First Report of Potato Virus H on Solanum muricatum in China.

Solanum muricatum, commonly known as pepino, pepino dulce, or tree melon, is a perennial shrub well known for its attractive, sweet, flavorful fruits and is frequently cultivated as an annual. It has gained increasing popularity in China and is grown as a cash crop in many provinces. S. muricatum belongs to the family Solanaceae and is closely related to tomato, eggplant, and potato. In 2012, during a study of serological relationships between PVH and PVM on potatoes, potato virus H (PVH) was detected serendipitously in symptomless pepino plantlets in Beijing, grown from tissue culture stocks. PVH is a recently discovered carlavirus reported from potato plants from Huhhot, Inner Mongolia Autonomous Region. Since then, it was found on potatoes in Yunnan, Hebei, Liaoning, Heilongjiang, and Xinjiang provinces. PVH induces mild symptoms with a slight leaf curl in systemic leaves, but most often it is almost symptomless or latent on potatoes (2). To confirm the presence of PVH on S. muricatum, surveys were conducted in 2012 and 2013 in Gansu, Yunnan, and Guangxi provinces and Beijing. Fruits and leaves were collected randomly from pepino plants displaying no obvious symptoms. For PVH detection, a combination of RT-PCR, genome sequencing and serological assays were used. RNAs extracted from fruits and leaves were amplified using RT-PCR with primer pairs PVHCPF and PVHCPR (2), and extracted samples were probed by Western blotting with the specific polyclonal antiserum against PVH (2). Among the 50 plants randomly collected, fruits and leaves of nine plants tested positive for PVH. Subsequently, an RT-PCR product of the expected size (2.6 kb) encompassing the triple-gene block, the capsid protein gene, and the cysteine-rich protein gene, was amplified with a specific primer pair (PVHB1F 5'-TGATGGAATTTACAAAAAC-3' and PVHUR 5'-CTTATGCGCATCTATCAATC-3'), and then cloned into pMD19-T (TaKaRa, Dalian, China) and sequenced (PVH-Pepino with GenBank Accession No. KF546312). Further sequence comparison showed that PVH-Pepino shared 91 to 98% nucleotide sequence identity in the genes mentioned above with those of the reported potato isolates PVH-Ho and PVH-YN (HM584819 and JQ904630, respectively). PVH-Pepino shared deduced amino acid identity of 98 to 99% in CP gene with PVH-Ho and PVH-YN, respectively, but only shared 57 to 67% amino acid identities with other reported carlaviruses (1,2). Thus, latent infection of PVH on S. muricatum was confirmed. To our knowledge, this is the first report of S. muricatum as a natural host of PVH. Our results suggest that PVH, as a new member of the genus Carlavirus, has a wider host range than originally expected. Potatoes and pepinos are crops widely grown in China. The fact that no symptoms were expressed by PVH in pepino plants (symptomless carrier) and only mild symptoms expressed by PVH in diseased potatoes makes detection and remediation of this disease more difficult. Although this finding does not show that PVH is economically important to pepino, this cannot be excluded in the presence of other viruses (2). References: (1) A. King et al. Page 881 in: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, London, 2011. (2) Y. Y. Li et al. PLOS ONE 8(6):e69255, 2013.

A new tospovirus causing chlorotic ringspot on Hippeastrum sp. in China.

A new tospovirus, HCRV 2007-ZDH, was isolated from a Hippeastrum sp. plant displaying necrotic and chlorotic ringspot symptoms in Yunnan province. This virus isolate was characterized based on particle morphology and RNA sequences analyses. Quasi-spherical, enveloped particles measuring about 70-100 nm, typical of tospoviruses, were observed in sap and cells of the infected plants. Transmission studies by inoculating this isolate mechanically to Hippeastrum sp. confirmed that 2007-ZDH is the causal agent of the chlorotic ringspot disease of Hippeastrum sp. The complete sequence of S RNA of 2007-ZDH was 2,744 nucleotides in length, sharing 74.4 % nucleotide identity with Tomato yellow ring virus (TYRV) isolate tomato (AY686718). The S RNA encoded a non-structural protein (NSs) (444 aa, 50.4 kDa) and the nucleocapsid (N) protein (273 aa, 30.1 kDa).The deduced NSs protein shared amino acid identities of 78.6, 76.3, and 74.9 % with that of TYRV, IYSV, and PolRSV, respectively. The deduced N protein shared amino acid identities of 86.1, 84.7, and 70.0 % with that of PolRSV, TYRV, and IYSV, respectively. These results suggest that the chlorotic ringspot virus belongs to a new tospovirus species, for which the name Hippeastrum chlorotic ringspot virus (HCRV) is proposed.

