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In-field management of Potato virus Y (PVY) faces challenges caused by the changing availability and environmental acceptability of chemical agents to control aphid vectors of the virus and by proliferation of PVY strains with different symptoms and rates of spread. From 2018 to 2020, foliar spray treatments were compared in field experiments in New Brunswick, Canada, to measure effectiveness at reducing spread of PVYO, PVYN:O, and PVYNTN strains. Mineral oil, insecticide, combined oil and insecticide spray, and a biopesticide (i.e., LifeGard WG) were compared. Insecticide-only and mineral oil–only treatments were not effective, but several combined oil and insecticide treatments and biopesticide treatments significantly reduced PVY spread. The biopesticide was proportionately more effective with recombinant PVYN:O and PVYNTN strains, possibly by exciting the plant’s hypersensitive resistance response, caused naturally only in cultivar ‘Goldrush’ by PVYO. Pesticide residue analysis showed that mineral oil increased the retention of pyrethroid insecticide in the potato foliage longer than with insecticide applied alone, which may explain the beneficial synergistic effect of combined sprays for reducing PVY spread. Tuber yields were generally unchanged in chemical insecticide treatments but were slightly lower in biopesticide treatment. The cost per PVY treatment was competitive across all effective treatments, including biopesticide; however, there was some revenue loss from lower yield with the biopesticide. This biopesticide is certified organic, however, and thus a small premium on the price for organic production could offset this yield deficit.

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus Potyvirus infecting potato crops worldwide. Host resistance offers an economical and effective means for the control or management of these viruses. In this study, 20 potato clones were screened for their resistance against PVA and PVY by mechanical or graft inoculation assay, and were explored for the relationship between extreme resistance genes Ra and Ry by the detection of molecular markers linked to Ryadg, Rysto, and Rychc. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 (susceptible to both PVY and PVA) and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained. Phenotyping of the F1 and BC1 populations by graft inoculation with PVA showed segregation ratios of 3:1 and 1:1 (resistant:susceptible), respectively. These results suggest that two independent loci control ER against PVA in Barbara: one confers ER to both PVA and PVY and the other confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.

The genus Coleviroid, family Pospiviroidae, comprises six known viroids, all infecting Plectranthus scutellarioides (Coleus blumei; coleus). In 2017, a novel viroid-like RNA sequence that shares ca. 65% identity with Coleus blumei viroid 1 (CbVd-1) was identified in a coleus cultivar infected by multiple coleviroids. Further sequence and secondary structure analyses are consistent with the discovery of a seventh viroid in the genus Coleviroid: tentatively named "Coleus blumei viroid 7" (CbVd-7). The viroid appears to be the product of a natural recombination event between CbVd-1 and Coleus blumei viroid 5. We prove CbVd-7 to be infectious and in turn demonstrate the ability of all known coleviroid left- and right-arm segments to recombine. With a length of 234 nucleotides, this is the smallest viroid described to date.

In this study, a set of duplex reverse transcription PCR (RT-PCR)–mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f. sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected with a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, RNA-CP, and RNA-TGB) were analyzed together with the existing Sss primers via real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by HRM analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV or Sss was found in 63 to 100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Of the soil samples collected from PMTV-infested fields, 63 to 83% and 100% led to PMTV and Sss infections in the bait tobacco plants, respectively, whereas no PMTV- or Sss-infected plants were obtained from soil samples collected from PMTV- and Sss-free fields.

In-field management of

The basic information on gene action and inheritance of quantitative traits, which is necessary to develop future breeding programme, is not widely studied in pigeonpea because it is a long duration crop and widely affected by stresses (biotic & abiotic). Therefore, present study was conducted among six generations in five pigeonpea crosses to know significance of additive-dominance model, gene action involved in inheritance of quantitative traits. These six population (P1, P2, F1, F2, BC1 and BC2) of five cross combinations of pigeonpea were studied during Kharif, 2014-15 for plant height, number of primary and secondary branches, pods plant-1, pod length, seeds pod-1, 100-seed weight and Seed yield plant-1.Significant deviation of scale(s) (A, B, C & D) from zero in all the crosses for most of the traits were evidence of presence of epistasis and hence further subjected to six parameter model to estimate the main gene effects (additive [ ] and dominance [hˆ] ) and their digenic interactions (additive × additive [iˆ] , additive × dominance [ ˆj] and dominance × dominance [lˆ] ). The estimates of six parameter model revealed that both additive and dominant gene effects were important in all the crosses for almost all the traits. However, the relative contribution of dominant gene effects was much higher than additive gene effects for plant height, pods plant-1, seed pod-1 and seed yield plant-1. Higher frequency of duplicate type of epistasis also confirms the prevalence of dominance gene effects for above traits except for seed yieldplant-1.

Potato virus Y (PVY) exists as several strains with distinct symptomology and tuber yield effects in different potato varieties. Recently, new recombinant strains have proliferated and dominated local populations around the world. In this study, PVYO, PVYN:O, PVYN-Wi, and PVYNTN strains were tracked across Canada from 2014 to 2017, showing rapid evolution of populations away from the traditionally dominant PVYO to recombinants PVYN-Wi (western Canada) and PVYNTN (eastern Canada). Simultaneously, 30 potato varieties were inoculated with PVYO, PVYN:O, and PVYNTN in controlled greenhouse experiments. Foliar symptoms of primary (mechanical inoculation mimicking aphid infection) and secondary (tuber seedborne) infection were cataloged, and tuber yield measured. On average, and generally similar in primary and secondary infection, symptom expression and yield reduction were most severe with PVYO, followed by PVYN:O and PVYNTN. Strong mosaic symptoms were most commonly expressed with PVYO infection, and only seen with PVYN:O or PVYNTN in 15 and 3 varieties, respectively. Across variety–strain combinations, yield reduction was correlated with symptom severity, most strongly in PVYO-infected plants (e.g., AC Chaleur, Beljade, Envol, Norland, and Pacific Russet), and four varieties exhibited tuber necrotic ringspot disease with PVYNTN (AC Chaleur, Envol, Pacific Russet, and Yukon Gold).

