bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Curtoviruses Associated with Curly Top Disease of Tomato in California and Monitoring for These Viruses in Beet Leafhoppers
Authors: Chen Li-Fang , Brannigan Kelly , Clark Rod , Gilbertson Robert L.
Published: December 2009

Curly top disease is caused by a complex of curtoviruses (family Geminiviridae), and it continues to plague tomato production in California. To better understand the etiology of curly top of tomatoes in California, polymerase chain reaction (PCR)-based methods were developed and used to characterize the curtoviruses involved, and to monitor for these viruses in the beet leafhopper vector, Circulifer tenellus. From 2002 to 2008, 86 processing and fresh market tomato fields in the Central Valley of California were surveyed for the incidence of curly top symptoms. Representative samples with curly top symptoms were collected from the surveyed fields, as well as from another 24 fields. The incidence of curly top symptoms in most fields ranged from trace (<1%) to low (1 to 5%); however, in 2002, 2003, and 2008, some fields had medium (5 to 20%) or high (>20%) incidences. PCR with general and species-specific primers was used to establish that the predominant species associated with tomato curly top disease were Beet mild curly top virus (BMCTV) and, to a lesser extent, Beet severe curly top virus (BSCTV). The incidence, relative amount, and species of curly top virus in leafhoppers, collected at monthly intervals by the California Department of Food and Agriculture (CDFA) personnel during 2003 to 2008, was also determined. The predominant species detected were BMCTV and BSCTV. The highest incidences of curly top in tomato fields were associated with high populations of leafhoppers (e.g., in 2003 when populations were two times greater than average) having high incidences and levels of curly top virus early in the growing seasons (e.g., March to May 2003 and 2008). Detection of curly top virus in leafhoppers early in the growing season was consistent with acquisition of virus from reservoir hosts in the foothills. However, continued detection of curly top virus in leafhoppers throughout the growing season and development of curly top in late-planted fresh market tomatoes were consistent with the presence of inoculum sources (e.g., weeds or crop plants) in the agricultural areas of the Central Valley. Geographical locations or “hotspots” having higher proportions of curly top virus–positive leafhoppers were identified, which may reveal areas having high concentrations of curly top virus reservoir hosts. The application of these molecular tools has provided new insight into curly top of tomato in California, and may lead to improved curly top management.
January Temperatures Predict Tobacco Blue Mold Severity: Evidence for Local Source and Long-Distance Transport of Inoculum in Connecticut
Authors: LaMondia J. A.
Published: December 2009

The tobacco blue mold pathogen, Peronospora tabacina, has been periodically reintroduced to the Connecticut River Valley cigar wrapper tobacco (Nicotiana tabacum) area of Connecticut and Massachusetts. Once introduced, there is a greater likelihood of disease in following years. Blue mold occurred from 1937 to 1956, 1979 to 1981, and most recently from 1996 to 2008. Disease severity was evaluated and rated annually from 1979 to 2008, and was correlated (r = 0.84; P = 0.002) with January temperatures when the pathogen was present in moderate amounts the previous year (severity rating >1). The date of the first report of disease was negatively correlated with disease severity the previous year (r = –0.63) and February temperatures (r = –0.83). Blue mold severity was not correlated with the date of the first disease, the previous year's disease severity, or rainfall amount or frequency after introduction of the pathogen. January temperatures may be used to predict the need for early-season fungicide applications to control disease from local overwintering inoculum following moderate to high blue mold severity. In years following little or no disease, forecasts of long-distance transport will continue to be a valuable tool to predict the risk of long-distance reintroduction and the need for fungicide application based on exposure.
First Report of Damping-Off Caused by Rhizoctonia solani AG-4 on Mediterranean Fan Palm in Italy
Authors: Polizzi G. , Aiello D. , Castello I. , Guarnaccia V. , Vitale A.
Published: December 2009

Mediterranean fan palm (Chamaerops humilis L.), one of just two autochthonous European palms, is native to the western Mediterranean Region in southwestern Europe and northwestern Africa. It can be found growing wild in the Mediterranean area. In Europe, this species is very popular as an ornamental plant. In March 2009, a widespread damping-off was observed in a stock of approximately 30,000 potted 1-month-old plants of C. humilis cv. Vulcano in a nursery in eastern Sicily. Disease incidence was approximately 20%. Disease symptoms consisted of lesions at the seedling shoot (plumule). Stem lesions were initially orange, turned brown, and followed by death of the entire plumule or eophyll. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently isolated from lesions when plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 μg/ml. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Mycelium was branched at right angles with a septum near the branch and a slight constriction at the branch base. Hyphal cells removed from cultures grown at 25°C on 2% water agar were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11 on 2% water agar in petri plates (3). Anastomosis was observed only with tester isolates of AG-4, giving both C2 and C3 reactions (2). One representative isolate obtained from symptomatic tissues was deposited at the Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (CBS No. 125095). Pathogenicity tests were performed on container-grown, healthy, 1-month-old seedlings. Twenty plants of C. humilis cv. Vulcano were inoculated near the base of the stem with two 1-cm2 PDA plugs from 5-day-old mycelial cultures. The same number of plants served as uninoculated controls. Plants were incubated in a growth chamber and maintained at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Symptoms identical to those observed in the nursery appeared 5 days after inoculation and all plants died within 20 days. No disease was observed on control plants. A fungus identical in culture morphology to R. solani AG-4 was consistently reisolated from symptomatic tissues, confirming its pathogenicity. To our knowledge, this is the first report in the world of R. solani causing damping-off on Mediterranean fan palm.