Molecular Characterization of Stolbur Group Subgroup E (16SrXII-E) Phytoplasma Associated with Potatoes in China.

Phytoplasmas have been reported from more than 70 plant species in China, most of which are from woody plants and very few are from potatoes (Solanum tuberosum). During the growing seasons of 2005 through 2011, potato disease surveys were conducted in seed and commercial fields in Yunnan Province and Inner Mongolia. Potato plants displayed symptoms of curled, yellowish and purplish leaves, shortened internodes, aerial tuber formation, and few small malformed underground tubers. Although the location of the fields surveyed each year varied, the disease seems to have become increasingly prevalent. In Yunnan, disease incidence was 5 to 24% in 2005 and 15 to 100% in 2010 and 2011. In Inner Mongolia, disease incidence in seed potato fields was 5 to 15% in 2006 and 25 to 50% in 2011. Total DNA was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.25-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, HhaI, HpaI, KpnI, MseI, and TaqI. Comparing the RFLP patterns of samples with previously published phytoplasma strains, the phytoplasmas matched patterns of the stolbur group, subgroup E (16SrXII-E) (1). In addition, the PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (2). The nested PCR product was cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska Fairbanks and GENEWIZ (South Plainfield, NJ). Nucleotide sequences (GenBank Accession No. EU293841) were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to 'Candidatus Phytoplasma fragariae' with RFLP patterns identical to group 16SrXII-E. To our knowledge, this is the first molecular characterization of the stolbur group phytoplasmas associated with potato disease in China. The potato is becoming increasingly important in China. The impacts of stolbur on potato yield losses, disease distributions, and insect vectors are currently under investigation. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of Aster Yellows (16SrI-B) Associated with Potatoes in Alaska.

During a disease survey conducted in 2009 in Alaska, one potato plant (Solanum tuberosum) with symptoms characteristic of aster yellows, such as apical leaves rolling inward, leaves turning yellow or purple, and presence of aerial tubers, was found in a commercial field. Total DNA was extracted from leaves, stems, and roots of the symptomatic and symptomless plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. A nested PCR was carried out with the first round primer pair P1/P7 followed by second round primer pair R16F2n/R16R2 (1,3). An approximate 1.2-kb PCR product was amplified from the symptomatic plant, but not symptomless plants. The PCR products from R16F2n/R16R2 were digested using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. The restriction fragment length polymorphism (RFLP) patterns were compared with those from known phytoplasma strains (1) and they matched the patterns for aster yellows subgroup B (16SrI-B). After P1/P7 amplification, the nested PCR product of primer pair P1A/16S-SR (2) was purified with a MiniElute Gel Extraction kit (Qiagen), sequenced by GENEWIZ (South Plainfield, NJ), and the sequence data analyzed by iPhyClassifier software (4). The results indicated that the sequence (GenBank Accession No. HQ599231) had 99.65% similarity to 'Candidatus Phytoplasma asteris' reference strain (GenBank Accession No. M30790). The RFLP similarity was identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC 005303). To our knowledge, this is the first report on the molecular identification of aster yellows phytoplasma associated with potatoes in Alaska. The source of the phytoplasma and pathway of disease transmission is currently under investigation. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of Clover Proliferation Group Phytoplasmas (16SrVI-A) Associated with Purple Top Diseased Potatoes (Solanum tuberosum) in China.

Phytoplasma diseases on potatoes are not well understood and have gone largely undetected in China. During the growing seasons of 2005 through 2010, potato disease surveys were conducted in seed and commercial fields in Yunnan Province. Samples were also harvested from three seed potato production areas in the Inner Mongolia Autonomous Region in 2007 and 2010. Disease incidence in these fields ranged from 15 to 85%. Plants displayed symptoms of branch proliferation, aerial tuber formation, upward rolling yellowish and purplish apical leaves, and extremely short stolen or chain tubers (irregular-shaped tubers). Total DNA from 250 samples was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.2-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, MseI, HhaI, HinfI, HpaII, KpnI, RsaI, and TaqI. The RFLP patterns of 120 of the 250 samples matched patterns of the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) phytoplasma (1). In addition, the nested PCR product of P1A/P7A (2) following P1/P7 amplification was cloned and sequenced (GenBank Accession No. HQ609490). Nucleotide sequences were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to 'Candidatus Phytoplasma trifolii' with RFLP patterns identical to group 16SrVI-A. To our knowledge, this is the first report of the CP group phytoplasmas associated with purple top diseased potatoes in China. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Molecular Characterization of 'Candidatus Phytoplasma asteris' Associated with Aster Yellows-Diseased Potatoes in China.

Potatoes (Solanum tuberosum) are one of the most important crops in China following rice, wheat, and corn. Aster yellows phytoplasma appeared to be widespread in China; it was found to cause diseases on alfalfa, oranges, peaches, periwinkles, bamboo (1), and cactus (4). However, scant information of this pathogen on potatoes is available except for a few short reports published during the 1950s. During the potato disease surveys conducted from 2005 to 2010 in Yunnan and Inner Mongolia, 10 to 35% of potato plants exhibited symptoms of yellowing or purpling of apical leaves, with the top leaves rolling inward and aerial tubers formation. Total DNA was extracted from midveins of leaves and roots of 125 diseased and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A nested PCR was carried out with the first round primer pair P1/P7 followed by the second round primer pair R16F2n/R16R2 (2,3). A PCR product of approximately 1.2 kb was amplified from diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting a 1.2-kb product using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. Comparing the RFLP patterns with previously published phytoplasma strains (2), aster yellows phytoplasma found on potato plants in Yunnan and Inner Mongolia belong to group I, subgroup B (16SrI-B). The PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (3) and P1A/16S-SR (3). The nested-PCR products from P1A/P7A and P1A/16S-SR were cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska-Fairbanks and GENEWIZ (South Plainfield, NJ). The nucleotide sequence (GenBank Accession No. HQ599228) was analyzed by iPhyClassifier software and had 99.53% sequence identity to the reference strain (GenBank Accession No. M30790) for 'Candidatus Phytoplasma asteris'. The RFLP similarity is identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC_005303). To our knowledge, this is the first report revealing the molecular characteristics of a phytoplasma associated with aster yellows-diseased potatoes in China. References: (1) H. Cai et al. Plant Prot. 31:38, 2005. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) W. Wei et al. Plant Dis. 91:461, 2007.

First Report of Kidney Bean Little Leaf Disease Associated with 16SrII Group Phytoplasma in China.

Kidney bean (Phaseolus vulgaris) is an important cash crop in China. It is widely grown in the Yunnan Province in the southwest region. In November of 2008, a new disease was observed on kidney bean plants grown in Yuanmou County. Affected plants displayed symptoms that included numerous twisted lateral shoots with abundant, tiny trifoliate leaves that were approximately one-tenth the size of healthy leaves. Some affected leaves, which were slightly distorted and oblong to oval, were evident on diseased plants and appeared pale green. On plants with little leaf symptoms, flowers were poorly formed, withered or aborted, and no pods were present. Surveys conducted in areas affected by disease revealed the presence of the disease in approximately 10% of the plants. DNA was extracted from 0.1 g of petioles and midveins harvested from both diseased and symptomless plants with the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Plants were evaluated for phytoplasma infection by a nested PCR assay with phytoplasma specific ribosomal operon primer pair P1/Tint followed by R16F2/R16R2 (2,3). An rDNA product of approximately 1,250 bp was amplified from seven of seven diseased plants, whereas no products were amplified from symptomless plants or a negative control devoid of DNA. Digests of nested PCR products (approximately 200 ng) with endonucleases AluI, ScaI, or EcoRI revealed no differences in restriction fragment length polymorphism (RFLP) among diseased plants. The amplicon was cloned and sequenced (GenBank Accession No. GQ336993). Comparison of in silico RFLP profiles with published profiles showed that kidney bean little leaf phytoplasma is a member of peanut witches' broom group 16SrII. Blast analysis of the kidney bean little leaf phytoplasma 16S rDNA sequence revealed that this strain is most similar (99.0%) to Syringa oblata yellows phytoplasma (Accession No. FJ263629) and to other phytoplasmas classified as group 16SrII members. Previously, phytoplasmas identified as 16SrII strains have been reported as probable cause of cactus witches' broom (1) and crotalaria witches' broom (4) in China. To our knowledge, this is the first report of a 16SrII phytoplasma infecting the kidney bean in China. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) I.-M Lee et al. Phytopathology 83:834, 1993. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996 (4) Z. H. Wang et al. Plant Pathol. 57:364, 2008.

First Report of Leaf Smut on Fescue (Festuca rubra) Caused by Urocystis agropyri in Interior Alaska.

In June and early July of 2003 and July of 2007, a smut disease was found on fescue (Festuca rubra L.) at the Fairbanks Golf and Country Club and vicinities, Interior Alaska. Diseased plants were pale green-to-slightly yellow and stunted. The lesions were long, narrow, yellowish green streaks parallel to the veins of leaves and sheaths. At a later stage, sori developed under the epidermis and the leaves and sheaths turned silvery gray. Rupture of the epidermis resulted in the release of masses of black spores that covered the leaves, especially along and within infected sheaths. Microscopic observation showed spore balls consisting of one-to-four, dark reddish brown teliospores surrounded by small, hyaline-to-light brown sterile cells. The spore balls were globose to elongate and 16 to 32 × 18 to 38 µm. The teliospores were globose, dark reddish brown, and 7.5 to 20 µm in diameter. Urocystis agropyri is recognized by R. W. Smiley et al. (2) and J. D. Smith et al. (3) as occurring on turf grasses; K. Vánky (4) restricts this species to Elymus spp. and recognizes U. ulei as the valid species infecting Festuca spp, including F. rubra. On the basis of host symptoms and morphology of spores, the smut found on F. rubra in Alaska fits better with published descriptions of U. agropyri (Preuss) Schrot (2,3), however, it likely is not conspecific with flag smut of wheat. For molecular verification of this identification, DNA was isolated from teliospores with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Primers were designed for amplification of the large subunit ribosomal gene based on the only two published sequences of U. ranunculi (Lib.) Moesz (GenBank Accession No. AF009879) and U. colchici (Schltdl.) Rabenh (GenBank Accession No. AF009878) (1). A 400-bp PCR product was amplified by the forward primer 3'-GCATTGTAAACTCAGAAGTGTTATCCG-5' and reverse primer 5'-TTCCCTAAACCTATATCCGGCG-3'. Nucleotide sequence of the PCR product (GenBank Accession No. AY547283) shared 97.7% homology with U. ranunculi and 98.2% with U. colchici. Comparison of nucleotide sequences will be more meaningful in the identification of species of Urocystis when additional sequences are published. To our knowledge, this is the first report of U. agropyri from turfgrass in Alaska. References: (1) D. Begerow et al. Can. J. Bot. 75:2045, 1997. (2) R. W. Smiley et al. Compendium of Turfgrass Diseases. 3rd ed. The American Phytopathological Society, St Paul, MN, 2005. (3) J. D. Smith et al. Page 189 in: Fungal Diseases of Amenity Turf Grassess. E. & F. N. Spon, London, 1989. (4) K. Vánky. Page 308 in: European Smut Fungi. Gustav Fischer Verlag, NY. 1994.

Expression of an Engineered Cecropin Gene Cassette in Transgenic Tobacco Plants Confers Disease Resistance to Pseudomonas syringae pv. tabaci.

ABSTRACT A chimeric gene fusion cassette, consisting of a secretory sequence from barley alpha-amylase joined to a modified cecropin (MB39) coding sequence and placed under control of the promoter and terminator from the potato proteinase inhibitor II (PiII) gene, was introduced into tobacco by Agrobacterium-mediated transformation. Transgenic and control plants reacted differently when inoculated with tobacco wildfire pathogen Pseudomonas syringae pv. tabaci at various cell concentrations. With control plants (transformed with a PiII-GUS [beta-D-glucuronidase] gene fusion), necrosis was clearly visible in leaf tissue infiltrated with bacterial inoculum levels of 10(2), 10(3), 10(4), 10(5), and 10(6) CFU/ml. With MB39-transgenic plants, however, necrosis was observed only in the areas infiltrated with the two highest levels (10(5) and 10(6) CFU/ml). No necrosis was evident in areas infiltrated with bacterial concentrations of 10(4) CFU/ml or less. Bacterial multiplication in leaves of MB39-transgenic plants was suppressed more than 10-fold compared to control plants, and absence of disease symptom development was associated with this growth suppression. We conclude that the pathogen-induced promoter and the secretory sequence were competent elements for transforming a cecropin gene into an effective disease-control gene for plants.