PAPER

Game-Changing Insights and Advanced Testing Strategies for HLVd in Cannabis: Sample Collection and Beyond

Introduction

Hop Latent Viroid (HLVd) is a significant pathogen and the biggest threat to cannabis growers worldwide. At MyFloraDNA, we are acutely aware of the industry’s need for robust and reliable research for the early detection and mitigation of the disease. Our scientists are at the forefront of developing high throughput, sensitive and cost-effective technologies for early disease detection, such as Hop Latent Viroid and other economically important pathogens. HLVd spread in the infected plant is not uniform; therefore, the age and plant tissue material is critical for disease diagnostics. There is a growing debate on the most suitable plant material for the early detection of HLVd. Recent reports suggest roots are best for HLVd detection, but these studies examined limited plant numbers. Isolation and sampling of root material are impractical for large grow facilities and may result in cross-contamination if not handled appropriately.
MyFloraDNA collaborated with one of the largest and most prestigious cannabis cultivation facilities to identify the suitable plant material for HLVd detection using a proprietary and sensitive detection method. Using >5000 plants and 600 tissue samples, we conducted the most extensive analysis to compare different tissue types by plant age to perform the most comprehensive HLVd research published up to now. The publication detailing our findings is available for download here.
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What is a viroid

Viroids are the tiniest known infectious RNA molecules, they do not contain protein and mainly cause diseases in plants (Diener, 1971). They depend on certain components of the host cell, specifically the DNA-based RNA polymerase and processing enzymes, to reproduce and cause disease (Semancik, 2003). One such viroid, known as HLVd, is a small, circular RNA molecule made up of 256 building blocks called nucleotides. It includes a common central region (CCR) and a terminal conserved hairpin (TCH) (Puchta et al., 1988; Adkar-Purushothama et al., 2023).
Figure 1. Viroids structures. Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs - Scientific Figure on ResearchGate.
Figure 2. Rolling circle model for Avsunviroid replication (adapted from Clark et al., 2019 (69)).

Demystifying HLVd in Cannabis:

In Cannabis, the viroid is associated with the “dudding” or “duds” disease of Cannabis, which is one of the most devasting cannabis diseases (sy. Hemp) (Cannabis sativa, Cannabis indica and Cannabis ruderalis). The infected plants can remain asymptomatic for extended period. The diseased plants are stunted, brittle, less vigorous producing low quality and quantity of trichomes and flowers. The plants show significantly reduced amounts of THC, cannabinoids and terpenes. Once one plant is infected, the disease can spread quickly to other plants (Adkar-Purushothama et al., 2023, Warren et al., 2019, Singh and Singh 2021, Bektas et al., 2019).

Understanding the characteristics and behavior of HLVd in Cannabis is crucial to effectively combat its impact on plant health and yield. HLVd, as an infectious circular RNA molecule, sets itself apart from viruses through its unique replication process, which exclusively occurs within plants. Unlike viruses, HLVd lacks a protein envelope and consists solely of RNA and are the tiniest known entities of their kind (three times smaller than viruses).

One of the significant challenges posed by HLVd is its latent nature. The infected plants may not exhibit visible symptoms for extended periods and can silently spread when there is direct contact between infected and healthy plants.

Image 1: Infected plant on the right, showing stunted growth.

Moreover, HLVd can be transmitted in various ways (Hadidi et al., 2022; Punja, 2023). Some of the primary modes of transmission include: Contaminated tools and equipment. Pests and even human touch. Root contact. Recycled water containing root cells. Seed transmission. Cuttings and plant tissue. As cannabis professionals, we must recognize that HLVd represents a formidable threat to our industry. To address these challenges and provide cultivators with reliable information, MyFloraDNA has conducted extensive research on HLVd detection, and the results are discussed in the article.

Testing Technologies

There are many techniques to detect viroids. These include bioassay, nucleic acid hybridization (Owens and Diener, 1981), return-polyacrylamide gel electrophoresis (Schumacher et al. 1986), RT-qPCR (Boonham et al., 2004, Chandelier et al., 2010, Mascia et al., 2010), and reverse transcription loop-mediated isothermal amplification (RT-LAMP; Notomi et al., 2000, Tsutsumi et al., 2010).