References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.
First Report of Phytophthora sansomeana Causing Wilting and Stunting on Corn in Ohio
Authors: Zelaya-Molina L. X. , Ellis M. L. , Berry S. A. , Dorrance A. E.
Published: December 2009

During the spring of 2004, corn seedlings with symptoms of wilting and stunting were observed in corn fields with emergence problems in Madison and Brown counties, Ohio. Phytophthora isolates were recovered from sections of root tissue of diseased seedlings placed on dilute V8 media amended with pentachloronitrobenzene, iprodione, benlate, neomycin sulfate, and chloramphenicol. Colonies were rosaceous on potato dextrose agar, with a growth rate of 5 mm per day. Homothallic isolates with paragynous antheridia were observed on lima bean agar (LBA); oogonia were 35 to 50 μm in diameter. Sporangia were ovoid to obpyriform, nonpapillate, with an average size of 49 × 30 μm. Pathogenicity was tested on corn seeds using a petri dish assay with 3-day-old cultures on LBA and a sand-cornmeal cup test amended with inoculum from 7-day-old cultures on LBA (1). After 1 week in the petri dish assay, the seeds failed to germinate completely and were covered with white, fungal-like, aerial mycelia and the pathogen was recovered from brown discolored radicle roots. In the cup assay, 2-week-old seedlings developed the same symptoms observed in the field; the pathogen was also isolated from brown discolored roots. In both assays, no symptoms developed in the noninoculated controls. Both pathogenicity tests were repeated two times. Genomic DNA was extracted from mycelia of two isolates and the internal transcribed spacer (ITS) region was amplified and sequenced using ITS6/ITS4 primers (2). Both isolates had identical ITS sequences (GenBank Accession No. GQ853880). A BLAST search of the NCBI database showed 100% homology with the sequence of the haplotype isolate of Phytophthora sansomeana (Accession No. EU925375). P. sansomeana is a new species characterized principally by a large oogonial diameter (37 to 45 μm), rapid growth rate (7 to 10 mm/day), and an ITS sequence falling in Cooke's clade 8 (4). Pathogenicity tests, morphological characteristics, and the ITS sequence analysis indicate that P. samsomena is the causal agent of the symptoms observed on corn seedlings. P. sansomeana has been reported as a pathogen of soybean in Indiana, Douglas-fir in Oregon, and weeds in alfalfa fields in New York (4). To our knowledge, this is the first report of P. sansomeana infecting corn in Ohio, albeit other isolates have previously been recovered from soybean in the state. There are four previous reports of Phytophthora spp. affecting corn in the United States and Mexico (3). Crop rotation will have little effect in management of this pathogen since corn and soybean are produced in the same fields continuously throughout the state.

References: (1) K. E. Broders et al. Plant. Dis. 91:727, 2007. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN. 1989. (4) E. M. Hansen et al. Mycologia 101:129, 2009.
First Report of the Peach Brown Rot Fungus Monilinia fructicola Resistant to Demethylation Inhibitor Fungicides in New Jersey
Authors: Burnett A. , Lalancette N. , McFarland K.
Published: December 2009

Reduced sensitivity and resistance of Monilinia fructicola to demethylation inhibitors (DMIs; fungicide group 3) have been previously found in stone fruit orchards in Georgia, South Carolina, Ohio, and New York (2). Resistance development is a major concern because of the importance of DMIs for brown rot management. Eleven single-spore isolates, originally collected during 2006 from separate commercial peach (Prunus persica) orchards in southern New Jersey, were removed from cold storage (5°C) in early 2008 and examined in vitro for resistance to the DMI propiconazole (Orbit 3.6EC; Syngenta Crop Protection, Inc., Greensboro, NC). After 19 months at 5°C, isolate 7 was inhibited 53.4% in growth on potato dextrose agar (PDA) amended at the discretionary dose of 0.3 μg/ml propiconazole; inhibition of the remaining isolates ranged from 81.4 to 100%. Inhibition values were based on two replications of eight colonies per isolate performed after incubation at 25°C for 4 days. Because of the previously reported relationship between duration of cold storage and propiconazole sensitivity, isolate 7 was tentatively deemed resistant (1). To confirm the in vitro results, isolates were grown at 25°C for 7 days on cellophane over PDA. Genomic DNA was isolated from mycelium with the DNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA). PCR with primers INS65-F and INS65-R was conducted on a GeneAmp thermal cycler (Applied Biosystems, Inc., Foster City, CA) as described previously to amplify a 65-bp region named ‘Mona’ associated with DMI resistance (2). PCR products were separated via electrophoresis on 0.8% agarose gel. The primers amplified a 376-bp fragment from isolate 7 and a 311-bp fragment from all other isolates, thus indicating the presence of Mona in isolate 7. Restriction fragment length polymorphism analysis using the BsrBI enzyme, specific to a single restriction site within Mona, was conducted on the amplified fragments from all isolates. Electrophoresis results showed digestion of the 376-bp fragment from isolate 7 into 140-bp and 236-bp fragments, thereby confirming the presence of Mona; none of the 311-bp fragments from the remaining isolates were cut by BsrBI. Although economic loss from brown rot has not been reported in New Jersey, these results show that propiconazole-resistant strains have been detected since 2006 and it is most likely that resistant strains of the pathogen are still present in commercial peach orchards. To combat this risk, current brown rot control recommendations are incorporating quinone outside inhibitors (QoIs; fungicide group 11) and carboxamides (fungicide group 7) into control programs as a resistance management strategy. More extensive sampling is planned to ascertain the prevalence and location of resistant strains.

