Plant Dis Research Articles

First Report of Soybean Pod and Stem Blight Caused by Diaporthe phaseolorum var. sojae in Taiwan.

Genera, Diaporthe and Phomopsis, from an important pathogenic complex of soybean (Glycine max) throughout the world, cause reductions in plant stands, yield, and seed health and quality (1). In a study of the Diaporthe/Phomopsis complex in Taiwan in March 2008, four stem samples with black fruiting structures in linear rows on senescent soybean were collected from the research fields at AVRDC, Shanhua, Tainan, Taiwan. Unidentified fungal isolates were obtained by surface disinfection of infected stems and plating excised tissues on potato dextrose agar (PDA). Colonies of the isolates showed ropelike white mycelia with yellowish tonalities. Small and scattered black stromata were observed frequently in the medium. Mutic pycnidia were found solitarily or aggregated in conidiomata on PDA plates after 10 days of incubation at 24°C with a 12-h photoperiod with near-UV light. All isolates produced α-conidia that measured 8.78 × 3.32 (7.00 to11.00 × 3.00 to 4.00) μm, and sporadically, β-conidia of 30.58 × 0.85 (26.00 to 33.00 × 0.60 to 1.20) μm. Perithecia were not observed in the collected stem samples or the fungal cultures on PDA. Restriction fragment length polymorphism patterns of the PCR products amplified by ITS4 and ITS5 primers for all isolates were identical to the patterns reported for Diaporthe phaseolorum var. sojae (3). Thus, all isolates were identified as D. phaseolorum var. sojae on the basis of morphologic and genetic characteristics (2,3). Pathogenicity was confirmed through inoculations during the V2 growth stage of soybean seedlings by atomizing conidial suspensions (1 × 107 α-conidia/ml) of each isolate on soybean seedlings as well as by injecting the inoculum into soybean stems separately. Four plants in each of three replications were inoculated for each method and six noninoculated plants served as controls. Plants were incubated in a growth chamber at 25°C and maintaining relative humidity at 100% by a humidifier for 48 h in darkness; thereafter, plants were maintained in the greenhouse at temperatures ranging from 23 to 34°C. Seven days after inoculation, red-brown leaf spots and coalescent lesions were observed on seedlings atomized by inoculum suspensions, as well as brown lesions and black pycnida in linear rows observed on plants inoculated by stem injection. No symptoms were observed on noninoculated plants. The fungal isolates obtained from inoculated soybeans were morphologically identical to those used as inoculum. The pathogenicity test was repeated twice. To our knowledge, this is the first explicit report identifying the causal agent of soybean pod and stem blight in Taiwan. The vouchers of infected specimens are available at AVRDC-The World Vegetable Center. Reference: (1) R. P. Mulrooney. Plant Dis. 70:600, 1988. (2) R. N. Pioli et al. Phytopathology 93:136, 2003. (3) A. W. Zhang et al. Phytopathology 88:1306, 1998.

Tiny Terrors: The Soybean Aphid

known how these other hosts may playa role in the over-wintering of the fungus, and if they will provide a broader geographic base than that currently known for kudzu. Because the introduction of soybean rust is relatively new to the continental United States, much remains unknown about the potential in-season and overwintering distribution of the fungus. It seems likely, as it is with most invasive species, that it may take a decade to fully understand the distribution of the fungus and its capacity to cause economic losses to the U.S.soybean crop. References Cited Harmon, P. F.,M. T.Momol, J. J. Marios, H. Dankers, and C. L.Harmon. 2005. Asian soybean rust caused by Phakopsora pachyrhizi on soybean and kudzu in Florida. Online. Plant Health Progress:doi:10.1094/PHP2005-0613-01-RS. Hartman, G. L., R. A. Hines, C.D. Faulkner, T. N. Lynch, and N. Pataky. 2007. Late season occurrence of soybean rust caused by Phakopsora pachyrhizi on soybean in Illinois. Plant Dis. 91: 466. Killgore, E.,and R.Heu. 1994. First report of soybean rust in Hawaii. Plant Disease 78: 1216. Lynch, T.N., J. J. Marios, D. L.Wright, P. F.Harmon, C.L. Harmon, M. R. Miles, and G. L. Hartman. 2006. First report of soybean rust caused Phakopsora pachyrhizi on Phaseolus species in the United States. Plant Dis. 90: 970. Miles, M.R.,G.L.Hartman, and R.D.Frederick. 2003. Soybean rust: Is the U.S.crop at risk? [Online] http://www.apsnet.org/online/feature/rust Ono, Y.,P. Buritica, and J. F.Hennen. 1992. Delimitation of Phakopsora, Physopella and Cerotelium and their species on Leguminosae. Mycol. Res. 96: 825-850. Schneider, R.W.,C.A Hollier, H. K.Whitman, M.E.Palm, J.M.McKemy, J. R.Hernandez, L.Levy,and R.DeVries-Paterson. 2005. First report of soybean rust caused by Phakopsora pachyrhizi in the continental United States. Plant Dis. 89: 774. Sinclair, J. B., and G.L.Hartman, [Eds.] 1995. Proceedings of the soybean rust workshop, 9-11 Aug.1995, pp. i-vii, 68. National Soybean Research Laboratory, Urbana, IL.

First Report of Crown Gall Caused by Agrobacterium tumefaciens on Euphorbia esula/virgata in Europe.