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus

The genus Coleviroid, family Pospiviroidae, comprises six known viroids, all infecting Plectranthus scutellarioides (Coleus blumei; coleus). In 2017, a novel viroid-like RNA sequence that shares ca. 65% identity with Coleus blumei viroid 1 (CbVd-1) was identified in a coleus cultivar infected by multiple coleviroids. Further sequence and secondary structure analyses are consistent with the discovery of a seventh viroid in the genus Coleviroid: tentatively named "Coleus blumei viroid 7" (CbVd-7). The viroid appears to be the product of a natural recombination event between CbVd-1 and Coleus blumei viroid 5. We prove CbVd-7 to be infectious and in turn demonstrate the ability of all known coleviroid left- and right-arm segments to recombine. With a length of 234 nucleotides, this is the smallest viroid described to date.

Potato virus Y (PVY) is an economically important disease agent in potatoes with worldwide distribution. It is mainly transmitted vegetatively by tubers and between plants by an aphid vector. PVY management is primarily through reducing viral inoculum in seed tubers and other sources and controlling transmission by aphids. Seed certification programs are designed to comprehensively test and restrict levels of PVY within a region, enforcing low levels of PVY in marketed seed lots and allowing growers informed choice to plant low-PVY crops. Sanitation techniques on the farm can also reduce volunteer, weed, and neighboring field sources of PVY. Major techniques to reduce aphid-mediated PVY spread include mineral oil foliar sprays, especially combined with insecticides. Spray timing is important, starting early after first plants emerge, continuing weekly through season to vine-kill, with additional sprays during rapid growth and periods of aphid abundance. Other practices for PVY reduction include avoiding mechanical transmission by field equipment, planting crop borders or intercropping as a barrier or sink for PVY, breeding PVY-resistant potato varieties, and roguing symptomatic plants from fields. Major remaining challenges in combatting PVY are management complacency, proliferation of PVY strains circumventing resistance and roguing, and informal trade of untested potatoes.

In this study, a set of duplex reverse transcription PCR (RT-PCR)-mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector

Potato virus Y (PVY) transmission was studied in six commercial potato fields in 2015 and 2016 in New Brunswick, Canada. Plants emerged from PVY-positive tubers, or PVY-free plants that were artificially inoculated with PVY shortly after emergence, were used as PVY inoculum plants in the study. In all trials, equal numbers of PVY inoculum plants from each of three strains common in the region, PVYO, PVYN:O and PVYNTN, were used. PVY inoculated into tractor-traffic rows showed 2 to 7 times as much PVY spread to previously virus-free plants by the end of the season (up to 48.5% in one tractor row, compared to a maximum of 16.3% in a distant control row unaffected by tractor traffic). Evidence supporting a hypothesis that tractor traffic enhances PVY transmission through aphid disturbance was observed by PVY spread in both directions along the rows, not biased in the direction of tractor travel, and that the ratio of spread of the three strains was nearly indentical in control and tractor rows. However, the lack of spread to immediately adjacent rows, and statistically significant spatial pattern matching the circumference of tractor wheels specific to each field support the hypothesis that direct mechanical transmission of wounded plant sap could also be a factor in the enhanced PVY transmission.

Potato virus Y (PVY) is a major cause of yield and quality loss in potato crops worldwide. Recently, populations of PVY strains have shifted dramatically toward recombinant strains such as PVYNTN and PVYN:O. A 2010 to 2016 survey of PVY strains in commercial fields of New Brunswick (NB), Canada, and five field trials tracking PVY spread in NB and Manitoba, were conducted to study the current status of PVY strains and their relative rates of spread. In NB, PVYO dropped from 82% of infections in 2010 to 14% in 2016, replaced mostly by PVYNTN (64%) and PVYN:O (22%).In field trials with Russet Burbank and Gold rush varieties, PVYNTN spread most effectively compared to PVYN:O and PVYO. Strain-specific PVY spread varied with the potato variety, possibly due to selective PVYO resistance in Goldrush, mostly expressed at the plant-to-plant transmission level with little difference in transduction to tubers in infected plants. Relevance of in-field differences in spread of strains to changes in regional PVY populations, and potential mechanisms responsible, are discussed.

Eight intraspecific (four each of mungbean x mungbean and urdbean x urdbean) and 24 interspecific (eight each of mungbean x urdbean, mungbean x ricebean and urdbean x ricebean) crosses were made at five different dates of ten days intervals i.e., September 11-20, 21-30, October 1-10, 11-20 and 21-30 during kharif/late kharif seasons, 2015 to obtain a total of 32 crosses. The per cent crossability at five different dates indicated that it was the highest during October 1-10 in both intra - (mungbean x mungbean, 72.27%, urdbean x urdbean, 55.55%) and interspecific (mungbean x urdbean, 43.24%, mungbean x ricebean, 16% and urdbean x ricebean, 9.92%) crosses. Similarly, the percent ovule fertility recorded was also the highest during October 1-10 in both intra - (mungbean x mungbean, 90.00%, urdbean x urdbean, 83.33%) and interspecific (mungbean x urdbean, 75.00%, mungbean x ricebean, 71.42% and urdbean x ricebean, 60.00%) crosses. Optimum temperature (~22˚C) , followed by low humidity (49.0) and comparatively enhanced sunshine (>8.5 hours) might be responsible for accelerating the highest pod sett during October 1-10 and thus may be suggested as appropriate period for obtaining highest pod setting in Vigna species studied.

