First Report of Streptomyces europaeiscabiei Causing Common Scab on Potato in Finland.

Common scab is an important disease in potato (Solanum tuberosum) caused by Streptomyces spp. In Finland, morphological and physiological characterization (5) and comparison of the 16S rRNA gene sequences have suggested that Streptomyces scabies and S. turgidiscabies are the main causal species (2), but occurrence of S. europaeiscabiei has not been previously studied. In September 2011, potato tubers of cvs. Fambo, Melody, Puikula, Rosamunda, Victoria, and Van Gogh showing symptoms of common scab were collected from 10 fields in southern (Lammi), eastern (Liperi), and western Finland (Isokyrö, Kalajoki, Tyrnävä). Symptoms included superficial, raised, and pitted corky lesions ranging from 3 to 10 mm in diameter. Isolations were made from scab lesions on water agar. Colonies phenotypically characteristic of Streptomyces were transferred to glucose yeast malt extract agar (GYM) after 7 days. Pure cultures were obtained through subsequent transfers to fresh GYM medium, on which the strains produced golden brown colonies and white to grey spores. DNA was extracted from bacteria using the E.Z.N.A. SP Plant DNA Mini Kit (Omega Bio-Tek, Inc.). Primers developed for the 16S rRNA gene sequences (4) were used to detect S. scabies and S. turgidiscabies by PCR. Of the 14 strains recovered, nine were assigned to S. scabies, three to S. turgidiscabies, and two remained unidentified. However, S. scabies and S. europaeiscabiei cannot be distinguished by the 16S rRNA gene sequence, but the ITS1 region of the 16S operon sequence amplified by PCR is cleaved by Hpy99I in S. scabies but not in S. europaeiscabiei (1). Altogether, 18 strains were tested, including nine obtained in 2011 and seven Finnish and two Swedish strains isolated and assigned to S. scabies in the mid-1990s (2,4,5). The ITS1 sequence of S. scabies type strain, ATCC49173, was cleaved with Hpy99I, in contrast to all other strains that were consequently assigned to S. europaeiscabiei. To further confirm the identity of the Finnish strains, sequences of atpD, recA, and rpoB genes from three strains (one from 1995, two from 2011) (GenBank Accession Nos. KJ802471 to 79) were found to be 100, 99.8, and 100% identical, respectively, to corresponding S. europaeiscabiei type strain CFBP 4497 sequences (3). Pathogenicity of the S. europaeiscabiei strains isolated in 2011 was confirmed using radish seedling assay (1). All strains prevented or reduced the growth of radish seedlings (cv. French Breakfast) or caused severe necrosis in repeated experiments. No symptoms were observed on the seedlings grown on oat meal agar without bacteria. The pathogenicity of the S. europaeiscabiei strains isolated in the mid-1990s was confirmed using minituber assays (4,5). In addition, two of these strains were tested in a glasshouse experiment and two in a radish seedling assay and shown to be pathogenic. The results suggest that S. europaeiscabiei is an important cause of potato common scab in Finland. In the earlier studies, S. europaeiscabiei may have been mistaken for S. scabies, as the restriction analysis of the ITS1 region was not done. References: (1) R. Flores-Gonzáles et al. Plant Pathol. 57:162, 2008. (2) J. F. Kreuze et al. Phytopathology 89:462, 1999. (3) D. P. Labeda. Int. J. Syst. Evol. Microbiol. 61:2525, 2011. (4) M. J. Lehtonen et al. Plant Pathol. 53:280, 2004. (5) P. Lindholm et al. Plant Dis. 81:1317, 1997.

Improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase.

RNA silencing limits transient expression of heterologous proteins in plants. Co-expression of viral silencing suppressor proteins can increase and prolong protein expression, but highly efficient silencing suppressors may stress plant tissue and be detrimental to protein yields. Little is known whether silencing suppression could be improved without harm to plant tissues. This study reports development of enhanced silencing suppressors by engineering the helper component proteinase (HCpro) of Potato virus A (PVA). Mutations were introduced to a short region of HCpro (positions 330-335 in PVA HCpro), which is hypervariable among potyviruses. Three out of the four HCpro mutants suppressed RNA silencing more efficiently and sustained expression of co-expressed jellyfish green fluorescent protein for a longer time than wild-type HCpro in agroinfiltrated leaves of Nicotiana benthamiana. Leaf tissues remained healthy-looking without any visible signs of stress.

First Report of Five Viruses Infecting Potatoes in Tanzania.