Hypertrophy and hyperplasia resembling crown galls were found on roots of Euphorbia esula/virgata at a single site in east-central Hungary in 2005. E. esula/virgata, known as leafy spurge in North America, is an invasive species causing substantial economic losses to the value of grazing lands in the Northern Great Plains of the United States and is the target of biological control. E. esula/virgata is widely distributed throughout Eurasia and is found on ditch banks, along roadsides, and in other noncultivated areas in its native range. Large galls on roots resembling crown gall were first noted in 1992 on plants collected for phylogenetic studies from three locations in east-central Hungary. One of these sites was relocated during a 2005 survey and galls were collected from infected plants. Galls were diced and incubated overnight in tubes containing 3 ml of sterile water at room temperature (20 to 25°C). The supernatant was streaked onto plates of potato dextrose agar (PDA), medium 1A, medium 2E, and Roy/Sasser medium. After 7 days, colonies were picked and streaked and subsequently purified on PDA. Of 104 isolates used to inoculate three sunflower plants each (by puncturing roots just below the soil line with a sterile dissecting needle holding a drop of fluid matrix containing bacterial cells), 35 caused galls. Thirty-three isolates were randomly selected from the 104 and used to inoculate three tomato plants each at the soil line. Seventeen caused galls, including two isolates that did not cause galls on sunflower. Finally, none of 20 randomly selected isolates caused galls on kalanchoe plants (Kalanchoe blossfeldiana). Three isolates, which formed the largest galls on sunflower, were used to inoculate five plants of E. esula/virgata growing in a 1:1:1 (peat/sand/Bozeman silt loam) potting mix. The tests were repeated. Galls were visible on inoculated plants within 6 weeks. Diagnostic biochemical tests done prior to and after reisolation indicated that the causal agent was Agrobacterium tumefaciens, which differed from A. rhizogenes in the production of alkali from litmus milk, a positive reaction for the ferric ammonium citrate and 3-ketolactose tests, and negative reactions for tests to detect the production of acid from erythritol and alkali from malonic, l-tartaric, and mucic acid. The three isolates of A. tumefaciens from E. esula/virgata had identical sequences and clustered most closely (99.8 to 99.9% similarity) with five isolates of A. tumefaciens from Tibet and Japan on the basis of cluster analysis using 16S rRNA sequences. Crown gall of E. esula/virgata has also been found in Montana and western North Dakota, and isolates were identified as A. tumefaciens biovars 1 and 2 (1) (the latter is now known as A. rhizogenes). To our knowledge, this the first report of crown gall on E. esula/virgata in Europe. Reference: (1) A. J. Caesar. Plant Dis. 78:796, 1994.

First Report of Alfalfa mosaic virus in Viburnum lucidum in Spain.

Viburnum sp. is an ornamental shrub widely used in private and public gardens. It is common in natural wooded areas in the Mediterranean Region. The genus includes more than 150 species distributed widely in climatically mild and subtropical regions of Asia, Europe, North Africa, and the Americas. In January 2007, yellow leaf spotting in young plants of Viburnun lucidum was observed in two ornamental nurseries in the Mediterranean area of Spain. Symptoms appeared sporadically depending on environmental conditions but normally in cooler conditions. Leaf tissue from 24 asymptomatic and five symptomatic plants was sampled and analyzed by double-antibody sandwich (DAS)-ELISA with specific polyclonal antibodies against Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany) and Alfalfa mosaic virus (AMV) (SEDIAG S.A.S, Longvic, France). All symptomatic plants of V. lucidum were positive for Alfalfa mosaic virus (AMV). The presence of AMV was tested in the 29 samples by one-step reverse transcription (RT)-PCR with the platinum Taq kit (Invitrogen Life Technologies, Barcelona, Spain) using primers derived from a partial fragment of the coat protein gene of AMV (2). The RT-PCR assays produced an expected amplicon of 700 bp in the five symptomatic seropositive samples. No amplification product was observed when healthy plants or a water control were used as a template in the RT-PCR assays. One PCR product was purified (High Pure PCR Product Purification Kit; Roche Diagnostics, Mannheim, Germany) and directly sequenced (GenBank Accession No. EF427449). BLAST analysis showed 96% nucleotide sequence identity to an AMV isolate described from Phlox paniculata in the United States (GenBank Accession No. DQ124429). This virosis has been described as affecting Viburnum tinus L. in France (1). To our knowledge, this is the first report of natural infection of Viburnum lucidum with AMV in Spain, which might have important epidemiological consequences since V. lucidum is a vegetatively propagated ornamental plant.

First Report of Sclerotinia sclerotiorum on African Daisy (Osteospermum sp.) in Italy.

African daisy (Osteospermum sp.) is an ornamental plant grown in the winter as a potted plant in Liguria (northern Italy) and is generally marketed in early-to-late spring in Italy and central and northern Europe. In the winter of 2006, stem lesions, general chlorosis, wilt, and plant death occurred in a greenhouse nursery. Affected plants were characterized by the presence of soft, watery tissues. Necrotic tissues were covered with a white, cottony mycelium. During periods of high humidity, black sclerotia differentiated within the mycelium. To recover the pathogen, diseased stem tissue was surface sterilized for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 100 mg/l of streptomycin sulfate. Sclerotinia sclerotiorum (Lib.) de Bary (2) was consistently isolated from affected tissue. Sclerotia produced on PDA measured 1.3 to 3.1 × 1.5 to 3.5 mm in diameter. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and then sequenced. BLASTn analysis (1) of the 633 bp showed a 100% identity with S. sclerotiorum. The nucleotide sequence was assigned GenBank Accession No. EU 556701. Pathogenicity of two isolates obtained from infected plants was confirmed by inoculating 10 80-day-old plants grown in 14-cm-diameter pots. Inoculum consisted of 1 cm2 of mycelium excised from a 10-day-old PDA culture of each isolate and placed on the soil surface around the base of each plant. Ten plants were inoculated with each isolate and 10 noninoculated plants served as controls. Plants were maintained in a greenhouse under shade at temperatures of 10 to 22°C (average 19°C), in high relative humidity (>90%), and were watered as needed. The trial was conducted twice. All inoculated plants developed leaf yellowing within 12 days of inoculation. White, cottony mycelium and black sclerotia developed on stems and at the base of all inoculated plants, which eventually wilted. Control plants remained symptomless. S. sclerotiorum was reisolated from the stems of inoculated plants. This disease has been reported on an Osteospermum sp. in the United States (3) and Argentina (4). To our knowledge, this is the first report of white mold on an Osteospermum sp. in Italy as well as in Europe. Currently, the economic importance of this disease is limited.