Alfalfa mosaic virus (AMV) was identified as the causal agent of internal tuber necrosis in the potato cultivar Innovator in New Brunswick, Canada. Further pathological characterization of the isolate (designated as isolate CaM) was performed on six potato cultivars and one breeding clone. Upon mechanical inoculation, four cultivars (Innovator, Yukon Gold, Rochdale Gold-Dorée, and Shepody) showed needle-sized necrotic spots and increasing calico symptoms on new leaves, whereas the remaining cultivars only developed calico symptoms on new leaves. All tubers of CaM-infected Innovator and Shepody plants developed sporadic internal necrotic spots, as did ca. 23 and 8% tubers of CaM-infected Yukon Gold and Rochdale Gold-Dorée, respectively. Sequence analysis of the CP gene of CaM with AMV isolates from potato, all presumed belonging to the “non-necrotic” strain and retrieved from GenBank, indicated that CaM shared >97.1% sequence identity with all but four Egyptian isolates. At the complete genome level, phylogenetic analysis of all available sequences demonstrated that RNA 1 and RNA 3 can be grouped into three major clades each, whereas RNA 2 can be clustered into two clades. CaM and Ca175-1, an AMV isolate that was deemed non-necrotic in a previous study, had different phylogenetic clade patterns, indicating different RNA 1-RNA 2-RNA 3 haplotypes: IA-I-IB (CaM) versus Ca175-1 (IB-II-IA). Despite the difference in haplotype composition, CaM and Ca175-1 induced similar levels of internal necrosis in tubers of Innovator and its parent Shepody. The results suggest that the internal necrosis in AMV-infected tubers depends on potato cultivar rather than on AMV strain/haplotype, and CaM is just a “regular” isolate of AMV.

The complete sequence of a strawberry vein banding virus (SVBV) isolate collected in Nova Scotia, Canada, and designated NS8, was determined. The 7,856-nucleotide circular double-stranded DNA genome contains seven open-reading frames (ORFs), which is consistent with other SVBV isolates and other members of the genus Caulimovirus. Comparison of NS8 with other whole-genome sequences retrieved from databases revealed that NS8 shares the highest sequence similarity (96.5% identity) with isolate China (accession number HE681085) and the lowest (88.3% identity) with clone pSVBV-E3 (accession number X97304). Despite the overall high sequence similarity between NS8 and China, the coat protein encoding ORF IV of NS8 shares only 90.9% sequence identity with the China isolate. Phylogenetic analysis at the complete-genome level placed NS8 and all Chinese isolates in one clade and clone pSVBV-E3 in a separate clade. Interestingly, phylogenetic analysis of all available ORF IV sequences, including those retrieved from databases and newly sequenced samples in this study from Canada, revealed three distinct clades. All Canadian isolates grouped together as one clade, pSVBV-E3 and several others from Europe, Egypt and the USA grouped as a second clade, and isolates from China formed a third clade. These results demonstrate that SVBV is more divergent than previously reported.

In the 2014 and 2015 crop seasons, the efficacies of different types, rates and combinations of mineral oil and insecticide foliar sprays for reducing Potato virus Y (PVY) spread were tested in controlled field trials in New Brunswick (NB), Canada. Experimental plots were planted with certified PVY-free Goldrush, supplemented with known virus-infected seed to raise PVY inoculum to 2.3% and 3% at the beginning of the 2014 and 2015 seasons, respectively. Treatments consisted of mineral oil-only sprays at different application rates, insecticide-only sprays of differing numbers, and several combined mineral oil and insecticide spray regimes, all compared to a no-spray control treatment. PVY spread to 18% (2014) and 22% (2015) of initially virus-free plants in no-spray control plots, with significant reductions observed in PVY spread in several treatments. Greatest PVY reductions, as low as 4% (2014) and 12% (2015), were in combined mineral oil and insecticide spray treatments, followed by oil-only sprays; while insecticide-only sprays did not significantly reduce PVY spread. As well as measuring PVY spread to marked test plants and randomly collected post-harvest tuber sample from the plots, exhibited similar treatment pattern for PVY incidence. Multiple logistic regression modeling confirmed the relative efficacy of combined oil and insecticide sprays for reducing PVY spread, while accounting for variable inoculum and aphid factors. Modeling also highlighted the importance of planting low-PVY seed initially, and of early application of foliar sprays. Local best management practice recommendations for reduction of in-field PVY spread were discussed.

Molecular markers reported to be linked to extreme resistance (ER) against Potato virus Y (PVY) were evaluated in potato germplasm. YES3-3A and YES3-3B, markers linked to Rysto, were detected in ‘Barbara’ and its three descendants that exhibit ER to PVY; RYSC3, a marker linked to Ryadg, was detected in breeding clones NY121 and NY123. Assessment of RYSC3 as a marker for selection for Ryadg-mediated ER validated its efficacy in identification of selections with ER to PVY.

In this study, on-farm within-season spread of Potato virus Y (PVY) was measured in 56 seed, processing and tablestock potato fields in New Brunswick, Canada between 2010 and 2014. These represented 13 potato varieties, managed by 16 growers employing a wide range of PVY control techniques. Many aspects of management techniques were quantified, as well local aphid abundances and climatological data. PVY spread, measured in leaves and tubers (by ELISA and RT-PCR), was tracked through the season in individually marked plants. Over the five seasons under study, on-farm PVY spread overall has declined substantially. Across all 56 fields, however, it has varied widely, from 10 fields not showing any spread during the crop season, up to as high as 76 % spread in one 2012 field. Factors correlated with increased PVY spread included seed-borne PVY inoculum planted in the field, aphid abundance early in the season, and to a lesser degree temperatures in July and over the preceding winter. Factors correlated with decreased spread included numbers of foliar mineral oil and insecticide sprays (especially of the lambda-cyhalothrin and flonicamid types), later crop planting and earlier first spraying dates. A mid-season leaf test for PVY was shown to be strongly indicative of ultimate PVY spread at harvest, and may prove a useful test in advising growers. A set of locally-relevant best management practices based on these results is discussed.

