Ecology and Epidemiology of Wheat Curl Mite and Mite-Transmissible Viruses in Colorado and Insights into the Wheat Virome
Summary (2 min read)
Wheat Curl Mite and Plant Tissue Collection
- Plants were examined using a dissecting microscope for the presence of WCMs.
- Plants were grown in gallon pots with 2-3 plants per pot in Promix HP © soil.
- Additionally, leaf tissues were tested for presence of WSMV, TriMV and HPWMoV.
Virus Detection and Quantification
- Total RNA was extracted from approximately 40 mg homogenized leaf tissues obtained from various sources described above, lysed in Trisure® (Bioline Meridian Bioscience) using Direct-zol® RNA Purification Kit (Zymo Research, CA, USA) according to the manufacturer's recommendations.
- The quantity of RNA was approximated using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, MA, USA) and stored at -80°C until virus quantification.
- To detect and quantify WSMV and TriMV, approximately 50 ng of RNA was used in a previously published qRT-PCR duplex assay (Price et al. 2010) with the TaqMan® RNA-to-Ct™ 1-step kit (Applied Biosystems™, ThermoFisher Scientific) on a QuantStudio™3 Real-Time PCR system (Applied Biosystems™, ThermoFisher Scientific).
- Field samples with CQ values above the lower detection limit, as defined by the standard curves described below, were considered to be positive for the specified virus.
- To detect HPWMoV, complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the Verso® cDNA Synthesis Kit (ThermoFisher Scientific).
Wheat Virome Analysis
- Four leaf tissue samples that previously tested positive for WSMV from Larimer county., positive for WSMV and TriMV from Bent county., positive for WSMV and HPWMoV from Phillips county., and positive for WSMV and TriMV from Kit Carson county.
- After all filtering operations, an average of 0.25x10 6 reads (3%) remained per dataset.
- Then, analysis of SNPs for the viruses from assembled virome data were performed using the Tablet software program to determine genetic diversity.
- Lastly, the raw sequence data was deposited in the NCBI Sequence Read Archive (SRA) repository under submission number SUB7870854.
- Phylogenetic analysis of the complete sequences of the nucleoprotein encoding RNA3 segments of members of both groups of HPWMoV and three variants all from the Phillips county sample, shows version Colorado RNA3C (MT762120) is similar to isolates from OH/TX or group A (Fig. 5 ).
Wheat Germplasm and Virulence Test
- A natural infection of wheat streak mosaic virus was observed in the Colorado State University Irrigated Variety Performance Trial (IVPT) at Burlington, CO, in 2019.
- The trial included 24 different genotypes (released varieties and experimental lines), planted in a randomized complete block design with three replications.
- To quantify virus titer in wheat varieties, 10 leaf samples were collected on June 21, 2019.
- All ten leaves were stacked, and a small section of tissue was cut from the center of the stack.
Virus Occurence in Colorado
- Survey of WCM-transmitted viruses revealed the presence of all three economicallyimportant viruses in Colorado, WSMV, TriMV and HPWMoV (Fig. 2 ).
- Of the 40 symptomatic samples tested, 38 were positive for one or more WCM-transmitted viruses.
- WSMV was found in all surveyed counties (Fig. 2.
Identification of Virus Genotypes
- To determine the genetic variability among WSMV isolates in Colorado, the authors sequenced a portion of the WSMV-NIb region and performed phylogenetic analyses with isolates from other wheat producing states in the U.S. and other regions of the world.
- Interestingly, an isolate, Larimer county 1, was 100% similar to a Kansas isolate (MK318278) that was collected from a wheat variety carrying the Wsm2 virus resistance gene that is known to confer resistance to WSMV (Fellers et al. 2019) .
- The isolate Larimer county 2 collected from same location as the Larimer county 1 isolate, appeared to be genetically distinct from other isolates in the U.S.
- In contrast, to the genetic diversity in WSMV isolates, there was limited variability in TriMV isolates collected in Colorado compared to that of other sequenced isolates.
- Phylogenetic analysis reveals two distinct groups of the HPWMoV isolates among available HPWMoV nucleoprotein sequences as observed by previous studies (Stewart 2016) .
Virus Resistance in Variety Trial
- Wheat varieity trial included a combination of 24 public and private varieties and experimental lines.
- Seed companies with entries in the variety trials included AgriMaxx Wheat, AgriPro Syngenta, Dyna-Gro Seed, Limagrain Cereal Seeds, and WestBred Bayer.
- The gerplasm included varieties with no known resistance, a single resistance marker to either WCM (WCM6D) or WSMV (Wsm2), and one variety, Guardian, with both resistance markers, WCM6D and Wsm2 (Table 4 ).
Discussion
- Mite-vectored wheat viruses continue to cause significant yield losses in Colorado.
- Wheat virome analysis confirmed the presence of known viruses such as WSMV, TriMV and HPWMoV, but also revealed presence of several mycoviruses and a novel Ixeridium yellow mottle virus 2 from Colorado wheat samples.
- The genetic diversity of WSMV has been evaluated among various isolates in the U.S. and from around the world by sequencing the coat protein (CP) (Robinson and Murray 2013) and more recent whole genome sequencing (Schubert et al. 2015) .
- There have been reports of increasing mite populations and virus infection in resistant varieties (Tatineni and Hein 2018) .
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