Tomato severe rugose virus: Another Begomovirus Causing Leaf Deformation and Mosaic Symptoms on Potato in Brazil.

Over the past 10 years, Tomato yellow vein streak virus (ToYVSV) has been a major begomovirus in the main solanaceous crop region of Campinas, São Paulo, which includes counties of Sumaré, Monte Mor, Elias Fausto, and Indaiatuba. The top leaves of potato plants (Solanum tuberosum) having deforming mosaic symptoms (dms), which includes a yellow mosaic or mottling on distorted and deformed leaflets, were associated with this geminivirus (4). Recently, a table potato crop (cv. Agata) from Sumaré, with a record of a few or no white flies (Bemisia tabaci), during the winter season of June to September 2006 had 5 to 7% dms, suggestive of seed potato tuber borne virus infection. Double-antibody sandwich (DAS)-ELISA for Potato virus Y (PVY), Potato leaf roll virus (PLRV), Potato virus X (PVX), and Potato virus S (PVS) (SASA kits and protocols, Edinburgh, Scotland) gave negative results for four field collected potato plants showing dms. Bioassays (mechanical transmission from potato leaf extracts in phosphate buffered saline, 1:5 w/v) with test plants of Nicotiana tabaccum cvs. Turkish and TNN, Gomphrena globosa, Chenopodium quinoa, Datura metel, Solanum tuberosum, and a Physalis sp. were all negative. Inoculated D. stramonium developed symptoms resembling ToYVSV infection including vein clearing and mild mottling on new leaves 2 to 3 weeks postinoculation. Using primers PAC1v1978/PAV1c715 for begomovirus detection (3), the predicted PCR amplified fragment of 1,320 bp was obtained from leaf DNA extracted from all four of the dms field potato plants, as well as the inoculated and symptomatic D. stramoniium test plants. Sequence analysis indicated 100% nt identity among the 1.3-kb PCR fragments obtained from potato and D. stramonium infected plants. Sequences of 96 cloned amplicons (pGEM-T Easy Kit; Promega, Madison, WI) from symptomatic plants in the Sumaré potato field were 98 to 99% identical to Tomato severe rugose virus (ToSRV). BLAST analysis of a consensus sequence (Sequencher 3.1; Gene Codes Corporation, Ann Arbor, MI) revealed more than 95 and 99% identity with ToSRV isolates from Uberlandia (Accession No. AY029750) and Goias (Accession No. DQ207749), respectively. The DNA-based phylogenetic dendrogram confirmed the highest similarity with ToSRV and the lowest similarity with ToYVSV (72%), which was located in another cluster. These results indicate that ToSRV was the causal agent producing dms in potato plants from Sumaré. Therefore, similarly to ToYVSV (4), potato dms can be caused by ToSRV. Preliminary tests revealed that ToSRV was transmitted via seed tubers. Thus, it is of concern for seed potato certification in Brazil, especially in the major seed-potato-producing state of Santa Catarina where an outbreak of ToSRV was recently reported in tomato crops (1). Although ToSRV has been identified in other solanaceous crops in Brazil, especially tomato (Lycopersicon esculentum) and sweet pepper (Capsicum annum) (2), to our knowledge, this is the first report of ToSRV in potato in Brazil. Reference: (1) A. T. M. Lima et al. Fitopatol. Bras. 31:224, 2006. (2) D. N. Nozaki et al. Summa Phytopathol. 33:93, 2007. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) J. A. C. Souza-Dias et al. CultivarHF 5(26):22, 2004.

First Report of Rice yellow mottle virus in Rice in The Gambia.

Rice yellow mottle virus (RYMV) of the genus Sobemovirus is a major biotic constraint to rice (Oryza sativa) production in Africa. First reported in Kenya during 1966, RYMV was later found in most countries in Africa where rice is grown (1). In countries in westernmost Africa (The Gambia, Guinea-Bissau, Mauritania, and Senegal), plants with leaf yellowing and mottling symptoms were observed, but RYMV was never isolated. Rice is the staple food in The Gambia. In 2006, four samples were collected from local rice varieties in the Kuntaur Region in the center of The Gambia. Mechanical inoculation with leaf extracts from all samples caused typical yellow mottle symptoms on the susceptible rice varieties BG90-2, Bouaké 189, and IR64. RYMV was detected in the four samples collected by ELISA with polyclonal antisera (2). The 720-nt coat protein gene was amplified for each isolate by reverse-transcriptase-PCR with primers 5'-CAAAGATGGCCAGGAA-3' (sense) and 5'-CTCCCCCACCCATCCCGAGAATT-3' (antisense) (2). The RT-PCR products were directly sequenced (EMBL Accession Nos. AM765810, AM765811, AM765812, and AM765813) and then aligned using ClustalW with a pool of RYMV coat protein sequences from West African isolates (EMBL Accession Nos. AJ279905, AJ279901, AJ885137, AJ885124, and AJ279935). Phylogenetic reconstruction by maximum-likelihood with PAUP indicated that the isolates from The Gambia formed a monophyletic group with over 97% nucleotide identity and are closely related to isolates of other countries in West Africa (Burkina Faso, Côte d'Ivoire, Guinea, Mali, and Sierra-Leone) with 91 to 94% identity. Detection of RYMV in The Gambia indicates that RYMV is present in westernmost Africa, which is referred to as the 'rice belt' of Africa, and shows that RYMV is widely distributed from eastern Africa (Tanzania) to the western part of the continent.

