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Citation: Adkar-Purushothama, C.R.;
Sano, T.; Perreault, J.-P. Hop Latent
Viroid: A Hidden Threat to the
Cannabis Industry. Viruses 2023,15,
681. https://doi.org/10.3390/
v15030681
Academic Editor: Jeanmarie Verchot
Received: 13 February 2023
Revised: 25 February 2023
Accepted: 3 March 2023
Published: 4 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
viruses
Review
Hop Latent Viroid: A Hidden Threat to the Cannabis Industry
Charith Raj Adkar-Purushothama 1,* , Teruo Sano 2and Jean-Pierre Perreault 1
1RNA Group, Department of Biochemistry and Functional Genomics, Universitéde Sherbrooke,
Sherbrooke, PQ J1E 4K8, Canada
2Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan
*Correspondence: charith.adkar@usherbrooke.ca
Abstract:
Hop latent viroid (HLVd) is the biggest concern for cannabis and hop growers worldwide.
Although most HLVd-infected plants remain asymptomatic, research on hops has demonstrated a
decrease in both the
α
-bitter acid and terpene content of hop cones, which affects their economic
value. The HLVd-associated “dudding” or “duds” disease of cannabis was first reported in 2019 in
California. Since then, the disease has become widespread in cannabis-growing facilities across North
America. Although severe yield loss associated with duds disease has been recorded, little scientific
information is available to growers in order to contain HLVd. Consequently, this review aims to
summarise all of the scientific information available on HLVd so as to be able to understand the effect
of HLVd on yield loss, cannabinoid content, terpene profile, disease management and inform crop
protection strategies.
Keywords:
hop latent viroid; cannabis disease; HLVd; viroid; duds; dudding; cannabis pathogen;
hop viroid; viroid disease
1. Introduction
Since its initial detection in 2019 in California, the “duds” disease of cannabis has be-
come the most devastating cannabis disease (syn. Hemp) (Cannabis sativa,
Cannabis indica
,
Cannabis ruderalis) in cannabis-growing areas. The causative agent of this disease was
found to be hop latent viroid (HLVd). A survey conducted in 2021 by the Dark Heart
Nursery Research that involved 200,000 tissue tests concluded that 90% of cannabis–
growing facilities in California were contaminated with HLVd. Furthermore, the re-
searchers noted that the HLVd infection could significantly affect the plants’ vigor and
yield. It has been estimated that it could cause losses of up to USD 4 billion annually for
the cannabis industry (https://www.cannabisbusinesstimes.com/article/cannabis-hop-
latent-viroid-infections-dark-heart-nursery-crop-loss/ [accessed on 13 February 2023]).
HLVd has been detected throughout the cannabis-growing facilities of North America
(https://nova-analyticlabs.com/nova-labs-tests-hlvd-and-two-other-diseases/ [accessed
on 13 February 2023] https://hempindustrydaily.com/this-elusive-pathogen-is-damaging-
hemp-nationwide-heres-how-to-fight-it/?cn-reloaded=1 [accessed on 13 February 2023]).
Another report indicated that infected crops could suffer anywhere from a 50%–70% loss
in tetrahydrocannabinol (THC) content, thus considerably lowering their commercial
value (https://stratcann.com/2021/04/01/the-hop-latent-viroids-warning-shot-to-the-
canadian-cannabis-industry/ [accessed on 13 February 2023]).
2. Viroids
To date, viroids are the smallest known infectious agents that are predominantly
known to cause disease in plants. The term “viroid” was proposed by Theodor Diener in
1971 to describe a protein-free, small (50–80 times smaller than the smallest viral genomes),
low molecular weight, plant pathogenic RNA molecule [
1
]. The potato spindle tuber viroid
(PSTVd) was the first viroid species to be identified and characterized [
2
]. Specifically,
Viruses 2023,15, 681. https://doi.org/10.3390/v15030681 https://www.mdpi.com/journal/viruses
Viruses 2023,15, 681 2 of 11
PSTVd was isolated from Irish potatoes showing degenerative disease in North America.
The existence of “viroids” was further supported by the subsequent discovery of citrus
exocortis viroid (CEVd) from citrus plants exhibiting citrus exocortis disease [
3
]. Since
then, more than 50 viroid/viroid-like RNAs have been isolated from different host plants,
including both mono- and dicotyledons. Advancements in plant pathogen diagnosis
technology, new viroids and their distribution in new geographic areas are constantly being
reported [
4
]. Viroid disease symptoms in the host plant greatly depend on both the viroid
variant and the host plant cultivar. For instance, the PSTVd variant intermediate (PSTVd-I
GenBank accession No. AY937179) induces severe disease symptoms in tomato cultivar
Rutgers, but it is asymptomatic in tomato cultivar Moneymaker, even though all plants
were cultivated under identical environmental conditions. On the other hand, two viroid
variants of the same viroid species can induce different disease symptoms on the same
plant cultivar. For example, PSTVd-I and PSTVd-RG1 (GenBank accession No. U23058),
which differ by only three nucleotides, induce intermediate and severe disease symptoms
on tomato cultivar Rutgers, respectively.