References: (1) K. D. Cox et al. Phytopathology 97:448, 2007. (2) C.-X. Luo et al. Plant Dis. 92:1099, 2008.
First Report of Tobacco ringspot virus in Joe-Pye Weed (Eupatorium purpureum) in Mississippi
Authors: Sabanadzovic S. , Ingram D. M. , Lawrence A. M.
Published: December 2009

Eupatorium purpureum L. (joe-pye weed, sweet joe-pye weed, and sweetscented joe-pye weed) is a wildflower perennial plant native to the eastern United States. In May 2006, virus-like symptoms including systemic chlorosis, mottling, and downward rolling of leaf blades were observed in a joe-pye weed plant located on the Mississippi State University campus. Young symptomatic leaves were ground in 0.1 M phosphate buffer, pH 7.2, and the slurry was inoculated on leaves of several herbaceous hosts grown in a greenhouse. Systemic symptoms were observed 1 to 2 weeks postinoculation in Cucumis sativus (chlorotic spots followed by systemic ringspot and leaf deformation), Chenopodium quinoa (necrotic lesions/leaf deformation), Nicotiana benthamiana (mosaic/line patterns), and N. rustica (necrotic ring spots). Electron microscopy of partially purified preparations from infected joe-pye weed and cucumber plants revealed the presence of intact and empty isometric viral particles of approximately 30 nm in diameter resembling nepoviruses or comoviruses. The original joe-pye weed plant and artificially infected herbaceous plants were tested by ELISA (Agdia Inc., Elkhart, IN) for several nepoviruses/comoviruses and found to be positive for Tobacco ringspot virus (TRSV; genus Nepovirus, family Comoviridae). Total RNA extracted from the original virus source plant was reverse transcribed using oligodT primer and submitted to PCR with the primer set TRS-F (5′TATCCCTATGTGCTTGAGAG3′) and TRS-R (5′CATAGACCACCAGAGTCACA3′) designed from the published sequences in GenBank of the RNA 1 of Tobacco ringspot virus. A specific 766-bp PCR product was cloned and sequenced. Sequence analysis showed that the virus from the joe-pye weed shared 94% nucleotide identity (98% at amino acid level) with a “bud blight” isolate of TRSV (Accession No U50869) (2) in the sequenced genome portion and slightly lower (90 to 92%) with other sequenced isolates of the same virus, thus further confirming the identity of the virus. In 2008 and 2009, TRSV was detected in an additional 16 symptomatic specimens of the same host collected from six distinct locations in Mississippi. Our results show that E. purpureum is a new host for TRSV. Considering that the related plant species E. capillifolium (small dogfennel) was already reported as a host of TRSV in North Carolina (1), this suggests that these two common plants may represent additional reservoirs of this virus in the region.

References: (1) M. C. Rush and G. V. Gooding. Phytopathology 60:1756, 1970, (2) P. A. Zalloua et al. Virology 219:1, 1996.
First Report of Myrothecium Leaf Spot of Common Bean in China Caused by Myrothecium roridum
Authors: Zhao Y.-J. , Li B.-J. , Shi Y.-X. , Xie X.-W.
Published: December 2009

Common bean (Phaseolus vulgaris L.) is an economically important crop in China. In June 2008, a new foliar disease was observed on beans in Shunyi District, Beijing, China. The disease occurred in approximately 15% of the plants in a commercial field. Leaf spots were circular to irregular, reddish brown, zonate, and 8 to 20 mm in diameter. Black sporodochia with white tuffs were present on older lesions and black spore masses were present in moist weather. Ten isolates recovered from lesions produced white, floccose colonies and spore masses after 4 days on potato dextrose agar. The rod-shaped, hyaline conidia had rounded ends and averaged 6.8 × 2.5 μm. All characteristics were consistent with the description of Myrothecium roridum Tode ex Fr. (1). The internal transcribed spacer regions of one isolate were sequenced and deposited in GenBank (Accession No. GQ 381291). Sequences of the isolate from bean in China were 98% similar to sequences of M. roridum in GenBank. To determine pathogenicity, 30 healthy seedlings of common bean were inoculated by spraying a 1 × 105 conidia ml–1 suspension of M. roridum onto the foliage. Ten seedlings were sprayed with sterile water and served as controls. Plants were kept in a humid chamber at 27°C overnight and then placed in a growth chamber. After 6 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Koch's postulates. There is one report of M. roridum on soybean in Korea (2). To our knowledge, this is the first report of Myrothecium leaf spot on common bean in China.

