US (CA): "The virulent nature of HLVd in cannabis demands a novel approach"

A 2021 survey from Dark Heart Nursery Research concluded that 90% of cannabis facilities in California were infected with HLVd. "Hop latent viroid (HLVd) can cause substantial economic losses in plant yield and crop value for cannabis growers. Once infected, the viroid spreads rapidly through cannabis plants, leading to stunted growth, weaker flower smell, diminished flower quality, lower yield, and up to 50% reduction in cannabinoid and terpene production. The best way to control the disease is early detection to limit the spread of the viroid in grow facilities." Therefore, a recent study (conducted by the University of California, MyFloraDNA, and Glass House Farms) examined a new tool for detecting HLVd in the early stages of plant development: MFDetect.

The need for a new technology
The researchers explain that several technologies have been developed and used to detect viroids in plants. The methods include bioassay, nucleic acid hybridization, return-polyacrylamide gel electrophoresis, RT-qPCR, and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Yet, some of these methods have advantages as well as drawbacks. "For example, RT-qPCR is a very accurate method for pathogen detection; however, this technique is expensive, requiring sophisticated equipment, expert personnel to perform the test, and it lacks high throughput capability. The advantage of RT-LAMP is its capability for high throughput at low cost, and turnaround times can be rapid."

According to the researchers, the virulent nature of this viroid in cannabis and its high rate of transmissibility and spread across most of the gardens in California demand a novel approach. One that combines the robust and accurate detection of RT-qPCR and the low-cost and high throughput of RT-LAMP to allow containment of viroid spread within California and beyond.

Results
The study describes the MFDetect system, a technology developed by MyFloraDNA that relies on a unique set of primers and one-step RT-LAMP plus-qPCR for detecting HLVd in infected plants. "A set of oligonucleotides was designed using the program (Primer Explorer 3.0) to detect HLVd successfully. The optimized primer design and concentration in our reaction significantly improved the specificity and expanded the capacity to detect and inspect plant material. Most of the commercial techniques either rely on RT-qPCR, RT-LAMP, or other rapid RNA amplification techniques limited by throughput and are expensive compared to the MFDetect system," the researchers say.

"Because of the high sensitivity, MFDetect may be able to detect infection in the leaves at an early stage of growth before symptoms appear, which is currently a limitation in the industry," according to the study. "Our findings show that an accurate RNA extraction method with a highly sensitive RT-LAMP can highly improve the application of MFDetect for viroid detection in cannabis and hemp."

In the present study, with 600 samples comparing the age and tissue type, the researchers detected the viroid in the leaf samples collected from mother plants from 5–6-week-old cuttings. "In a subsequent experiment, we observed that leaf, petiole, and root tissue types are equally sensitive to HLVd detection with MFDetectTM and Taqman RT-qPCR. The ease of collecting tissue material and the higher viroid loads in the leaf material make it an ideal material for HLVd detection. While in leaf tissue, it was easy to extract RNA in the extraction buffer with a tissue-lyser, root, and petiole tissues were challenging. The root needed an extra cleaning step, while the petiole was difficult to grind. There is room to further improve the protocol for RNA extraction from those demanding tissues."

"Overall, MFDetect for detection of HLVd is rapid, precise, sensitive, and cost-effective," the researchers say. "Follow-up experiments are planned to further narrow the earliest plant growth stage, plant tissue type for detection, and the source of HLVd transmission (e.g., water, soil or seed) in the grow facilities. When combined with healthy cultivation practices and meristem culture, one can produce viroid-free planting material for nurseries and growers."

To read the complete study, go to www.mdpi.com