Potato (Solanum tuberosum L.) is an increasingly important food and cash crop in Tanzania (3). Potato production is concentrated in the southern highlands and mainly carried out by smallholder farmers. A certification system for seed potatoes does not exist in the country. Currently, there is little information about viruses infecting potatoes in Tanzania. In October through December 2011, occurrence of the most common, globally distributed potato viruses, Potato leaf roll virus (PLRV), Potato virus A (PVA), M (PVM), S (PVS), Y (PVY), and X (PVX) (1), was determined in 219 potato plants in 16 fields ranging from 0.2 to 1 ha. Potato crops, 1 to 3 months old, consisted sometimes of mixtures of varieties identified as Arika, Chekundu, Kagiri, Kiazi, Kikondo, Sasamka, or Tigoni by farmers, but could not be independently confirmed. The fields were located in the regions of Mbeya (Kawetele, Kikondo, Umalia, Uyole) and Rungwe (Mwakaleli) ~100 km apart in the southern highlands. Virus-like symptoms observed in most fields included yellowish-green mosaic, leaf rolling, and veinal necrosis. Symptoms in tubers were not studied. Leaves from 10 symptomatic and three symptomless plants were sampled from each field and tested by double antibody sandwich (DAS)-ELISA (1) at ARI-Uyole. Virus-specific antibodies and negative and positive controls were used according to the supplier's instructions (Science and Advice for Scottish Agriculture, Edinburgh, United Kingdom). PVS and PLRV were detected in 55% and 39% of the samples, respectively, and in all fields sampled. PVX and PVM were found in most fields and in 14% and 5% of the samples, respectively. PVA and PVY were only detected in two localities. Co-infection with PVS and PLRV was detected in 14% of the tested plants. Mixed infections involving three or four viruses were detected in 5% of the plants. A total of 20 samples, which were collected from Uyole and Mwakeleli and found to be ELISA-positive for one or several viruses, were pressed on FTA cards (GE Healthcare, Buckinghamshire, United Kingdom), transported to University of Helsinki, and analyzed by reverse-transcription PCR (2) using virus-specific primers designed to amplify the coat protein (CP) encoding region. All ELISA-positive samples tested positive by reverse transcriptase (RT)-PCR. Four and five samples ELISA-negative for PVX or PVA, respectively, were positive when tested by RT-PCR, suggesting that the actual incidence of these viruses may be higher than detected by DAS-ELISA. The PCR products from three to five samples per virus were sequenced without cloning, which reconfirmed detection of PLRV, PVA, PVS, PVX, and PVM (GenBank Accession Nos. KC866618 through KC866622, respectively) and revealed few if any differences among isolates of the viruses. The CP sequences were compared with viruses from other countries and continents (4). CP similarities suggested that viruses might have been introduced to Tanzania through potato trade or through introducing new cultivars without adequate indexing for viruses. These results suggest the need for the development of virus control schemes in potato crops, including the nascent, domestic certified seed potato production in Mbeya. References: (1) G. Loebenstein et al., eds. Virus and Virus-Like Diseases of Potatoes and Production of Seed Potatoes. Kluwer, Dortrecht, Netherlands, 2001. (2) J. Ndunguru et al. Virol. J. 2:45, 2005. (3) J. Rahko. Potato Value Chain in Tanzania. Univ. Helsinki, Finland, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Report of Raspberry leaf blotch virus in Raspberries in Finland.

