The experimental transmission of hop latent viroid and its elimination by low temperature treatment and meristem culture

Two aspects of hop latent viroid (HLVd) relevant to control were examined: the production of viroid-free plants from infected material and transmission of HLVd in the field. Plants free from HLVd were obtained by a combination of storing infected source plants at low temperature (2–4^oC in the dark) for several months followed by meristem culture using small explants. A total of 77 plants of six cultivars and male pollinator clones were grown from meristems and 28 of these were free from HLVd. Tests showed that the cutting of stems (mimicking the use of tools) was more effective than abrasion (mimicking natural plant to plant contact) for the mechanical transmission of HLVd between hop plants. When field-grown test plants were inoculated, infection occurred more commonly in May before plants had grown large enough for significant contact between neighbouring plants than later in the season. The aphid Phorodon humuli could not be shown to transmit HLVd.
These results indicate that all hop varieties and pollinator clones can be made available to the industry free from HLVd and that the chances of infection can be reduced by avoiding early-season cultural operations that cut into hop shoots.     

The distribution and spread of hop latent viroid within two commercial plantings of hop (Humulus lupulus)

The distribution and spread of hop latent viroid (HLVd) in two adjacent plantings of the hop cultivars Omega and Wye Challenger have been studied for three seasons. The planting of cv. Omega was heavily infected at the start of the survey and spread was rapid; the density of infection was lower in the planting of cv. Wye Challenger and spread was much slower. It is not known whether the difference in rate of spread was a varietal effect or because of the higher density of infection in the Omega planting. The distribution of known infections in 1991 suggests that plant-to-adjacent-plant spread, either by contact or on tools, does occur. However, the overall distribution of infection and the occurrence of new infections not adjacent to existing ones, suggests that this is not the only means of transmission; whether a vector is involved is not known.     

Effect of viruses on agronomic and brewing characteristics of four hop (Humulus lupulus) cultivars in Australia

The effect of Hop latent virus (HpLV), Hop mosaic virus (HpMV), and Prunus necrotic ring spot virus [PNRSV (apple, A, and intermediate, I, serotypes)], on the survival of softwood cuttings, the vigour (height) of early season growth, cone yield, and the levels of brewing organic acids in mature plants, was assessed in four hop (Humulus lupulus) cultivars in Tasmania, Australia. Virus infections were associated with an increase in the mortality of softwood cuttings following propagation. In all cultivars, height of early growth was a poor indicator of the effect of viruses on cone yield and levels of brewing organic acids (alpha and beta acids). In cv. ‘Nugget’, infection by the virus combinations studied was not associated with reductions in cone yield, however plants infected by PNRSV‐I, in 2000, had 11% lower alpha acids and 7% lower beta acids. In ‘Opal’, infection by HpLV and HpMV were the most deleterious to cone yield, however the effect of HpMV was ameliorated when in combination with PNRSV‐I. Reductions in alpha and beta acid content were attributable only to mixed infections of HpLV + HpMV in combination with either serotype of PNRSV. In ‘Pride of Ringwood’, yield loss was mostly attributable to HpMV and to a lesser extent, HpLV. Some ameliorations in cone yield loss occurred in plants containing a mixed infection between HpMV and HpLV or either of the ilarvirus serotypes. Both of the ilarviruses and HpMV caused reductions in alpha acid content. In ‘Victoria’, cone yield loss was mostly attributable to combinations of viruses such as HpLV + PNRSV‐I and HpLV + HpMV. The deleterious effect of HpLV + HpMV was ameliorated by PNRSV‐A and to a lesser extent, PNRSV‐I. Infection by the virus combinations studied did not significantly affect alpha and beta acid levels in either year. Results suggested the effect of viruses and their combinations differed between cultivars and varied between seasons. This information, when combined with knowledge of the rates of virus re‐infection, can be used to recommend control strategies for the Australian hop industry.     