The complete genome comprising three genomic RNAs of three Canadian and two Chinese isolates of Potato mop-top virus were sequenced and analysed. Two open reading frames (ORFs) were found in RNA1 of 6.1 kb, encoding a readthrough RNA-dependent RNA polymerase (RdRp). A coat protein (CP)-readthrough protein was encoded by RNA2 of 3.1 kb. Four ORFs that encoded the triple gene block proteins (TGBps) and a cysteine-rich protein were found in RNA3 (2.9 kb) of the Chinese isolate ‘Yunnan’; whereas in the remaining isolates (three Canadian isolates and the Chinese isolate ‘Guangdong’), only three ORFs encoding TGBps were observed in RNA3. A single nucleotide mutation of A2462 to G2462 abolished the start codon ‘AUG’ for the fourth putative ORF in RNA3 of these isolates. Based on phylogenetic and sequence similarity analysis of these isolates as well as those reported by others at the complete RNA sequence level, each of RNA1, RNA2 and RNA3 could be divided into at least two groups. In Canadian isolates ‘Ch9’, ‘Ch10’ and ‘Ch20’ and Chinese isolate ‘Guangdong’, all genomic RNAs belonged to group A; and in Chinese isolate ‘Yunnan’, all of its RNA belonged to group B. Interestingly, in Swedish isolate ‘Sw’, RNA1 and RNA2 belonged to group A while RNA3 belonged to group B. A duplex RT-PCR for differentiating groups A and B of RNA3 was developed and evaluated. All PMTV samples collected in Guangdong, China, and New Brunswick, Canada, possessed a RNA3 belonging to group A; whereas the samples collected in Yunnan, China, possessed a RNA3 belonging to group B.

Surveys of commercial and seed potato fields for virus diseases (1998 to 2002) in Manitoba established that Potato virus Y (PVY) is of concern in seed potato production. To determine the prevalence of PVY strains, PVY-infected tubers identified by reverse transcription-polymerase chain reaction (RT-PCR) from surveys (2000 to 2001) were grown for symptom expression and strain characterization by strain-specific RT-PCR, bioassays, and serological assays. Of the samples collected (2000 to 2001) and tested by RT-PCR, 4.0% contained PVY. Further analysis of the PVY-positive samples by a duplex RT-PCR facilitating the simultaneous detection of common (PVYO) and tobacco veinal necrosis strains (PVYN/NTN) indicated that 37.5% contained PVYO and 63.5% contained PVYN-type isolates. Analysis of the PVYN-type samples using three monoclonal antibodies (MAbs) showed that all reacted with only the PVYO MAbs and not with the PVYN-specific MAb. Partial nucleotide sequences of both ends of PVY-RNA showed that the PVYN-type isolates resembled those reported in 1996 from Manitoba. These isolates are designated as PVYN:O. In view of the increased incidence of PVYN:O in one production area, seed tubers imported from other provinces of Canada and the neighboring United States were analyzed for PVYN:O. The PVYN:O was detected in imported seeds from Minnesota, Montana, and North Dakota.

A field isolate of Potato virus Y (PVY) was collected in New Brunswick, Canada in 2007 due to unusual symptoms observed on different potato cultivars. To unveil the PVY strain identity, tobacco and potato bioassays, PVY

The responses of 14 potato cultivars to five Potato virus Y (PVY) isolates belonging to four strains (ordinary [PVYO], tobacco veinal necrosis [PVYN], N:O group [PVYN:O], and nonrecombinant potato tuber necrotic [PVYNTN]) were studied in primary and secondary infections. For the primary infection experiments, foliage symptoms were monitored daily after mechanical inoculation with a PVY isolate until harvest; and, for the secondary infection experiments, foliage symptoms were monitored regularly from plant emergence until harvest. Tuber symptoms (namely, tuber necrotic ringspots) were checked at harvest and monthly postharvest for up to 4 months. In both infections, symptoms varied significantly depending on potato cultivar and virus strain or isolate. In primary infections, local lesions occurred on inoculated leaves of ‘AC Chaleur’, ‘Eramosa’, ‘Goldrush’, ‘Jemseg’, ‘Katahdin’, ‘Ranger Russet’, and ‘Yukon Gold’ after inoculation with PVYO isolates, followed by systemic necrosis on latterly emerged uninoculated leaves. In contrast, plants of ‘CalWhite’, ‘La Rouge’, ‘Red LaSoda’, ‘Russet Burbank’, ‘Russet Norkotah’, and ‘Superior’ did not exhibit any visible symptoms on inoculated leaves but developed mild to severe mosaic on latterly emerged leaves after infection with PVYO isolates. In all cultivars, near-symptomless to mild mosaic was induced by PVYN and mild to severe mosaic by PVYN:O. PVYNTN induced mild to severe mosaic in plants of all cultivars except AC Chaleur, ‘Cherokee’, and Yukon Gold, which developed visible systemic necrosis. Necrotic ringspots were observed in tubers of PVYNTN-infected plants of AC Chaleur, Cherokee, and Yukon Gold. The tuber symptoms were also incited by PVYN-Jg on Cherokee. In secondary infections, the symptoms were generally more severe than primary infections even though the symptom types did not alter. As in the greenhouse, a clear symptom severity pattern (PVYO-FL > PVYO-RB > PVYNTN-Sl > PVYN:O-Mb58 > PVYN-Jg) was observed in AC Chaleur, Cherokee, Eramosa, Goldrush, Jemseg, Katahdin, Ranger Russet, and Yukon Gold in the field.

Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 were also performed. The efficacy of the HRM assays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.

Potato virus Y (PVY) undergoes continuous nucleotide mutation and genome recombination, leading to novel strains with varying pathogenicity on host plants. Little is known about how tomato plants respond to infections with different PVY strains, especially the emerging strains such as PVYNTN and PVYN:O, and in combination with other viral species such as Potato virus X (PVX). In this study, the response of tomato (cultivar Rutgers) plants to single and mixed infections with PVY and PVX was investigated. Plants infected singly with PVYO, PVYN:O, PVYN or PVYNTN developed mosaic symptoms and leaf deformation; whereas plants infected with PVX developed mild local lesion and varying degrees of mosaic and leaf deformation symptoms. Mixed PVX+PVY infections induced more severe symptoms, which include severe local and systemic leaf necrosis, leaf drop and severe leaf deformation, compared to single infections with PVX or PVY, indicating PVX-PVY synergism. The level of PVX-PVY synergism was affected significantly by PVY strains, with PVYNTN being the greatest effector. Quantitative analysis of viral titre indicated that mixed PVX+PVY infections elevated PVX level and reduced PVY level. A comparative transcriptional analysis of 46 key genes involved in signal molecule synthesis and signal transduction using NanoString technology revealed diverse gene expression profiles in both single and mixed infections. Two hierarchical expression clusters, one up-regulated and the other down-regulated, were observed during the infections. In general, mixed infections led to more genes being differentially regulated than single infections. The expression alteration of genes involved in oxidative stress and salicylic acid (SA)-pathways is particularly noteworthy, as the mixed infections, especially PVX+PVYNTN infections, led to a further elevation of reactive oxygen species (ROS) and SA producing abilities and a reduction of ROS scavenging ability over single infections.

Aphids are viral vectors in potatoes, most importantly of Potato virus Y (PVY), and insecticides are frequently used to reduce viral spread during the crop season. Aphids collected from the potato belt of New Brunswick, Canada, in 2015 and 2016 were surveyed for known and novel mutations in the Na-channel (para) gene, coding for the target of synthetic pyrethroid insecticides. Specific genetic mutations known to confer resistance (kdr and skdr) were found in great abundance in Myzus persicae (Sulzer) (Hemiptera: Aphididae), which rose from 76% in 2015 to 96% in 2016. Aphids other than M. persicae showed lower frequency of resistance. In 2015, 3% of individuals contained the resistance mutation skdr, rising to 13% in 2016 (of 45 species). Several novel resistance mutations or mutations not before reported in aphids were identified in this gene target. One of these mutations, I936V, is known to confer pyrethroid resistance in another unrelated insect, and three others occur immediately adjacent and prompt similar chemical shifts in the primary protein structure, to previously characterized mutations associated with pyrethroid resistance. Most novel mutations were found in species other than M. persicae or others currently tracked individually by the provincial aphid monitoring program, which were determined by cytochrome C oxidase I (cox1) sequencing. Through our cox1 DNA barcoding survey, at least 45 species of aphids were discovered in NB potato fields in 2015 and 2016, many of which are known carriers of PVY.

The transmission of PVY strains, PVYO, PVYN:O, and PVYNTN via tuber cutting and plant wounding was investigated in PVY susceptible cultivars Shepody and Russet Norkotah. For the tuber cutting experiment, after one infected tuber was cut with a knife, four uninfected tubers were cut sequentially with the same knife without disinfecting it between the cuts. In the plant wounding experiments, wounds were induced in the healthy and infected plants of Shepody by bouncing, brushing, hammering, squeezing, and Carborundum rubbing treatments. These treatments allowed exchange of sap between the healthy and infected plants. Results demonstrated that seed cutting did not transmit PVY, whereas plant wounding treatments caused varying levels of PVY transmission, depending on the wounding treatment. Plant bouncing was the least effective whereas hammering was the most effective.

Potato cultivar Exploits and breeding clone F87084 have been considered resistant to Potato virus Y (PVY). To further explore the degree of resistance and whether the resistance is strain specific, these materials, together with cultivar Rochdale Gold-Doree and breeding line F02010, were investigated for their response to different PVY strains including PVYO, PVYN:O, PVYNTN and PVYN. Both F02010 and Rochdale Gold-Doree were readily infected with all tested PVY strains after either mechanical or graft inoculation, indicating susceptibility of the materials to PVY. F87084 was unable to be infected by any of the tested PVY strains as no ELISA-detectable level of PVY were found in plants after either mechanical or graft inoculation, demonstrating extreme resistance in F87084 to all strains of PVY tested. Exploits was infected with PVYN:O/PVYNTN/PVYN after either mechanical or graft inoculation, indicating susceptibility of the cultivar to these PVY strains. The cultivar was also infected readily with PVYO after graft inoculation. However, despite induction of local lesions on the inoculated leaves, mechanical inoculation with PVYO may or may not lead to systemic symptoms and ELISA-detectable level of PVY, depending on temperature. At low temperature (e.g., 22 °C), no visible systemic symptoms or ELISA-detectable level of PVY was found in the plants; whereas at high temperature (e.g., 30 °C), systemic symptoms and high level of PVY were detected in the plants. These results demonstrate that Exploits possesses temperature-dependent hypersensitive resistance to PVYO. Analysis of a segregating population of F87084 × F02010 revealed that the ER in F87084 is controlled by a dominant resistance gene.