Raspberry (Rubus idaeus L.) is a valuable and widely grown softfruit that is a host for 40 viruses and virus-like agents, of which many are not characterized at the molecular level. Recently, Raspberry leaf blotch virus (RLBV, putative emaravirus species) was described from raspberries (cv. Glen Ample) in the United Kingdom and Serbia. Plants displayed conspicuous yellow blotches on leaves and abnormal development of leaf hairs in the corresponding areas of the abaxial side (3). Similar symptoms were observed in 'Glen Ample' grown in protective plastic tunnels and open fields in the main berry growing area in eastern Finland in June 2011. In three farms, leaves were sampled from symptomatic and symptomless plants of 'Glen Ample' and also cv. Polka displaying no symptoms. Total RNA was extracted using CTAB reagent. Equal amounts of RNA were pooled from 13 samples and subjected to small-RNA (sRNA) deep sequencing (Fasteris SA, Plan-les-Ouates, Switzerland) to detect viruses without advance information (2). Contigs were built on 21- to 24-nt sRNA reads using Velvet. Contigs larger than 50 nt were used to search homologous sequences in GenBank by BLAST and significant similarity (up to 99%) was observed with RLBV RNA3 and RNA4. Mapping sRNA reads to the genome of RLBV (1) by MAQ resulted in significant coverage of RNA1 (16%), RNA2 (37%), RNA3 (46%), RNA4 (65%), and RNA5 (27%). cDNA was synthesized on RNA of one symptom-expressing plant using random hexamer primers and the cDNA tested by PCR with a forward primer (RLBV-F 5'-TCAAATCCACTTGCATAGAACC-3', nt 723 to 744) and reverse primer (RLBV-R1 5'-CCTCAAACCTTGCAAACACA-3', nt 1,318 to 1,337) designed according to the nucleocapsid (NP) gene of the Scottish RLBV isolate (3). The sequence of the amplified partial NP gene (576 nt; GenBank Accession No. JQ684678) was 92.8% and 94.8% identical to the Scottish isolate at nt and amino acid levels, respectively. The forward primer RLBV-F and a new reverse primer (RLBV-R2 5'-GCCGAAAGTCAAACCTGGTG-3', nt 943 to 962) were used to test additional plants for RBLV and to make a probe (198 nt) to detect RLBV using digoxigen-labeled sense and antisense RNA probes, as described for European mountain ash ringspot associated virus (1). RLBV was detected in all tested plants of 'Glen Ample' with yellow leaf blotch symptoms in the three farms, but not in any symptomless plants of 'Glen Ample' and 'Polka.' The sense probes gave strong signals, in contrast to the antisense probes, which gave only weak or no detectable signals in the virus-positive plants, consistent with the negative RNA strand of RLBV being encapsidated in virus particles. The results show RLBV is associated with severe, distinct, and characteristic symptoms in raspberries of cv. Glen Ample grown in plastic tunnels and open fields in Finland and has an apparent negative impact on plant growth and yield. Our observations in 2011 also suggest that the incidence of diseased plants is much higher in plastic tunnels than in open fields, perhaps because the conditions for the vector of RLBV (raspberry leaf and bud mite, Phyllocoptes gracilis Nalepa) (1) are more favorable in plastic tunnels. These results clarify the etiology of raspberry leaf blotch disease in Finland and emphasize the need to inspect raspberry planting materials for RLBV for better control of the disease. References: (1) A. K. Kallinen et al. Phytopathology 99:344, 2009. (2) J. F. Kreuze et al. Virology 388:1, 2009. (3) W. J. McGavin et al. J. Gen. Virol. 93:430, 2012.

Detection of Viruses in Sweetpotato from Honduras and Guatemala Augmented by Deep-Sequencing of Small-RNAs.

Sweetpotato (Ipomoea batatas) plants become infected with over 30 RNA or DNA viruses in different parts of the world but little is known about viruses infecting sweetpotato crops in Central America, the center of sweetpotato domestication. Small-RNA deep-sequencing (SRDS) analysis was used to detect viruses in sweetpotato in Honduras and Guatemala, which detected Sweet potato feathery mottle virus strain RC and Sweet potato virus C (Potyvirus spp.), Sweet potato chlorotic stunt virus strain WA (SPCSV-WA; Crinivirus sp.), Sweet potato leaf curl Georgia virus (Begomovirus sp.), and Sweet potato pakakuy virus strain B (synonym: Sweet potato badnavirus B). Results were confirmed by polymerase chain reaction and sequencing of the amplicons. Four viruses were detected in a sweetpotato sample from the Galapagos Islands. Serological assays available to two of the five viruses gave results consistent with those obtained by SRDS, and were negative for six additional sweetpotato viruses tested. Plants coinfected with SPCSV-WA and one to two other viruses displayed severe foliar symptoms of epinasty and leaf malformation, purpling, vein banding, or chlorosis. The results suggest that SRDS is suitable for use as a universal, robust, and reliable method for detection of plant viruses, and especially useful for determining virus infections in crops infected with a wide range of unrelated viruses.

Simultaneous virus-specific detection of the two cassava brown streak-associated viruses by RT-PCR reveals wide distribution in East Africa, mixed infections, and infections in Manihot glaziovii.

