Honey Bee Pathogen Multiplex Panel
The Honey Bee Pathogen Panel is currently offered to consumers on a fee for service basis. This test panel was developed by NAGC as a tool used to screen for the health of honey bees. NAGC’s pathogen panel is specific for the following 11 viral and bacterial pathogens: Acute Bee Paralysis Virus, Black Queen Cell Virus, Kashmir Bee Virus, Lake Sinai Virus 1 and 2, American Foulbrood, Chronic Bee Paralysis Virus, Deformed Wing Virus, Israeli Acute Bee Paralysis Virus, Slow Bee Paralysis Virus and European Foulbrood. This multi-target panel has been shown to be highly specific for each target using the unique DNA sequence from each pathogen. In addition to the cost savings, the utilization of a multiplex panel shortens the turnaround time which, in turn, heightens the throughput capacity of the laboratory.
In parallel to developing the multiplex panel for quick and reliable testing, NAGC has also conducted a 10-week study exploring storage conditions of honey bees to determine the acceptable parameters for storage, transit and detection. The preliminary data suggests that the pathogens were still detectable after 10 weeks stored at room temperature (~70°F) without any decrease in sensitivity. These samples were compared to samples stored at the colder storage recommendations of -20°C and 4°C. NAGC also investigated higher than room temperature storage and those conditions are not recommended. They hope this new data will help support less stringent guidelines for testing and hopefully open the opportunity to testing for beekeepers that may not have access to cold transit shipping conditions.
This project was funded by the National Corn Growers Association and the North Dakota Department of Agriculture.
Chantawannakul P., L. Ward, N. Boonham and M. Brown. 2006. A scientific note on the detection of honeybee viruses using real-time PCR (TaqMan) in Varroa mites collected from a Thai honeybee (Apis mellifera) apiary. Journal of Invertebrate Pathology 91: 69-73.
de Miranda, J.R., G. Cordoni and G. Budge. 2010. The Acute bee paralysis virus-Kashmir bee virus-Israeli acute paralysis virus complex. Journal of Invertebrate Pathology 103: S30-S47.
Roetschi A., H. Berthoud, R. Kuhn and A. Imdorf. 2008. Infection rate based on quantitative real-time PCR of Melissococcus plutonius, the causal agent of European foulbrood, in honeybee colonies before and after apiary sanitation. Apidologie 36: 362-371.
Runckel, C., M.L. Flenniken, J.C. Engel, J.G. Ruby, D. Ganem, R. Andino and J.L. DeRisi. 2011. Temporal analysis of the honey bee microbiome reveals four novel viruses and seasonal prevalence of known viruses, Nosema and Crithidia. PLoS ONE 6:e20656.
Ryba S., D. Titera, M. Haklova and P. Stopka. 2009. A PCR method of detecting American Foulbrood (Paenibacillus larvae) in winter beehive wax debris. Veterinary Microbiology doi:10.1016/j.vetmic.2009.05.009.
Tentcheva, D. L. Gauthier, N. Zappulla, B. Dainat, F. Cousserans, M.E. Colin and M. Bergoin. 2004. Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Applied and Environmental Microbiology 70: 7185-7191.
Chronic Wasting Disease
Chronic wasting disease (CWD) is a naturally occurring transmissible spongiform encephalopathy (TSE) affecting members of the cervid species, including white-tailed and mule deer, wapiti and moose. As with other TSEs, including scrapie of sheep, transmissible mink encephalopathy, bovine spongiform encephalopathy (BSE or mad cow disease) and variant/sporadic Creutzfeldt-Jakob disease (CJD) in humans, CWD is characterized by identifying an abnormally folded protein. The cellular prion protein that is responsible for causing the disease is identified through many labor intensive steps to detect the misfolded protein in the animal. This structural change renders it resistant to degradation, which has led to the epidemic of the disease spreading through waterways, feeding pastures and laterally to other cervid animals in the area.
Currently, the only recognized method by the USDA-APHIS is a technique that requires a brain biopsy (Immunohistochemistry or IHC), thus each animal tested is no longer living.
NAGC is optimizing two methods to detect CWD which will be able to survey living animals in addition to non-living animals in an effort to get ahead of the disease. By testing asymptomatic animals and using preventative measures, the identification of the disease can be established before the possible transmission to other animals and animal resources (food and water).
Corn has become an increasingly important crop within the state of North Dakota, where it is currently grown in every county; though the productivity and risk of production varies considerably from region to region (Ransom, 2004). Stalk and ear rot diseases of corn can be caused by many fungi and bacteria. Most of these pathogens occur commonly in the fields and behave opportunistically by primarily infecting senescing, injured or stressed plants. Several fungal species in the genus Fusarium are responsible for diseases such as root rot, stalk rot, ear rot, seedling blight and sudden death syndrome (Wang et al., 2015). Ear rot affects grain quality, limits the use of certain cultivars, and causes concern about toxins (fumonisin and trichothecene) in corn used for feed.
Fusarium verticillioides is reported to be the primary fungus species that causes Fusarium ear rot in the United States, but two other Fusarium species (F. proliferatum and F. subglutanins) also infect corn and cause ear rot disease. In all three species, the disease symptoms are similar, but only F. verticillioides and F. proliferatum produce fumonisins (Beck et al., 2005). The most agriculturally important Fusarium species that produce trichothecenes are F. graminearum, F. culmorum, F. sporotrichioides, F. poae and F. equiseti (Bluhm, 2002; Jurado et al., 2005). All of these Fusarium species are also common fungal pathogens in cereals, particularly, head blight in small-grain crops. Thus, there is a strong need for rapid detection and identification of the Fusarium species, to provide guidance for corn producers on the use of fungicides either to pre-treat fields or as seed treatments.
Given the complex diversity of species and often confusing taxonomy, conventional methods to identify Fusarium are both labor intensive and time consuming (Bluhm et al., 2004; Demeke et al., 2005). As an alternative to conventional microbiological procedures in diagnosis, a molecular based technology offered by NAGC is a rapid and sensitive method to detect target DNA molecules.
NAGC has begun the development of a pathogen screening assay to detect Fusarium species in both seed and soil samples. The development of this high throughput, real-time PCR method would ultimately help establish a cost-effective monitoring regime for producers, which will help them proactively safeguard and manage their crops against future Fusarium outbreaks.
The NAGC collaborated with the USDA and university labs to identify Xanthomonas in corn. As the top corn producing country, there is a strong need to provide US farmers with reliable and early disease detection for a variety of pathogens, particularly for the recently confirmed Bacterial Leaf Streak Disease (BLSD). BLSD is caused by a bacteria, Xanthomonas vasicola pathovar vasculorum (Xvv), which was thought to be restricted to South Africa. The first reported detection of Xvv in the US was in Nebraska last year (2016) and further surveying has confirmed its presence in several other states (Minnesota, South Dakota, Iowa, Colorado, Illinois, Kansas, Texas and Oklahoma) with more being added (USDA-APHIS, 2016, August 29 and Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, 2016, August 26). Symptoms of BLSD are similar to other diseases, making visual diagnosis difficult to impossible.
Due to its recent introduction to the US, the epidemiology (control and spread) of BLSD is largely unknown, but it is likely that foliar fungicides typically used against gray leaf spot will be ineffective against this bacterial disease.
Through the USDA’s collaboration, the NAGC has been able to modify the initial assay to allow for a greater number of samples (high throughput) to be analyzed in a shorter period of time. The information gathered will allow for a better understanding of the transmission of the disease and protect producers from further spread within the US. There are only a few research labs that can support and are capable of developing high throughput assays using the latest molecular instrumentation that the NAGC possesses. This assay has the potential to screen other plants that may not yet be identified as alternative hosts (e.g. cotton, food crops or weeds) to the bacteria, which have been important sources in particular strains of Xanthomonas vasicola pathovars (Coutinho, et al., 2015).
Importantly, optimization of the assay will enable producers to test a variety of potential contaminated sources (farming equipment, seeds, soil), which can help market corn for exportation as well as assert liability of contamination from farming equipment that moves from field to field. Furthermore, this assay will provide a more reliable diagnosis, allowing producers to be more proactive in their management strategies.
This project was funded by the National Corn Growers Association and the North Dakota Corn Council.
Lang J., E. DuCharme, J. Ibarra Caballero, E. Luna, T. Hartman, M. Ortiz-Castro, K. Korus, J. Rascoe, T.A. Jackson, K. Broders, J. Leach. (2017). Detection and characterization of Xanthomonas vasicola pv. vasculorum (Cobb 1894) comb. nov. causing bacterial leaf streak of corn in the United States. Phytopathology. 2017 Jul 5. doi: 10.1094/PHYTO-05-17-0168-R.
