Occurrence of Viroids in Commercial Hop (Humulus lupulus L.) Production Areas of Washington State

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1 2007 Plant Management Network. Accepted for publication 23 September Published. Occurrence of Viroids in Commercial Hop (Humulus lupulus L.) Production Areas of Washington State Kenneth C. Eastwell, Associate Plant Pathologist, and Mark E. Nelson, Research Technologist Supervisor, Department of Plant Pathology, Washington State University, Prosser Corresponding author: Kenneth C. Eastwell. Eastwell, K. C. and Nelson, M. E Occurrence of viroids in commercial hop (Humulus lupulus L.) production areas of Washington State. Online. Plant Health Progress doi: /php rs. Abstract Viroid status was investigated in commercial hop (Humulus lupulus) plantings in three regions of intensive hop production in Washington State. Hop stunt viroid (HSVd), the causal agent of hop stunt disease, was detected for the first time in hop plants in North America. HSVd was detected in samples from 10 of the 33 hop gardens sampled and in 19 of 126 plant samples. Infection with HSVd was associated with chlorosis and reduced plant vigor. Reverse transcriptionpolymerase chain reaction amplification, sequence identity, and pathogenicity of hop extracts on cucumber seedlings confirmed the presence of HSVd. Apple fruit crinkle viroid (AFCVd), recently reported in hops in Japan, was not detected by RT-PCR in any samples. Hop latent viroid (HLVd), which is frequently associated with hop germplasm world-wide, was detected with high frequency (98 of 126 hop plants surveyed). Introduction The US is the world s second largest hop (Humulus lupulus L.) producing country and produced 26,200 metric tons of hops in Approximately 77% of the US production in 2006 originated on farms in the state of Washington, with Oregon (15%) and Idaho (8%) being the other states with major commercial hop production (17). Small plantings of hops occur east of the continental divide primarily for local use. Twenty-two major varieties and several minor varieties are grown commercially in the US. Hop plants are clonally propagated either by rooted cuttings or by rhizomes. Three viroids are currently known to infect hop. Hop stunt disease was first recognized as a serious impediment to hop production in Japan in the 1940s, and by the 1960s had spread throughout the major hop-producing areas of northern Japan (20). The causal agent of this disease was identified as Hop stunt viroid (HSVd) in 1977 (13). With the exceptions of rhizomes introduced from Japan into South Korea (9), hop stunt disease has not been widely described in other hop growing regions of the world. Consequently, most information regarding symptomatology is limited to the cultivars Kirin II and Shinshuwase commonly grown in Japan. Typical symptoms reported include stunting, leaf curling, and small cones (20). Stunting appears 3 to 5 years after established plants become infected, thereby facilitating the unwitting propagation of infected plants. Hop latent viroid (HLVd) was first identified in hop plants in 1987 (7,8). HLVd can be detected in new plantings soon after propagation and is widely distributed wherever hops are grown (1), including North America (6). The impact of HLVd on yield from most cultivars is relatively minor (2), and it is tolerated in most hop production areas because of the modest agronomic impact coupled with rapid spread. Unlike HSVd, HLVd has a very narrow host range limited primarily to H. lupulus, Humulus japonicus Sieb. & Zucc., and stinging nettle (Urtica dioica L.) in Europe (4). In 2004, Apple fruit crinkle viroid (AFCVd) was reported in commercial hop plants in Japan and preliminary information indicated that symptoms caused by this

2 viroid are reminiscent of those caused by HSVd (12). The distribution of AFCVd in hops grown outside of Japan is not known at this time. The only other known host for AFCVd is apple (Malus domestica L.) (3), and AFCVd has not been detected in apples grown in North America. Knowledge of the viroid profile within a hop growing region is critical for developing management practices to ensure industry sustainability. The objective of this study is to determine which viroids are present in Washington State hop production regions and to gain preliminary information on affected cultivars. These results provide the foundation for further investigations and surveys to determine distribution and impact of viroids on US hop production. Detection of Viroids in Washington Hop Production Areas In September 2004, leaf tissue was collected from 33 commercial hop gardens representing each of the three areas of concentrated hop production in Washington State. Because the primary