The expanding cassava brown streak disease (CBSD) epidemic in East Africa is caused by two ipomoviruses (genus Ipomovirus; Potyviridae), namely, Cassava brown streak virus (CBSV), and Ugandan cassava brown streak virus (UCBSV) that was described recently. A reverse transcription polymerase chain reaction (RT-PCR) based diagnostic method was developed in this study for simultaneous virus-specific detection of the two viruses. Results showed that CBSV and UCBSV are distributed widely in the highlands (> 1000 m above the sea level) of the Lake Victoria zone in Uganda and Tanzania and also in the Indian Ocean costal lowlands of Tanzania. Isolates of UCBSV from the Lake Victoria zone were placed to two phylogenetic clusters in accordance with their origin in Uganda or Tanzania, respectively. Mixed infections with CBSV and UCBSV were detected in many cassava plants in the areas surveyed. CBSV was also detected in the perennial species Manihot glaziovii (DNA-barcoded in this study) in Tanzania, which revealed the first virus reservoir other than cassava. The method for detection of CBSV and UCBSV described in this study has important applications for plant quarantine, resistance breeding of cassava, and studies on epidemiology and control of CBSD in East Africa.

Genetic diversity of Potato virus Y infecting tobacco crops in China.

Genetic variability of Potato virus Y (PVY) isolates infecting potato has been characterized but little is known about genetic diversity of PVY isolates infecting tobacco crops. In this study, PVY isolates were collected from major tobacco-growing areas in China and single-lesion isolates were produced by serial inoculation on Chenopodium amaranticolor. Most isolates (88%) caused systemic veinal necrosis symptoms in tobacco. Of these, 16 isolates contained a PVY(O)-like coat protein (CP) and PVY(N)-like helper component proteinase (HC-pro) and, in this respect, were similar to the PVY(N-Wi), PVY(N:O), and PVY-HN2 isolates characterized from potato in Europe, the United States, and China, respectively; two isolates contained a PVY(O)-like HC-pro and a PVY(N)-like CP; another two isolates had recombination junctions in the CP-encoding region. Both the HC-pro and CP of PVY were under negative selection as a whole; however, seven amino acids in HC-pro and six amino acids in CP were under positive selection. Selection pressures differed between the subpopulations of PVY distinguished by phylogenetic analysis of HC-pro and CP sequences. When PVY isolates from potato were included, no host-specific clustering of the PVY isolates was observed in phylogenetic and nucleotide diversity analyses, suggesting frequent spread of PVY isolates between potato and tobacco crops in the field.

First Report of Sweetpotato symptomless virus 1 and Sweetpotato virus A in Sweetpotatoes in Tanzania.

Sweetpotato (Ipomea batatas L.) is grown widely from tropical to temperate regions and is an important food security crop in tropical countries. In Africa, sweetpotato is infected by RNA viruses of many taxa (4), but DNA viruses, such as the genus Begomovirus (family Geminiviridae), infecting sweetpotatoes in the Americas have been reported only in Kenya (3). A caulimo-like DNA virus (family Caulimoviridae) has been detected in sweetpotatoes in Uganda (1). Recently, two novel badnaviruses (genus Badnavirus, family Caulimoviridae) and a new mastrevirus (genus Mastrevirus, family Geminiviridae) were discovered in a local sweetpotato cultivar maintained in a germplasm collection in Peru (2) but were not reported elsewhere. This study examined the possible existence of these novel viruses in landrace sweetpotato varieties grown in Tanzania. Nine landrace sweetpotato varieties and one introduced cultivar (NIS 91 from the International Potato Centre, Peru) were sampled from six regions of Tanzania. DNA was extracted (2) and amplified by PCR using primers (MastvKF: 5'-GACAGACCCCTAGGGTGA-3'; MastvsR 5'-ACTGCATATAGTACATGCCACA-3') designed in this study to amplify partial, putative movement and coat protein gene sequences of Sweetpotato symptomless virus 1 (SPSMV-1) (GenBank Accession No. FJ560945) (2). Products of the expected size were detected in seven samples (varieties Ex-London, Ex-Lyawaya, Gairo, Hombolo, Kagole white, Mbeya, and Shangazi) representing four regions surveyed (Dodoma, Mbeya, Morogoro, and Kagera). PCR products from five samples were sequenced (396 nt; GenBank Accession Nos. HQ316938 to HQ316942) and found to be identical to each other and the isolate described originally in Peru (2). Amplification with primers (BadnaBKF: 5'-CAAATTAGGAGGCAGATAAATG-3'; BadnaBsR: 5'-GGTCTTCTTATGTTCCACCTT-3') designed in this study according to the sequence of Sweetpotato virus B (SPBV-B) (GenBank Accession No.FJ560944) resulted in products of the expected size in three samples (varieties Ex-Lyawaya, Gairo, and Hombolo collected in Mbeya, Morogoro, and Dodoma, respectively) that were positive also for SPSMV-1. Sequences of the products (787 nt; HQ316935 to HQ316937) were nearly identical (99.4%). They were 96.8 to 96.9% similar to a region (nts 830-1616) of Sweetpotato virus A (SPBV-A; FJ560943) (2), whereas they were only 83.2 to 83.6 % similar to the corresponding region (1,486 to 2,272 nt) of SPBV-B (FJ560944) (2). No virus was detected in cv. NIS 91. All plants sampled exhibited mild mottling or mosaic symptoms, but a contribution to the symptoms by other untested viruses cannot be excluded because few of the large number of sweetpotato viruses have been studied in Africa (4). To our knowledge, this is the first report of SPSMV-1 and SPBV-A outside South America and in sweetpotatoes grown in the field. The results show that the two viruses are distributed widely in local sweetpotato varieties in Tanzania, which suggests that they may be found in other sweetpotato-growing areas where they have not been studied. While the yield losses caused by SPSMV-1 and SPBV-A remains to be studied, the data from this study are of practical importance in terms of regional and international exchange of sweetpotato germplasm. References: (1) V. Aritua et al. Plant Pathol. 56:324, 2007. (2) J. F. Kreuze et al. Virology 388:1, 2009. (3) T. Paprotka et al. Virus Res. 149:224, 2010. (4) F. Tairo et al. Mol. Plant Pathol. 6:199, 2005.

