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  Figure 1. Aerial view of tanoak mortality in Humboldt County, CA. Image courtesy of California Oak Mortality Task Force

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  Figure 1: AmplifyRP® assay used with Agdia’s AmpliFire isothermal fluorometer

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

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.

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.

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

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  Figure 1: Closeup of symptoms caused by Lettuce chlorosis virus (LCV) in field lettuce (Lactuca sativa L.) Image provided courtesy of G. J. Holmes.

  Agdia, Inc. (Elkhart, IN) is happy to announce the commercialization of a rapid, user-friendly, RNA-based assay, on their AmplifyRP^® XRT platform, for the detection of Lettuce chlorosis virus.   

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, 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 Caulimovirus, Dahlia 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.

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.

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, 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

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.

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 1. and 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.).

Rhizoctonia 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.

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, 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 virus, Chrysanthemum 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, 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, 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, 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, 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.

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.

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 Xanthomonas: X. euvesicatoria, X. gardneri, X. 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. euvesicatoria, X. gardneri, X. perforans, X. vesicatoria, X. axonopodis pv. citri, Xanthomonas campestris pv. campestris, Xanthomonas hortorum pv. pelargonii, X. oryzae pv. oryzae and X. fragrariae.

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

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

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

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

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

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

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

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

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

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 20^th 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. invicta, S. 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, 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).