References: (1) M. Fitton et al. CMI Mycol. Pap. No. 253, 1970. (2) K. J. Yum et al. Plant Pathol. J. 6:313, 1990.
First Report of Fruit Rot Disease of Mango Caused by Botryosphaeria dothidea and Neofusicoccum mangiferae in Taiwan
Authors: Ni H. F. , Liou R. F. , Hung T. H. , Chen R. S. , Yang H. R.
Published: December 2009

Mango (Mangifera indica L.) is an economically important fruit crop in the tropical and subtropical areas of the world. In southern Taiwan, mango is grown on 18,000 ha of hilly land mainly located in Tainan, Kaohsiung, and Pingtung. Tons (180,000) of mango with a value of NT$6.6 billion (US$206 million) are produced annually. In 2008, mango fruit rot disease was observed 1 week after harvest on 30 to 72% of stored mangoes collected from seven orchards in southern Taiwan. The initial symptom was a small, brown lesion and rot symptoms advanced progressively. Two predominant fungi were isolated from the margin of lesions on acidified potato dextrose agar (PDA with lactic acid, pH 3.8). Isolates of each fungal type were transferred to 2% water agar containing sterilized pine needles and exposed to near UV light to induce sporulation. For the first fungus, conidia obtained from pycnidia were ovate, one-celled, and hyaline, with an average length and width of 12.93 ± 0.93 × 6.98 ± 0.40 μm and an average length/width ratio of 1.85. To confirm the identity of the fungus, PCR amplification by universal primers, ITS1/ITS4, and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421486). It showed a sequence identity of 100% with Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A. J. L. Phillips) (GenBank Accession No. AY615185). For the second fungus, conidia obtained from pycnidia were fusiform, one-celled, and hyaline, with an average length and width of 22.87 ± 1.32 × 6.42 ± 0.46 μm and a length/width ratio of 3.53. The ITS sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421485). It showed a sequence identity of 100% with Botryosphaeria dothidea (Moug.: Fr.) Ces & De Not.) (GenBank Accession No. AY 786321). To test pathogenicity, four mango fruits were wounded with a sterile needle, inoculated with mycelium agar plugs (0.5 mm in diameter) excised from separate monoconidial cultures, and incubated in a plastic box with a 100% relative humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites of each fungus 2 days postinoculation. In control experiments, sterile agar plugs were placed on the wounded mango fruits. These fruits remained completely free from symptoms throughout the experiment. The pathogen was reisolated from the lesions of inoculated fruits and identified as N. mangiferae and B. dothidea, thus fulfilling Koch's postulates. N. mangiferae and B. dothidea have been reported on mango trees in Australia and South Africa (1). To our knowledge, this is the first report of these fungi causing fruit rot of mango in Taiwan.

References: (1) B. Slippers et al. Mycologia 97:99, 2005.
First Report of Grapevine leafroll-associated virus 4 and 5 in Grapevines in China
Authors: Pei G.-Q. , Dong Y.-F. , Zhang Z.-P. , Fan X.-D.
Published: December 2009

Grapevine leafroll disease (GLD) is one of the most important diseases of grapevines worldwide. Nine serologically distinct viruses in the Closteroviridae family are associated with GLD. Previous studies reported that Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, and -7 were present in grapevines in China with GLRaV-1 and -3 the predominant viruses associated with GLD (1). To confirm if GLRaV-4 and -5 were also present in China, 36 dormant canes from individual vines of 29 cultivars that showed GLD leaf symptoms during the growing season were collected from the germplasm collection plot of the Research Institute of Pomology, Chinese Academy of Agricultural Sciences. Total RNA extracted by a silica capture protocol (2) from phloem-enriched bark of 36 samples was tested separately for GLRaV-4 and -5 by reverse transcription (RT)-PCR using virus-specific primers. Primers LR4F (5′-ACATTCTCCACCTTGTGCTTTT-3′) and LR4R (5′-CATACAAGCGAGTGCAATTAC-3′) (4) were used to amplify a 321-bp fragment corresponding to a partial region of the HSP70 gene from GLRaV-4. One sample from cv. Autumn Royal was infected by GLRaV-4. The amplicon was cloned and a single clone was sequenced (GenBank Accession No. GQ246624) that showed 99% nucleotide identity with a corresponding region of a GLRaV-4 isolate from the United States (Accession No. AF039553). Since antiserum specific to GLRaV-4 was unavailable, a second pair of primers, LR4CP-F (5′-GGTGTCCAGCGCTTCCAA-3′) and LR4CP-R (5′-GCCAGAGAAGCATCGTAA-3′), was designed on the basis of the sequence of GLRaV-4 from Chile (Accession No. EU746620) that amplified a 300-bp fragment specific to the coat protein gene of GLRaV-4. The amplicon was cloned and a single sequence (Accession No. GQ479041) was compared with a corresponding nucleotide sequence of GLRaV-4 from Chile (Accession No. EU746621) showing 99% identity. A sample from cv. Malaga Rose was positive when tested by ELISA with antibodies specific to GLRaV-5 (Neogen Europe, Ltd. Scotland, UK) and this was confirmed by amplification of a 690-bp fragment corresponding to the GLRaV-5 coat protein gene using virus-specific primers LR5F (5′-CCCGTGATACAAGGTAGGACA-3′) and LR5R (5′-CAGACTTCACCTCCTGTTAC-3′) (3). The amplicon was cloned and a single clone was sequenced (Accession No. GQ246625) that showed 95% nucleotide identity with the CP gene sequence of GLRaV-5 from Argentina (Accession No. EU815935). To our knowledge, this is the first report of GLRaV-4 and -5 in grapevines in China. Confirmation of these viruses in China is very important for producing virus-free plants and this information also will be helpful in developing a multiplex RT-PCR assay to simultaneously detect multiple GLRaVs and helpful with studies on the molecular variability of these viruses.