Evolution of cassava brown streak disease-associated viruses.

Cassava brown streak disease (CBSD) has occurred in the Indian Ocean coastal lowlands and some areas of Malawi in East Africa for decades, and makes the storage roots of cassava unsuitable for consumption. CBSD is associated with Cassava brown streak virus (CBSV) and the recently described Ugandan cassava brown streak virus (UCBSV) [picorna-like (+)ssRNA viruses; genus Ipomovirus; family Potyviridae]. This study reports the first comprehensive analysis on how evolution is shaping the populations of CBSV and UCBSV. The complete genomes of CBSV and UCBSV (four and eight isolates, respectively) were 69.0-70.3 and 73.6-74.4% identical at the nucleotide and polyprotein amino acid sequence levels, respectively. They contained predictable sites of homologous recombination, mostly in the 3'-proximal part (NIb-HAM1h-CP-3'-UTR) of the genome, but no evidence of recombination between the two viruses was found. The CP-encoding sequences of 22 and 45 isolates of CBSV and UCBSV analysed, respectively, were mainly under purifying selection; however, several sites in the central part of CBSV CP were subjected to positive selection. HAM1h (putative nucleoside triphosphate pyrophosphatase) was the least similar protein between CBSV and UCBSV (aa identity approx. 55%). Both termini of HAM1h contained sites under positive selection in UCBSV. The data imply an on-going but somewhat different evolution of CBSV and UCBSV, which is congruent with the recent widespread outbreak of UCBSV in cassava crops in the highland areas (>1000 m above sea level) of East Africa where CBSD has not caused significant problems in the past.

Involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems.

The nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. This chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (RNP) particles, and to counteract plant host defense responses. Plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. Investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections.

First Report of European mountain ash ringspot-associated virus in Sorbus aucuparia from Eastern Karelia, Russia.