Aphid Resistance in Soybeans
In 2008, the economic loss for the soybean industry due to the presence of aphids was estimated to be approximately 4 billion US dollars annually (Kim et al, 2008). Aphid infestation can decrease soybean yield as high as 50% (Wang et al., 1994; Ragsdale et al., 2007). High aphid populations can reduce crop production directly when their feeding causing severe damage such as stunting, leaf distortion, and reduced pod set (Sun et al., 1990). Although proper use of insecticides can greatly reduce the damaging effects of aphids on soybean yield, this approach is costly (∼33 U.S. dollars/hectare), detrimental for the environment and can lead to the development of insecticide resistant aphids. In addition, this practice could also adversely affect the population of insects that normally prey on aphids (Ragsdale et al., 2007).
Aphis glycines, and a close relative A. gossipii, are the only aphid species found colonizing soybean in the Unites States (Hill et al., 2004). To date, four soybean aphid biotypes are now known in relation to resistance genes. Depending on the type of soybean aphid, there are different resistance gene combinations from the soybean plant that offer tolerance to these pests.
The use of soybean lines naturally resistant to aphids is another management approach to control soybean aphids. By selecting the varieties that are resistant to the biotypes of aphids most prevalent in ND, farmers will decrease aphid damage and decrease yield losses due to aphids. Farmers will decrease insecticide applications reducing costs and impact on the environment.
Scientists at the NAGC have optimized high throughput assays to screen soybean varieties for Rag1, Rag2, Rag3 genes. From the genetic work of our collaborator, Dr. Brian Diers (University of Illinois-Urbana) and research from Dr. Dechun Wang (Michigan State University), identification of genetic markers linked to the Rag genes have made it possible to rapidly screen new soybean varieties for aphid resistance.
This project was funded in part by the North Dakota Soybean Council.
Zhang S., Z. Zhang, C. Bales, C. Gu, C. DiFonzo, M. Li, Q. Song, P. Cregan, Z. Yang and D. Wang. 2017. Mapping novel aphid resistance QTL from wild soybean, Glycine soja 85-32. Theoretical and Applied Genetics, 1-12.
Phytophthora root and stem rot (PRSR), caused by the soil-borne fungus Phytophthora sojae is a common disease found throughout the United States. PRSR has been ranked as a leading destructive soybean disease reportedly causing an annual loss of over 44 million bu from 1996-2009 (Koenning and Wrather, 2010; Wrather and Koenning, 2009).
The NAGC has developed a test that allows for the identification of the Phytophthora pathogen, allowing ND soybean producers to quickly identify the Phytophthora pathogen. An accurate course for remediation can then be determined. It can also be an indicator of what soybean producers might expect in a particular field the following year.
This project was funded in part by the North Dakota Soybean Council.
Catal, M., F. Erler, D. Fulbright and G. Adams. 2013. Real-time quantitative PCR assays for evaluation of soybean varieties for resistance to the stem and root rot pathogen Phytophthora sojae. European Journal of Plant Pathology, 137, 859-869.
Clavibacter michiganensis subsp. nebraskensis (Cmn), the causal organism of Goss’s Wilt, can infect a corn crop in any developmental stage, whether through wounds or transmitted directly through seed. Once the bacteria has colonized a crop, the surface-infested crop residue later becomes the main source of inoculum for future outbreaks of Goss’s wilt and leaf blight.
NAGC has optimized a test for Goss’s Wilt with high throughput capabilities that was very recently developed by a group from the University of MN (McNally et. al., 2016), which utilized a PCR-based assay to detect, identify and quantify the causal agent of Goss’s Wilt (Cmn). Utilizing the Goss’s Wilt assay, NAGC is able to provide an accurate, sensitive and affordable method for producers and researchers to assess the potential for developing Goss’s Wilt as a pre-screen of seed before purchase, for detecting the organism in field samples to assess the bacterial lode in the field before planting, as well as detecting the organism on symptomatic plant samples.
This project was funded in part by the North Dakota Corn Utilization Council.
McNally, R.R, C.A. Ishimaru and D.K. Malvick. 2016. PCR-mediated detection and quantification of the Goss’s Wilt pathogen Clavibacter michiganensis subsp. nebraskensis via a novel gene target. Phytopathology 126:1465-1472.
NAGC Fee Schedule
Honey Bee Testing
Multiplex Group 1……………………………………………………$50/Sample
Multiplex Group 2……………………………………………………$50/Sample
Any Single Pathogen Test………………………………………..$50/Sample
Bulk Pricing (full screen)……………………………………………$50/Sample
Plant Tissue/Residue & Seeds……………………….$45 to $90/Sample
Soil………………………………………………………………$40 to $120/Sample