To date, 44 species of viroids have been characterized and classified into either the
Avsunviroidae or the Pospiviroidae families based on the structure of the mature viroid
RNA, the central conserved region (CCR), any self-cleaving activity (i.e., ribozyme), the
subcellular site of replication in the host plant, the mode of replication and the enzymes
involved in that replication [5]. There are five members in the family Avsunviroidae. All of
these viroid species replicate in chloroplasts through a symmetric rolling-circle mechanism
and exhibit self-cleavage activity. All of the remaining 39 viroid species are grouped under
the family Pospiviroidae. Members of the Pospiviroidae family replicate in the host’s nucleus
through an asymmetric rolling-circle mechanism and are characterized by the presence of
five structural/functional domains (the terminal left (TL), pathogenicity (P), central (C),
variable (V) and terminal right (TR) domains).
3. Hop Latent Viroid
In 1987, Pallas et al. reported the presence of viroid-like RNA in nucleic acid preparations
from two of the three commercial hop varieties (Humulus lupulus L., family, Cannabaceae)
grown in the León region of Spain. Although this RNA molecule had the same size as the
avocado sunblotch viroid (ASBVd), its physical and biological properties differed from
those of all previously characterized viroids [
6
]. Since this viroid-like RNA showed a faster
electrophoretic mobility than the hop stunt viroid (HSVd; 297-nt in size), it was tentatively
identified as “hop viroid-like RNA fast” (HV-f). In order to evaluate the possible infection
of hops by HV-f, a survey was conducted in 17,000 hectares of hop gardens in Hallertau,
Germany, the main German hop-growing region. This survey revealed the presence of
HV-f in all of the hop cultivars examined [
7
]. Since HV-f did not induce any visible disease
symptoms in hops, it was tentatively named “hop latent viroid” (HLVd). Comprehensive
worldwide surveys revealed the presence of HLVd in most of the hop cultivars tested [
7
].
Although HLVd-infected hop plants are symptomless, infection significantly reduces both
yield and either the
α
-bitter acid or the essential oil content in the hop cone, thus reducing
its market value [
8
]. Subsequently, HLVd has been detected in 90% to 100% of the tested
hop germplasms in European countries [9].
Both biochemical and molecular biology analyses revealed that HLVd is a covalently
closed, circular RNA of 256-nt. Thermodynamically, HLVd folds into a rod-like secondary
structure, with 65.5% of the residues being internally base-paired (see below). Although
HLVd had a CCR, it lacked an oligo (A) stretch in the upper part of the left domain in its
secondary structure. HSVd, which is known to cause stunting disease in hops, exhibited
45% sequence similarity with HLVd, while PSTVd was found to be 51% identical with
it. The closest sequence identity found with HLVd was coconut cadang-cadang viroid
(CCCVd; 54%), while ASBVd (36%) was least similar [
7
]. Based on structural features such
as the rod-like conformation, the presence of the five functional domains, the absence of
any self-cleavage activity, the presence of both a terminal conserved hairpin (TCH) and a
Viruses 2023,15, 681 3 of 11
CCR identical to that of members of the other species of the genus, HLVd was placed in the
genus Cocadviroid of the family Pospiviroidae (Table 1).
Table 1. Characteristic features of HLVd.
Features Characteristics
Structure of mature viroid RNA Rod-like
Central conserved region (CCR) Present
Terminal conserved hairpin (TCH) Present
Self-cleaving activity (i.e., ribozyme) absent
Replication site in the host Nucleolus
Mode of Replication Asymmetric rolling circle
Enzymes involved in replication DNA-dependent RNA polymerase II
4. Host Range and Transmission of HLVd
Until mid-2019, HLVd was known to infect only three natural hosts, specifically the
commercial hop (Humulus lupulus), the Japanese hop (Humulus japonicus Sieb. and Zucc.)
(Figure 1) and the stinging nettle (Urtica dioica L.) [
10
]. Heat-generated HLVd variants (i.e.,
thermomutants) can infect what otherwise are considered as non-susceptible species such
as tomato (Solanum lycopersicum) and Nicotiana benthamiana [
11
]. In 2019, two independent
groups reported the detection of HLVd in stunted cannabis plants in the USA [12,13].
Viruses 2023, 15, 681 3 of 11
activity, the presence of both a terminal conserved hairpin (TCH) and a CCR identical to
that of members of the other species of the genus, HLVd was placed in the genus Cocadvi-
roid of the family Pospiviroidae (Table 1).
Table 1. Characteristic features of HLVd.
Features Characteristics
Structure of mature viroid RNA Rod-like
Central conserved region (CCR) Present
Terminal conserved hairpin (TCH) Present
Self-cleaving activity (i.e., ribozyme) absent
Replication site in the host Nucleolus
Mode of Replication Asymmetric rolling circle
Enzymes involved in replication DNA-dependent RNA polymerase II
4. Host Range and Transmission of HLVd
Until mid-2019, HLVd was known to infect only three natural hosts, specifically the
commercial hop (Humulus lupulus), the Japanese hop (Humulus japonicus Sieb. and Zucc.)
(Figure 1) and the stinging nettle (Urtica dioica L.) [10]. Heat-generated HLVd variants (i.e.,
thermomutants) can infect what otherwise are considered as non-susceptible species such
as tomato (Solanum lycopersicum) and Nicotiana benthamiana [11]. In 2019, two independent
groups reported the detection of HLVd in stunted cannabis plants in the USA [12,13].
Figure 1. Symptoms (leaf distortion and vein yellowing) on Japanese hop plants infected with HLVd.
With the globalization of agriculture, viroids have become widely distributed in both
new environments and new geographical areas. For example, HLVd was introduced to Bra-
zil from the USA through hop germplasm [14]. HLVd can be transmitted over long distances
and can be introduced into hop gardens by infected propagative materials. HLVd transmits
within the hop yards mechanically, by grafting and vegetative propagation, and by contam-
inated tools or machinery [15]. HLVd transmission by either pollen or seed has been re-
ported as being either low or non-existing, respectively [10,16]. Despite the new infections
away from the infected hop yards are common, to date insect transmission of HLVd is not
known [17].
5. HLVd-Associated Symptoms in Hops
Although HLVd-infected hop plants are symptomless, infection significantly reduces
both cone yield and either the bitter acid or the essential oil content in the hop cones of sensi-
tive cultivars [8]. Currently, HLVd has been reported in commercial hop yards in Australia,
New Zealand, China, Japan, Republic of Korea, Russia, Slovenia, Poland, Germany, the Czech
Republic, Hungary, France, Spain, Portugal, the United Kingdom, South Africa, the USA
and Brazil [15]. However, due to the asymptomatic nature of the HLVd infection in most
Figure 1.
Symptoms (leaf distortion and vein yellowing) on Japanese hop plants infected with HLVd.
With the globalization of agriculture, viroids have become widely distributed in both
new environments and new geographical areas. For example, HLVd was introduced to
Brazil from the USA through hop germplasm [
14
]. HLVd can be transmitted over long
distances and can be introduced into hop gardens by infected propagative materials. HLVd
transmits within the hop yards mechanically, by grafting and vegetative propagation, and
by contaminated tools or machinery [15]. HLVd transmission by either pollen or seed has
been reported as being either low or non-existing, respectively [
10
,
16
]. Despite the new
infections away from the infected hop yards are common, to date insect transmission of
HLVd is not known [17].
5. HLVd-Associated Symptoms in Hops
Although HLVd-infected hop plants are symptomless, infection significantly reduces
both cone yield and either the bitter acid or the essential oil content in the hop cones
of sensitive cultivars [
8
]. Currently, HLVd has been reported in commercial hop yards
in Australia, New Zealand, China, Japan, Republic of Korea, Russia, Slovenia, Poland,
Germany, the Czech Republic, Hungary, France, Spain, Portugal, the United Kingdom,
South Africa, the USA and Brazil [
15
]. However, due to the asymptomatic nature of the
Viruses 2023,15, 681 4 of 11
HLVd infection in most of these hop cultivars, these HLVd infections have long gone
unnoticed. HLVd symptoms are evident only by comparison of infected susceptible and
healthy hop cultivars. In susceptible cultivars such as ‘Omega’, HLVd-associated symptoms
include chlorosis, slow growth and the presence of fewer and smaller cones [
8
]. HLVd also
affects both nursery production and breeding programs since it significantly reduces both
the rooting and the establishment of softwood cuttings [10].
6. Effect of HLVd on the Terpene and Essential Oil Contents
Despite the absence of any characteristic symptoms in somatic tissues, HLVd induces
physiological changes that affect both qualitatively and quantitatively the metabolites of the
lupulin secretory glands and of the essential oils, thus indicating that the HLVd infection
is not a truly latent one [
18
]. In HLVd-infected plants, the yield loss can be as low as 8%
for the Wye Challenger cultivar, and as high as 37.5% for the Slovenian hop cultivar [
8
,
19
].
The reductions in
α
-bitter acid were 15 and 30% for the cultivars Wye Challenger and
Omega, respectively. Both cultivars showed increased
β
-bitter acid and oil contents [
18
].
A higher amount of
β
-bitter acid and oil content leads to the early maturation of the hop
cones [
8
]. The effect of HLVd on the
α
-bitter acid content was genotype-dependent. This
reduction in
α
-bitter acids ranged from 20% to 50% within English hop cultivars [
17
]. Other
hop cultivars in the Czech Republic also showed signification reductions in the
α
-bitter
acid content in HLVd-infected plants (ex. Saaz, 40% reduction; Premiant, 40% reduction;
Aurora, 18% reduction; and, Sybilla, Marynka, Pulawski and Magnat, from 11% to 23%
reduction) [19].
HLVd is also known to affect both the oil and the terpene profiles of hops. For instance,
HLVd infection increased the levels of monoterpenes such as myrcene and both
α
- and
β
-pinene by 29% to 41.6% as compared to healthy plants [
20
]. On the contrary, HLVd
infection decreased the levels of sesquiterpenes such as
β
-caryophyllene,
α
-humulene,
α
-
copaene,
γ
-muurolene,
β
-bisabolene,
γ
-cadinene and
δ
-cadinene by 13 to 29%. The possible
influence of some oxidative-reduction processes that are activated by the viroid-caused
pathogenesis was assumed to be involved in the accumulation of terpenes alcohols such
as geraniol and methylgeranate, as well as in the reduction in the levels of the majority of
the ketones detected in the spectra of the essential oils [
20
]. However, these changes in the
composition of the essential oils present in the hop cones are both genotype-dependent and
cultivar-specific [
21
]. For instance, the linalool content in the cones of infected plants was
found to be higher for cultivars Sybillla, Lubelski and Pulawski, but lower for cultivars
Marynka and Magnat. The methylgeranate content was found to be lower in the infected
plants from all cultivars [
20
]. In terms of the importance of the HLVd infection of hops with
economic importance, even the slightest differences in either the content or composition in
α
-bitter acid,
β
-bitter acid, oil content and in the terpene profile can change the resulting
beer’s aroma [10]
7. HLVd Disease in Cannabis
HLVd disease in Cannabis plants is loosely described as “duds” or “dudding disease”
(https://stratcann.com/insight/the-hop-latent-viroids-warning-shot-to-the-canadian-ca-
nnabis-industry/ [accessed on 13 February 2023]). Like in hops, only a few cultivars of
cannabis show HLVd-associated symptoms, implying that both symptom expression and
disease severity are cultivar genotype dependent. In 2014, cannabis growers started an on-
line thread discussing the “dudding disease” symptoms in cannabis plants (https://www.
thcfarmer.com/threads/what-to-do-with-duds.64342/ [accessed on
13 February 2023
]).