Mountain ash (Sorbus aucuparia L.) is a tree that is native to northern Europe. It was recently found to be infected with European mountain ash ringspot-associated virus (EMARaV) in Germany and Finland (2,3). EMARaV is not transmitted mechanically and no vector is known, but it is related to negative-sense RNA viruses transmitted by eriophyid mites (3) and represents a new viral species of the genus Emaravirus, which is not assigned to any family. In Finland, EMARaV is common, widely distributed, and detected in all tested ringspot disease-affected and symptomless mountain ash trees (2). Ringspot symptoms occur in mountain ash also in Sweden (west from Finland). In this study, ringspot-affected mountain ash trees were found in Ustreka, which marks the eastern edge of the geologically defined Baltic Shield and the eastern geobotanical borderline of Fennoscandia in eastern Karelia, Russia (1). This border zone can be recognized by changes in vegetation, including occurrence of Siberian larch (Larix sibirica Ledeb.), which does not belong to Fennoscandian flora. Three young mountain ash trees (4 to 5 years old and 2 to 3 m tall) displayed symptoms characteristic of EMARaV at Lake Tsumba (61°50'58″N, 37°45'34″E) in August 2009. EMARaV was detected in collected leaves by reverse transcription-PCR using virus-specific primers as described (2). The nucleocapsid protein genes (944 nucleotides) of the three isolates (Rus1, Rus2, and Rus3) were 97 to 99% identical (GenBank Accession Nos. GU563317, GU563318, and GU563319). On the basis of phylogenetic analysis, the Russian isolates, and three previously characterized isolates from Finland (Ris60, Ris61, and Kuo 12) formed a distinct cluster separate from the remaining 14 previously characterized isolates from Finland and one isolate from Germany. Division of EMARaV to two genetically distinguishable groups was realized for the first time. EMARaV-infected mountain ash trees suffer from chlorosis and growth reduction of varying severity, which impairs their value as an ornamental and for carpentry. To our knowledge, this is the first report in Russia on definite identification of a virus and virus disease in an economically important native plant of northern Europe in the natural habitat as far east as the geobotanical border zone of Fennoscandia. References: (1) T. Ahti and M. Boychek. The Botanical Journeys of A. K. Cajander and J. I. Lindroth to Karelia and Onega River in 1898 and 1899. Finnish Museum of Natural History, Helsinki, Finland, 2006. (2) A. K. Kallinen et al. Phytopathology 99:344, 2009. (3) N. Mielke and H. P. Muehlbach. J. Gen. Virol. 88:1337, 2007.

Detection, distribution, and genetic variability of European mountain ash ringspot-associated virus.

European mountain ash ringspot-associated virus (EMARAV) was recently characterized from mountain ash (rowan) (Sorbus aucuparia) in Germany. The virus belongs tentatively to family Bunyaviridae but is not closely related to any classified virus. How commonly EMARAV occurs in ringspot disease (EMARSD) affected mountain ash trees was not reported and was investigated here. Virus-specific detection tools such as reverse transcription-polymerase chain reaction and dot blot hybridization using digoxigenin-labeled RNA probes were developed to test 73 mountain ash trees including 16 trees with no virus-like symptoms from 16 districts in Finland and Viipuri, Russia. All trees were infected with EMARAV. Hence, EMARAV is associated with EMARSD and can also cause latent infections in mountain ash. Symptom expression and the variable relative concentrations of viral RNA detected in leaves showed no correlation. Infectious EMARAV was detected also in dormant branches of trees in winter. Subsequently, genetic variability, geographical differentiation, and evolutionary selection pressures were investigated by analyzing RNA3 sequences from 17 isolates. The putative nucleocapsid (NP) gene sequence (944 nucleotides) showed little variability (identities 97 to 99%) and was under strong purifying selection. Amino acid substitutions were detected in two positions at the N terminus and one position at the C terminus of NP in four isolates. The 3' untranslated region (442 nucleotides) was more variable (identities 94 to 99%). Six isolates from a single sampling site exhibited as wide a genetic variability as isolates from sites that were hundreds of kilometers apart and no spatial differentiation of populations of EMARAV was observed.

Interactions and biocontrol of pathogenic Streptomyces strains co-occurring in potato scab lesions.

AIMS: To test interactions between pathogenic strains of Streptomyces turgidiscabies, S. scabies and S. aureofaciens. To study biological control of S. turgidiscabies and S. scabies using the nonpathogenic Streptomyces strain (346) isolated from a scab lesion and a commercially available biocontrol agent (S. griseoviridis strain K61; 'Mycostop'). METHODS AND RESULTS: Pathogenic strains of S. turgidiscabies and S. aureofaciens inhibited growth of S. scabies in vitro, whereas strain 346 and S. griseoviridis inhibited the pathogenic strains and were subsequently tested for control of scab in the greenhouse and field. Strains 346 and K61 suppressed development of common scab disease caused by S. turgidiscabies in the greenhouse. Strain 346 reduced incidence of S. turgidiscabies in scab lesions on potato tubers in the field. CONCLUSIONS: Streptomyces turgidiscabies shows antagonism against S. scabies that occurs in the same scab lesions and shares the ecological niche in the field. Biocontrol of S. turgidiscabies is possible with nonpathogenic Streptomyces strains but interactions may be complicated. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces turgidiscabies may have potential to displace S. scabies under the Scandinavian potato growing conditions. Biological control of the severe potato scab pathogen, S. turgidiscabies, is demonstrated for the first time. The results can be applied to enhance control of common scab.