First Report of Anthracnose of Salal Caused by Colletotrichum acutatum in British Columbia

Gaultheria shallon Pursh (salal) is a native, Pacific Northwest forest understory plant produced by numerous commercial nurseries as a garden and landscape ground cover. Seed is sown in soilless media in greenhouse plug flats, and seedlings are transplanted into larger pots at 24 to 36 weeks. In October 2005, approximately 30% of 16-week-old G. shallon Pursh (salal) seedlings in a commercial greenhouse in coastal British Columbia, Canada were observed to be dying back. Black, water-soaked, cankers girdled the stems and petioles resulting in seedling necrosis. Cankers extended into the base of affected leaves, and irregular, black leaf spots with concentric rings were observed. A Colletotrichum sp. was isolated on potato dextrose agar (PDA) + 0.005% streptomycin. In May 2006, 160 20-week-old salal seedlings (two- to five-leaf stage) in four plug cell flats of 40 seedlings each were inoculated with a hand atomizer with a mycelial and spore suspension of approximately 1 × 108 CFU/ml (dilution plating) in 100 mL sterile dH2O; 120 seedlings (four flats of 30 seedlings each) were sprayed with water alone. Flats were covered with black plastic for 48 h and then placed randomly in a shaded greenhouse at 14 to 22°C with overhead watering. The percentage of necrotic seedlings with black, water-soaked cankers and leaf spots in the inoculated flats at 1 to 8 weeks was 3.8, 7.6, 8.2, 16.4, 44.9, 62.0, 79.7, and 81.6, consecutively; versus 2.5% at 8 weeks in the noninoculated flats. Infection of the noninoculated seedlings was presumably due to spore splash from irrigation. Both the original isolate used as inoculum and colonies reisolated on PDA from inoculated, surface-sterilized, symptomatic tissues were identified as Colletotrichum acutatum J.H. Simmonds based on morphology (2) and PCR using a species-specific primer (1). Hyaline, aseptate, fusiform conidia, measuring 8 to 16 × 2.5 to 4 μm, were produced in acervuli without setae on plants and in culture. The teleomorph (Glomerella) stage was not observed. To our knowledge, this is the first report of an anthracnose disease of salal.

Detection of the "Candidatus Liberibacter americanus" in phloem vessels of experimentally infected cataranthus roseus by scanning electron microscopy

Presence of Huanglongbing (HLB) disease in citrus orchards in the state of Sao Paulo has been known since 2004. Its identification was made by molecular techniques which revealed the presence of two forms of the bacteria “Candidatus Liberibacter”. One previously described in Asia as Ca. L. asiaticus and the other a novel type, Ca. L. americanus (Coletta-Fo et al., Plant Dis. 88:1382. 2004; Teixeira et al., Plant Dis. 89:107. 2005). Transmission electron microscopy confirmed the presence of characteristic bacteria in the phloem vessels of affected sweet orange leaves (Tanaka et al., Fitopatol. Bras. 31:99. 2006). Attempts to observe these bacteria by scanning electron microscopy (SEM) to understand their three dimensional morphology were hindered by the extremely low concentration at which they usually occur in citrus plants. On the other hand, Ca. L. americanus was successfully transferred to the ornamental periwinkle (Cataranthus roseus (L.) G. Don.) using dodder (Cuscuta sp.) as previously reported for other Ca. Liberibacter (Garnier & Bove, Proc. Conf. IOCV 12: 212. 1993). Infected plants developed chlorotic symptoms and contained higher concentrations of the bacterium as evaluated by molecular techniques and transmission electron microscopy. For in situ observations of Ca. L. americanus under SEM, midribs from symptomatic leaves and young stems were fixed with aldehyde, infiltrated with glycerol, immersed in liquid nitrogen and fractured with a scalpel, and the pieces post-fixed in 0.1% OsO4 for 24 h, dehydrated in acetone, dried at the critical point, gold coated by sputtering and examined in a LEO 435 VP scanning electron microscope. In the vascular region of the infected leaves and stems, exposed by the fracture of frozen tissues, many phloem vessels contained bacterial cells, interpreted as Ca. L. americanus, in varied numbers. Some vessels were obliterated by aggregated bacterial cells (Fig. 1 A). Bacteria were typically baciliform and elongated, 2-3 μm long with rounded ends, 200-300 nm wide and with a smooth outer surface (Fig. 1 B). These values match those obtained in sectioned bacterial cells. Such bacterial cells were absent in the phloem vessels of healthy, uninoculated periwinkle plants. This report is the first description of Candidatus Liberibacter by scanning electron microscopy. RESUMO Deteccao de Candidatus Liberibacter americanus em vasos de floema de Catharantus roseus infectados experimentalmente, utilizando microscopia eletronica de varredura “Candidatus Liberibacter americanus”, agente causal do Huanglongbing (HLB) foi visualizado pela primeira vez ao microscopio eletronico de varredura infectando vasos do floema de plantas de vinca (Catharantus roseus) experimentalmente infectadas utilizando-se Cuscuta sp. no Brasil.

First Report of Race 8 of Downy Mildew, Caused by Peronospora farinosa f. sp. spinaciae, of Spinach in the United States.