References: (1) Y. Dong et al. China Fruits 6:9, 2005. (2) X. Foissac et al. Acta Hortic. 550, 37, 2001. (3) X. Good and J. Monis. Phytopathology 91:274, 2001. (4) F. Osman et al. J. Virol. Methods 141:22, 2007.
First Report of Tobacco rattle virus Associated with Corky Ringspot in Potatoes Grown in North Dakota
Authors: David N. , Mallik I. , Gudmestad N. C.
Published: December 2009

Tobacco rattle virus (TRV) belongs to the genus Tobravirus and causes a stem mottle of potato (Solanum tuberosum) foliage and necrotic arcs and rings in tubers referred to as corky ringspot. This virus is generally transmitted by a number of species of stubby-root nematode. The virus is widespread and has been reported in California, Colorado, Florida, Idaho, Michigan, Oregon, Washington, Minnesota, and Wisconsin (2). In the spring of 2009, we received potato tubers of cv. Russet Burbank with internal necrotic arcs very similar to those caused by TRV from potato storages located in Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions of two tubers from each location using the Total RNA Isolation kit (Promega Corp., Madison WI). These extracts were tested for TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 (Primer A) and identical to nucleotides 6113 to 6132 (Primer B) within the 3′ terminus of TRV-SYM RNA-1 (GenBank Accession No. X06172) (3). The expected 463-bp amplicons from two separate tuber samples from each county were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. The sequences obtained from the four clones at both locations were found to be identical to each other and were 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession Nos. EU315226.1 and AF055912.1, respectively). Since sequences from all four clones were identical, only one of the sequences was submitted to Genbank (Accession No. GQ223114) and thus represents a consensus sequence. The extracts also tested positive in RT-PCR with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8-kbp amplicon (1). No evidence was found by RT-PCR for several other viruses that cause tuber necrosis in potato (Potato mop top virus, Tomato spotted wilt virus, Alfalfa mosaic virus, and tuber necrosis strains of Potato virus Y). The virus was mechanically transmitted by inoculating sap from symptomatic tubers from both counties to tobacco cv. Samsun NN, which showed typical bright yellow patches and spots on leaves 2 weeks postinoculation. TRV was confirmed in tobacco by RT-PCR from total RNA extracted from tobacco leaves with both sets of the aforementioned primers. To our knowledge, this is the first report of TRV in North Dakota and the first report of corky ringspot disease of potato in this state.

References: (1) J. M. Crosslin et al. Virus Res. 96:99, 2003. (2) N. C. Gudmestad et al. Plant Dis. 92:1254, 2008. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992.
First Report of Southern Blight on Silverbush (Convolvulus cneorum) Caused by Sclerotium rolfsii in Italy
Authors: Polizzi G. , Aiello D. , Guarnaccia V. , Parlavecchio G. , Vitale A.
Published: December 2009