Molecular Characterization of Sweet potato feathery mottle virus (SPFMV) Isolates from Easter Island, French Polynesia, New Zealand, and Southern Africa.

Strains of Sweet potato feathery mottle virus (SPFMV; Potyvirus; Potyviridae) infecting sweet-potato (Ipomoea batatas) in Oceania, one of the worlds' earliest sweetpotato-growing areas, and in southern Africa were isolated and characterized phylogenetically by analysis of the coat protein (CP) encoding sequences. Sweetpotato plants from Easter Island were co-infected with SPFMV strains C and EA. The EA strain isolates from this isolated location were related phylogenetically to those from Peru and East Africa. Sweetpotato plants from French Polynesia (Tahiti, Tubuai, and Moorea) were co-infected with SPFMV strains C, O, and RC in different combinations, whereas strains C and RC were detected in New Zealand. Sweetpotato plants from Zimbabwe were infected with strains C and EA and those from Cape Town, South Africa, with strains C, O, and RC. Co-infections with SPFMV strains and Sweet potato virus G (Potyvirus) were common and, additionally, Sweet potato chlorotic fleck virus (Carlavirus) was detected in a sample from Tahiti. Taken together, occurrence of different SPFMV strains was established for the first time in Easter Island, French Polynesia, and New Zealand, and new strains were detected in Zimbabwe and the southernmost part of South Africa. These results from the Southern hemisphere reflect the anticipated global distribution of strains C, O, and RC but reveal a wider distribution of strain EA than was known previously.

Distinguishing bacterial pathogens of potato using a genome-wide microarray approach.

A set of 9676 probes was designed for the most harmful bacterial pathogens of potato and tested in a microarray format. Gene-specific probes could be designed for all genes of Pectobacterium atrosepticum, c. 50% of the genes of Streptomyces scabies and c. 30% of the genes of Clavibacter michiganensis ssp. sepedonicus utilizing the whole-genome sequence information available. For Streptomyces turgidiscabies, 226 probes were designed according to the sequences of a pathogenicity island containing important virulence genes. In addition, probes were designed for the virulence-associated nip (necrosis-inducing protein) genes of P. atrosepticum, P. carotovorum and Dickeya dadantii and for the intergenic spacer (IGS) sequences of the 16S-23S rRNA gene region. Ralstonia solanacearum was not included in the study, because it is a quarantine organism and is not presently found in Finland, but a few probes were also designed for this species. The probes contained on average 40 target-specific nucleotides and were synthesized on the array in situ, organized as eight sub-arrays with an identical set of probes which could be used for hybridization with different samples. All bacteria were readily distinguished using a single channel system for signal detection. Nearly all of the c. 1000 probes designed for C. michiganensis ssp. sepedonicus, c. 50% and 40% of the c. 4000 probes designed for the genes of S. scabies and P. atrosepticum, respectively, and over 100 probes for S. turgidiscabies showed significant signals only with the respective species. P. atrosepticum, P. carotovorum and Dickeya strains were all detected with 110 common probes. By contrast, the strains of these species were found to differ in their signal profiles. Probes targeting the IGS region and nip genes could be used to place strains of Dickeya to two groups, which correlated with differences in virulence. Taken together, the approach of using a custom-designed, genome-wide microarray provided a robust means for distinguishing the bacterial pathogens of potato.

Natural wild hosts of sweet potato feathery mottle virus show spatial differences in virus incidence and virus-like diseases in Uganda.

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of sweetpotato. An East African strain of SPFMV incites the severe 'sweetpotato virus disease' in plants co-infected with Sweet potato chlorotic stunt virus and threatens subsistence sweetpotato production in East Africa; however, little is known about its natural hosts and ecology. In all, 2,864 wild plants growing in sweetpotato fields or in their close proximity in Uganda were observed for virus-like symptoms and tested for SPFMV in two surveys (2004 and 2007). SPFMV was detected at different incidence in 22 Ipomoea spp., Hewittia sublobata, and Lepistemon owariensis, of which 19 species are new hosts for SPFMV. Among the SPFMV-positive plants, approximately 60% displayed virus-like symptoms. Although SPFMV incidence was similar in annual and perennial species, virus-like diseases were more common in annuals than perennials. Virus-like diseases and SPFMV were more common in the eastern agroecological zone than the western, central, and northern zones, which contrasted with known incidence of SPFMV in sweetpotato crops. The data on a large number of new natural hosts of SPFMV detected in this study provide novel insights into the ecology of SPFMV in East Africa.