Downy mildew, caused by Peronospora farinosa f. sp. spinaciae, is the most economically important disease of spinach (Spinacia oleracea) in the United States and the European Union. In the United States, 23,000 ha of spinach, with a crop value of approximately $170 million, were grown during 2005 (1; http://www.nass.usda.gov/index.asp ). Additionally, per capita, fresh-market spinach consumption has increased 214% in the past decade (1; http://www.nass.usda.gov/index.asp ). Increased demand for fresh-market spinach has led to changes in spinach production practices such as higher planting densities and year-round production. There are currently 10 described races (races 1 to 10) of P. farinosa f. sp. spinaciae. Race 8 was recovered from the Netherlands in 2004 (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]), but has not been previously identified in the United States. In February 2007, several commercial fresh-market spinach fields in central Arizona were severely affected with downy mildew. Symptoms consisted of bright yellow leaf lesions ranging in size from 1 to 3 cm in diameter that supported dense purple sporulation of the pathogen on the corresponding abaxial leaf surface. Affected fields were primarily planted with spinach cv. Parrot, which is reported to be resistant to races 1 to 7 and 9. As much as 32 ha were affected and disease incidence reached as high as 25 to 30%. An isolate (PAR1) of the pathogen was obtained and used to inoculate a standard set of 10 differential spinach cultivars for race identification as previously described (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]). Briefly, a spore suspension (1 × 105 sporangia per ml) was misted onto test plants; plants were then incubated in a dew chamber (20°C, 100% relative humidity) for 24 h and maintained in a greenhouse. Inoculation tests were conducted at least twice at each of two different locations (Arkansas and California), with each test including two replications of 15 plants per differential cultivar. The selective development of downy mildew on specific differentials indicated that the isolate was race 8 (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]). To our knowledge, this is the first report of race 8 in the United States. Since there are a number of commercial spinach cultivars available with resistance to race 8, the economic impact of this race in the United States is expected to be low if resistant cultivars are grown (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]). Reference: (1) R. N. Acharya and I. Molina. NFAPP Newsl. Second Quarter, 2005.

Iris yellow spot virus in Onion Seed Crops in South Africa.

In December 2006, symptoms typical of iris yellow spot caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) were observed on scapes (seed stalks) in an onion (Allium cepa L.) seed crop in the Klein Karoo of the Western Cape Province, South Africa. Symptoms included diamond-shaped chlorotic or necrotic lesions on the scapes, some of which had 'green-islands' with nested diamond-shaped lesions, as well as indistinct, circular to irregular, chlorotic or necrotic lesions of various sizes. At the time symptoms were observed, approximately 5% of the scapes had lodged as a result of extensive lesions resembling those caused by IYSV. The crop was 2 to 3 weeks from harvest. Symptomatic tissue from two plants (two samples from one plant and four samples from the other plant) was tested for IYSV by reverse-transcriptase (RT)-PCR. Total RNA was extracted from symptomatic scape tissue with the SV Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer's instructions. First strand cDNA was synthesized with the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas Inc., Hanover, MD), followed by PCR amplification with primers IYSV-For (TGG YGG AGA TGY RGA TGT GGT) and IYSV-Rev (ATT YTT GGG TTT AGA AGA CTC ACC), which amplify the nucleocapsid (NP) gene of IYSV. An amplicon of expected size (approximately 750 bp) was observed for each of the symptomatic plants assayed and was sequenced. Comparison of the sequence (GenBank Accession No. EF579801) with GenBank sequences revealed 95% sequence identity with the NP gene of IYSV GenBank Accession No. EF419888, with eight amino acid differences. The known geographic distribution of IYSV in onion bulb or seed crops has increased rapidly in recent years in many areas of the world (1). To our knowledge, this is the first confirmation of IYSV in South Africa. Approximately 6,100 ha of onion bulb crops are grown annually in South Africa in the Western Cape, Kwazulu Natal, Limpopo, and Northern Cape provinces, and 600 ha of onion seed crops are grown primarily in the semi-arid regions of the Western Cape. Examination of an additional 10 onion seed crops in the Klein Karoo during January 2007 revealed the presence of iris yellow spot in three more crops at approximately 5% incidence in each crop. The four symptomatic crops had all been planted as bulb-to-seed crops, using vernalized bulbs produced on the same farm. This suggests that IYSV may have been disseminated into the seed crops on the vernalized bulbs, either as infected bulb tissue or in viruliferous thrips on the bulbs. Reference: (1) D. H. Gent et al. Plant Dis. 90:1468, 2006.

Shoot Blight Caused by Diplodia pinea on Afghan and Austrian Pines in Texas.

Shoot blight was observed on ornamental Afghan (Pinus eldarica) and Austrian pines (P. nigra) at several sites in metropolitan Dallas, TX in the summer of 2005. Shoots were stunted, cankered, often resinous, sometimes curled or crooked at the tips, and bore brown needles that often had been killed before full elongation. Pycnidia on necrotic needles, stems, and cones of each host species yielded conidia characteristic of the fungus Diplodia pinea. Individual conidia and hyphal tip transfers produced pure cultures confirmed as D. pinea using a species-specific PCR assay (1), which allows differentiation from the similar pine shoot blight pathogen D. scrobiculata. Five isolates (three from Afghan pine and two from Austrian pine) were tested for pathogenicity by inoculation of potted 1-year-old Afghan pine seedlings obtained from the Texas Forest Service Nursery. Elongating terminal shoots were wounded by removing a needle pair approximately 2 cm below the shoot apex. A 4-mm-diameter plug cut from an actively growing culture on water agar (WA) was placed fungus side down on the wound. Noncolonized WA plugs were placed on similarly wounded control seedlings. Nonwounded control seedlings also were used. Parafilm was wrapped around the shoots to hold the agar plugs in place and was removed 2 weeks later. Each treatment was applied to four seedlings. Five weeks after inoculation, 9 of the 20 inoculated seedlings (including at least one inoculated with each isolate) exhibited dieback of shoot tips. One wounded control seedling exhibited slight tip dieback, no other nonwounded or wounded control seedlings developed symptoms. Segments of shoots were harvested, surface disinfested, and incubated on WA to determine the presence of the pathogen. The pathogen was reisolated from 11 of the 20 inoculated seedlings but not from any control seedlings. To our knowledge, this is the first report of D. pinea as a cause of shoot blight of Afghan pine and the first substantiated report of the occurrence of D. pinea in Texas. Although widely distributed in much of eastern North America, reports of the presence of D. pinea in the other southern gulf coast states of Alabama, Louisiana, and Mississippi, as well as the western states of Colorado, New Mexico, and Utah, are lacking. Reference: (1) D. R. Smith and G. R. Stanosz. Plant Dis. 90:307, 2006.