objective was to determine if previously unreported viroids might be present, collections were not necessarily random and specifically included both apparently healthy and unthrifty plants when observed. If plants uniformly appeared healthy in a given garden, arbitrary samples were collected from within that garden. Samples from twelve known and several unidentified cultivars were tested for HLVd, HSVd, and AFCVd by reverse transcription-polymerase chain reaction (RT-PCR). RNA for analysis was extracted and purified using the RNeasy Plant Mini kit (Qiagen, Valencia, CA). Approximately 0.5 g of leaf tissue was collected directly into grinding bags (Agdia, Elkhart, IN) and stored at 4 C until processed. For extraction, 2 ml 0.43 M 2-mercaptoethanol was added, and samples were ground with a hand held tissue homogenizer (Agdia); 150 µl extract was transferred to 450 µl RLT buffer (Qiagen) and mixed vigorously. Samples were heated to 56 C for 3 min and the suspension transferred to QIA shredder columns (Qiagen). The remainder of the protocol followed the manufacturer s recommendations. A one-tube duplex RT-PCR was developed for the detection of HLVd and HSVd using viroid specific primers and a separate RT-PCR assay was performed for detection of AFCVd using primers that react with a wide range of apscaviroids (Table 1). RT-PCR was performed using the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). First strand synthesis was performed at 60 C for 30 min. PCR was initiated by activating the enzyme at 94 C for 2 min, followed by 40 cycles of melting at 94 C for 15 sec, annealing at 59 C for 30 sec, and extension at 68 C for 1 min. A final extension at 68 C for 10 minutes was performed. Products were resolved by electrophoresis through a 3% agarose gel (GTG NuSieve Agarose, Lonza Rockland, Atlanta, GA); a molecular size marker (100 bp ladder, Invitrogen) was used for reference (Fig. 1). Higher molecular size bands frequently appear in assays for HSVd and HLVd (Fig. 1, lanes 3 to 6). Cloning and sequencing of these products confirmed that they represent multimeric amplification products arising because of the circular nature of the viroid genomes. Table 1. Primer sequences used to detect viroids in total RNA extracted from hop plants. Viroid detected AFCVd* HLVd HSVd Primer sequences 5 -CTGGTTGGGACCGCTGGGAC-3 5 -GACTCGTCGTCGACGAAGGGTC-3 5 -CCACCGGGTAGTTTCCAACT-3 5 -ATACAACTCTTGAGCGCCGA-3 5 -GCCCCGGGGCTCCTTTCTCAGGTAAG-3 5 -GGCAACTCTTCTCAGAATCC-3 Product size (bp) * These primers for AFCVd amplify sequences from a range of apscaviroids, including Pear blister canker viroid

3 Fig. 1. Amplification products from reverse transcription-polymerase chain reactions (RT-PCR) were analyzed to detect viroids in hop leaves. Duplex RT-PCR was used to detect Hop latent viroid (HLVd) and Hop stunt viroid (HSVd) (lanes 1 to 6). Lanes: 1, water control; 2, healthy hop seedling; 3, Tettnanger hop plant infected with HLVd; 4, Golden Light Peach from Korea (positive control for HSVd); 5, Glacier hop plant infected with HLVd; and 6, Glacier hop plant infected with HLVd and HSVd. A separate RT-PCR was used to detect Apple fruit crinkle viroid (AFCVd) (lanes 7 to 11). Lanes: 7, water control; 8, healthy hop seedling; 9, pear sample infected with the apscaviroid Pear blister canker viroid; 10, Glacier hop plant infected with HLVd; and 11, Glacier hop plant infected with HLVd and HSVd. The size in base pairs of molecular size markers (MW) are indicated on the left. Hop stunt viroid was detected in ten of the 33 hop gardens sampled in the 2004 evaluation. Infected plants were detected among 8 of 12 cultivars sampled (Table 2). Subsequent testing of other hop gardens revealed infected plants representing the remaining four cultivars (data not shown). Samples in which HSVd was detected in 2004 originated from all three areas where hop production is concentrated in Washington (Table 3). The detection of HSVd in many cultivars and in all major Washington production areas suggests that it may have been present in the industry but undetected for a significant period of time. The same samples were also assayed for the other viroids known to infect hop plants. As previously reported (6), HLVd is widely distributed in Washington hop production (Table 2). HLVd was detected in all three growing regions (Table 3) and in all hop gardens sampled with the exception of one garden planted to Galena and one garden planted to Tettnanger. AFCVd was not detected in any of the hop samples collected (Tables 2 and 3). Additional testing is necessary to determine if this pathogen may be present but at a frequency that is too low to be detected in the limited numbers of samples assayed during this initial screening process.

4 Table 2. Plants in hop production gardens of Washington State tested by reverse transcription polymerase chain reaction for Apple fruit crinkle viroid (AFCVd), Hop latent viroid (HLVd), and Hop stunt viroid (HSVd). Hop cultivar No. of plants tested No. of samples that tested positive AFCVd HLVd HSVd Centennial Galena Horizon Willamette Zeus Millenium Cluster Columbus/Tomahawk Sterling Tettnanger Glacier Cascade Unidentified cultivars TOTAL Table 3. Geographical distribution of viroids detected in hop production gardens of Washington State for plants assayed by reverse transcription-polymerase chain reaction for Apple fruit crinkle viroid (AFCVd), Hop latent viroid (HLVd), and Hop stunt viroid (HSVd). Hop production region No. of samples tested No. of samples that tested positive AFCVd HLVd HSVd Moxee Valley Toppenish Creek Area Lower Yakima Valley TOTAL Pathogenicity of Hop stunt viroid Isolated from North American Hop Plants In spring 2005, a newly planted garden of Glacier in the Toppenish Creek area exhibited bright yellow leaves dispersed through the basal foliage of the bines (Fig. 2), moderate epinasty, and a delay in climbing towards the top wire relative to adjacent plantings of Glacier. In addition, pale yellow speckling developed along the major veins of older leaves (Fig. 3). This hop garden was not sampled in the 2004 survey so a RT-PCR assay for the viroids was conducted in May Extracts from 20 of 20 symptomatic Glacier plants yielded an amplicon consistent with the presence of HSVd whereas none of the extracts from 20 asymptomatic Glacier plants yielded corresponding amplification products. Amplicons from three reactions were sequenced to confirm that they were derived from HSVd (see below). All forty extracts yielded amplicons consistent with HLVd indicating that both symptomatic and asymptomatic plants were infected with this viroid. None of the hop plants yielded positive results for AFCVd. The symptomatic plants continued to exhibit suppressed growth and canopy development throughout the growing season as revealed by false color infra-red aerial photography conducted 1 week before commercial harvest (Fig. 4). The visible retardation of growth was specifically associated with plants in which HSVd was detected by RT-PCR.

5 Fig. 2. Early growth of young Glacier hop plants infected with Hop stunt viroid exhibit bright yellow foliage at the base of the plant. The discoloration became less pronounced during the growing season but infected plants continue to exhibit poor growth. Fig. 3. Prominent yellow speckling develops along the veins of older leaves of the sensitive hop cultivar Glacier resulting from infection of Hop stunt viroid. Fig. 4. Aerial photograph of a Glacier hop garden reveals distinct differences in canopy development. The hop garden was photographed with a false color infra-red camera 1 week before commercial harvest. Red is indicative of vegetation while turquoise indicates no vegetative cover. The canopy of rows 1 to 7 is extensive with very little soil exposure; these are rows of Glacier in which Hop stunt viroid (HSVd) was not detected. Rows 8 to 34 reveal reduced red intensity that indicates weak and sparse canopy development relative to that of rows 1 to 7. These rows were planted with rhizomes of Glacier from a different source and the plants that developed were infected with HSVd. Both portions of the hop garden were planted at the same time and grown using the same practices. The only consistent difference in virus or viroid content between the plantings is the presence or absence of HSVd. (Photo courtesy of Washington State University Center for Precision Agricultural Systems).