Potato plants that exhibited mosaic symptoms were collected in Xiangxi, Hunan province, China. Multiplex RT-PCR screening for common viruses revealed the presence of potato virus A (PVA) in these samples. ELISA with virus-specific antibodies confirmed infection by PVA in the plants. Rod-shaped virions of ~750 nm in length and ~13 nm in width were observed by transmission electron microscopy. One virus isolate (designated PVA-Hunan) was subjected to molecular characterization. The viral genome consisted of 9,567 nucleotides, excluding the poly(A) tail, and encoded a polyprotein of 3,059 amino acids. A second characteristic potyvirus open reading frame (ORF), pretty interesting Potyviridae ORF (pipo), was located at nucleotides 2,834-3,139. The isolate shared 84 % to 98 % and 93 % to 99 % sequence identity with other PVA isolates at the nucleotide and amino acid level, respectively. Phylogenetic analysis demonstrated that, within the PVA group, PVA-Hunan clustered most closely with the Finnish isolate Her, then with isolates 143, U, Ali, M and B11. The isolate TamMV stood alone at a separate branch. However, scanning of complete genome sequences using SimPlot revealed 99 %-sequence identity between PVA-Hunan and TamMV in the 3′-proximal end of the genome (~nt 9,160 to the 3′end) and a 50 %-94 % (average ~83 %) identity upstream of nt 9,160. In contrast, 98 % identity between PVA-Hunan and isolates M and B11 was detected for nucleotides 1 to ~9,160, but only ~94 % for the 3′-proximal region, suggesting a genome recombination event (RE) at nt 9,133. The recombination breakpoint also was identified by the Recombination Detection Program (RDP). The RE was further confirmed by analysis of the CP gene, where the apparent RE was located.

The current-season spread of Potato virus Y (PVY) was monitored in 19 fields under various management practices in New Brunswick, Canada, through the 2011 and 2012 growing seasons. The focus of this study was to evaluate the role of seedborne PVY inoculum, aphid vector abundance, and the numbers, timing, and types of insecticide and mineral oil sprays, and to confirm the reliability and forecasting capacity of midseason PVY testing. In each field, 100 to 110 virus-free plants were identified shortly after emergence and were assessed four times from early July to early September (after top-kill) with enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR) to track PVY spread. In addition, tubers harvested during development in August and after top-kill were grown-out in the greenhouse for ELISA testing. PVY spread to selected virus-free plants varied widely, ranging from 0 to 76.2% across all studied fields. Of the 19 fields over two seasons, 10 fields were planted with no detectable seedborne PVY, and they showed 0 to 8.7% (mean 2.9%) PVY spread by harvest. The remaining nine study fields with 0.9 to 5.8% seedborne PVY showed 1 to 76.2% (mean 15.2%) PVY spread by harvest. PVY spread was detected in most fields during midseason testing with ELISA and RT-PCR; all tests correlated well with final PVY rates after top-kill, though RT-PCR detection in developing tubers was most sensitive and correlated. Logistic regression modeling was used to identify major factors in PVY spread, including seedborne PVY, early-season aphid abundance, and the numbers of insecticide and mineral oil sprays. The best-fitting model, constructed using these factors as well as a measurement of July PVY incidence (ELISAJuly), strongly explained PVY spread by harvest, with the most significant management factor being the number of mineral oil sprays supplemented with insecticide used during the growing season. A similar model fitted without the ELISAJuly did not adequately predict ultimate PVY spread. The analysis suggests that mineral oil alone was effective at lowering PVY spread, and more effective when combined with insecticide, particularly when used early in the season. No evidence was found for differences in PVY spread across the eight cultivars used or across the range of mineral oil application rates, whereas some evidence was found for differences in the effectiveness of different insecticide types.

Post-harvest screening of potato ‘Kennebec’ revealed a Potato virus Y (PVY) incidence of 15.8%, a rate that is unusually high for a cultivar possessing a high level of field resistance to the virus. Randomly selected tubers were planted in a field plot and the resulting plants were monitored. Approximately 16% of plants developed symptoms ranging from mild mosaic symptoms to severe necrosis/rugosity/stunting. ELISA and RT–PCR analysis revealed that infections with Potato virus S (PVS), Potato virus X (PVX) and PVY, mostly in mixed-infections, occurred commonly in 14 sampled plants. Two strains, namely the common strain (PVY°) and the recombinant tuber necrotic strain (PVYNTN) were identified in the PVY-positive plants. In general, mild mosaic was associated with infections with PVX and PVS; intermediated mosaic was associated with PVS and PVYNTN infections; whereas severe leaf deformation/necrosis/drop symptoms were associated with PVYNTN and PVX co-infections, or with PVY° and either PVS or PVX ...

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

International audience; The management of Potato virus Y (PVY) in potato crops poses a con-tinual challenge due to the non-persistent mode of transmission of the virus and the propagation of seed potato tubers over several generations in the field. While PVY- resistant cultivars remain the most efficient way to protect potato crops against PVY, a vast majority of cultivars grown do not display significant resistance to PVY. Due to the short time period for PVY transmission by non-colonising aphid vectors, efficient control of PVY relies on preventing aphids landing on a crop and on adopting precautionary measures by ensuring that crops are grown in areas of low aphid and low virus pressure and limiting field generation. Prophylactic mea-sures such as roguing and early haulm destruction limit PVY spread but are not efficient alone. Among all existing control methods, spraying potato crops with mineral oils can offer significant protection against PVY spread, but their efficacy do vary in field conditions. The combination of several control methods such as mineral oil treatments, crop borders, intercropping, straw mulching or insecticide treatments can increase protection. These emphasise the importance of controlling virus through appropriate monitoring methods and crop management enforced by seed certification schemes through the use of ‘clean’ input seed and, when possible, the segregation of seed and ware crops to minimise the risk of virus transmission. This chapter presents and discusses the most widely used techniques of control and management of PVY, their effectiveness and their mode of action. This chapter also presents the history, objectives and principles of seed potato certification schemes and their role in minimising the spread of viruses within potato crops worldwide.

