Disease Notes: March 3, 2005

Viral Etiology of Diseases Detected in Melon in Guatemala. C. Jord, M. I. Font, and P. Martnez-Culebras, Departamento de Ecosistemas Agroforestales, Universidad Politcnica de Valencia, Cno. de Vera s/n, 46022 Valencia, Spain; and J. Tello, Departamento Produccin Vegetal, Universidad de Almera, Caada de San Urbano s/n 04120. Almera, Spain. Plant Dis. 89:338, 2005; published on-line as DOI: 10.1094/PD-89-0338A. Accepted for publication 30 November 2004.

At the beginning of 1999, 30 melon (Cucumis melo L.) plots on several farms (1,500 ha) in the Zacapa Valley of Guatemala were visited, and melon plants with two different symptomologies were observed. One group of plants exhibited stem necrosis at the crown level, and less frequently, small necrotic spots on leaves. Some plants exhibited necrosis of veins and yellow areas that evolved into interveinal necrosis and often expanded into large necrotic interveinal lesions. Roots were poor and lacked secondary rootlets. In some cases, wilt and plant death were detected. Affected plants appeared as localized patches in various areas of the plots on farms that were visited. Double-antibody sandwich enzymelinked immtinosorbent assay (DAS-ELISA) serological analyses were carried out with 34 symptomatic plants. In these plants, a mixture of the crown and root were analyzed with two repetitions and two lots of different Melon necrotic spot virus (MNSV) polyclonal antisera (Loewe No. 07097 and Sanofi No. 70217). All 34 plants were positive for this virus. These results were confirmed using reverse transcription-polymerase chain reaction (RT-PCR) with specific MNSV primers (1). Spores of Olpidium bornovanus, the vector of MNSV, were seen on all ELISA-positive plants after staining rootlets with potassium hydroxide and neutralization with hydrochloric acid. In the same fields, another group of melon plants showed yellowing, curling, and mottling of leaves. Leaves collected from five symptomatic plants gave positive results in triple-antibody sandwich- ELISA using a Tomato yellow leaf curl begomovirus antiserum (DSMZ AS- 0421 and DSMZ AS-0546/2). In 2001, these results were confirmed using PCR with degenerate primers that amplify the core region of most begomovirus coat protein genes (P. Martnez-Culebras, M. I. Font, and C. Jord, unpublished). A 560-bp DNA fragment was amplified in these symptomatic melon samples. Three of the PCR products were sequenced and each showed 99% identity with the Melon chlorotic leaf curl virus isolate from Guatemala (GenBank Accession No. AF325497). Only one mixed infection of MNSV and MCLCV was found. During the four years subsequent to 1999, the number of melon plants showing both types of symptoms has increased. This study provides information on the current status of virus diseases in melon crops in Guatemala, and to our knowledge, this is the first report of MNSV in Guatemala.

Reference; (1) B. Gosalvez et al. J. Virol. Methods 113:87.

First Report of Downy Mildew of Opium Poppy Caused by Peronospora arborescens in Spain. B. B. Landa and M. Montes-Borrego, College of Agriculture (ETSIAM), University of Crdoba (UCO), P.O. Box 3048, 14080 Crdoba, Spain; F. J. Muoz-Ledesma, Alcaliber S.A., Ctra. Carmona-El Viso del Alcor, km 1.8, Carmona (Sevilla), Spain; and R. M. Jimnez-Daz, ETSIAM-UCO, and Institute of Sustainable Agriculture, CSIC, P.O. Box 4084, 14080 Crdoba, Spain. Plant Dis. 89:338, 2005; published on-line as DOI: 10.1094/PD-89-0338B. Accepted for publication 13 October 2004.

Opium poppy (Papaver somniferum) is an economically important pharmaceutical crop in Spain with approximately 7,400 ha cultivated annually. In the spring of 2004, severe attacks by a new foliar disease were observed approximately 500 km apart in commercial opium poppy fields in the Castilla-La Mancha and Andalusia regions of central and southern Spain, respectively. The incidence of affected fields ranged from 40 to 50%, and incidence of diseased plants ranged from 20 to 30%. Initial disease symptoms included irregularly shaped, chlorotic-to-light yellow leaf lesions (ranging in size from 0.5 to 4 cm). Affected tissues curled, thickened, and became deformed and necrotic as disease developed. Lesions expanded in size and often coalesced, eventually giving rise to large necrotic areas in leaves or death of entire leaves. In wet weather or conditions of high relative humidity, a dense felt of sporangiophores with sporangia was produced on the abaxial leaf surface and occasionally on the adaxial surface. Microscopic observations revealed sporangiophores branching dichotomically at least four to six times, ending with sterigmata bearing single sporangia. Sporangia were hyaline, elliptical to spherical in shape, and measured 18 to 24 14 to 18 m (average 19 1.2 15 1.6 m). Occasionally, oospores formed in necrotic leaf tissues. Oospores were dark brown (the surface was irregularly ridged) and measured 36 to 46 m in diameter (average 39 4.4 m). The oospore wall was 3 to 11 m thick. On the basis of the observed morphological features of six symptomatic plant samples from fields at Castilla-La Mancha and Andalusia regions, we identified the pathogen as Peronospora arborescent (1). Pathogenicity was confirmed by inoculating 4- to 6-week-old opium poppy plants (cv. nigrum) with an isolate collected from a field in Ecija, Andalusia. seed of test plants was surface disinfested and germinated under sterile conditions. Plants were sprayed with a suspension of 1 to 5 10^sup 5^ sporangia per ml in sterile distilled water. Plants sprayed with sterile water served as controls. There were five replicate plants per treatment. Plants were enclosed in sealed plastic bags and kept in the dark for 24 h. This was followed by incubation in a growth chamber at 21C, 60 to 90% relative humidity, and a 12-h photoperiod (fluorescent light: 360 E.m^sup -2^.s^sup -1^). After 5 to 7 days, typical downy mildew symptoms developed in inoculated plants. All control plants remained symptomless. Sporulation by the pathogen on symptomatic leaves occurred when affected plants were sprayed with water, enclosed in sealed plastic bags, and incubated at 21C in the dark for 24 h. To our knowledge, this is the first report of P. arborescent infecting opium poppy in Spain. Infestations of poppy weeds (Papaver rhoeas) and wild Papaver somniferum were also observed in affected opium poppy fields, which may bear importance in the epidemiology of the disease as alternative hosts for inoculum increase and survival of P. arborescent under field conditions.