The genus Convolvulus has more than 200 species that are encountered in temperate to tropical climates all around the world. Convolvulus cneorum L., also known as silverbush, is a perennial shrub native to southern Europe (Sicily and Croatia) with dense, silver foliage and masses of large, circular, white flowers. During July of 2009, a widespread blight was observed on approximately 10% of 12,000 4-month-old potted silverbush plants. The plants were obtained from cuttings and produced by a commercial nursery in eastern Sicily, Italy. Symptomatic plants initially had sunken, tan lesions at the ground level that developed into typical southern blight. Circular and crescent-shaped patches were observed on the masses of weeds on the surface of the containers where silverbush were grown. At the soil line, white mycelia and small (1 to 2 mm in diameter), brown, spherical sclerotia with internally differentiated rind, cortex, and medulla characteristic of Sclerotium rolfsii Sacc. were observed. Crown and stem lesions were surface disinfested (1% NaOCl) for 1 min, rinsed in sterile water, and placed on potato dextrose agar. Isolation consistently yielded colonies of Sclerotium rolfsii (teleomorph Athelia rolfsii (Curzi) Tu & Kimbrough) with typical sclerotia produced within 6 to 7 days (2). Pathogenicity tests were performed on 20 plants by placing 10 sclerotia obtained from 10-days-old cultures in the soil below the crown portion on each of 5-month-old healthy cuttings of silverbush. The same number of plants served as noninoculated controls. All plants were maintained in a growth chamber at 25 ± 1°C and enclosed for 7 days in polyethylene bags. Plants were then moved to a greenhouse where temperatures ranged from 24 to 28°C. Symptoms of southern blight developed after 7 to 20 days on all inoculated plants. Control plants remained symptomless. S. rolfsii was reisolated from symptomatic plants. S. rolfsii was reported for the first time in Sicily in 2004 in an ornamental nursery (1). To our knowledge, this is the first report in the world of S. rolfsii on silverbush and it is the first outbreak of southern blight on Convolvulus species. The high susceptibility of silverbush to the pathogen could be a limiting factor for the cultivation in nursery of this indigenous wildflower plant.

References: (1) G. Polizzi et al. Plant Dis. 88:310, 2004. (2) Z. K. Punja and A. Damiani. Mycologia 88:694, 1996.
First Report of Powdery Mildew Caused by Leveillula taurica on Giant Blazing Star (Mentzelia laevicaulis)
Authors: Sampangi R. K. , Glawe D. A. , Barlow T. , Mohan S. K.
Published: December 2009

Mentzelia laevicaulis (Dougl. ex Hook.) Torr. & Gray (Loasaceae; common names are giant blazing star and smoothstem blazing star) is widely distributed throughout western North America in sites ranging from lowland deserts to mountainous areas in Canada and the United States. During a plant disease survey in June 2007 in the Owyhee Mountains, Canyon County, Idaho, leaves of M. laevicaulis displaying whitish, mycelial growth were collected from plants growing on stream banks and gravelly road embankments. Diseased leaves exhibited localized, chlorotic discolorations associated with signs of a powdery mildew. Of approximately 20 plants at the site, 50% were infected. White mycelia and conidia were more abundant on the adaxial leaf surfaces than on the abaxial surfaces. Severely diseased leaves were buckled and slightly twisted. By August and through September, sporulation was greatest on mature plants, and lowermost leaves were completely covered with flocculose, dense, white mycelia. Dimorphic conidia were lanceolate or cylindrical and measured (44-) 46 to 67 (-71) × (14-) 14.5 to 20 (-21) μm. DNA was extracted and PCR was used to amplify the internal transcribed spacer (ITS) region as described previously (2), except that primers ITS 5 and P3 (4) were used. The resulting 633-bp sequence (GenBank Accession No. GQ860947) included a 616-bp region identical to a sequence reported previously for Leveillula taurica (Lév.) Arnaud from eastern Washington (GenBank No. AY912077), as well as ITS regions from L. taurica previously reported from Iran (GenBank No. AB048350) (2) and Australia (GenBank No. AF 073351) (2). Based on the ITS sequence, the present fungus grouped within Khodaparast et al. (3) Clade 1 that included L. taurica strains they distinguished from other, superficially similar species of Leveillula. On the basis of morphological and sequence data, the fungus was determined to be L. taurica (1,3). A voucher specimen was deposited in the Mycology Herbarium (WSP) at Washington State University. To our knowledge, this is the first report of a named powdery mildew species from a member of the Loasaceae. The only previous report of a powdery mildew on a loasaceous host was an undetermined Oidium sp. on a species of Mentzelia (1). The discovery of L. taurica on a previously unknown native host species is further evidence that this introduced pathogen is becoming endemic to the Pacific Northwest. Native host species, such as M. laevicaulis, may play a role in the epidemiology of powdery mildew diseases caused by L. taurica on crop and ornamental species in the Pacific Northwest.

References: (1) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (2) D. A. Glawe et al. Mycol. Prog. 4:291, 2005. (3) S. A. Khodaparast et al. Mycol. Res. 105:909, 2001. (4) S. Takamatsu and Y. Kano. Mycoscience 42:135, 2001.
First Report of Kidney Bean Little Leaf Disease Associated with 16SrII Group Phytoplasma in China
Authors: Dong J. H. , Zhang L. , McBeath J. H. , Zhang Z. K.
Published: December 2009

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 Blight of Common Bean Caused by Phytophthora capsici in Connecticut
Authors: LaMondia J. A. , Li D. W. , Vossbrinck C. R.
Published: December 2009