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Ry

Significant progress has been made in recent years in understanding pathological, serological and molecular properties of various strains of PVY and the aphid-mediated transmission. PVYO and PVYN appear to be the basic strain groups. Through genome recombination between these two groups, progeny groups whose genome possess one (e.g., PVYN:O or PVYN-Wi) to three (e.g., recombinant PVYNTN or European-PVYNTN) recombinant joints of PVYO and PVYN emerged. PVYN:O causes PVYN-like veinal necrosis in tobacco, but reacts to PVYO-specific antibody. PVYNTN causes potato tuber necrotic ringspot diseases in sensitive potato cultivars, and PVYN-like necrosis in tobacco plants, and reacts to PVYN-specific antibody. Through single nucleotide mutation(s), non-recombinant PVYNTN (or North American PVYNTN) also emerged from PVYN. It is also noteworthy that PVYN isolates originated from North America and Europe may have evolved separately; and to date most recombinant strains appear to be progenies of Eu-PVYN and PVYO. Several RT-PCR-based methodologies have been developed to characterize and detect various strains of PVY. A field survey revealed that PVYN:O has become a predominant strain in Manitoba and neighbouring states in USA. Moreover, three distinct variant groups inciting severe, intermediate and mild veinal/petiole/stem necrosis, respectively, on tobacco plants were observed within the PVYN:O isolates collected in Manitoba. Pathological and molecular diversity within PVYO strain group were revealed in New Brunswick, represented by PVYO-FL as a severe variant and by PVYO-RB as a mild variant. Studies on the transmission of PVY by various species of aphids revealed that aphid behavior plays an important role in the vector-mediated transmission. Application of mineral oil on the growing crop, especially in combination with use of crop borders, reduces aphid-mediated PVY transmission. Based on recent PVY research studies, Bartlett Superior 70 Oil was approved in 2011 for application to potato crops in Canada.

A TaqMan real-time reverse transcription-PCR (real-time RT-PCR) procedure was developed, optimized, and compared with other routine methods to detect Potato virus Y (PVY) in dormant potato tubers. Three pairs of primers and probes were designed and evaluated for their suitability to facilitate the real-time RT-PCR detection of PVY for all strain groups including PVYO, PVYN, PVYN:O (= PVYN-Wi) and PVYNTN. Among the primer and probe combinations tested, the combination PVY-1 produced the lowest threshold cycle (Ct) value of 25.75. The procedure was further optimized by adjusting various parameters including primer/probe concentration, reaction volume, amplification cycles, and master mixes from different sources. The real-time RT-PCR was then employed to detect PVY from dormant tubers of different cultivars and potato fields, and the results were compared with those obtained from conventional RT-PCR, enzyme-linked immunosorbent assay (ELISA) on sprouts and grow-out testing. Out of 1,069 single-virus infected (PVY only) tubers tested, both formats of RT-PCR detected PVY in 52 samples, ELISA on sprouts in 45, ELISA on leaves in 54 and visual observations in 53. However, in 61 multiple-virus infected tubers tested, both formats of RT-PCR, and ELISA on both sprouts and leaves detected a similar number of positives, thus, making all the methods equally sensitive. Considering that ELISA requires sprouting of dormant potato tubers for PVY testing, grow-out testing takes approximately 6–8 weeks to obtain results, and conventional RT-PCR needs post-PCR processing, real-time RT-PCR offers a speedy alternative for large scale detection of PVY from dormant tubers. The method is therefore recommended for testing of PVY in potato tubers on a commercial scale in a diagnostic laboratory.

The strain diversity and variant population of Potato virus Y (PVY) was investigated in potato lots with high PVY incidence in 2009 in New Brunswick, Canada, using reverse transcription (RT)-PCR, enzyme-linked immunosorbent assay (ELISA) and biological assays. Twenty lots with PVY infection rates >3 % were chosen following post-harvest testing for further investigation. Assays using multiplex RT-PCR revealed the existence of the common (PVYO), the recombinant N:O (PVYN:O) and the recombinant (European) potato tuber necrosis (Eu-PVYNTN) strains. PVYO was the predominant strain while PVYN:O and Eu-PVYNTN were widespread in these potato lots. PVYN:O and Eu-PVYNTN, either present alone or mixed with PVYO, were found in 19 and 13 of the 20 lots, respectively. Neither the tobacco veinal necrosis strain (PVYN) nor the non-recombinant (North American) PVYNTN was detected in the potatoes. Among the PVYO strain group, PVYO-Oz/-FL variant type was the predominant followed by the uncharacterized PVYO and the PVYO-139/-RB types. Biological assays of representative PVY positive-samples using tobacco and Yukon Gold were consistent with the findings of RT-PCR and serological assays.

A field isolate of Potato virus Y (PVY) was collected in New Brunswick, Canada in 2007 due to unusual symptoms observed on different potato cultivars. To unveil the PVY strain identity, tobacco and potato bioassays, PVYO and PVYN-specific antibody-based enzyme-linked immunosorbent assays, and reverse-transcription polymerase chain reaction (PCR)-based genotyping were carried out. All the assays demonstrated that the isolate, designated as PVYO-FL in this study, belonged to the PVYO strain group. Greenhouse tests with the potato cvs. FL 1533 and Jemseg confirmed the severe nature of infection by PVYO-FL. The complete genome sequences of PVYO-FL and PVYO-RB, the latter a mild PVYO isolate, were determined. BLAST analysis revealed that the two isolates shared 97 and 98% sequence identities at the nucleotide and polyprotein levels, respectively. Further BLAST analysis unveiled that PVYO-FL shared 99.7% nucleotide sequence identity with PVYO-Oz, an isolate reported in New York, United States, whereas the PVYO-RB isolate shared 99.2% sequence identity with PVYO-139, a PVYO isolate reported in New Brunswick, Canada. A phylogenetic tree of available, full-length sequences of PVY isolates demonstrated two subgroups within the PVYO branch, one clustered with PVYO-RB and the other with PVYO-FL. Group-specific sense primers for differentiation of the two subgroups were developed and evaluated. A limited survey of potato tubers collected from a field plot at the Potato Research Centre, Agriculture and Agri-Food Canada, using the newly developed PCR primers, indicated that 65.3 and 2.4% of the PVYO-positive tubers were infected with PVYO isolates belonging to the PVYO-FL and PVYO-RB subgroups, respectively. Assessment of the pathogenicity of three representative isolates from each subgroup on the potato cv. Jemseg demonstrated that severe and mild symptoms were induced by the PVYO-FL-like and PVYO-RB-like isolates, respectively.