The association of HLVd infection with symptomatic plants was confirmed in 2019 by
two independent
teams using high-throughput sequencing technology and subsequent
bioassays [
12
,
13
]. In susceptible cultivars HLVd induces symptoms (Figure 2) such as
shorter internodal spacing, smaller leaves, stunting, malformation (outwardly horizon-
tal plant structure), chlorosis, brittle stems, reduced vigor, lower water intake, reduced
flower mass and trichomes [
12
,
13
]. At the flowering stage, susceptible plants typically
Viruses 2023,15, 681 5 of 11
show smaller and looser buds, weaker flower smell and less trichome production. This
effect is reflected in both the yield and loss of quality that includes up to a 50% re-
duction in both cannabinoid and terpene production (https://www.plantcelltechnology.
com/blog/everything-you-should-know-about-hop-latent-viroid-hplvd/ [
accessed on
13 February 2023
]). A survey conducted in 2021 revealed that approximately 90% of all
cannabis-growing facilities in California tested positive for HLVd, and 30% of the plants
in each facility showed symptoms of the viroid’s infection (https://docs.google.com/
document/d/1G5Lwz14F5-baVGveMe_0yGnam7z1E0qQZJ8YAcardFw/edit# [
accessed on
13 February 2023]). Since its first detection in California, HLVd did not take much time to
find its way to British Columbia, Canada. Now, HLVd is prevalent in Canadian cannabis-
growing facilities. This clearly illustrates the severity of HLVd disease and its threat to the
cannabis industry in North America.
Viruses 2023, 15, 681 5 of 11
in both the yield and loss of quality that includes up to a 50% reduction in both cannabinoid
and terpene production (https://www.plantcelltechnology.com/blog/everything-you-should-
know-about-hop-latent-viroid-hplvd/ [accessed on 13 February 2023]). A survey conducted
in 2021 revealed that approximately 90% of all cannabis-growing facilities in California tested
positive for HLVd, and 30% of the plants in each facility showed symptoms of the viroid’s
infection (https://docs.google.com/document/d/1G5Lwz14F5-baVGveMe_0yG-
nam7z1E0qQZJ8YA
cardFw/edit# [accessed on 13 February 2023]). Since its first detection in California, HLVd
did not take much time to find its way to British Columbia, Canada. Now, HLVd is preva-
lent in Canadian cannabis-growing facilities. This clearly illustrates the severity of HLVd
disease and its threat to the cannabis industry in North America.
Figure 2. HLVd associated symptoms in susceptible cannabis plants.
8. Comparison between HLVd Isolated from Hops and Cannabis
Due to the absence of a protein coat around the viroid genome, its secondary structure
plays a significant role in determining its ability to invade the host plant, survival and path-
ogenesis. Therefore, understanding the secondary structure of HLVd is paramount to un-
derstanding the host-viroid relationship. Due to the non-coding nature of viroids, they re-
cruit a host DNA-dependent RNA polymerase during replication. Specifically, nucleus-rep-
licating viroids such as HLVd use DNA-dependent RNA polymerase II [22]. Since it is an
abnormal condition for DNA-dependent RNA polymerase II to use RNA as a template,
the resulting replication is error-prone [23]. The sequence variants created by the replica-
tion of a master sequence during this process are called “quasi-species” [24]. Analysis of
high-throughput sequencing data obtained from PSTVd-infected plants revealed the pres-
ence of “quasi-species” of the members of the family Pospiviroidae [25,26]. However, the
sequence analysis of HLVd-infected hop plants revealed only a small number of sequence
variants as compared to what has been seen with other viroid species [7,27].
In order to understand both the sequence variation and adaptation of HLVd to cannabis
plants, the HLVd-type species isolated from hops (GenBank Acc. No.: NC_003611) and all of
the HLVd sequences isolated from cannabis plants were compared. In order to achieve this
goal, all of the HLVd sequences isolated from cannabis plants that are available in NCBI
were retrieved, and only the full-length sequence (256-nt) was considered for the analysis.
Warren et al. [13] reported two distinct HLVd isolates, specifically isolates Can1 (GenBank
Acc. No.: MK876285) and Can2 (GenBank Acc. No.: MK876286). The Can1 isolate showed a
100% sequence similarity with the HLVd-type species, while the Can2 isolate had one mis-
match. Specifically, at nucleotoide 225, the uracil (U) was mutated to adenine (A) in isolate
Figure 2. HLVd associated symptoms in susceptible cannabis plants.
8. Comparison between HLVd Isolated from Hops and Cannabis
Due to the absence of a protein coat around the viroid genome, its secondary structure
plays a significant role in determining its ability to invade the host plant, survival and
pathogenesis. Therefore, understanding the secondary structure of HLVd is paramount to
understanding the host-viroid relationship. Due to the non-coding nature of viroids, they
recruit a host DNA-dependent RNA polymerase during replication. Specifically, nucleus-
replicating viroids such as HLVd use DNA-dependent RNA polymerase II [
22
]. Since it is
an abnormal condition for DNA-dependent RNA polymerase II to use RNA as a template,
the resulting replication is error-prone [
23
]. The sequence variants created by the replication
of a master sequence during this process are called “quasi-species” [
24
]. Analysis of high-
throughput sequencing data obtained from PSTVd-infected plants revealed the presence of
“quasi-species” of the members of the family Pospiviroidae [
25
,
26
]. However, the sequence
analysis of HLVd-infected hop plants revealed only a small number of sequence variants as
compared to what has been seen with other viroid species [7,27].
In order to understand both the sequence variation and adaptation of HLVd to cannabis
plants, the HLVd-type species isolated from hops (GenBank Acc. No.: NC_003611) and all
of the HLVd sequences isolated from cannabis plants were compared. In order to achieve
this goal, all of the HLVd sequences isolated from cannabis plants that are available in
NCBI were retrieved, and only the full-length sequence (256-nt) was considered for the
analysis. Warren et al. [
13
] reported two distinct HLVd isolates, specifically isolates Can1
(GenBank Acc. No.: MK876285) and Can2 (GenBank Acc. No.: MK876286). The Can1
isolate showed a 100% sequence similarity with the HLVd-type species, while the Can2
Viruses 2023,15, 681 6 of 11
isolate had one mismatch. Specifically, at nucleotoide 225, the uracil (U) was mutated
to adenine (A) in isolate Can2 as compared to the HLVd-type species. Hereafter, this
point mutation will be referred to as U225A. However, the sequence of the Can2 isolate
was 100% identical to an HLVd isolate retrieved from a commercial hop garden in China
(GenBank Acc. No.: EF613183). Interestingly, both isolates have also been reported from
HLVd-infected cannabis plants located elsewhere in the USA. More specifically, HLVd
reported from Delta county (CO, USA) was found to be 100% similar to the Can1 isolate,
and the Can2 isolate matched with an HLVd isolated from both Santa Barbara (CA, USA)
and Boulder counties (CO, USA) [
12
,
28
]. This indicates the presence of at least
two HLVd
sequence variants infecting cannabis.
In order to understand the effect of U225A on HLVd structure, the secondary structure
of the HLVd Can2 isolate was predicted using the RNAfold WebServer (http://rna.tbi.
univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi [accessed on 30 November 2022]) and
was compared with the secondary structure of the HLVd-type species. The
∆
G values for
both the Can2 isolate and the HLVd-type species were found to be
−
95.30. The single
nucleotide change (U225A) did not affect the structure of the Can2 isolate as compared
to that of the type species. Although a single point mutation at position 225 did not alter
the secondary structure, it is interesting to note that this change is located within the
lower pathogenicity domain (Figure 3). Previously, we have isolated a variant of PSTVd
from Dahlia (PSTVd-D; GenBank Acc. No.: AB623143) that induces mild symptoms on
tomato cultivar Rutgers as compared to what has been seen with PSTVd-I. At the genomic
level, these two differed by nine nucleotides [
29
]. Through mutagenic studies, it was
demonstrated that a change in one nucleotide in the pathogenicity domain of PSTVd-D
is crucial in both disease symptoms attenuation and in the escape from the host’s defense
mechanism in the tomato cultivar Rutgers [
30
]. Similarly, the HSVd isolated from hops
(HSVd-hop type) adapts to grapevines by the presence of five point mutations [
31
]. Hence,
it would be interesting to study the effect of the single nucleotide change observed in the
pathogenicity domain of the HLVd isolated from cannabis on the host’s transcriptome, on
the disease’s severity and on its adaptability to the host plant.
Viruses 2023, 15, 681 6 of 11
Can2 as compared to the HLVd-type species. Hereafter, this point mutation will be referred
to as U225A. However, the sequence of the Can2 isolate was 100% identical to an HLVd
isolate retrieved from a commercial hop garden in China (GenBank Acc. No.: EF613183).
Interestingly, both isolates have also been reported from HLVd-infected cannabis plants
located elsewhere in the USA. More specifically, HLVd reported from Delta county (Colo-
rado, USA) was found to be 100% similar to the Can1 isolate, and the Can2 isolate matched
with an HLVd isolated from both Santa Barbara (California, USA) and Boulder counties (Col-
orado, USA) [12,28]. This indicates the presence of at least two HLVd sequence variants in-
fecting cannabis.
In order to understand the effect of U225A on HLVd structure, the secondary structure
of the HLVd Can2 isolate was predicted using the RNAfold WebServer (http://rna.tbi.uni-
vie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi [accessed on 30 November 2022]) and was
compared with the secondary structure of the HLVd-type species. The ΔG values for both the
Can2 isolate and the HLVd-type species were found to be −95.30. The single nucleotide
change (U225A) did not affect the structure of the Can2 isolate as compared to that of the type
species. Although a single point mutation at position 225 did not alter the secondary structure,
it is interesting to note that this change is located within the lower pathogenicity domain
(Figure 3). Previously, we have isolated a variant of PSTVd from Dahlia (PSTVd-D; GenBank
Acc. No.: AB623143) that induces mild symptoms on tomato cultivar Rutgers as compared to
what has been seen with PSTVd-I. At the genomic level, these two differed by nine nucleotides
[29]. Through mutagenic studies, it was demonstrated that a change in one nucleotide in the
pathogenicity domain of PSTVd-D is crucial in both disease symptoms attenuation and in
the escape from the host’s defense mechanism in the tomato cultivar Rutgers [30]. Simi-
larly, the HSVd isolated from hops (HSVd-hop type) adapts to grapevines by the presence
of five point mutations [31]. Hence, it would be interesting to study the effect of the single
nucleotide change observed in the pathogenicity domain of the HLVd isolated from can-
nabis on the host’s transcriptome, on the disease’s severity and on its adaptability to the
host plant.
Figure 3. Secondary structure prediction of the HLVd-type species. The boxed regions are the terminal
conserved hairpin (TCH) and Central Conserved Regions (CCR). The arrow indicates the U225A point
mutation observed in the HLVd isolated from a commercial hop gardens of China.
9. Control of HLVd Disease
The basis of viroid control is the production of viroid-free propagative materials. There
are several layers of control measures that are involved. The first and foremost is the pre-
vention provided by testing all of the incoming cannabis plants and cannabis products that
could potentially act as a carrier for HLVd. Hence, it is good practice to test the plants and
products for potential HLVd contaminants before bringing them into the facility. Once the
plants are received, a 30-day quarantine is critical in order to properly evaluate for the
presence of plant pathogens in incoming plant material. Given that HLVd infection is
asymptomatic in many cannabis cultivars, it is recommended to test any new plant during
the third week of quarantine, and certainly before sending the newly received plants into
production. Although there is no chemical treatment available for controlling HLVd dis-
ease, methods such as meristem tip culture [32], thermotherapy [33] and cold treatment [34]
have been found to reduce viroid titer significantly. Meristem culturing of shoot tip success-
fully eliminated HLVd from two infected hop cultivars [35]. However, combining cold treat-
ment (incubating plant materials at 2 to 4 °C in the dark for periods ranging from 8 to 21
Figure 3.
Secondary structure prediction of the HLVd-type species. The boxed regions are the
terminal conserved hairpin (TCH) and Central Conserved Regions (CCR). The arrow indicates the
U225A point mutation observed in the HLVd isolated from a commercial hop gardens of China.
9. Control of HLVd Disease
The basis of viroid control is the production of viroid-free propagative materials.
There are several layers of control measures that are involved. The first and foremost is the
prevention provided by testing all of the incoming cannabis plants and cannabis products
that could potentially act as a carrier for HLVd. Hence, it is good practice to test the plants
and products for potential HLVd contaminants before bringing them into the facility. Once
the plants are received, a 30-day quarantine is critical in order to properly evaluate for
the presence of plant pathogens in incoming plant material. Given that HLVd infection is
asymptomatic in many cannabis cultivars, it is recommended to test any new plant during
the third week of quarantine, and certainly before sending the newly received plants into
production. Although there is no chemical treatment available for controlling HLVd disease,
methods such as meristem tip culture [
32
], thermotherapy [
33
] and cold treatment [
34
] have
been found to reduce viroid titer significantly. Meristem culturing of shoot tip successfully
eliminated HLVd from two infected hop cultivars [
35
]. However, combining cold treatment
Viruses 2023,15, 681 7 of 11
(incubating plant materials at 2 to 4
◦
C in the dark for periods ranging from 8 to 21 months)
and the meristem culture of tips of less than 0.5 mm in size was found to be more effective
in eliminating HLVd [
34
]. Thermotherapy involving incubating plants at 36
◦
C for
14 days
rapidly decreased viroid titer, while the original titer was restored after six months under
field conditions. The decrease in HLVd titer was correlated with the induction of an RNA
silencing mechanism that resulted in the cleaving of the viroid RNA [
33
]. Unlike other
viroid species, HLVd is known to produce negligible levels of mutated variants under
standard cultivation conditions. However, thermotherapy resulted in a significant increase
in the number of sequence variants detected in the HLVd population. All mutated cDNAs
were infectious and evolved into complex progeny populations that contained low levels
of the molecular variants. These thermomutants were found to be infectious to both
tomato and Nicotiana benthamiana plants, both of which are a non-host for the HLVd-type
species [
18
]. Although both cold and heat treatments could potentially reduce HLVd titer,
subsequent meristem tissues are required to completely eliminate all of the viroid [10].
10. Detection and Management of HLVd Infection
As discussed elsewhere, HLVd primarily spreads via vegetative cuttings, contaminated
tools and by other mechanical means within either the hop yards or the indoor cannabis-
growing facility. Since there are no control measures available for viroid-associated diseases,
HLVd spread can only be prevented by employing a multilayer management system
that includes, but is not limited to, the timely identification and removal of infected
plants. The absence of visible symptoms associated with HLVd challenges the ability of
growers to contain these viroids. Biological indexing is not practical for HLVd due to its
limited host range and lack of symptomatology. However, if a susceptible host plant is
available, graft inoculation was found to be more effective than rub-inoculation for the
infection of hop plants with the hop latent viroid [
36
]. Due to these difficulties, and to the
laborious procedure of biological indexing, molecular detection methods such as dot-blot
hybridization, RT-PCR and RT-qPCR can be employed for routine diagnosis [
27
]. Since
viroid distribution is uneven in the host plant, it is important to take multiple leaf samples
from the lower to the upper stem, covering both old and new leaves. Although partial
replanting following the removal of diseased and adjacent plants can be effective, better
control has been achieved by removing all plants in a yard, followed by replanting with
certified viroid-free material [
10
]. Roguing for viroid-infected hops required that all plants,
including rhizomes, be destroyed by treating with urea and chloropicrin in the autumn in
order to ensure that the disease is not carried over to the succeeding crop [
10
]. However,
in the case of cannabis (which is grown indoors), incineration or treating with bleach
could effectively eliminate the liberation of HLVd into the environment. Studies on HSVd
demonstrated that it could be found in hop residue; however, leaves and cones were found
to lose their infectivity within three months when left to be weather-beaten [
37
]. That said,
HLVd can survive longer in plant material (including the cannabis flower) if it is stored
at lower than room temperature. This suggests the chance of HLVd transmission through
cannabis products that are either transferred between the producers or are purchased in
the market.
The removal of viroid-infected plants, and the sanitation of equipment, tools and of
the vicinity of the viroid-infected plants are critical to checking the spread of the viroid
in both fields and in indoor growth facilities. Heating blades at 160
◦
C for 10 min was
found to be effective, while doing so at 140
◦
C was not. These measures have seen the
virtual elimination of HSVd from the Kirin Brewery yards in Japan [
10
]. Among the
different heat and chemical treatments tested for tool sanitation against viroids, the most
reliable and widely used one is an aqueous solution of at least 5% household bleach
(sodium hypochlorite, minimum 1% available chlorine) [
15
]. However, the treatment of
greenhouse tools with 10% regular Clorox bleach (an active ingredient of 5.25% sodium
hypochlorite [NaOCl]) for 10 s was found to be effective against the transmission of PSTVd
in tomatoes [
38
]. The strong corrosive effect on both the greenhouse structure and tools,
Viruses 2023,15, 681 8 of 11
and the potential phytotoxic effect on plants, should be considered before employing bleach
as a sanitizer. Another alternative is Virkon S, which has already been proven effective
against both human and animal viral pathogens [
39
,
40
]. Although it is relatively expensive
and corrosive, it is the most promising disinfectant against viroids and viruses infecting
greenhouse-grown tomatoes when it is used at a 2% concentration (20 g/L) [
38
]. In addition
to testing and sanitation procedures, it is important to maintain both high standard cultural
practices and high personal hygiene practices. This includes, but is not limited to, regular
handwashing and the changing of personal protective equipment (PPR) between the hop
yards or cultivation rooms.
11. Viroid Species Known to Infect Hops
Aside from HLVd, at least three other viroid species are known to infect hops. As
hops and cannabis belong to the same family, there is a significant risk that these viroids
can also adapt and infect cannabis under ideal environmental conditions. Such a scenario
is not uncommon for viroids. For instance, PSTVd is known to cause disease in potato
and tomato plants, both of which belong to the family Solanaceae. HSVd is one of the very
important viroids infecting hops. HSVd was first discovered in Japanese hop fields, with
typical symptoms, including stunting, leaf curling, small cone formation and a substantial
reduction of alpha-acid content, being observed after 3–7 years of infection, [
41
]. HSVd has a
very broad host range and has been reported to be the causal agent of diseases such as citrus
cachexia, cucumber pale fruit, peach and plum dapple fruit and hop stunt [
42
]. Due to its
quasi-species nature, HSVd can quickly adapt to grapevines, which serve as a reservoir for
the viroid [
43
]. HSVd was reported to infect hops and stone fruits in the USA and Canada,
respectively [
44
,
45
]. The apple fruit crinkle viroid (AFCVd), first identified as the causal
agent of apple fruit crinkle disease, induces stunting in hops that resembles the HSVd-
associated symptoms observed in Japan [
41
] and also infects persimmon asymptomatically.
Among all hop-infecting viroids, the citrus bark cracking viroid (CBCVd) is the most
aggressive, and symptoms appear after one year of infection. In hops, symptoms include
leaf down curling, severe bine stunting, a reduction in cone size, dry root rotting after the
first dormancy and complete plant dieback in 3–5 years [
46
]. However, it is interesting to
note that the infection of hop by CBCVd alone is not observed in either nature or in hop
fields [
47
]. In other words, CBCVd is found only in hop plants already infected with HLVd.
Experimentally, the hop plants co-inoculated with CBCVd and HLVd plants showed more
severe disease symptoms than those inoculated with only either CBCVd or HLVd [
47
]. It
should be noted that both HLVd and CBCVd belong to the same viroid genus, namely
Cocadviroid. Figure 4illustrates HSVd-, AFCVd- and CBCVd-associated symptoms on
hop, respectively.
Viruses 2023,15, 681 9 of 11
Viruses 2023, 15, 681 8 of 11
Although it is relatively expensive and corrosive, it is the most promising disinfectant
against viroids and viruses infecting greenhouse-grown tomatoes when it is used at a 2%
concentration (20 g/L) [38]. In addition to testing and sanitation procedures, it is important
to maintain both high standard cultural practices and high personal hygiene practices.
This includes, but is not limited to, regular handwashing and the changing of personal
protective equipment (PPR) between the hop yards or cultivation rooms.
11. Viroid Species Known to Infect Hops
Aside from HLVd, at least three other viroid species are known to infect hops. As hops
and cannabis belong to the same family, there is a significant risk that these viroids can also
adapt and infect cannabis under ideal environmental conditions. Such a scenario is not un-
common for viroids. For instance, PSTVd is known to cause disease in potato and tomato
plants, both of which belong to the family Solanaceae. HSVd is one of the very important
viroids infecting hops. HSVd was first discovered in Japanese hop fields, with typical symp-
toms, including stunting, leaf curling, small cone formation and a substantial reduction of
alpha-acid content, being observed after 3–7 years of infection, [41]. HSVd has a very broad
host range and has been reported to be the causal agent of diseases such as citrus cachexia,
cucumber pale fruit, peach and plum dapple fruit and hop stunt [42]. Due to its quasi-spe-
cies nature, HSVd can quickly adapt to grapevines, which serve as a reservoir for the viroid
[43]. HSVd was reported to infect hops and stone fruits in the USA and Canada, respectively
[44,45]. The apple fruit crinkle viroid (AFCVd), first identified as the causal agent of apple
fruit crinkle disease, induces stunting in hops that resembles the HSVd-associated symp-
toms observed in Japan [41] and also infects persimmon asymptomatically. Among all hop-
infecting viroids, the citrus bark cracking viroid (CBCVd) is the most aggressive, and symp-
toms appear after one year of infection. In hops, symptoms include leaf down curling, severe
bine stunting, a reduction in cone size, dry root rotting after the first dormancy and complete
plant dieback in 3–5 years [46]. However, it is interesting to note that the infection of hop by
CBCVd alone is not observed in either nature or in hop fields [47]. In other words, CBCVd
is found only in hop plants already infected with HLVd. Experimentally, the hop plants co-
inoculated with CBCVd and HLVd plants showed more severe disease symptoms than
those inoculated with only either CBCVd or HLVd [47]. It should be noted that both HLVd
and CBCVd belong to the same viroid genus, namely Cocadviroid. Figure 4 illustrates
HSVd-, AFCVd- and CBCVd-associated symptoms on hop, respectively.
Figure 4. The disease symptoms observed in hop plants infected with HSVd included curled droop-
ing leaf, while those infected with AFCVd showed both shortened internodes and leaf down curling
Figure 4.
The disease symptoms observed in hop plants infected with HSVd included curled drooping
leaf, while those infected with AFCVd showed both shortened internodes and leaf down curling (the
yellowing of the leaves is not a symptom, but rather is a cultivar characteristic). Lastly, those infected
with CBCVd showed leaf chlorosis.
12. Conclusions and Prospective
Although HLVd infection was first reported in hop plants in 1988, both the scientific
community and stakeholders prioritized research on HSVd which caused more severe
visible disease symptoms in infected hop plants than did HLVd. However, scientific
data on the negative effects of HLVd on both the
α
-bitter acid and terpene contents in
both symptomatic and asymptomatic hop collected over the years have attracted the
attention of researchers in recent years. The effect of HLVd on asymptomatic hop plants
is supported by recent transcriptomic studies conducted on hop plants co-inoculated
with HLVd and CBCVd aimed at understanding the increased aggravation of the CBCVd-
induced disease symptoms in the presence of HLVd [
47
]. Although two HLVd sequence
variants were detected in cannabis plants, it is not clear whether the identified sequence
variants are the result of viroid quasi-species or true dominant HLVd variants (refer to
Adkar-Purusothama et al., [26]
for details on viroid quasi-species). Hence, further studies
are required in order to understand whether or not both of the HLVd sequence variants are
able to infect and induce disease symptoms in cannabis plants. If so, what are the HLVd
sequence variant-specific disease symptoms on the susceptible cannabis plant and HLVd
variant-specific effect on the cannabinoids. These studies will help in understanding the
HLVd “master sequence” that cause the initial infection in cannabis plants, and in the study
of the HLVd-cannabis host interaction. Additionally, it would be interesting to understand
the role of the U225A mutation located in the lower pathogenicity domain in both HLVd’s
pathogenicity and its adaptability to cannabis plants. Since HSVd, AFCVd and CBCVd
are all infectious to hop plants, it is worth conducting a bioassay on cannabis plants and
preparing to contain them all if any infection is found.
To date, HLVd-resistant cannabis cultivars are not known. Meristem tissue culture
is the only effective control method via which infected plants can be saved. This process
is both laborious and expensive. Although tissue-cultured plantlets are viroid-free, it is
important to understand that they are not viroid-resistant. Hence, following preventive
measures in order to avoid an insurge of HLVd into a given growth environment is crucial in
HLVd-associated disease management. However, for sustainability reasons, it is important
to find a practical long-term solution by employing strategies such as the control of HLVd
Viruses 2023,15, 681 10 of 11
infection by breeding HLVd-resistant plants, by cross-protection and by developing RNA
interference-mediated resistance in the plants.
Author Contributions:
Conceptualization, C.R.A.-P. and J.-P.P.; methodology, C.R.A.-P.; software,
C.R.A.-P.; validation, T.S. and J.-P.P.; resources, T.S. and J.-P.P.; writing—original draft preparation,
C.R.A.-P.; writing—review and editing, C.R.A.-P., T.S. and J.-P.P. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The RNA group is supported by a grant from the Universitéde Sherbrooke.
J.-P.P. holds the Research Chair of the Universitéde Sherbrooke in RNA Structure and Genomics and
is a member of the Centre de Recherche du CHUS.
Conflicts of Interest: The authors declare that there is no conflict of interest.
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