Some effects of hop latent viroid on two cultivars of hop (Humulus lupulus) in the UK

Some effects of infection by hop latent viroid (HLVd) on two commercial plantings of hop, one each of two high alpha-acid cultivars, are described. The cultivar Omega was severely affected: yield of cones and individual cone weights from infected plants were both lower than from uninfected plants by c. 35% and the alpha-acids content (as assessed by HPLC) of the cones c. 30% lower. Wye Northdown was less severely affected: cone weight was 8% less (though this was not statistically significant) and alpha-acids content lower by c. 15%. In both cultivars beta-acids were higher in infected plants which, together with differences in other chemical components, suggested that cones on infected plants had matured, or were maturing, earlier than those on uninfected plants. These effects, together with the previous finding that HLVd occurs frequently in some cultivars (Barbara, Morton & Adams, 1990), suggest that HLVd is a potentially important constraint on hop production in the UK and should be eliminated if possible.     

Efficient and stable transformation of hop (Humulus lupulus L.) var. Eroica by particle bombardment

To the best of our knowledge, this is the first accurate and reliable protocol for hop (Humulus lupulus L.) genetic transformation using particle bombardment. Based on the highly productive regeneration system previously developed by us for hop var. Eroica, two efficient transformation protocols were established using petioles and green organogenic nodular clusters (GONCs) bombarded with gusA reporter and hpt selectable genes. A total of 36 hygromycin B-resistant (hyg^r) plants obtained upon continuous selection were successfully transferred to the greenhouse, and a first generation group of transplanted plants was followed after spending a complete vegetative cycle. PCR analysis showed the presence of one of both transgenes in 25 plants, corresponding to an integration frequency of 69.4% and an overall transformation efficiency of 7.5%. Although all final transformants were GUS negative, the integration frequency of gusA gene was higher than that of hpt gene. Petiole-derived transgenic plants showed a higher co-integration rate of 76.9%. Real-time PCR analysis confirmed co-integration in 86% of the plants tested and its stability until the first generation, and identified positive plants amongst those previously assessed as hpt ⁺ only by conventional PCR. Our results suggest that the integration frequencies presented here, as well as those of others, may have been underestimated, and that PCR results should be taken with precaution not only for false positives, but also for false negatives. The protocols here described could be very useful for future introduction of metabolic or resistance traits in hop cultivars even if slight modifications for other genotypes are needed.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

 A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions. Received: 10 March 1997 / Revision received: 12 November 1997 / Accepted: 22 November 1998     

Population Structure and Genetic Diversity within Peach latent mosaic viroid Field Isolates from Peach Showing three Symptoms

To gain insight into the molecular basis of field isolates inducing the symptoms of leaf yellowing, discolouration along leaf sides and leaf mosaic, six isolates from peach showing the three different symptoms in the field were studied by single-strand conformation polymorphism analysis and nucleotide sequence analysis. Results revealed that each Peach latent mosaic viroid (PLMVd) isolate is composed of a population of genetically related variants (haplotypes), one being predominant with frequencies from 32% to 57%, and most of others having a low frequency of 4–5%. Each predominant haplotype was sequenced, as well as some non-predominant haplotypes selected randomly for comparative purposes. In each isolate, sequence alignment among the predominant and non-predominant haplotypes demonstrated that the predominant haplotype had the least variation with others among them, and its sequence was identical to the consensus sequence, which inflected that the predominant haplotype displayed a wide representative of sequence for others in a PLMVd isolate. The similarities and genetic distance between the predominant sequences from peach showing the same symptoms were higher and smaller, respectively, than that with different symptoms; they were more than 98.8% and <1%, respectively, between the predominant sequences with same symptomatic source, and were <98.5% and more than 1%, respectively, between the predominant sequences with different symptomatic source. Some particular variations were indicated for these isolates, and it revealed that the isolates with the symptom of discolouration along leaf sides on their source peach trees had a G or U in position 169 nt, and the isolates with the symptom of leaf yellowing had U and C in 115 and 116 nt, respectively, and the isolates with the symptom of leaf mosaic showed diversity at (3 nt: delete C; 5 nt: A and 54 nt: U).     

Random amplified polymorphic DNA (RAPD) markers in hop, Humulus lupulus: level of genetic variability and segregation in F1 progeny

The level of genetic variation in 24 hop genotypes was studied using the recently developed technique for producing random amplified polymorphic DNAs (RAPDs). Of the 60 primers screened, eight produced polymorphic RAPD bands, 38 produced bands that were monomorphic for all genotypes and 19 did not produce any amplification product. It appeared that the level of polymorphism among the genotypes was generally low. Three of the primers, A11, A17 and C9, were used to determine the stability and segregation of RAPD markers in five families with a total of 182 F₁ progeny. The segregation ratios of these markers in the f₁ progeny suggested that they were inherited in a Mendelian manner. RAPD markers were stable and may be useful for the construction of linkage maps in hop.     