6 The utility of the biological assay for detecting HSVd was also evaluated. In May 2005, extracts from leaves of infected Glacier in the Toppenish Creek area and from an infected Horizon plant in the Moxee Valley identified in the 2004 survey were mechanically inoculated onto the cotyledons of National Pickling cucumber seedlings. These source plants were infected with HSVd and HLVd. Plants were evaluated 30 days post inoculation after being maintained in a glass greenhouse at 30 C without supplemental lighting. Nine of nine cucumber plants inoculated with hop extract from RT-PCR positive plants exhibited epinasty and greatly reduced internode length. Cucumber plants inoculated with extracts from hop plants that were negative for HSVd by RT-PCR, and cucumber plants inoculated with buffer only did not exhibit these symptoms. RT-PCR confirmed the presence of HSVd in the symptomatic cucumber plants (data not shown). Strain Identities of Hop stunt viroid and Hop latent viroid To confirm the identity of RT-PCR products obtained from the 2004 and 2005 field sample analysis, amplicons from representative reactions were cloned and sequenced. A minimum of six clones from each of three independent RT- PCR reactions of isolates from select gardens were analyzed. The sequences obtained from the Horizon (Moxee Valley) and Glacier (Toppenish Creek) samples are identical to HSVd isolate hkfki previously reported from hop in Japan (GenBank GI: ). In 2006, samples were collected from a commercial garden of the cultivar Cascade located in the lower Yakima Valley; samples from this planting were not included in the initial tests conducted in Most plants in the garden exhibited poor growth and reduced yield relative to adjacent plantings of Cascade. Four out of five samples yielded HSVd-specific amplicons in the RT-PCR assay and the sequences of these amplicons are identical to the hkfki isolate of HSVd. However, a second genotype of HSVd was also obtained from one of the five plants in this Cascade garden. The sequence of this second genotype is 98% identical to a grape isolate of HSVd (GenBank GI: ). Studies at the latter site continue to determine if the grape isolate of HSVd was a surface contamination from nearby viticultural operations or an active infection of the hop plant. It is postulated that HSVd in hop plants may have originated in grapevines in Japan (11). HSVd has a wide host range (10) and strain identification may provide evidence for the source of HSVd in North American hop plants. Amplicons from the HLVd RT-PCR assay of samples obtained during the 2004 survey were cloned and sequenced. The consensus sequences from Nugget (Moxee Valley) and Glacier (Toppenish Creek) are identical to the original HLVd sequence reported by Puchta et al. (8) (GenBank GI: ). This is consistent with the sequence of HLVd previously identified in Washington State (6). Based on results reported here, at least two viroids are widely dispersed in hop plantings of Washington State. Isolates detected are identical or very similar to isolates for which the sequences have been reported in other hop production areas. Disease Management Testing procedures are available for detection of HSVd in order to minimize propagation of infected plants, and to assist in identifying and removing infected plants from established gardens. A cucumber bioassay can be used to identify hop plants infected with HSVd (18). Mechanical inoculation of cucumber leads to the development of severely stunted plants when grown at 30 C for 30 days. Certain cucumber cultivars such as Cucumis sativus L. Suuyou also develop leaf epinasty and curling (18). However, most diagnoses are performed either by molecular hybridization assays or by RT-PCR assays. Laboratory methods are advantageous in that results can be obtained within 2 days compared to the 30- day incubation period required for the bioassay. HSVd is a newly recognized pathogen in North America that poses a serious threat to hop production. With the detection methods presented herein, and with the knowledge that HSVd is already present in the Washington hop industry, measures should be initiated to minimize further spread of the viroid.

7 HSVd was detected in all three major hop production regions of Washington. Surveys are needed to obtain a more accurate assessment of the distribution and frequency of this viroid in the state and to determine if a weed host can serve as a reservoir for HSVd in the North American environment. The occurrence and distribution of HSVd in other regions of North America remains to be determined. The long latent period of infection before visible symptoms appear may have contributed significantly to the inadvertent propagation and distribution of infected material in Washington. A certification program was established in Japan to help provide a reliable source of viroid-free hop plants replacing HSVd-infected hop gardens and for garden renovation and has made significant progress in reducing the incidence of hop stunt disease (9,19). In Washington State, a rootstock program exists for the development and distribution of propagation material to nurseries certified by the Washington State Department of Agriculture. The existence of this program and its ability to serve regional needs will be a critical element in the management of HSVd. Since the primary mode of transmission of HSVd is through mechanical means (14,20), consideration must be given to the potential spread of the viroid by equipment used in farm operations. This is particularly important in moving equipment between infected and non-infected hop yards. Treatment of tools with a variety of aggressive chemical has been shown to inactivate viroids (14,15,16). However, additional research is needed to determine appropriate methods and/or chemicals to reduce the likelihood of viroid transmission in hop field operations. HLVd is highly prevalent in samples tested from all regions sampled in Washington. There are no acute disease symptoms associated with the presence of this viroid but more detailed analyses will be required to evaluate the impact of HLVd on production of contemporary hop varieties. Although AFCVd is not detected in the current assessment of hop plants, it is prudent to maintain routine testing for this viroid. The severity of infection by AFCVd on hop plants in Japan approaches the crop losses incurred by HSVd (12). AFCVd induces fruit symptoms not recorded in the US on several apple cultivars. However, on the cultivar Red Delicious, studies suggested that AFCVd may be the causal agent of blister bark disease (3). This disease occurs in North America but is rare (unpublished observation), and the association between AFCVd and the North American blister bark disease has not been confirmed (5). However, continued vigilance for the introduction of AFCVd into North American hop production areas is warranted, and perhaps a combined survey of both apples and commercial hop production areas in Washington State should be considered. Acknowledgments PPNS #0462, Department of Plant Pathology, College of Agricultural, Human, and Natural Resource Sciences Agricultural Research Center Project No. 0290, Washington State University, Pullman , USA. The contributions to this work provided, in part, by the Washington Hop Commission, Hop Research Council, and Busch Agricultural Resources are gratefully acknowledged, as is the technical assistance of Carolina de Lasa Andres, Keri Druffel, and Lorraine Seymour. Literature Cited 1. Barbara, D. J., and Adams, A. N Hop latent viroid. Pages in: Viroids. A. Hadidi, R. Flores, J. W. Randles, and J. S. Semancik, eds. CSIRO Publ., Collingwood, Australia. 2. Barbara, D. J., Morton, A., Adams, A. N., and Green, C. P Some effects of hop latent viroid on two cultivars of hop (Humulus lupulus) in the UK. Ann. Appl. Biol. 117: Ito, T., Kanernatsu, S., Koganezawa, H., Tsuchizaki, T., and Yoshida, Y Detection of a viroid associated with apple fruit crinkle disease. Ann. Phytopathol. Soc. Japan 59: Knabel, S., Seigner, L., and Wallnöfer, P. R Nachweis des Hop latent viroids (HLVd) mit der Polymerase-Kettenreaktion (PCR). Gesunde Pflanzen 51:

8 5. Koganezawa, H., and Ito, T Apple fruit crinkle viroid. Pages in: Viroids. A. Hadidi, R. Flores, J. W. Randles, and J. S. Semancik, eds. CSIRO Publ., Collingwood, Australia. 6. Nelson, M. E., Klein, R. E., and Skrzeczkowski, L. J Occurrence of hop latent viroid (HLVd) in hops in Washington State. Phytopathology 88:S Pallas, V., Navarro, A., and Flores, R Isolation of a viroid-like RNA from hop different from Hop stunt viroid. J. Gen. Virol. 68: Puchta, H., Ramm, K., and Sänger, H The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Res. 16: Sano, T Hop stunt viroid. Pages in: Viroids. A. Hadidi, R. Flores, J. W. Randles, and J. S. Semancik, eds. CSIRO Publ., Collingwood, Australia. 10. Sano, T., Hataya, T., Terai, Y., and Shikata, E Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan. J. Gen. Virol. 70: Sano, T., Mimura, R., and Ohshima, K Phylogenetic analysis of hop and grapevine isolates of Hop stunt viroid supports a grapevine origin for hop stunt disease. Virus Genes 22: Sano, T., Yoshida, H., Masafumi, G., Monma, T., Kawasaki, H., and Ishizaki, K Characterization of a new viroid strain from hops: Evidence for viroid speciation by isolation in different host species. J. Gen. Plant Pathol. 70: Sasaki, M., and Shikata, E On some properties of hop stunt disease agent, a viroid. Proc. Jpn. Acad. Ser. B. 53: Sasaki, M., Fukamizu, K., Yamamoto, K., Ozawa, T., Kurokawa, M., and Kagami, Y Epidemiology and control of hop stunt disease. Pages in: Proc. of the Intn'l Workshop on Hop Virus Dis., Rauischholzhausen A. Eppler, ed. Deutsche Phytomedizinische Gesellschaft, Ulmer Verlag, Stuttgart, Germany. 15. Takahashi, T Diagnosis and control of hop stunt disease. Agric. Hortic. 54: Takashashi, T., and Yaguchi, S Strategies for preventing mechanical transmission of hop stunt viroid: Chemical and heat inactivation on contaminated tools. Z. Pflanzenkr. Pflanzenschutz 92: USDA National Agricultural Statistics Service (USDA-NASS) National hop report, 2006 hop production. Online. USDA-NASS, Washington, DC. 18. Yaguchi, S., and Takahashi, T Response of cucumber cultivars and other Cucurbitaceous species to infection by Hop stunt viroid. Phytopath. Z. 109: Yaguchi, S., and Takahashi, T Survival of Hop stunt viroid in the hop garden. Phytopath. Z. 109: Yamamoto, H., Kagami, Y., Kurokawa, M., Nishimura, S., Ukawa, S., and Kubo, S Studies on hop stunt disease in Japan. Rep. Res. Lab. Kirin Brewery Co. Ltd. 16:49-62.