Tomato chlorotic dwarf viroid (TCDVd) manually inoculated to transgenic (cv.‘Desiree’) potato plants containing antimicrobial cationic peptides failed to develop symptoms in above ground plant parts, but infected tubers were symptomatic. Plants from the infected tubers (second generation plants) emerged as either severely stunted (bushy stunt isolate, BSI) or tall and symptomless. Molecular characterization of BSI isolates showed TCDVd sequence variants 95 to 98% identical to TCDVd sequences from the database, while a viroid variant identical to TCDVd type isolate (acc # AF162131) was cloned from symptomless plants. The TCDVd BSI variants had novel U165C, GU177-178AA, and UCAC181-184CUUU nucleotide substitutions in the terminal right (TR) domain of the viroid molecule. The cloned viroid cDNAs of the BSI were infectious to experimental (cv. ‘Sheyenne’) tomato plants causing stunted plants with profuse auxiliary shoots. Visual evaluation of the susceptibility of the BSI to 18 potato and 21 tomato cultivars revealed severe symptoms in most cultivars of both species. The progeny variants accumulating in each potato and tomato cultivar exhibited the same novel TR domain in most cultivars, with only a slight variation in a few. The severity of the stunting symptoms induced by TCDVd from BSI isolates in both potato and tomato cultivars has not been noted previously with other TCDVd isolates and, as such, it is proposed that this new isolate be recognized as a distinct genotype. Emergence of this type of sequence variant in commercial fields or commercial tomato greenhouses could potentially cause relevant losses in both crops.

Pink rot caused by Phytophthora erythroseptica is found in most major potato-growing regions of the world. The pathogen can survive for many years in soil by means of oospores which are disseminated from diseased potato tissues. The ability to detect the pathogen in soil could be a valuable management tool that enables growers to plan control strategies depending on the presence of pathogen propagules in a particular field. However, soils are one of the most challenging environmental matrices to obtain microbial DNA that will support PCR. A method was developed that combined traditional baiting technique with PCR methods to detect P. erythroseptica in infested soil samples. Hairy nightshade (Solanum sarrachoides Sendt.) and bitter nightshade (Solanum dulcamara L.) two leaf stage (TLS) seedlings and cotyledon leaves successfully baited P. erythroseptica from zoospore suspensions, artificially inoculated soils and naturally infested soils. The pathogen was detected in the bait tissue with PCR methods. PCR increased the precision of the bait test. However, time was still required for the pathogen to infect and develop on the bait tissues. Although P. erythroseptica was detected from some bait plants only after 2 days of incubation, 10 days of incubation produced consistent results across the replicates with hairy and bitter nightshade cotyledon leaves and TLS seedlings.

In-season chemical or optical measures of crop N status can be effective tools in optimizing potato fertilizer N management. The feasibility of using a gene expression as an alternative approach for early detection of potato nitrate deficiency was examined using three potato cultivars (Shepody, Russet Norkotah, and Red Pontiac) with abundant (7.5 mM NO3), limited (0.75 mM NO3) or deficient (0 mM NO3) nitrate supply in nutrient culture over a 7 d period. RNA was extracted from the last fully expanded leaf and quantified using realtime RT-qPCR. Reduced nitrate supply had no measurable effect on shoot dry weight or leaf chlorophyll concentration, but decreased petiole nitrate concentration. Under deficit nitrate supply, down-regulation of nitrate reductase and nitrite reductase was measured within 3 days for all cultivars, and down-regulation of asparagine synthetase was measured in two cultivars. Nitrate supply had no effect on expression of ammonium transporter. In this experimental system, plant gene expression markers detected a reduction of nitrate supply prior to measureable reductions in plant growth or in N status measured using common chemical or optical methods.

Pink rot of potatoes caused by Phytophthora erythroseptica is known to infect all underground potato (Solanum tuberosum) tissues such as roots, stolons, tubers and basal stems. Potato leaves and stems do not normally exhibit symptoms except when extensive infection of underground plant parts results in plant wilt, chlorosis and necrosis. This is the first study to investigate the spread of P. erythroseptica in above-ground potato tissues using both traditional isolation and PCR methods originally developed to detect the pathogen in tubers. P. erythroseptica was detected in 66% of leaf and stem tissue samples originated from artificially-inoculated and naturally-infected tubers of ‘Yukon Gold’ and ‘Shepody’ by PCR methods. However, it was only recovered in pure cultures from 38% of stem and leaf tissue samples. The pathogen was also detected in leaf and stem tissues and aerial tubers of plantlets grown in potting mixtures infested with P. erythroseptica. Pure cultures of P. erythroseptica obtained from stem and leaf tissues, together with the 95% of the detection with the real-time PCR assay and 45% with the conventional PCR assay confirmed the viability of the pathogen in above-ground potato tissues. Furthermore, the pathogen was detected in progeny tubers and stolons produced by the infected potato plants. P. erythroseptica was also detected in a few samples of debris taken from naturally senesced above-ground potato tissue after harvest.

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.