References: (1) S. M. Francis. No. 686 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1981.

First Incidence of Plum Pox Virus on Apricot Trees in China. M. Navratil and D. Safarova, Faculty of Science, Palaeky University in Olomouc, Slechtitelu 11, 783 71 Czech Republic; R. Karesova, Research and Breeding Institute of Pomology, Holovousy v Podkrkonosi, 508 01 Horice, Czech Republic; and K. Petrzik, Institute of Plant Molecular Biology, Academy of Sciences, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic. Plant Dis. 89:338, 2005; published on-line as DOI: 10.1094/PD-89-0338C. Accepted for publication 13 December 2004.

Plum pox disease, caused by Plum pox virus (PPV), is the most severe virus disease of plums, apricots, and peaches. The disease causes heavy losses for fruit growers and the international trade of propagation materials and fresh fruits. PPV was first reported in Bulgaria in 1917 (1). It is now widespread in Europe and has been reported from Cyprus, Syria, Egypt, India, Kazakhstan, Chile, the United States, and Canada. Leaves on symptomatic apricot trees (Prunus armeniaca cvs. Hong Mei and Bai Mei and a selected genotype) in the Hunan Province of China showed typical yellow rings and diffused chlorotic spots. Samples from three suspected trees were repeatedly analyzed using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELlSA) and reverse transcription- polymerase chain reaction (RT-PCR) in the summers of 2001-2003. PPV was detected in leaves, bark, and leaf buds of all three trees using ELISA with polyclonal and monoclonal antibodies provided by M. Navratil, Palaeky University, Olomouc, Czech Republic (3). The results were confirmed using RT-PCR amplification of a 243-bp of the coat protein gene with a PPV-specific primer pair (2). BLAST analysis of two RT-PCR product sequences (GenBank Accession Nos. AY750961 and AY795603) showed 100% homology to multiple sequences of the PPV-D strain (GenBank Accession Nos. X81080, AF440743, and AF401295). The third sequence (GenBank Accession No. AY795602) had a C at position 112 rather than the T found in the other sequences. The ELISA, RT-PCR, and sequence results indicate that PPV-D was present in the apricot trees. To our knowledge, this is the first indication of PPV occurrence in China. This sporadic incidence of PPV on apricot trees requires addressing problems with the occurrence and spread of plum pox diseases in China and starting an eradication program.

References: (1)D. Alanasoff. Annu. Univ. Sofia Fac. Agron. et Sylvie. 11:49, 1932. (2) T. Candresse et al. Phytopathology. 88:198, 1998. (3) I. Hilgert et al. Hybridoma. 12:215, 1993.

First Report of Barley as Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B, and Ribosomal Protein Subgroup rpI-B in Lithuania. L. Urbanaviciene and R. Jomantiene, Fitovirus Laboratory, Institute of Botan\y, LT-08406 Vilnius, Lithuania; and R. E. Davis, Molecular Plant Pathology Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705. Plant Dis. 89:339, 2005; published on- line as DOI: 10.1094/PD-89-0339A. Accepted for publication 2 December 2004.

Numerous diseased plants of barley (Hordeum vulgaris L.) exhibiting twisted, abnormally thin and yellowed awns, reduced spikelets, and general stunting and yellowing were observed in fields in the Vilnius and Kaisiadorys regions of Lithuania. The possible association of a phytoplasma with the disease, termed barley deformation (BaDef), was assessed using polymerase chain reaction (PCR). Three phytoplasma universal primer pairs (P1/P7, R16F2n/R16R2, and rpFl/rpRI) (1,2,4) were employed to amplify ribosomal (r) RNA gene (rDNA) and ribosomal protein (rp) gene sequences. Template DNA extractions and PCR (direct and nested) were conducted as previously described (4). Although DNA was amplified in PCRs containing template extracted from diseased plants, no amplification was observed in PCRs containing DNA from symptomless plants sampled from the same fields. The BaDef phytoplasma was identified and classified according to Lee et al. (4) through restriction fragment length polymorphism (RFLP) analysis of 1.2-kbp 16S rDNA amplified in the PCR primed by primer pair R16F2n/R16R2 and analysis of the 1.2-kbp rp gene sequences amplified in PCR primed by primer pair rpFl/rpRI. On the basis of collective RFLP patterns of amplified 16S rDNA and rp gene sequences, the BaDef phytoplasma was classified as a member of group 16SrI (group I, aster yellows phytoplasma group), subgroup B (16SrI-B), and rp subgroup rpI-B. Ribosomal protein subgroup B was distinguished from other rp subgroups on the basis of the presence of a recognition site for Hpall. The 1.8-kbp rDNA product of PCR primed by P1/P7 and the 1.2- kbp rpFl/rpRI PCR product were cloned and sequenced, and the sequences were deposited in GenBank under Accession No. AY734453 for the BaDef 16S rDNA and Accession No. AY735448 for the BaDef rp gene sequence. Previously, only oat proliferation (OatP) phytoplasma, a member of subgroup 16SrI-A, had been characterized in a cereal crop (Avenu sativa L.) in Europe (3); BaDef is another phytoplasmal disease threatening cereal crops in the region.

References: (1) S. Deng and D. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) D. E. Gundersen and I. M. Lee. Phylopathol. Mediterr. 35:144, 1996. (3) R. Jomantiene et al. Plant Dis. 86:443, 2002. (4) I. M. Lee et al. Im. J. Sysl. Bacteriol. 48:1153, 1998.

First Report of Canola Blackleg Caused by Pathogenicity Group 4 of Leptosphaeria maculons in Manitoba. Y. Chen and W. G. D. Fernando, Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Plant Dis. 89:339, 2005; published on- line as DOI: 10.1094/PD-89-0339B. Accepted for publication 13 December 2004.

Leptosphaeria maculons (Desmaz.) Ces. & de Not., causal agent of blackleg of canola (Brassica napus L.), was initially placed in several pathogenicity groups (PG) on the basis of the interaction phenotypes (IP) of L. maculans isolates on the differential canola cvs. Westar (W), Glacier (G), and Quinta (Q) (4). PGl isolates are weakly virulent and PG2, PG3, and PG4 isolates are highly virulent. In Manitoba, the L. maculans population consists mainly of PG2 isolates (virulent on W and avirulent on G and Q), a few PGl isolates (avirulent on W, G, and Q), and PGT (virulent on W and Q, but avirulent on G) (3). Since the blackleg fungus is known to have a high level of evolutionary potential, the oilseed Pathology Laboratory at the University of Manitoba, Winnipeg, Canada, examines the pathogenic variability of L. maculans isolates from the Canadian Prairies and North Dakota each year. During 2002, the presence of PG3 (virulent on W and G and avirulent on Q) was reported in Manitoba (1). During 2003, a canola field located at La Riviere, Manitoba, 200 km southwest of Winnipeg, was found to be severely affected by blackleg. Stubble from this field was arbitrarily collected in mid-April 2004, and 98 singlepycnidia pure cultures were obtained by isolating fungi from surface-sterilized (2% sodium hypochlorite), infested residue, cultured on V8 agar at room temperature under cool-white florescent light for 24 h. Pycnidiospores were harvested after 14 days of incubation using the Miracloth filtering method (1). PG testing was performed using the three differential cultivars in the greenhouse. Known PG2, 3, and 4 isolates, 86-12, Liffole6, and PL30.2, respectively, were included as positive controls. For each of the 98 isolates, 12 7-day-old cotyledons of each differential cultivar grown in Metro Mix were wound-inoculated with 10 l of a pycnidiospore suspension (1 10^sup 7^ per ml) (1). Inoculated plants were maintained in the greenhouse (16/21C night/day and a 16-h photoperiod with cool-white florescent light). The experiment was repeated three times. Disease severity on cotyledons was assessed 12 days after inoculation with a O to 9 scale (0 to 2 = resistant; 3 to 6 = intermediate; and 7 to 9 = susceptible). Of the 98 isolates tested, five were PGI, 51 were PG2, 24 were PG3, 13 were PGT, and five were PG4. The isolates classified as PG4 gave IP reactions of 7 to 9, 7 to 9, and 6.6 to 8.2, on W, G, and Q, respectively. PG3 was reported one year ago, but highly virulent isolates belonging to PG4 have not been previously detected in Manitoba. To our knowledge, this is the first report of the occurrence of PG4 isolates of L. maculons, and the first report of PG4 causing canola blackleg in Manitoba. The appearance of PG4 may be evidence of pathogen population changes occurring under high- selection-pressure exerted by resistance genes in commercial cultivars (2), or through importation of PG4 isolates with canola seed.

References: (1) W. G. D. Fernando and Y. Chen. Plant Dis. 87:1268, 2003. (2) B. J. Hewlett. Can. J. Plant Pathol. 26:245, 2004. (3) M. Keri et al. Can. J. Plant Palhol. 23:199, 2001. (4) A. Mengistu el nl. Plant Dis. 75:1279, 1991.

Reservoir Weed Hosts for Turnip mosaic virus in Iran. Sh. Farzadfar, Plant Virology Department, Plant Pests and Diseases Research Institute, P.O. Box 19395-1454, Tehran, Iran; K. Ohshima, Laboratory of Plant Virology, Saga University, P.O. Box 840-8502, Saga, Japan; R. Pourrahim, Plant Virology Department, Plant Pests and Diseases Research Institute, Tehran, Iran; A. R. Golnaraghi, Plant Protection Department, Science and Research Campus, Islamic Azad University, P.O. Box 14515-775, Tehran, Iran; S. Sajedi, Botany Department, Plant Pests and Diseases Research Institute, Tehran, Iran; and A. Ahoonmanesh, Plant Pathology Department, Esfahan University of Technology, Esfahan, Iran. Plant Dis. 89:339, 2005; published on-line as DOI: 10.1094/PD-89-0339C. Accepted for publication 15 December 2004.

During the summer of 2003, weed samples of Rapistruiii ntgosuin and Sisymbrium loeselii showing severe mosaic, malformation, and stunting were collected from cauliflower fields in Tehran Province of Iran. Using double-antibody sandwich enzyme-linked immunosorbent assay (DASELlSA) with specific polyclonal antibodies, the samples were tested for the presence of Beet western yellows virus, Cauliflower mosaic virus, Radish mosaic virus, Turnip crinkle virus, Turnip mosaic virus (TuMV) (DSMZ, Braunschweig, Germany), Cucumber mosaic virus, and Tobacco mosaic virus (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). Leaf extracts were used for mechanical inoculation and they produced chlorotic local lesions on Chenopodium amaranticolor, necrotic lesions on leaves and shoot apex necrosis on Nicotiana glutinosa, leaf deformation, mosaic, and stunting on Petunia hybrida, and severe mosaic, distortion, and stunting on Brassica rapa. These symptoms were similar to those that were described previously for TuMV (4). ELlSA results showed that the original leaf samples and inoculated indicator plants reacted positively with TuMV antibodies, but not with antibodies for any of the other viruses listed above. Also, reverse transcription- polymerase chain reaction of total RNA extracted from the collected leaf samples using the universal primers for potyviruses (3) resulted in the amplification of two fragments of the expected sizes, approximately 700 and 1,700 bp. TuMV, a member of the genus Potyvirus in the family Potyviridae, is transmitted by aphids in a nonpersistent manner (4). This virus is geographically widespread with a wide host range that can infect 318 species in 156 genera of 43 plant families including, Brassicaceae, Chenopodiaceae, Asteraceae, Cucurbitaceae, and Solanaceae (2,4). R. rugosum and S. loeselii, two annual or biennial plants in the Brassicaceae family, were common and widely distributed in the fields surveyed. The presence of TuMV-infected weed hosts in cauliflower fields may impact disease management strategies. TuMV was first observed on stock plants (Matthiola sp.) in Iran (1). To our knowledge, this is the first report of natural occurrence of TuMV on weed hosts in Iran.

References: (1)M. Bahar et al. Iran. J. Plant Pathol. 21:11, 1985. (2) J. R. Edwardson and R. G. Christie. The potyvirus group. FIa. Agric. Exp. Stn. Monogr. Ser. No. 16, 1991. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) J. A. Tomlinson. Turnip mosaic virus. No. 8 in: Descriptions of Plant Viruses. CMI/AAB, Surrey, England, 1970.

First Report of Pythium Root Rot of Rau Ram (Polygonum odoratum). E. N. Rosskopf and C. B. Yandoc, USDA, ARS, USHRL, Fort Pierce, FL; B. Stange and E. M. Lamb, IRREC, University of Florida, Fort Pierce; and D. J. Mitchell, University of Florida, Gainesville. Plant Dis. 89:340, 2005; published on-line as DOI: 10.1094/PD-89-0340A. Accepted for publication 17 December 2004.

Polygonum odoratum (= Persicaria odorata), known as rail ram or sang hum, is native to southeastern Asia \and is a common herb in Vietnamese cuisine (1). It has been studied most extensively for its aromatic compound content (2). In Florida, rau ram commonly is grown hydroponically in greenhouses using large, cement beds with recirculated water. The plants form dense mats from which new growth is trimmed for market. During January of 2002, a severe dieback was observed in one production house in Saint Lucie County, FL. Plants with less severe symptoms were yellowed and stunted. Roots of symptomatic plants were largely decayed with root symptoms beginning as a tip necrosis. The cortex of severely affected roots slipped off easily, leaving a stringy vascular system. Plating of symptomatic tissue from 20 randomly selected plant samples was performed with multiple general and selective media including potato dextrose agar, corn meal agar with pimaricin, ampicillin, rifampicin, and pentachloronitroben/.ene (PARP) (3). All colonies produced were identified as Pythium helicoides Drechsler on the basis of sporangial, oogonial, and antheridial characteristics (4). Isolates had proliferous, obovoid, papillate sporangia, and were homothallic with smooth-walled oogonia and thick-walled, aplerotic oospores. Multiple antheridial attachments per oogonium were common with the antheridium attached along its entire length. Pathogenicity tests were conducted using P. odoratum plants grown from commercial transplants. Two tests were performed. Each test was conducted using eight inoculated and eight control plants. In the first test, plants were maintained in ??-cm pots immersed in sterilized pond water for the duration of the test. Plants were inoculated with five 7- 70- mm sections of freshly growing mycelial culture per plant using 10- day-old cultures of Pythium helicoides grown on water agar. Chlorosis was observed at approximately 2 months after inoculation. Root necrosis was observed in inoculated plants approximately 5 months after inoculation. This test was performed in the greenhouse with temperatures ranging from 20 to 30C. The second test was performed in growth chambers at 35 to 40C. Plants were maintained in 10-cm pots immersed in Hoagland’s solution and were inoculated with four 6-mm plugs per plant. Symptoms were observed on inoculated plants at this temperature within 1 week of inoculation. No chlorosis or root decay was observed in noninoculated, immersed plants. The pathogen was reisolated from inoculated, symptomatic tissue. To our knowledge, this is the first report of root rot of P. odoratum caused by Pythium helicoides.

References: (I)R. E. Bond. Herbarist 55:34, 1989. (2) N. X. Dung et al. J. Essent. oil Res. 7:339, 1995. (3) M. E. Kannwischer and D. J. Mitchell. Phytopathology 68:1760, 1978. (4) A. J. van der Plaats- Nitcrink. Monograph of the Genus Pythium. Vol. 21, Studies in Mycology. Centraalbureau voor Schimmelcutltures, Baarn, The Netherlands, 1981.

Occurrence of Mosaic Caused by Cucumber mosaic virus in Lobelia Hybrids in France and Italy. L. Cardin, INRA, URIH Phytopathologie, BP167, F-06903 Sophia-Antipolis cedex, France; and B. Delecolle and B. Moury, INRA, Station de Pathologie Vgtale, Domaine St Maurice, BP94, F-84143 Montfavet cedex, France. Plant Dis. 89:340, 2005; published on-line as DOI: 10.1094/PD-89-0340B. Accepted for publication 8 December 2004.

In 2002, mosaic symptoms associated with yellowish ringspots were observed on leaves of a hybrid of lobelia (Lobelia spp.) grown in a public garden in Alsace (France). In 2003, similar symptoms were observed in Lobelia laxiflora in the Hanbury botanical garden (La Mortola, Italy) and the botanical garden of Nice (France). Cucumber mosaic virus (CMV) was identified in samples collected from the three locations on the basis of the following: symptoms exhibited by a host range of inoculated plants previously described (1); the observation of isometric particles (approximately 30 nm) with an electron microscope in crude sap preparations from inoculated plants and semipurified extracts of claytonia perfoliata; and the positive reaction in double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) to antibodies raised against CMV (2). In double- immunodiffusion analysis, each isolate was shown to belong to the group II strains of CMV (4). In these experiments, no differences were observed among the isolates collected. To test if CMV was responsible for the symptoms observed in Lobelia spp., an isolate from Alsace was grown in Xanthi-nc tobacco plants following isolation from local lesions on Vigna unguiculata and then mechanically inoculated to L. speciosa cv. Compliment mix (10 plants), L. siphilitica (10 plants), L. inflata (Indian tobacco) (10 plants), L. erinus cvs. Crystal and Empereur Guillaume (5 plants), L. erinus pendula cvs. Saphyr and Cascade (5 plants), L. laxiflora (10 plants), and L. gemrdii cv. Vedrariensis (5 plants) and grown in a hydroponic system. Eight weeks postinoculation, all plants except L. laxiflora exhibited systemic mosaic and chlorotic ringspot symptoms on leaves and resulted in strong DAS-ELISA reactions for CMV, whereas mock-inoculated controls remained symptomless and virus free. Symptoms were particularly severe on L. siphilitica and L. speciosa, but mild on L. inflata and L. gerardii. Foliar mosaic symptoms appeared only 6 months postinoculation in 7 of 10 inoculated L. laxiflora plants. Only these plants were CMV positive using DAS-ELISA. No symptoms were observed in flowers of any plants infected with CMV. CMV has been previously reported in other species of the family Lobeliaceae including L. cardinalis, L. erinus, L. gracilis, and L. tenuior following natural or experimental infection (3) but Koch’s postulates were not completed. This study validates that CMV is responsible for mosaic diseases in Lobelia spp., and shows that hybrids from L. cardinalis such as L. speciosa and L. gerardii also are susceptible to CMV. Mosaic symptoms in L. siphilitica and L. speciosa are particularly damaging to their ornamental quality. Moreover, perennial plants such as L. laxiflora can be sources of CMV contamination by aphid transmission.

References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) J. C. Devergne and L. Cardin. Ann. Phytopalhol. 7:225, 1975. (3) L. Douine et al. Ann. Phylopalhol. 11:439, 1979. (4) M. J. Roossinck. J. Virol. 76:3382, 2002.

Impatiens necrotic spot virus in Greenhouse-Grown Potatoes in New York State. K. L. Perry, L. Miller, and L. Williams, Department of Plant Pathology, 334 Plant Science Bldg. Cornell University, Ithaca, NY 14853. Plant Dis. 89:340, 2005; published on-line as DOI: 10.1094/ PD-89-0340C. Accepted for publication 9 December 2004.

Impatiens necrotic spot virus (INSV; genus Tospovirus) was detected in experimental greenhouse-grown potatoes (Solanum tuberosum) and Nicotiana benthamiana in New York State in July and August of 2003 and 2004. Potato leaves exhibiting necrotic lesions with a concentric pattern similar to those induced by Tomato spotted wilt virus (1) were observed on cvs. Atlantic, Huckleberry, NY115, and Pentland Ivory. The presence of INSV was confirmed using double- antibody sandwich enzyme-linked immunosorbent assay and a rapid ‘ImmunoStrip’ assay (Agdia, Inc., Elkhart, IN). INSV-specific sequences were amplified from total RNA extracts using reverse transcription-polymerase chain reaction with ‘Tospovirus Group" primers (Agdia, Inc.) and two independently amplified DNAs were sequenced. A common sequence of 355 nucleotides (GenBank Accession No. AY775324) showed 98% identity to coding sequences in an INSV L RNA. The virus was mechanically transmitted to potato and N. benthamiana and could be detected in asymptomatic, systemically infected potato leaves. Stems nodes and leaves were removed from infected potato plants, and sterile in vitro plantlets were established (2). None of the regenerated in vitro plantlets of cvs. Pentland Ivory (6 plantlets) or NY115 (5 plantlets) were infected with INSV. Two of ten regenerated cv. Atlantic plantlets initially tested positive, but INSV could not be detected after 6 months in tissue culture. In vitro tissue culture plantlets could not be established from infected cv. Huckleberry plants, even though they were consistently obtained from uninfected plants. Infected greenhouse plants were grown to maturity and the tubers harvested, stored for 6 months at 4C, and replanted in the greenhouse. INSV could not be detected in plants from 26 cv. Huckleberry, 4 cv. NY115, or 4 cv. Atlantic tubers. Although this isolate of INSV was able to systemically infect potato, it was not efficiently maintained or transmitted to progeny tubers. This might explain why INSV has not been reported as a problem in potato production. Lastly, in both years, dying N. benthamiana provided the first sign of a widespread greenhouse infestation of INSV in a university facility housing ornamental and crop plants. INSV induced a systemic necrosis in N. benthamiana, and this host may be useful as a sensitive ‘trap’ plant indicator for natural infections in greenhouse production.

References’. (I)T. L. German. Tomato spoiled will virus. Pages 72- 73 in: Compendium of Potato Diseases. W. R. Stevenson et al., eds. The American Phylopathological Society, St. Paul, 2001. (2) S. A. Slack and L. A. Tufford. Meristem culture for virus elimination. Pages 117-128 in: Fundamental Methods of Plant Cell, Tissue and Organ Culture and Laboratory Operations. O. L. Gamborg and G. C. Philips, eds. Springer-Velag, Berlin, 1995.

A New Begomovirus Causes Tomato Leaf Curl Disease in Baja California Sur, Mexico. R. J. Holgun-Pea and R. Vzquez-Jurez, Centre de Investigaciones Biolgicas del Noroeste, La Paz, B.C.S. 23000, Mexico; and R. F. Rivera-Bustamante, Centre de Investigacin y de Estudios Avanzados del IPN, Irapuato, Guanajuato 36500, Mexico. Plant Dis. 89:341, 2005; published on-line as DOI: 10.1094/PD-89- 0341A. Ac\cepted for publication 5 December 2004.

More than 10,000 ha of tomatoes are grown in the field and greenhouses on the Baja California Peninsula of Mexico. Information about the etiology of geminivirus-like diseases affecting tomato crops in all horticultural regions in the area has been difficult to obtain and assess. From 2001 through 2003, stunting, foliar discoloration, reduced leaf size, and leaf crumpling symptoms were observed and analyzed in one large area of tomato plantings in El Carrizal (near the city of La Paz in Baja California Sur). This leaf curl disease resembled that caused by China del tomate virus and has been observed at levels of incidence ranging from 60 to 90%. DNA isolated from symptomatic plants was analyzed using DNA hybridizaton and polymerase chain reaction (PCR) amplification of the 5′ regions of the replication and coat protein genes, including the intergenic region (3). Comparisons of the nucleotide sequence (GenBank Accession No. AY339619) with corresponding sequences in GenBank resulted in 84.2% identity with Tomato mild mottle virus and 61.7% with Tomato severe leaf curl virus; both isolates originate from Central America. The relatively low nucleotide sequence identities from its closest relatives suggested that the virus may be a new begomovirus species of unambiguous American ancestry. In a phylogenetic analysis using PAUP 4.0 software, the Baja California isolate clustered in a separate group from other Mexican sequences. Moreover, the iteron (iterative sequences motifs associated in virus replication) arrangements (1) are unique among known New World begomoviruses, but identical to analogous elements from a tobacco- infecting begomovirus from China. On the other hand, it is well known that there are interactions between geminiviruses in mixed infections in some horticultural areas of Mexico (2). To determine the identity of the putative geminivirus involved in the disease, we used selected restriction enzyme (EcoRI, HinaIII and XbA) analysis and PCR with specific primers. No evidence of mixed infections with other geminiviruses was obtained. DNA fragments of the expected size (1.1 kb) showed different digestion patterns compared with other well-characterized geminiviruses isolated from Mexico such as China del tomate virus, Pepper huasteco yellow vein virus, Tomato leaf curl Sinaloa virus, and Pepper golden mosaic virus. Epidemiological, experimental, and natural host range studies indicated that the Baja California isolate has a relatively narrow host range infecting tomatoes, peppers (Capsicum annuum L.), and Peruvian apple (Nicandra physalodes L.). Reproduction of characteristic leaf curling symptoms in tomato seedlings infected with viruliferous whiteflies (Bemisia tabaci Genn.) and inoculated biolistically using infectious DNA (0.5 g/ml) as inoculum were obtained. Koch’s postulates were completed using PCR and DNA hybridization to confirm virus identity. These results confirm that the Baja California isolate is different from other begomoviruses isolated from Mexico. The virus is tentatively named Tomato chino Baja California virus (ToChBCV), genus Begomovirus, family Geminiviridae.

References: (1) G. R. Arguello-Astorga et al. Arch. Virol. 146:1465, 2001. (2) J. Mendez-Lozano et al. Phytopathology 93:270, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

First Report of Fusarium oxysporum Causing Yellows on Sugar Beet in the Red River Valley of Minnesota and North Dakota. C. E. Windels and J. R. Brantner, University of Minnesota, NW Research and Outreach Center, Crookston 56716; and C. A. Bradley, Department of Plant Pathology, and M. F. R. Khan, Department of Soil Science, North Dakota State University, Fargo 58105. Plant Dis. 89:341, 2005; published on-line as DOI: 10.1094/PD-89-0341B. Accepted for publication 8 December 2004.

In 2002, somel sugar beet (Beta vulgaris L.) fields in the Red River Valley (RRV) of Minnesota and North Dakota had symptoms characteristic of Fusarium yellows (4). In 2004, [asymptotically =]% of fields in the RRV had symptomatic plants. Interveinal yellowing of older leaves typically began in mid-July and as the disease progressed, younger leaves turned yellow. Sometimes, one side of the leaf was yellow or necrotic while the other side remained green. As leaves died, they remained attached to the crown. Transverse sections of roots revealed a light gray-brown discoloration of the vascular tissue but no external rotting of roots. Isolations from 35 symptomatic roots collected in eight fields yielded 25 isolates identified as F. oxysporum (from single conidia grown on homemade potato dextrose agar and carnation leaf agar) (3). Pathogenicity was determined by dipping roots of 5-week-old sugar beet plants (cv. ACH 9363) in a suspension of 10^sup 4^ conidia per ml for 8 min (12 isolates, 10 to 12 plants per isolate). Plants were planted in Cone- tainers (3.8 cm diameter 21 cm; Stuewe and Sons, Inc. Corvallis, OR) containing sterile soil. Three known cultures of F. oxysporum Schlecht. emend. Snyd. & Hans. f. sp. betae Stewart (= F. conglutinans var. betae Stewart [41) also were included (13 and 216c from L. Hanson, USDA-ARS, Fort Collins, CO; 0-1122 from The Pennyslvania State University Fusarium Research Center). The control was sterile water. Plants were placed in a greenhouse at 24 to 27C with natural light supplemented with illumination from high- pressure sodium-vapor lamps for 16 h daily and lightly fertilized biweekly to avoid chlorosis from nutrient deficiency. After 6 to 7 weeks, plants were rated for disease on a 0 to 4 scale: 0 = no disease; 1 = slight to extreme plant stunting, leaves may be wilted; 2 = chlorotic leaves, some with necrosis at margins; 3 = tap root dried and brown to black in color, leaves dying; and 4 = plant dead (1). The experiment was repeated. Disease severity differed between trials, but all isolates of F. oxysporum and F. oxysporum f. sp. betae resulted in dis ease ratings statistically (P < 0.05) greater than that of the water control. In Trial 1, isolates of F. oxysporum averaged a rating of 2.1 (range of 1.8 to 3.3) and F. oxysporum f. sp. betae averaged 2.1 (range of 2.0 to 2.2) compared with 0.1 for the water control. One isolate of F. oxysporum had a statistically higher rating than did the cultures of F. oxysporum f. sp. betae. In Trial 2, isolates of F. oxysporum averaged a rating of 3.3 (range of 2.7 to 3.7) and F. oxysporum f. sp. betae averaged 3.1 (range of 2.7 to 3.4) compared with 0.2 for the water control. Cultures of F. oxysporum (8 of 12) resulted in ratings statistically higher than that of the least pathogenic culture of F. oxysporum f. sp. betae. Cultures of F. oxysporum and F. oxysporum f. sp. betae recovered from inoculated plants were identical to those used to inoculate plants. To our knowledge, this is the first report of F. oxysporum f. sp. betae on sugar beet in the Red River Valley of Minnesota and North Dakota. The disease has been reported in California, Colorado, Montana, Nebraska, Oregon, Texas, and Wyoming (1,2).

References: (I) R. A. Cramer et al. J. Phytopathol. 151:352, 2003. (2) O. A. Fisher and J. S. Gerik. Phytopathology 84:1098, 1994. (3) P. E. Nelson el al. Fusarium Species: An illustrated Manual for Identification. The Pennsylvania State University Press. University Park, 1983. (4) D. Stewart. Phytopathology 21:59, 1931.

First Report of a Leaf Spot and Stem Canker Caused by Myrothecium rorldum on Watermelon in the United States. K. W. Seebold, Jr., D. B. Langston, Jr., and R. C. Kemerait, Jr., Department of Plant Pathology, University of Georgia, Coastal Plain Experiment Station, Tifton 31793; and J. E. Hudgins, University of Georgia Cooperative Extension Service, Bainbridge 31717. Plant Dis. 89:342, 2005; published on-line as DOI: 10.1094/PD-89-0342A. Accepted for publication 7 December 2004.

Myrothecium roridum Tode:Fr, pathogenic to a number of cucurbit species, causes fruit rots, cankers on crowns and stems, and leaf spots. Hosts include cantaloupe and honeydew (Cucurbita melo) and cucumber (Cucumis sativus) ( 1,3). In June 2004, following a period of heavy rainfall, numerous round-to-oblong, brown lesions with concentric rings were observed on leaves of watermelon (Citrullus lanatus) cv. Desert King at the Blackshank Farm in Tifton, GA. Disease was localized in the field and severity was low (<5% of leaf area affected). No symptoms were observed on fruit. Sections of tissue were removed from the margin between healthy and diseased tissue and plated on acidified, 25% potato dextrose agar (aPDA). A small plug of agar and mycelium were removed from colonies that emerged from lesions and were transferred to aPDA. Isolated colonies were characterized by a white, floccose mycelium with concentric, dark green-to-black rings of sporodochia bearing viscid masses of conidia. Conidia were cylindrical with rounded ends and measured 6 to 8 1.5 to 2.5 m. The features of the fungus were consistent with the description of Myrothecium roridum (1,2). Pathogenicity tests were conducted in a temperature-controlled greenhouse. Twenty-five watermelon plants (cv. Desert King) were inoculated with a conidial suspension of M. roridum (5 10^sup 5^ conidia per ml) plus 0.1% vol/ vol Tween 20. Inoculum was applied on leaves and stems until runoff with a hand-held mister, and plants were placed in a dew chamber for 72 h. Ten plants were sprayed with sterile, distilled water to serve as controls. Inoculated and noninoculated control plants were removed from the dew chamber and maintained at 25 to 28C. Symptoms appeared 8 days after inoculation and were characterized by round, dark lesions with concentric rings; noninoculated plants were symptomless. Sections of symptomatic tissue were plated, and M. roridum was reisolated. Although M. roridum is a common pathogen of melons and cucumber, to our knowledge, this is the first field report of a lea\f spot caused by M. roridum on watermelon in the United Slates. No further occurrences of the disease on watermelon have been observed in Georgia since the initial discovery of M. roridum in 2004; however, losses could be potentially severe if widespread infection of fruit were to occur.

Reference: (1) B. D. Brulon. Craler RoI. Pages 49-50 in: Compendium of Cucurbit Diseases. T. A. Zitter et al., eds. The American Phytopathological Society, St. Paul, MN, 1996. (2) M. B. Ellis. Page 552 in: Demaliaceous Hyphomycetes. CAB International, Wallingford, UK, 1971. (3) D. F. Farr et al. Page 809 in: Fungi on Plants and Plant Products in the United Stales. The American Phytopathological Society, St. Paul, MN, 1989.

Occurrence of Stem-Pitting Strains of Citrus tristeza virus in Croatia. S. Cerni, D. Skoric, and M. Krajacic, Department of Biology, Faculty of Science, University of Zagreb, Marulicev trg 9a, HR-IOOOO Zagreb, Croatia; Z. Gatin, Institute for Adriatic Crops and Karst Reclamation, Put Duilova II, HR-21000 Split, Croatia; and C. Santos, V. Martins, and G. Nolasco, CDCTPV, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. Plant Dis. 89:342, 2005; published on-line as DOI: 10.1094/PD-89-0342B. Accepted for publication 9 December 2004.

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 4330′N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Ncrctva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field- infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kastela, Split, Metkovic (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTVl and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision.

References: (1) G. Nolasco et al. Eur. J. Plant Palhol. 108:293, 2002. (2) A. Roy el al. Arch. Viral. 148:707, 2003. (3) A. Saric and I. Dulic. Agric. Conspectus Sd. 55:171, 1990. (4) G. Suastika et al. J. Gen. Plant Pathol. 67:73, 2001.

First Report of Narcissus mosaic virus Infecting Crocus spp. Cultivars in the Netherlands. R. Miglino, A. Jodlowska, and A. R. van Schadewijk, Flower Bulb Inspection Service (BKD), Research Laboratory, Zwartelaan 2, 2161 AL, Lisse, the Netherlands. Plant Dis. 89:342, 2005; published online as DOI: 10.I094/PD-89-0342C. Accepted for publication 6 December 2004.

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pickwick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzymelinked immunosorbent assay (ELISA) and reverse transcriptionpolymerase chain reaction (RT- PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle vims (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5′-AGC ATG GCG CCA TCT TGT GAC TG-3′) was located upstream in the conserved viral replicase-encoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5′-CTG AAG TCA CAA TGG GTG AAG AA-3′) was located downstream at position 3969-3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus.

References: (1) M. G. Bellardi and A. Pisi. Inf. Filopalol. 37:33, 1987. (2) A. F. L. M. Derks. Crocus spp. Pages 260-264 in: Virus and Virus-like Diseases of Bulbs and Flower Crops. G. Loebenstein et al., eds. Wiley publishers, West Sussex, UK, 1995. (3) S. A. Langeveld el al. J. Gen. Virol. 72:1531, 1991.

First Report of Leaf Spot on Japanese Plum Caused by an Alternaria sp. in Korea. Youngjim Kim, Division of Biotechnology, The Catholic University of Korea, Puchon 420-743, Korea; Hyang Burm Lee, Mycology Research Laboratory, School of Biological Sciences, Seoul National University, Kwanak-gu, Seoul 151-747, Korea; and Seung Hun Yu, College of Agriculture, Chungnam National University, Daejeon 305-674, Korea. Plant Dis. 89:343, 2005; published on-line as DOI: 10.1094/PD-89-0343A. Accepted for publication 26 January 2005.

Japanese plum (Prunus salicina Lindley) is a deciduous tree in the family Rosaceae. In Korea, this plant is widely distributed in orchards as an important stone fruit as well as in gardens as an ornamental tree because of their abundant white blossoms. Every September to November since 2001, leaf spots were observed on Japanese plum in a garden in Cheongyang, Chungnam District, Korea. Early symptoms consisted of small, brown spots that were 2 to 5 mm in diameter. Later, the leaf lesions became circular or irregular, dark brown, expanded to 15 mm in diameter, and resulted in discoloration with necrosis on twisted leaves that was followed by defoliation. In November, older lesions sometimes appeared blackish brown as sporulation occurred on the lesions. The causal fungus was isolated from diseased leaves and cultured on potato dextrose agar. A culture has been placed in the CABI Herbarium (IMI Accession No. 387139). Conidial dimension averaged 34 12 m. On the basis of morphological characteristics of conidia and conidiophores, the causal fungus was identified as a small spored species of Alternaria as described by E. G. Simmons (1). Pathogenicity tests were conducted by inoculating slightly wounded and nonwounded leaves with a conidial suspension adjusted to 1 10^sup 6^ conidia/ml. Four leaves per each experiment were either wounded or not and inoculated with a spore suspension. The eight leaves were placed in a moist chamber at 25C. After 6 to 10 days, small brown spots appeared on 87% of the wounded and nonwounded leaves. Control leaves sprayed with distilled water did not develop any symptoms. The causal fungus was consistently reisolated from the leaf spot\s. Results from pathogenicity tests were similar in a repeated test. It is possible that small-spored Alternaria spp. isolates are host specific (2). Eight Altemaria spp., including A. alternate:, A. tennis, A. tenuissima, and A. citri, have been found to cause black spot on fifteen Primus spp. in China, Japan, Hong Kong, Libya, Mexico, Australia, and the United States (2). Further studies on the host- specific toxin production, geographical distribution, and host ranges for the species of Alternaria isolated from Japanese plum are in progress. To our knowledge, this is the first report of leaf spot on Japanese plum (P. salicina) caused by a small-spored Alternaria sp. in Korea.

References: (1) E. G. Simmons. Mycolaxon 55:79, 1995. (2) K. Inoue and H. Nasu. J. Gen. Plant Palhol. 66:18, 2002.

Copyright American Phytopathological Society Mar 2005