Infection with Rhizoctonia solani induces defense genes and systemic resistance in potato sprouts grown without light.

Rhizoctonia solani is an important soilborne and seedborne fungal pathogen of potato (Solanum tuberosum). The initial infection of sprouts prior to emergence causes lesions and may be lethal to the sprout or sprout tip, which results in initiation and compensatory growth of new sprouts. They emerge successfully and do not suffer significant damage. The mechanism behind this recovery phenomenon is not known. It was hypothesized that infection may induce pathogen defense in sprouts, which was investigated in the present study. Tubers were sprouted in cool and moist conditions in darkness to mimic conditions beneath soil. The basal portion of the sprout was isolated from the apical portion with a soft plastic collar and inoculated with highly virulent R. solani. Induction of defense-related responses was monitored in the apical portion using microarray and quantitative polymerase chain reaction techniques at 48 and 120 h postinoculation (hpi) and by challenge-inoculation with R. solani in two experiments. Differential expression of 122 and 779 genes, including many well-characterized defense-related genes, was detected at 48 and 120 hpi, respectively. The apical portion of the sprout also expressed resistance which inhibited secondary infection of the sprouts. The observed systemic induction of resistance in sprouts upon infection with virulent R. solani provides novel information about pathogen defense in potato before the plant emerges and becomes photosynthetically active. These results advance our understanding of the little studied subject of pathogen defense in subterranean parts of plants.

Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy.

Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae) and Sweet potato feathery mottle virus (SPFMV; Potyviridae) interact synergistically and cause severe diseases in co-infected sweetpotato plants (Ipomoea batatas). Sweetpotato is propagated vegetatively and virus-free planting materials are pivotal for sustainable production. Using cryotherapy, SPCSV and SPCSV were eliminated from all treated single-virus-infected and co-infected shoot tips irrespective of size (0.5-1.5mm including 2-4 leaf primordia). While shoot tip culture also eliminated SPCSV, elimination of SPFMV failed in 90-93% of the largest shoot tips (1.5mm) using this technique. Virus distribution to different leaf primordia and tissues within leaf primordia in the shoot apex and petioles was not altered by co-infection of the viruses in the fully virus-susceptible sweetpotato genotype used. SPFMV was immunolocalized to all types of tissues and up to the fourth-youngest leaf primordium. In contrast, SPCSV was detected only in the phloem and up to the fifth leaf primordium. Because only cells in the apical dome of the meristem and the two first leaf primordia survived cryotherapy, all data taken together could explain the results of virus elimination. The simple and efficient cryotherapy protocol developed for virus elimination can also be used for preparation of sweetpotato materials for long-term preservation.

Antimycin A-producing nonphytopathogenic Streptomyces turgidiscabies from potato.

AIM: To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions. METHODS AND RESULTS: In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium. CONCLUSIONS: Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.

Molecular Genetic Characterization of Sweet potato virus G (SPVG) Isolates from Areas of the Pacific Ocean and Southern Africa.

Sweet potato virus G (SPVG, genus Potyvirus, family Potyviridae) was detected in sweetpotato (Ipomoea batatas) storage roots sold in the local markets and storage roots or cuttings sampled directly from farmers' fields. Using serological and molecular methods, the virus was detected for the first time in Java, New Zealand, Hawaii, Tahiti, Tubuai, Easter Island, Zimbabwe, and South Africa, and also in an imported storage root under post-entry quarantine conditions in Western Australia. In some specimens, SPVG was detected in mixed infection with Sweet potato feathery mottle virus (genus Potyvirus). The coat protein (CP) encoding sequences of SPVG were analyzed for 11 plants from each of the aforementioned locations and compared with the CP sequences of 12 previously characterized isolates from China, Egypt, Ethiopia, Spain, Peru, and the continental United States. The nucleotide sequence identities of all SPVG isolates ranged from 79 to 100%, and amino acid identities ranged from 89 to 100%. Isolates of the same strain of SPVG had nucleotide and amino acid sequence identities from 97 to 100% and 96 to 100%, respectively, and were found in sweetpotatoes from all countries sampled except Peru. Furthermore, a plant from Zimbabwe was co-infected with two clearly different SPVG isolates of this strain. In contrast, three previously characterized isolates from China and Peru were phylogenetically distinct and exhibited <90% nucleotide identity with any other isolate. So far, the highest genetic diversity of SPVG seems to occur among isolates in China. Distribution of SPVG within many sweetpotato growing areas of the world emphasizes the need to determine the economic importance of SPVG.