Phytophthora capsici Leonion was first identified on pepper (Capsicum annuum L.) and is widespread on solanaceous and cucurbitaceous crops. It was first documented on Phaseolus lunatus L. in Delaware in 2002 (1), followed by reports on snap beans (Phaseolus vulgaris L.) in Michigan in 2003 (2), and on Long Island, NY in 2008 ( http://vegetablemdonline.ppath.cornell.edu/ NewsArticles/Bean_phytoJune09.html ). In 2009, we observed snap and wax beans in commercial production with water-soaked lesions on foliage, stems, and pods. Twelve to sixteen hectares were affected in the flood plain of the Connecticut River in central Connecticut. Weather conditions had been warm and very wet. Lesions displayed white mycelia and sporangia. P. capsici was isolated from surface-sterilized tissue on potato dextrose agar (PDA) and malt extract agar. Hyphal tips were subcultured onto V8 media for further analysis. To confirm Koch's postulates, two isolates were tested for pathogenicity against bean (cv. Valentino) and pepper (cv. Cayenne) by placing colonized PDA plugs or PDA alone next to the crown or in stem branches. Symptoms similar to those observed in the field on bean and pepper developed on inoculated plants and the pathogen was reisolated. Controls did not develop disease. Sporangia of P. capsici growing on V8 medium were ellipsoid, ovoid, pyriform, but occasionally irregular, papillate, and 54.0 ± 5.7 × 31.1 ± 4.7 μm (n = 31) with a length/width (L/W) ratio of 1.8 ± 0.3. The papillae were 5.4 ± 0.9 μm (n = 31) and the pedicels were 24.5 ± 12.6 × 3.0 ± 1.0 μm. Sporangia collected from bean plants were smaller with longer pedicels; the sporangia were 44.9 ± 9.1 × 26.0 ± 2.8 μm with a L/W ratio of 1.7 ± 0.2; papillae were 4.6 ± 1.0 μm; and the pedicels were 49 ± 20.0 × 2.8 ± 0.9 μm (n = 20). To confirm the identity of our isolate genetically, DNA was extracted from one P. capsici isolate and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified and sequenced (GenBank Accession No. GU011684). The ITS sequence was identical to sequences of P. capsisci in GenBank and confirmed our identification of this new isolate as P. capsici. To our knowledge, this is the first report of P. capsici infecting Phaseolus vulgaris in Connecticut and New England.

References: (1) C. R. Davidson et al. Plant Dis. 85:886, 2002. (2) A. J. Gevens et al. Plant Dis. 92:201, 2008.
Sustained oscillations in an evolutionary epidemiological model of influenza A drift
Authors: Magal Pierre , Ruan Shigui
Published: December 2009

Understanding the seasonal/periodic reoccurrence of influenza will be very helpful in designing successful vaccine programmes and introducing public-health interventions. However, the reasons for seasonal/periodic influenza epidemics are still not clear, even though various explanations have been proposed. In this paper, we study an age-structured type evolutionary epidemiological model of influenza A drift, in which the susceptible class is continually replenished because the pathogen changes genetically and immunologically from one epidemic to the next, causing previously immune hosts to become susceptible. Applying our recently established centre manifold theory for semi-linear equations with non-dense domain, we show that Hopf bifurcation occurs in the model. This demonstrates that the age-structured type evolutionary epidemiological model of influenza A drift has an intrinsic tendency to oscillate owing to the evolutionary and/or immunological changes of the influenza viruses.
Pre-clinical and behavioural toxicity profile of PAMAM dendrimers in mice
Authors: Chauhan Abhay Singh , Jain Narender Kumar , Diwan Prakash Vamanrao
Published: December 2009

A toxicity study of different polyamidoamine (PAMAM) dendrimers in Swiss albino mice was performed with a clinical overview. In the current study, mice were treated with various G4 dendrimers (NH2and OH surface groups) at different dosage levels (low, medium and high) via the intraperitoneal route for 15 consecutive days, followed by a 15 day recovery period. The toxicity profile was investigated in mice for their general behaviour, feed intake, body weight, carbohydrate, lipid and protein metabolism, haematological parameters, histopathology and cell viability. No significant differences were observed in feed intake, body weight and organ weight among the various dendrimer-fed groups and control. In general, dendrimers have no significant effect on the carbohydrate and lipid metabolism. However, declined glucose levels in the high-NH2dose group indicates the interference of this dosage level with glucose metabolism. Although all the test groups showed activity in the normal range, the high-NH2group showed comparatively higher alanine aminotransferase and aspartate aminotransferase activity. Minor, but insignificant (p> 0.05), differences were observed in the red blood cells, haematocrit value and haemoglobin of the treated group when compared to the control group in a dose-dependent manner, but these recovered during the recovery period. There was no effect on other haematological parameters. Histopathological evaluation of dendrimer-treated groups did not reveal any abnormalities in the low- and medium-dose groups, but at a high dose level, toxicity was observed in the liver and kidney. However, after the recovery period, toxicity in the high dose level was not found. A cell viability study of cationic dendrimers has shown reduced cytotoxicity after the complexation with a guest molecule such as indomethacin. This study showed that dendrimers are not toxic, in general. The high dose of G4-NH2displayed some toxicity; however, that too was recovered after some time.
Evaluation of recombinant Brachyspira pilosicoli oligopeptide-binding proteins as vaccine candidates in a mouse model of intestinal spirochaetosis
Authors: Movahedi Abdolreza , Hampson David J.
Published: December 2009