Video in science. Protocol videos: the implications for research and society.

The Chinese folktale, Three Blind Men and an Elephant , is more than 2,000 years old. Three blind men approached an elephant and tried to describe it. The first man felt the elephant's ear and decided that it was like a fan. The second man touched the elephant's knee and decided that it was like a tree. The third man held the elephant's trunk and concluded that it was like a snake. They could not agree on what an elephant is really like (Kuo & Kuo, 1976). If each of the three men had felt the whole of the elephant, or at least experienced each of the three parts themselves, they would have gained a better idea of what animal they were trying to describe. Scientists, like the blind men, often have too narrow a focus when thinking about their field of research or particular topics. To obtain a larger, more complete picture—to understand what the ‘elephant’ is—they need as much information as possible. One way of achieving this is to take advantage of new technologies, such as video, and use it to communicate scientific methods, protocols and results. Here, I describe the possible advantages of using video to communicate scientific protocols instead of verbal descriptions, and highlight some further benefits of such an approach, in particular in educational and outreach programmes. Visual information is fundamental to communication. As humans, we have relied on our visual perception of the world to convey ideas, feelings and images ever since our ancestors started painting animals on cave walls thousands of years ago. Drawings, paintings, photography, film, television, analogue and digital video, computer‐generated images and the Internet have all further increased and enhanced the use of images to communicate information—not just in general, but specifically with regard to scientific ideas, results and communication. …

Identification of Tomato yellow leaf curl virus and Tomato mottle virus in Two Counties in Alabama.

During July 2005, approximately 23% of tomato plants (Solanum lycopersicum L. 'Sebring') in a commercial field in St. Clair County, Alabama showed symptoms of stunting, leaf deformation, mottling, and reduced leaf size, which resembled symptoms of Tomato yellow leaf curl virus (TYLCV). A high population of whiteflies (Bemisia tabaci) was observed in this field, and as the season progressed, 100% of the plants became symptomatic. During October 2006, similar symptoms in tomato were observed at low incidences (less than 10%) in a commercial greenhouse in Jefferson County. Two samples from St. Clair County and six from Jefferson County were collected and tested for the presence of a begomovirus by PCR using three pairs of primers, PAR1c496 and PAL1v1978, a degenerate primer pair designed to amplify regions of the begomovirus A component, PBL1v2040 and PCRc154, a degenerate primer pair that amplifies a hypervariable region of the begomovirus B component (3), and C473 and PTYC1v2406, which are specific to TYLCV (1,2). Primer pair PAR1c496 and PAL1v1978 produced two amplicons (1,360 and 1,159 bp) in all samples tested, which suggests the presence of a monopartite and bipartite begomovirus. Primer pair pBL1v2040 and PCRc154 produced a 678-bp amplicon that would be consistent with the presence of a bipartite begomovirus. Primer pair C473 and PTYC1v2406 produced an 850-bp amplicon that would be consistent with the presence of TYLCV. Sequence analysis revealed that the 1,360-bp amplicon had 98% sequence identity to isolates of TYLCV from Cuba (GenBank Accession No. AJ223505), the Dominican Republic (GenBank Accession No. (AF04715), Florida (GenBank Accession Nos. AF260331 and AY530931), Egypt (GenBank Accession No. AY594174), and Almeria (GenBank Accession No. AJ489258). The 1,159-bp amplicon had a 97 to 99% sequence identity to the A component of Tomato mottle virus (ToMoV) Florida (GenBank Accession Nos. L14460, EF028241, and M90495) and Puerto Rico (GenBank Accession No. AY965900). Each of the eight tomato samples were shown to be infected with TYLCV and ToMoV. Symptoms of plants infected with both viruses resembled those of TYLCV because the milder symptoms of ToMoV are masked in the field by the more severe symptoms of TYLCV. To our knowledge, this is the first report of ToMoV and TYLCV in the state of Alabama. Reference: (1) M. Ghanim et al. Virology 240:295, 1998. (2) M. K. Nakhla et al. Phytopathol. Mediterr. 32:163, 1993. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Sawadaea Powdery Mildew of Box Elder (Acer negundo) in Wisconsin

Box elder leaves affected by powdery mildew were collected from a single tree in an urban yard in the fall of 2006 in Madison, WI. As much as half of the area of the leaf blades, when viewed from above or below, was pale green-to-tan and necrotic. Mycelium was not visible to the eye on either the upper or lower leaf surfaces, but very sparse white mycelium was observed on the lower surface of leaves with the aid of a dissecting microscope. Chasmothecia were present singly or in groups of a few on the lower surface of leaves. Morphology of the chasmothecia, including simple and bifid appendages with uncinate to circinate apices, was sufficient to identify the pathogen to the genus Sawadaea (1). Other characters were not sufficiently distinct to make an identification of the species. However, DNA was extracted from the chasmothecia, and analysis of a 542-bp sequence of nuclear rDNA ITS (GenBank Accession No. EF122238) revealed a 100% match with the respective sequence obtained from Genbank for S. bicornis (Accession No. AB193380). A specimen from which these chasmothecia were obtained has been deposited in the U.S. National Fungus Collections (BPI 877328). S. bicornis is a European pathogen of maples, including box elder when grown there (1). Knowledge of the geographic distribution and hosts of Sawadaea powdery mildews in North America is extremely limited. S. bicornis was first reported in North America only recently, occurring in the states of Idaho and Washington on Norway maple (Acer platanoides) (3). The morphologically very similar European powdery mildew pathogen of maples, S. tulasnei, is known only from New York, Ohio, and Montreal, Canada (4), and an unidentified Sawadaea species on box elder has been reported from California (2). References (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart-New York, 1995. (2) S. Hirose et al. Mycol. Res. 109:912, 2005. (3) C. Nischwitz and G. Newcombe. Plant Dis. 87:451, 2003. (4) J. Weiland and G. Stanosz. Plant Dis. 90:830, 2006.