Comparative chemical attributes of native North American hop,Humulus lupulus var. lupuloides E. Small

The genetic diversity of 159 representative genotypes of native hop (Humulus lupulus var. lupuloides E. Small, Cannabaceae) from 34 selected populations was assessed by relative magnitudes and ranges of alpha acids (AA), beta acids (BA), and the cohumulone (CoH) component of alpha acids, with reference to temporal changes between 1989-1990 and 2001, and to the same attributes in American and European hop cultivars, principally H. lupulus var. lupulus L. Chemical profiles of these genotypes were generated by high pressure liquid chromatography (HPLC) of methanol extracts from their processed samples (cones). The alpha ratio (AR, alpha acids / alpha+beta acids) measured the degree to which alpha acids predominated in cone extracts. Synchronous ranges of AR and CoH were also selected for graphic portrayals of native hop genotypic diversity. Cones sampled and analyzed from eight populations that were accessible in both 1989 and 2001 were distinct in chemical attributes, indicating a succession of genotypes, and suggesting temporal cycling of H. lupulus var.lupuloides germplasm. The principal distinctions between the two sub-species were a markedly higher proportion of CoH (38-88% vs. 19-41%) in alpha acids of H. l. var. lupuloides, and generally higher concentrations of AA in cultivars of both American and European commercial hop cultivars, predominantly H. lupulus var. lupulus. All of the 159 native hop genotypes also contained detectable levels of xanthohumol and xanthogalenol, prenylflavonoids recently reported to have mammalian anti-cancer activity. Some native genotypes had previously exhibited natural repellence of insect and mite pests; thus H. lupulus var. lupuloides germplasm offers a diverse resource of underutilized and yet undefined biochemicals.     

Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing ~(32)p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a ~(32)P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.     

Genetic diversity of hop stunt viroid from symptomatic and asymptomatic citrus trees in Iran

Hop stunt viroid as the causal agent of cachexia disease has detected from citrus trees in different areas in Iran. Although cachexia has not been reported as a decline disease for citrus trees, it can impair crop quality and reduce plant yields. This study was undertaken to molecularly detect HSVd among different commercial citrus cultivars and determine genetic diversity of this viroid in Mazandaran province of Iran. Sampling was performed from symptomatic and symptomless citrus cultivars in Mazandaran province. HSVd specific primers were used for molecular detection. SSCP and sequencing were applied to assay HSVd genetic diversity. Results showed the detection of HSVd in all symptomatic Satsuma (25 out of 25), Clementine (25 out of 25), sweet lime (20 out of 20) and sweet orange cv. Valencia (7 out of 7), as well as, 31% (14 out of 22), 100% (12 out of 12) and 33% (5 out of 15) of page mandarin, lemon and grapefruit trees, respectively. 10 different HSVd genomes were identified by sequencing the SSCP profiles among which HSVd‐IR1 had the most frequency.     

Hop Latent Viroid (HLVd)-Caused Pathogenesis: Effects of HLVd Infection on Lupulin Composition of Meristem Culture-Derived Humulus lupulus

Season-dependent fluctuation of hop latent viroid in meristem tips enabled selection of viroid-free cultures from Osvald's clone 31, 72, 114, and cv. Premiant. These mericlones were used to evaluate effect of viroid infection on the composition of hop resins and essential oils in the first production year. Healthy plants were compared with naturally re-infected individuals under field conditions. On average, viroid infection decreased content of -bitter acids by 40 %. The content of -bitter acids, as well as the ratios of humulone/cohumulone and lupulone/colupulone was not influenced by viroid infection. The content of all monoterpenes was for 29, 37.4 and 41.6 % higher for myrcene, - and -pinene, respectively, in infected plants compared with the healthy controls. The contents of sesquiterpenes like -caryophyllen -humulene, -copaene, -muurolene, -bisabolene, -cadinene, and -cadinene decreased by 13.7, 13, 14, 18.5, 29, 21.7 and 18.5 %, respectively, due to viroid infection. The possible influence of some oxidative-reduction processes activated by viroid-caused pathogenesis was assumed to be involved in the accumulation of terpenes alcohols like geraniol and methylgeranate, and in the reduction of the contents of the majority of ketones detected in the spectra of essential oils.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid‐related compounds and possible transport proteins