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of humans, and various species of animals and birds, in which it may induce a mild colitis and diarrhoea. The aim of the current study was to evaluate the use of putative oligopeptide-binding proteins of B. pilosicoli as vaccine components. A partial genome sequence of B. pilosicoli porcine strain 95/1000 was subjected to bioinformatics analysis, and six genes predicted to encode oligopeptide-binding proteins were selected. Following a PCR-based distribution study of the genes across different strains of the spirochaete, they were amplified from B. pilosicoli human strain WesB and cloned in Escherichia coli. The recombinant histidine-tagged proteins were purified and subjected to in vitro and in vivo immunogenicity analysis. Recombinant products (P-1 and P-3) from two genes that were immunogenic and recognized by sera from pigs that had recovered from B. pilosicoli infections were tested in a mouse model of intestinal spirochaetosis. For each recombinant protein, groups of 12 C3H/HeJ mice were vaccinated subcutaneously with 100 μg protein emulsified in Freund's incomplete adjuvant, twice with a 2 week interval. Two weeks later the vaccinated and non-vaccinated control animals were challenged orally with B. pilosicoli strain WesB. Both proteins induced systemic and local colonic IgG antibody responses, and, following experimental infection, the cumulative number of colonization days was significantly (P<0.001) less in both groups of vaccinated mice compared to the control mice. There were significantly (P=0.012) fewer mice colonized in the group vaccinated with P-1 than in the non-vaccinated control group. The results suggest that oligopeptide-binding proteins may have potential for use as components of vaccines for B. pilosicoli.
Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ionization time of flight mass spectrometry analysis
Authors: Alispahic Merima , Hummel Karin , Jandreski-Cvetkovic Delfina , Nöbauer Katharina , Razzazi-Fazeli Ebrahim , Hess Michael , Hess Claudia
Published: December 2009

Rapid and reliable identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, has become increasingly important, since many of them are now recognized as human and/or animal pathogens. Matrix-associated laser desorption/ionization–time of flight (MALDI-TOF) MS has been shown to be a rapid and sensitive method for characterization of micro-organisms. In this study, we therefore established a reference database of selected Arcobacter, Helicobacter and Campylobacter species for MALDI-TOF MS identification. Besides the species with significance as food-borne pathogens – Arcobacter butzleri, Helicobacter pullorum, Campylobacter jejuni and Campylobacter coli – several other members of these genera were included in the reference library to determine the species specificity of the designed MALDI Biotyper reference database library. Strains that made up the reference database library were grown on Columbia agar, and yielded reproducible and unique mass spectra profiles, which were compared with the Bruker Biotyper database, version 2. The database was used to identify 144 clinical isolates using whole spectral profiles. Furthermore, reproducibility of MALDI-TOF MS results was evaluated with respect to age and/or storage of bacteria and different growth media. It was found that correct identification could be obtained even if the bacteria were stored at room temperature or at 4 °C up to 9 days before being tested. In addition, bacteria were correctly identified when grown on Campylosel agar; however, they were not when grown on modified charcoal cefoperazone deoxycholate agar. These results indicate that MALDI-TOF MS fingerprinting is a fast and reliable method for the identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, with applications in clinical diagnostics.
Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of ‘spoligotyping’ with new spacers and a microbead-based hybridization assay
Authors: Zhang Jian , Abadia Edgar , Refregier Guislaine , Tafaj Silva , Boschiroli Maria Laura , Guillard Bertrand , Andremont Antoine , Ruimy Raymond , Sola Christophe
Published: December 2009

The aims of the present study were to implement a microbead-based ‘spoligotyping’ technique and to evaluate improvements by the addition of a panel of 25 extra spacers that we expected to provide an increased resolution on principal genetic group 1 (PGG 1) strains. We confirmed the high sensitivity and reproducibility of the classical technique using the 43 spacer panel and we obtained perfect agreement between the membrane-based and the microbead-based techniques. We further demonstrated an increase in the discriminative power of an extended 68 spacer format for differentiation of PGG 1 clinical isolates, in particular for the East African–Indian clade. Finally, we define a limited yet highly informative reduced 10 spacer panel set which could offer a more cost-effective option for implementation in resource-limited countries and that could decrease the need for additional VNTR (variable number of tandem repeats) genotyping work in molecular epidemiological studies. We also present an economic analysis comparing membrane-based and microbead-based techniques.
Phosphoribosylpyrophosphate synthetase (PrsA) variants alter cellular pools of ribose 5-phosphate and influence thiamine synthesis in Salmonella enterica
Authors: Koenigsknecht Mark J. , Fenlon Luke A. , Downs Diana M.
Published: December 2009

Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of thepurFgene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present inSalmonella enterica. Here, we describe mutant alleles of the essentialprsAgene, which encodes PRPP synthetase (E.C.2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.