First Report of Cucumber mosaic virus in Viola hederacea in France and Italy.

Viola hederacea Labill. (Australian violet or trailing violet), family Violaceae, is native to eastern Australia and used for its ornamental quality in humid conditions, especially in terrariums. Mosaic and chlorotic ringspots associated with a mild crinkling on leaves of V. hederacea were observed in gardens in southeast France and La Mortola, Italy in 2001 and 2003, respectively. These symptoms were different from the small chlorotic spots reported in the same species in Queensland, Australia, from which a rhabdovirus was isolated (1). In samples collected from both locations, presence of Cucumber mosaic virus (CMV) was suspected on the basis of the symptoms exhibited by a range of inoculated plants (2) and the observation of isometric particles of approximately 30 nm with the electron microscope in sap preparations. This was confirmed by positive reactions with CMV polyclonal antiserum (3) in double-antibody sandwich-ELISA. Because of the viscosity of the V. hederacea extracts, 0.01% (w/v) of pectolyase in phosphate buffer was used to grind the samples and the homogenate was kept at room temperature for 2 h. In double-immunodiffusion analysis, each isolate was shown to belong to the group II of CMV. Tomato strains D and To were used as positive controls for CMV groups I and II, respectively. The virus was transmitted by aphids (Myzus persicae) in a nonpersistent manner from infected Nicotiana tabacum cv. Xanthi-nc plants to healthy Xanthi-nc plants. To determine if CMV was responsible for the symptoms observed, the French and Italian isolates were multiplied in Xanthi-nc tobacco plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 10 plants of V. hederacea. A systemic vein clearing developed on leaves 10 days after inoculation, followed by mosaic and crinkling. CMV was detected in each plant 3 weeks after inoculation. To our knowledge, this is the first report of CMV in V. hederacea in France and Italy.

Tomato rugose mosaic virus in Tomato Crops in São Paulo State, Brazil

Geminiviruses were of limited economic importance to tomato crops until the introduction of biotype B, when an increase in the incidence of begomovirus was reported (Ribeiro ., Arch. Virol. 148:281. 2003). In recent years, the occurrence of geminivirus in tomato causing a severe outbreak has been observed in the Campinas region, SP. In preliminary surveys (ToYVSV) turned out to be the most frequent begomovirus species, affecting about 70% of tomato crops. Occasionally, however, another possible begomovirus, inducing leaf crumple, has been noticed affecting tomato in that region (Colariccio ., Summa Phytopathol. 27:105. 2001). The present work aimed to identify the virus associated with the leaf crumple symptom. Thus, leaf samples from affected ‘Carmen’ tomato plants from Mogi Guacu, SP, showing growth reduction, green and yellow mosaic, besides leaf crumple and curling, were submitted to electron microscopy, mechanical, graft and transmissions, as well as DNA extraction for PCR using universal primers (Rojas ., Plant Dis. 77:340. 1993). Sequencing and phylogenetic analysis were performed to compare this virus with other geminivirus sequences deposited in the GenBank. Typical geminated particles were observed in negatively stained preparations from foliar veins. observation revealed the presence of hexagonally-packed crystalline array or of loose aggregates in the nuclei of phloem-infected parenchyma cells, with partial alteration of nucleoli into a granular structure. These cytopathic effects corresponded to those described for begomoviruses. The virus was transmitted by and graft, but not mechanically, like (ToRMV) from Minas Gerais State. A DNA-A fragment of about 1.2 kbp

First Report of Acidovorax avenae subsp. citrulli Infecting Edible Seed Watermelon (Citrullus lanatus var. lanatus) in China.

Edible seed watermelon, Citrullus lanatus var. lanatus, is a type of watermelon from which only the seeds are consumed. Between 2003 and 2005, a bacterial disease was discovered on this type of watermelon in several regions of Xinjiang Province in western China, with a diseased area of 40,000 ha. The average disease incidence on fruit and seedlings was approximately 30 and 20%, respectively. Symptoms were most noticeable on fruit. Lesions on the fruit rind were first noticeable as small pinpoint water-soaked areas. As the lesions rapidly enlarged, black, starshaped cracks formed in the rind, and the rind along with the flesh around the spots hardened. If no secondary invaders or saprophytes entered through the cracks, the inside flesh often disintegrated into dry stiff cavities. The fruit with necrotic spots became markedly malformed. The pathogen also infected seedlings. First symptoms on the seedling appeared as dark, water-soaked lesions on the underside and edge of the cotyledons. As the cotyledon expanded, the lesions become necrotic and often extended along the length of the midrib and eventually merged into large, black, withered areas. To determine the causal agent of the disease, 54 bacterial strains were isolated from cotyledons, fruits, or seeds of edible seed watermelon. By spray inoculation, these strains were inoculated onto edible seed watermelon cv. Xinzigua1 at the two-true-leaf stage with bacterial concentrations at 107 CFU/ml. All strains were strongly virulent to cv. Xinzigua1, which is planted in large areas in Xinjiang, by forming many water-soaked spots on cotyledons and true leaves. There were no symptoms in the control that was sprayed with sterile deionized water. Through biological and biochemical characteristics, including Gram reaction and tests for catalase, oxidase, and oxygen requirement, all strains were identified as Acidovorax avenae subsp. citrulli. To verify pathogen identification, specific polymerase chain reaction was also carried out. One set of 16S primers, WFB1/WFB2 for A. avenae subsp. citrulli, was used (1). A single unique band of approximately 360 bp was amplified for all strains tested. To our knowledge, this is the first report of A. avenae subsp. citrulli infecting edible seed watermelon. Reference: (1) R. R. Walcott and R. D. Gitaitis. Plant Dis.84:470, 2000.

First Report of Botrytis Gray Mold on Common Calla Lily in Buenos Aires, Argentina.

Common calla lily (Zantedeschia aethiopica (L.) Spreng., family Araceae) is an evergreen herbaceous South African ornamental plant that forms a tuft of fleshy-stalked, glossy, dark green leaves. At bloom during the summertime, large, funnel-shaped, waxy-white spaths that surround a bright yellow spadix form at the end of high stalks. In August 2003, large, irregular brown spots with a 3- to 4-mm yellow halo were observed on leaves of 10 plants growing near Japanese quince shrubs (Chaenomeles lagenaria (Loisel.) Koidz.) in Escobar, Buenos Aires. Debris of Japanese quince petals were attached to the center of the lesions with profuse sporulation of Botrytis cinerea Pers. (1). Pathogen spores were disposed on potato dextrose agar (PDA) and incubated at 22°C. Mycelium was initially whitish and turned gray with age. Black conidiophores bore botryose heads of hyaline, ellipsoid, unicellular conidia, gray in mass, 7.5 to 10.5 μm × 6.8 to 7.5 (average 9.2 to 7 μm). Black, irregular sclerotia formed at random in culture. Inoculum was prepared from 7-day-old cultures on PDA. Six flowering common calla lilies planted in 5-liter plastic pots were inoculated by spraying a suspension of 2.5 × 106 conidia per ml of sterile distilled water. Six healthy plants were sprayed with sterile distilled water. Each plant was covered with a transparent polyethylene bag for 3 days and kept at 21°C under a 12-h photoperiod. After a 12-day incubation period, leaves showed elliptic to irregular brown spots surrounded by yellow halos. Tiny round to irregular brown spots developed on flower spaths that finally blighted. Water-treated plants remained symptomless. Koch's postulates were fulfilled by pathogen reisolation from diseased organs. To our knowledge, this is the first report of B. cinerea on Z. aethiopica in Argentina. Infection efficiency of B. cinerea increases when inoculated petals are positioned on leaves (2), which has epidemiological importance in landscapes with association of plant species that are potential hosts of this pathogen. Reference: (1) M. V. Ellis and J. M. Waller. No. 431 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1974. (2) C. Sirjusingh et al. Plant Dis. 80:154, 1996.

Cowpea mild mottle virus infecting soybean crops in northwestern Argentina

Argentina is one of the leading soybean producing and exporting countries in the world. During the 2004/2005 cropping season, soybean production reached a record �8.� million tons. Although soybean is grown mainly in the central Pampas, it is also cultivated in the northwest of the country, where it plays a significant role in the local economy. Among the biotic factors affecting soybean yields in this region, fungal diseases account for the major economic losses (Wrather et al., Plant Dis. 81:107. 1��7). Nevertheless, diseases of viral etiology have also been recorded. Six different virus species have been detected infecting soybean in Argentina: Soybean mosaic virus (SMV), Alfalfa mosaic virus (AMV), Tobacco streak virus (TSV), Cucumber mosaic virus (CMV), Peanut mottle virus (PMV) and a geminivirus which has not been characterized yet. A survey conducted in Salta Province (NW Argentina) during late summer 2005 revealed the presence of soybean plants with systemic leaf chlorosis, distortion and stunting, which were negative in serological or hybridization tests to the viruses mentioned above. This work reports on the identification of the disease’s causal agent. Leaf dip preparations were made from leaves of symptomatic and asymptomatic (presumably healthy) soybean plants. Samples were ground in phosphate buffer pH 7 + 0.01% (w/v) sodium sulphite, stained with 2% uranyl acetate and examined under a JEOL 1220 electron microscope. Straight filamentous particles of about 6�0-650 nm, which resembled those of carlavirus, were consistently associated with the diseased plants, while no particles were observed in samples from asymptomatic soybeans. On this basis, plants were tested for Cowpea mild mottle virus (CPMMV, genus Carlavirus) by DAS-ELISA. Leaf samples were ground in extraction buffer (PBS pH 6.8 + 0.05% Tween 20 + 2% polyvinyl pyrrolidone) at a 1:5 (w/v) dilution. Specific polyclonal antisera (DSMZ GmbH, Germany) were used. Positive (supplied by DSMZ) and negative (healthy soybean) controls were included on each microtitre plate. After incubation with p-nitrophenyl phosphate at room temperature for 1 h, A405nm values greater than 0.�00 were obtained for the infected samples, compared with less than 0.100 for the healthy controls. CPMMV, a virus serologically unrelated to any other member of the genus, was first reported infecting Vigna unguiculata (L.) Walp. in Ghana (Brunt & Kenten, Ann. Appl. Biol. 74:67. 1�7�). Natural hosts of CPMMV include several species in the family Fabaceae. Its occurrence on soybean has been reported in southeast Asia, India and west Africa (Suryawanshi et al., Indian J. Virol. 5:12�. 1�8�). More recently, a disease causing stem necrosis of soybean in Brazil has also been attributed to CPMMV or a related carlavirus (Almeida et al., Fitopatol. Bras. �0:1�1. 2005). In Argentina, CPMMV has already been found on common bean (Phaseolus vulgaris L.) in Salta Province (RodriguezPardina et al., Australas. Plant Pathol. ��:12�. 2004), where large populations of the whitefly vector Bemisia tabaci Genn. may be responsible for the transmission of the pathogen to other crops. The possible spread of CPMMV to different soybean production regions, as well as its economic impact, still has to be determined.