Female flowers of hop (Humulus lupulus) are an essential source of terpenoid‐related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono‐ and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D‐LC‐MS/MS analysis identified 1015 proteins. Of these, most correspond to housekeeping and primary metabolism‐related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid‐derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra‐ and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes.     

Structure and inheritance of ribosomal DNA variants in cultivated and wild hop, Humulus lupulus L.

Genetic variation was assessed among cultivated and wild hop, Humulus lupulus, by restriction fragment length polymorphisms (RFLPs) of the ribosomal RNA genes (rDNA). Two rDNA length variants of 10.3 and 9.3 kbp represented by three phenotypes designated A, B and C were detected with XhoI. Restriction-site mapping showed that hop rDNA is structurally similar to those of most higher plants. A high level of homogeneity existed in rDNA repeat lengths among the diverse hop genotypes. Generally, phenotype A was predominant in wild and cultivated European and Asian genotypes; phenotype B in North American cultivars; while phenotype C was present only in native North American hop, providing a potential molecular marker for the identification of this germ plasm. The rDNA data provided genetic evidence for the separation of native and cultivated American genotypes and supports the hypothesis that North American hop cultivars are of hybrid origin from European and native American genotypes. The segregation of rDNA phenotypes in four F₁ families suggests that a single locus with two co-dominant alleles controls genetic variability for rDNA variants in hop.     

Identification of quantitative trait loci for resistance to Verticillium wilt and yield parameters in hop (Humulus lupulus L.)

Verticillium wilt (VW) can cause substantial yield loss in hop particularly with the outbreaks of the lethal strain of Verticillium albo-atrum. To elucidate genetic control of VW resistance in hop, an F₁ mapping population derived from a cross of cultivar Wye Target, with the predicted genetic basis of resistance, and susceptible male breeding line BL2/1 was developed to assess wilting symptoms and to perform QTL mapping. The genetic linkage map, constructed with 203 markers of various types using a pseudo-testcross strategy, formed ten major linkage groups (LG) of the maternal and paternal maps, covering 552.98 and 441.1 cM, respectively. A significant QTL for VW resistance was detected at LOD 7 on a single chromosomal region on LG03 of both parental maps, accounting for 24.2–26.0 % of the phenotypic variance. QTL analysis for alpha-acid content and yield parameters was also performed on this map. QTLs for these traits were also detected and confirmed our previously detected QTLs in a different pedigree and environment. The work provides the basis for exploration of QTL flanking markers for possible use in marker-assisted selection.     

Anti-inflammatory and anti-tumor-promoting effects of 5-deprenyllupulonol C and other compounds from Hop (Humulus lupulus L.)

A new phloroglucinol derivative, 5‐deprenyllupulonol C ( 1 ), along with four other phloroglucinol derivatives, 2 – 5 , five chalcones, 6 – 10 , four flavanones, 11 – 14 , two flavonol glycosides, 15 and 16 , and five triterpenoids, 17 – 21 , were isolated from the female inflorescence pellet extracts of hop (Humulus lupulus L.). Upon evaluation of these compounds against the Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Raji cells, twelve compounds, i.e., 1 – 4, 11 – 14, 17 – 19 , and 21 , showed potent inhibitory effects on EBV‐EA induction, with IC₅₀ values in the range of 215–393 mol ratio/32 pmol TPA. In addition, eleven compounds, i.e., 1 – 4, 6, 11, 12, 14, 17, 18 , and 20 , were found to inhibit TPA‐induced inflammation (1 μg/ear) in mice, with ID₅₀ values in the range of 0.13–1.06 μmol per ear. Further, lupulone C ( 2 ) and 6‐prenylnaringenin ( 14 ) exhibited inhibitory effects on skin‐tumor promotion in an in vivo two‐stage mouse‐skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter.