First Report of Curly Top Disease of Basil Caused by Beet severe curly top virus in California.

In August 2012, symptoms of stunted growth and leaf epinasty, crumpling, and yellowing, were observed in basil plants (Ocimum basilicum) grown in a shadehouse in Calipatria in the Imperial Valley of California. Populations of the beet leafhopper (Circulifer tenellus) carrying curtoviruses (genus Curtovirus, family Geminiviridae) were detected in the Imperial Valley in May 2012. Together, this suggested a curtovirus etiology for this virus-like disease of basil. Total DNA extracts were prepared from leaves of nine representative symptomatic plants (BA1 through 9) and used in the PCR with the general curtovirus primer pair, BGv377 and BGc1509 (1,2). This primer pair directed the amplification of the expected ~1.1 kb DNA fragments from extracts prepared from all nine plants, and not from equivalent extracts from symptomless plants. The sequences of 1.1 kb fragments amplified from four plants (BA1 through 4) were determined, and BLAST analyses revealed 99% nucleotide sequence identities among these sequences, and 98% identities with the homologous region (V2/CP) of Beet severe curly top virus-Cfh (BSCTV-Cfh; GenBank Accession No. U02311). A second primer pair (BGv981 5'-AACGGTCAGGCTATGCCGTCTAC-3' and BGc479 5'-GAAAGACCTCGCCTTCTTCTAGGG-3') was designed to amplify the remainder of the viral genome. The expected size ~2.4 kb fragments were amplified from the extracts of the BA1 through 9 plants, and the fragments from the BA1 and 2 plants were cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced. Using the sequences of the overlapping PCR-amplified fragments, the complete viral genome sequences of the BA1 and BA2 isolates were determined. The BA1 and BA2 sequences were 2,934 bp and were 99% identical to each other and to the sequence of BSCTV-Cfh (3). To confirm the infectivity of BSCTV in basil, the BSCTV-Cfh infectious clone, which originated from California, was used for agroinoculation and leafhopper transmission experiments in basil plants (cvs. Sweet aroma and Genovese). Basil plants agroinoculated with the BSCTV-Cfh clone developed stunted growth and leaf crumpling and curling symptoms, similar to symptoms observed in the symptomatic plants from the Imperial Valley. The presence of viral DNA in symptomatic plants was confirmed by PCR with the BGv377/BGc1509 primer pair. Basil plants inoculated with an empty vector control did not develop symptoms, nor was curtovirus DNA amplified from these plants by PCR. Beet leafhoppers were given a 48-h acquisition access period on BSCTV-Cfh-infected sugarbeet plants, followed by a 48-h inoculation access period on healthy basil plants. These plants developed curly top symptoms approximately 21 days after inoculation, indicating that BSCTV was transmitted to basil by the beet leafhopper. Together, these results establish that the cause of the disease symptoms in basil in the Imperial Valley of California was BSCTV. This is the first report of curly top disease in basil, which is the second member of the mint family (Lamiaceae) known to be infected by a curtovirus. The stunted growth induced in basil by BSCTV has the potential to cause yield and economic loss, particularly in open field or screenhouse production when beet leafhopper populations are high. References: (1) L-F. Chen et al. Plant Dis. 94:99, 2010. (2) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (3) D. C. Stenger. Mol. Plant-Micro. Interact. 7:154, 1994.

First Report of Alfalfa mosaic virus Infecting Basil (Ocimum basilicum) in California.

Basil (Ocimum basilicum L.) plants collected from three fields in Imperial County, CA in May, 2011 were found to be exhibiting yellowing, chlorotic sectors and spots on leaves, resulting in unmarketable plants. Dodder (Cuscuta spp.) was present in one of the fields, but was not visibly associated with symptomatic plants. Total nucleic acid was extracted from four symptomatic and three asymptomatic basil plants, as well as from the dodder plant with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Nucleic acid extracts were tested by reverse transcription (RT)-PCR for the presence of Alfalfa mosaic virus (AMV) using primers designed to amplify a 350-nt region of the AMV coat protein gene (3). RT-PCR produced bands of the expected size in extracts from all symptomatic plants and the dodder sample. No amplification was obtained from symptomless plants. A 350-nt band amplified from one plant was gel-extracted, sequenced (TACGen, Richmond, CA), and confirmed to be AMV by comparison to sequences available in GenBank (Accession No. K02703). Although serological tests on an initial basil sample were negative for AMV by ELISA using antiserum produced against AMV by R. Larsen, USDA-ARS, Prosser, WA (unpublished), AMV was confirmed by ELISA and RT-PCR in symptomatic Nicotiana benthamiana, N. clevelandii, and Malva parviflora plants following mechanical transmission from basil source plants. The fields with AMV infections were located at opposite ends of the production region from one another, indicating widespread dispersal of AMV in the region. All AMV positive plants were adjacent to alfalfa. Two additional basil plantings in shade houses open to the outside environment did not have AMV symptomatic plants and were also confirmed negative by RT-PCR, but these plantings were at the extreme north end of Imperial Valley agriculture and well away from any alfalfa fields. At the time the basil plantations were sampled for AMV, no aphids were found in any plantations, but during the several weeks prior to finding the AMV-positive plants, cowpea aphid, Aphis craccivora Koch; pea aphid, Acyrthosiphon pisum Harris; blue alfalfa aphid, Acyrthosiphon kondoi Shinji; and spotted alfalfa aphid, Therioaphis maculata Buckton were colonizing Imperial Valley alfalfa fields, producing winged adults. AMV is transmitted by at least 14 aphid species (1), and most aphid populations increase during the late spring in this important desert agricultural region. The acquisition of AMV by dodder suggests the parasitic plant may serve as a vector of AMV within basil fields, although further study will be necessary for clarification. Significant acreage of basil is grown in the Imperial Valley. This acreage is surrounded by extensive and increasing alfalfa production totaling 55,442 ha (137,000 acres) in Imperial County and representing a 21% increase in acreage over 2009 for the same region (2). To our knowledge, this is the first report of basil infected by AMV in California. The proximity of basil production to such a large alfalfa production region warrants the need for enhanced efforts at aphid management in basil production to reduce vector populations and reduce transmission to basil crops. References: (1) E. M. Jaspars and L. Bos. Alfalfa mosaic virus. No. 229 in: Descriptions of Plant Viruses. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, England, 1980. (2) C. Valenzuela. Imperial County California Crop and Livestock Report, 2010. (3) H. Xu and J. Nie. Phytopathology 96:1237, 2006.

Biology and Molecular Characterization of Cucurbit leaf crumple virus, an Emergent Cucurbit-Infecting Begomovirus in the Imperial Valley of California.

Cucurbit leaf crumple virus (CuLCrV) is an emergent and potentially economically important bipartite begomovirus first identified in volunteer watermelon plants in the Imperial Valley of southern California in 1998. Field surveys indicated that CuLCrV has become established in the Imperial Valley; and field plot studies revealed that CuLCrV primarily infects cucurbits, including cantaloupe, squash, and watermelon. Full-length DNA-A and DNA-B clones of an Imperial Valley isolate of CuLCrV were obtained by polymerase chain reaction (PCR) with overlapping primers. These clones were infectious in various cucurbits and common bean (cv. Topcrop); symptoms included stunted growth and leaf crumple, curl, and chlorosis. CuLCrV was not sap-transmissible, and immunolocalization and DNA in situ hybridization studies revealed that it is phloem-limited. A CuLCrV agroinoculation system was generated, and host range studies revealed differential susceptibility in cucurbits, with squash, watermelon, cantaloupe, and honeydew melon being most to least susceptible, respectively. Germplasm screening studies identified a number of resistant cantaloupe and honeydew melon cultivars. The genome organization of this CuLCrV isolate (CuLCrV-CA) is similar to other bipartite begomoviruses, and phylogenetic analysis placed CuLCrV in the Squash leaf curl virus (SLCV) cluster of New World bipartite begomoviruses. A CuLCrV-specific PCR test was developed which allows for differentiation from other begomoviruses, including SLCV.

First Report of Tomato yellow leaf curl virus Associated with Tomato Yellow Leaf Curl Disease in California.

Tomato yellow leaf curl disease caused by the whitefly-transmitted begomovirus (genus Begomovirus, family Geminiviridae) Tomato yellow leaf curl virus (TYLCV) is one of the most damaging diseases of tomato. TYLCV was introduced into the New World in the early 1990s and by the late 1990s, it was found in Florida (2). In 2005 and 2006, the virus was reported from northern Mexico (states of Sinaloa and Tamaulipas) (1) and subsequently from Texas and Arizona. In March 2007, tomato (Lycopersicon esculentum) plants growing in a greenhouse in Brawley, CA showed TYLCV-like symptoms including stunted upright growth, shortened internodes, and small upcurled leaves with crumpling and strong interveinal and marginal chlorosis. These plants also sustained high populations of whiteflies. Symptomatic tomato leaves and associated whiteflies were collected from inside the greenhouse. Leaf samples also were collected from symptomless weeds (cheeseweed [Malva parviflora] and dandelion [Taraxacum officinale]) outside of the greenhouse. Total nucleic acids were extracted from 41 symptomatic tomato leaf samples, seven samples of adult whiteflies (approximately 50 per sample), and six leaf samples each from cheeseweed and dandelion. PCR analyses were performed with the degenerate begomovirus primers PAL1v1978 and PAR1c496 (3) and a TYLCV capsid protein (CP) primer pair (4). The expected size of approximately 1.4-kbp and 300-bp DNA fragments, respectively, were amplified from extracts of all 41 symptomatic tomato leaves and adult whitefly samples; whereas the 300-bp DNA fragment was amplified from all six cheeseweed samples and four of the six dandelion samples. Sequence analysis of a portion of the AC1/C1 gene from the approximately 1.4-kbp fragment amplified from 12 tomato leaf samples and four whiteflies samples revealed 99 to 100% identity with the homologous sequence of TYLCV from Israel (GenBank Accession No. X15656). The putative genome of the California TYLCV isolate was amplified using PCR and an overlapping primer pair (TYBamHIv: 5'-GGATCCACTTCTAAATGAATTTCCTG-3' and TYBamHI2c: 5'-GGATCCCACATAGTGCAAGACAAAC-3'), cloned and sequenced. The viral genome was 2,781 nt (GenBank Accession No. EF539831), and sequence analysis confirmed it was a bona fide isolate of TYLCV. The California TYLCV sequence is virtually identical (99.7% total nucleotide and 100% CP amino acid sequence identity) to a TYLCV isolate from Sinaloa, Mexico (GenBank Accession No. EF523478) and closely related to isolates from China (AM282874), Cuba (AJ223505), Dominican Republic (AF024715), Egypt (AY594174), Florida (AY530931), Japan (AB192966), and Mexico (DQ631892) (sequence identities of 98.2 to 99.7%). Together, these results establish that TYLCV was introduced to California, probably from Mexico. Because the tomatoes in this greenhouse were grown from seed, and symptoms did not appear until after initial fruit set, the virus was probably introduced via viruliferous whiteflies. To our knowledge, this is the first report of TYLCV infecting tomato plants in California. References: (1) J. K. Brown and A. M. Idris. Plant Dis. 90:1360, 2006. (2) J. E. Polston et al. Plant Dis. 83:984, 1999. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) R. Salati et al. Phytopathology 92:487, 2002.

Evaluation of Cotton Germ Plasm for Resistance to the Whitefly and Cotton Leaf Crumple (CLCr) Disease and Etiology of CLCr in California's Imperial Valley.

Cotton (Gossypium hirsutum) entries were evaluated for resistance to the whitefly (Bemisia tabaci biotype B) and cotton leaf crumple (CLCr) disease during the 1999 to 2001 growing seasons in the Imperial Valley of California. Entries were evaluated for densities of whitefly adults and nymphs, and for CLCr, by visual rating and squash/dot blot hybridization analyses. Differences in whitefly densities were detected among entries, but none were highly resistant, nor was there any correlation with CLCr disease severity. Entries AP 4103 and AP 6101 had relatively low whitefly densities and were highly susceptible (high CLCr disease severity ratings and viral titers), whereas NK 2387C and DPX 1883 also had low whitefly densities but were highly resistant (no symptoms or detectable viral titers). Other entries showed moderate CLCr resistance, which was independent of whitefly density. Geminivirus DNA-A and DNA-B components were consistently detected in cotton leaves with CLCr symptoms by polymerase chain reaction (PCR) with degenerate begomovirus primers, and full-length DNA-A and DNA-B clones were obtained. Cotton seedlings inoculated with these cloned DNAs by particle bombardment developed CLCr symptoms, and progeny virus was whitefly-transmissible. Sequence analysis revealed that these clones comprised the genome of a California isolate of the bipartite begomovirus Cotton leaf crumple virus (CLCrV-CA). Thus, CLCr disease in the Imperial Valley is caused by CLCrV-CA, and cotton entries with high levels of resistance were identified.

Efficacy of CC traps and seasonal activity of adult Bemisia argentifolii (Homoptera: Aleyrodidae) in Imperial and Palo Verde Valleys, California.

Adult whitefly Bemisia argentifolii Bellows & Perring trap (CC trap) catches were compared with suction type trap catches. CC trap catches were significantly correlated to the suction trap catches. Higher numbers of B. argentifolii adults were caught in CC traps oriented toward an untreated, B. argentifolii-infested, cotton field as compared with traps oriented toward Bermuda grass fields, farm roads, or fallow areas. CC trap catches at five heights above ground (from 0 to 120 cm) were significantly related to each other in choice and no-choice studies. CC trap catches were low in the Imperial and Palo Verde Valleys from late October to early June each of 1996, 1997, and 1998. Trap catches increased with increasing seasonal air temperatures and host availability. Trap catches were adversely affected by wind and rain. Abrupt trap catch increases of 40- to 50-fold for 1-2 d in late June to early July followed by abrupt decreases in adult catches suggest migrating activity of adults from other nearby crop sources.

Use of CC traps with different trap base colors for silverleaf whiteflies (Homoptera: Aleyrodidae), thrips (Thysanoptera: Thripidae), and leafhoppers (Homoptera: Cicadellidae).

During 1996, 1997, and 1999, studies were conducted in cotton, sugar beets, alfalfa, yardlong bean, and peanut fields to compare insect catches in CC traps equipped with different trap base colors. The studies were conducted in southwestern United States, China, and India. The nine colors, white, rum, red, yellow, lime green, spring green, woodland green (dark green), true blue, and black, varied in spectral reflectance in the visible (400-700 nm) and near-infrared (700-1050 nm) portions of spectrum. Lime green, yellow, and spring green were the three most attractive trap base colors for silverleaf whitefly, Bemisia argentifolii Bellows & Perring, and leafhopper, Empoasca spp. adults. The three trap base colors were moderately high in the green, yellow, and orange spectral regions (490-600 nm), resembling the spectral reflectance curve of the abaxial (underleaf) surfaces of green cotton leaves. True blue and white were the most attractive trap base colors for western flower thrips, Frankliniella occidentalis (Pergande), adults. The true blue and white trap bases were moderately high in the blue spectral region (400-480 nm).

A New Bipartite Geminivirus (Begomovirus) Causing Leaf Curl and Crumpling in Cucurbits in the Imperial Valley of California.

During fall 1998, volunteer watermelons (Citrullus lunatus L. (Thunb.) Matsum. & Nakai) showing leaf curl, crumpling, and yellowing symptoms were found in a commercial honeydew melon (Cucumis melo L. subsp. melo Inodorus group) field in the Imperial Valley of California. The plants were infected with a begomovirus (family Geminiviridae, genus Begomovirus) based on (i) a positive response in squash blots probed with a general begomovirus DNA probe (1) and (ii) amplification of DNA-A (≈1.2 kb) and DNA-B (≈1.4 kb) fragments by polymerase chain reaction (PCR) with degenerate DNA-A (PAL1v1978/PAR1c496) and DNA-B (PBL1v2040/PBR1c970) primers, respectively (3). The DNA-A and -B fragments were cloned and sequenced (GenBank accession nos. AF224760 [DNA-A] and AF224761 [DNA-B]). The DNA-A and -B fragments had a nearly identical (99.5%) common region (CR) of 186 (DNA-A) and 187 (DNA-B) nucleotides, indicating they were from the same begomovirus. Database searches conducted with these sequences revealed no high degree of sequence identity (i.e., >90%) with other begomoviruses, including Squash leaf curl virus (SqLCV [2]) from southern California. The partial AC1 sequence (669 nt) was most identical to Tomato severe leaf curl virus (ToSLCV) from Guatemala (83%) and SqLCV (81%), the partial AV1 sequence (135 nt) was most identical to Tomato golden mosaic virus from Brazil (84%) and SqLCV (81%), and the CR was most identical to Squash yellow mottle virus from Costa Rica (81%), ToSLCV (81%), and SqLCV (77%). The partial BV1 sequence (465 nt) was most identical to Bean calico mosaic virus and SqLCV (72%), and the partial BC1 sequence (158 nt) was most identical to SqLCV (75%). Watermelon seedlings bombarded with a DNA extract from infected watermelon volunteers developed crumpling and distortion symptoms, whereas seedlings bombarded with gold particles alone developed no symptoms. Geminivirus infection in symptomatic seedlings was confirmed by PCR. These results suggest a new begomovirus caused the disease symptoms in the watermelon volunteers. Leaf crumpling and curling symptoms were not observed in spring melons in the Imperial Valley in 1999, but on 2 July and 17 August 1999, cantaloupe (C. melo L. subsp. melo Cantalupensis group), muskmelon (C. melo L. subsp. melo Cantalupensis group), and watermelon plants with leaf crumpling and yellowing were found. These plants were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments (97 to 98% identity for CR and partial AC1 sequence). A survey of fall melons, conducted 23 to 24 September 1999, revealed widespread symptoms of leaf curl and crumpling on new growth of muskmelon plants in all seven commercial fields examined (estimated incidence 25 to 50%) and on watermelon volunteers. No such symptoms were observed on leaves of honeydew melons. Symptomatic muskmelon and watermelon leaves, collected from eight locations throughout the Imperial Valley, were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments. Thus, a new begomovirus has emerged in the Imperial Valley; the name Cucurbit leaf crumple virus (CuLCrV) is proposed. References: (1) R. L. Gilbertson et al. Plant Dis. 75: 336, 1991. (2) S. G. Lazarowitz and I. B. Lazdins. Virology 180:58, 1991. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.