild mottle virus (PMMoV), Tobacco mild green mosaic virus (TMGMV), and Zucchini Green Mottle Mosaic Virus (ZGMMV).

Agdia’s ImmunoStrip® platform provides end-users with a high level of utility. ImmunoStrips® are fit for use in the lab or in the field by those having no previous diagnostic experience, and results are visualized within 30 minutes.  Increasing levels of market demand persists for field-deployable plant pathogen detection products and Agdia maintains they will continue to expand this area of their catalog.

Agdia Adds to Diagnostic Suite for ToBRFV with Release of a Rapid, RNA-based Isothermal Molecular Kit

Agdia, Inc. (Elkhart, IN) has added another test kit to their Tomato brown rugose fruit virus (ToBRFV) diagnostic suite. AmplifyRP® XRT for ToBRFV is the second of three ToBRFV diagnostic assays to be launched by Agdia in the first half of 2021. Their high-specificity ELISA assay for ToBRFV was released on January 7. An ImmunoStrip® lateral flow device for ToBRFV is expected to be released in the coming months (Q1 or Q2 of 2021.)

Agdia’s AmplifyRP® XRT for ToBRFV has been validated for use with tomato and pepper seeds and leaf. As a rapid, field-deployable molecular method requiring far less training than traditional PCR methods, this assay provides users with greater flexibility to deploy detection capabilities where they need it, when they need it. Use cases for this assay include:

  • Laboratory-based molecular diagnosis with crude or purified extracts with faster time-to-result than traditional PCR or qPCR methods. This assay can be used with Agdia’s AmpliFire® isothermal fluorometer or with most commonly-used real-time PCR machines.
  • In-field monitoring at remote production sites as a stand-alone assay.
  • Directly test ELISA extracts for molecular confirmation of serological screening results, allowing you to act immediately to address production issues.

Agdia’s AmplifyRP® XRT for ToBRFV is highly specific to ToBRFV and has been experimentally proven to detect isolates from around the world. No cross-reactivity was observed with high titer samples from other Tobamoviruses, including Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Zucchini Green Mottle Mosaic Virus (ZGMMV) and more.

Tomato brown rugose fruit virus is a resistance-breaking Tobamovirus that causes severe economic losses in solanaceous crops, including Solanum lycopersicum (tomato) and Capsicum spp. (pepper). It causes symptoms typical of Tobamoviruses that include mosaic and chlorosis on the leaves and discoloration and deformation of the fruit.  These symptoms decrease yield and render fruit unmarketable.

Tomato and pepper seeds, transplants and fruits from certain countries are subject to a USDA-APHIS Federal Import Order in the United States. Tomato brown rugose fruit virus has also been classified as a quarantine pathogen by EPPO (European and Mediterranean Plant Protection Organization).

Agdia Adds to Diagnostic Suite for ToBRFV with Release of a Rapid, RNA-based Isothermal Molecular Kit

Agdia, Inc. (Elkhart, IN) has added another test kit to their Tomato brown rugose fruit virus (ToBRFV) diagnostic suite. AmplifyRP® XRT for ToBRFV is the second of three ToBRFV diagnostic assays to be launched by Agdia in the first half of 2021. Their high-specificity ELISA assay for ToBRFV was released on January 7. An ImmunoStrip® lateral flow device for ToBRFV is expected to be released in the coming months (Q1 or Q2 of 2021.)

Agdia’s AmplifyRP® XRT for ToBRFV has been validated for use with tomato and pepper seeds and leaf. As a rapid, field-deployable molecular method requiring far less training than traditional PCR methods, this assay provides users with greater flexibility to deploy detection capabilities where they need it, when they need it. Use cases for this assay include:

  • Laboratory-based molecular diagnosis with crude or purified extracts with faster time-to-result than traditional PCR or qPCR methods. This assay can be used with Agdia’s AmpliFire® isothermal fluorometer or with most commonly-used real-time PCR machines.
  • In-field monitoring at remote production sites as a stand-alone assay.
  • Directly test ELISA extracts for molecular confirmation of serological screening results, allowing you to act immediately to address production issues.

Agdia’s AmplifyRP® XRT for ToBRFV is highly specific to ToBRFV and has been experimentally proven to detect isolates from around the world. No cross-reactivity was observed with high titer samples from other Tobamoviruses, including Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), Zucchini Green Mottle Mosaic Virus (ZGMMV) and more.

Tomato brown rugose fruit virus is a resistance-breaking Tobamovirus that causes severe economic losses in solanaceous crops, including Solanum lycopersicum (tomato) and Capsicum spp. (pepper). It causes symptoms typical of Tobamoviruses that include mosaic and chlorosis on the leaves and discoloration and deformation of the fruit.  These symptoms decrease yield and render fruit unmarketable.

Tomato and pepper seeds, transplants and fruits from certain countries are subject to a USDA-APHIS Federal Import Order in the United States. Tomato brown rugose fruit virus has also been classified as a quarantine pathogen by EPPO (European and Mediterranean Plant Protection Organization).

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