RT-qPCR is a standard, accurate method for finding pathogens, but it’s expensive and needs special equipment and skilled people to do the test. On the other hand, RT-LAMP is becoming more popular for detecting pathogens in plants, animals, and humans. It’s more affordable, quicker, and can handle many samples simultaneously (Bostan et al. 2004; Tseng. 2021). Both RT-qPCR and RT-LAMP have been used extensively to detect plant pathogens, and each has pros and cons. Considering the destructive nature of HLVd in Cannabis and its rapid dissemination across California, a more versatile and reliable detection method is needed. The new detection method that is sensitive, affordable, precise, and capable of handling thousands of samples with a quick turnaround time may be able to limit the spread of the disease in grow facilities.

Research Findings that Shape the Future

In an extensive research study, we compared the widely used TaqMan RT-qPCR method with a proprietary plant pathogen diagnostic assay, MFDetect™. This hybrid assay combines the robustness of RT-LAMP with the sensitivity of RT-qPCR, resulting in unparalleled accuracy and efficiency. Additionally, we explored the efficacy of testing various plant tissues, including leaves, petioles, and roots, to determine the optimal approach for HLVd detection. Based on the analysis of over 600 samples, we concluded that leaves as the most suitable plant tissue for accurate HLVd detection, regardless of the growth stage. The simplified sample collection process associated with leaf material streamlines real-world applications, enhancing overall efficiency.

MFDetect™

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Accessible and effective strategies for all cultivation facilities.

Translating Research into Action

The implications of HLVd for the cannabis industry cannot be underestimated. From the first official report of this viroid in Cannabis in California in 2019 by two separate studies (Bektas et al., 2019; Warren et al., 2019), the viroid has already spread to more than 90% of cultivation, as shown by findings from the research performed in 2021 by Dark Heart Nursery Research. According to a recent report, HLVd is a silent threat and probably the biggest problem for Cannabis farmers around the world (https://mjbizdaily.com/experts-sound-alarm-over-global-spread-of-cannabis-viroid/). It’s estimated that HLVd could cause the cannabis industry to lose up to $4 billion in crops every year (Adkar-Purushothama et al., 2023).

These studies have awakened consciousness about this viroid. Today, we face the challenge of developing research and technologies that support accessible strategies applicable to all growers and cultivation facilities, such as MFDetect™, to prevent the spread of the viroid and protect our people’s jobs and the industry.

Biosecurity Action Plans are Key to Guarantee Success

Drawing from our research findings and collective industry expertise, we recommend the following best practices to prevent and control HLVd:
Meticulous Equipment Maintenance: Thoroughly sterilize tools and equipment before working with new plants to minimize the risk of contamination.
Stringent Hygiene Measures: Cultivation managers and staff must rigorously adhere to strict hygiene protocols, including thorough hand and glove sterilization, to prevent HLVd transmission.
Download and print this poster to ensure your crew is aware of the crucial biosecurity measures they should follow both at work.
Comprehensive Testing Protocols: Conduct rigorous pathogen testing on clones and seeds to ensure they are free from HLVd infection before propagation.
Integrated Pest Management: Implement effective pest control measures, as pests can act as vectors for HLVd transmission.
Harnessing Tissue Culture Techniques: Consider incorporating tissue culture methods, which hold promise in eliminating HLVd from infected plants and increasing the likelihood of selecting HLVd-free clones.

Conclusion

We all strive for the growth of our industry, and working as a community is crucial to continuously elevate our standards and effectively address the current needs and challenges. It is imperative that we continuously expand our knowledge base and provide recommendations to protect cannabis crops, ensuring high-quality yields. Every grower and every plant matters. By embracing advanced testing strategies, such as utilizing MFDetect™ and prioritizing leaf tissue analysis, cultivators can confidently detect and manage HLVd infections. Through collaboration, cutting-edge advancements, and a steadfast commitment to biosecurity, we can safeguard the future of the cannabis industry and ensure the continued growth and success of cultivators worldwide. Please, don’t miss out on the full story. It’s crucial to gather firsthand information. Click here to download our complete research. Angel Fernandez Plant Geneticist CEO at MyFloraDNA

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