Recent CO2 levels promote increased production of the toxin parthenin in an invasive Parthenium hysterophorus biotype.

Recent carbon dioxide (CO2) concentrations promoted higher parthenin concentrations in an invasive Parthenium hysterophorus biotype. Mean concentrations of parthenin, an allelopathic and defensive sesquiterpene lactone, were 49% higher at recent (~400 ppm) than at mid-twentieth-century (~300 ppm) CO2 concentrations, but did not vary in a non-invasive biotype, suggesting that recent increases in atmospheric CO2 may have already altered the chemistry of this destructive weed, potentially contributing to its invasive success.

Suppresive plants as weed management tool: Managing Parthenium hysterophorus under simulated grazing in Australian grasslands.

Parthenium hysterophorus L. is among one of the most problematic invasive grassland weeds in Australia, and in many other countries around the world. It can reduce pasture and livestock production, natural community biodiversity, and negatively affect human and animal health. Sowing of selected suppressive pasture plants in parthenium weed infested grasslands has shown potential to improve efficacy of management. However, such species need to be tested for their ability to suppress weed growth under grazing conditions. The parthenium weed suppressive and fodder production capacity of six selected pasture species [purple pigeon grass (Setaria incrassata), buffel grass (Cenchrus ciliaris), butterfly pea (Clitoria ternatea), Kangaroo grass (Themeda triandra), bull Mitchell grass (Astrebla squarrosa) and Indian bluegrass (Bothriochloa pertusa)] was tested under no (0%), low (25%), moderate (50%) and heavy (75%) simulated grazing pressures in a grassland area of south-central Queensland, Australia. Purple pigeon grass, buffel grass and butterfly pea legume suppressed the growth of parthenium weed by >50% under low and moderate simulated grazing pressures, as well as generating moderate to high amounts of fodder biomass (up to 5.07 t ha-1 per year). Native species, Kangaroo grass and bull Mitchell grass both suppressed the parthenium weed's growth by >50% under low simulated grazing pressure, however, they generated low to moderate amounts of biomass, 1.83 t ha-1 and 2.7 t ha-1 per year, respectively. The sowing of selected suppressive pasture species in parthenium weed infested grasslands with low-to-moderate grazing pressure, assuming this corresponds closely with the simulated treatment, would provide an additional tool to the best practice weed management strategy as well as sustaining fodder production.

Risk factors for community-associated Clostridioides difficile infection in young children.

The majority of paediatric Clostridioides difficile infections (CDI) are community-associated (CA), but few data exist regarding associated risk factors. We conducted a case-control study to evaluate CA-CDI risk factors in young children. Participants were enrolled from eight US sites during October 2014-February 2016. Case-patients were defined as children aged 1-5 years with a positive C. difficile specimen collected as an outpatient or ⩽3 days of hospital admission, who had no healthcare facility admission in the prior 12 weeks and no history of CDI. Each case-patient was matched to one control. Caregivers were interviewed regarding relevant exposures. Multivariable conditional logistic regression was performed. Of 68 pairs, 44.1% were female. More case-patients than controls had a comorbidity (33.3% vs. 12.1%; P = 0.01); recent higher-risk outpatient exposures (34.9% vs. 17.7%; P = 0.03); recent antibiotic use (54.4% vs. 19.4%; P < 0.0001); or recent exposure to a household member with diarrhoea (41.3% vs. 21.5%; P = 0.04). In multivariable analysis, antibiotic exposure in the preceding 12 weeks was significantly associated with CA-CDI (adjusted matched odds ratio, 6.25; 95% CI 2.18-17.96). Improved antibiotic prescribing might reduce CA-CDI in this population. Further evaluation of the potential role of outpatient healthcare and household exposures in C. difficile transmission is needed.

Transmission of Squash vein yellowing virus to and From Cucurbit Weeds and Effects on Sweetpotato Whitefly (Hemiptera: Aleyrodidae) Behavior.

Since 2003, growers of Florida watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] have periodically suffered large losses from a disease caused by Squash vein yellowing virus (SqVYV), which is transmitted by the whitefly Middle East-Asia Minor 1 (MEAM1), formerly Bemisia tabaci (Gennadius) biotype B. Common cucurbit weeds like balsam apple (Momordica charantia L.) and smellmelon [Cucumis melo var. dudaim (L.) Naud.] are natural hosts of SqVYV, and creeping cucumber (Melothria pendula L.) is an experimental host. Study objectives were to compare these weeds and 'Mickylee' watermelon as sources of inoculum for SqVYV via MEAM1 transmission, to determine weed susceptibility to SqVYV, and to evaluate whitefly settling and oviposition behaviors on infected vs. mock-inoculated (inoculated with buffer only) creeping cucumber leaves. We found that the lowest percentage of watermelon recipient plants was infected when balsam apple was used as a source of inoculum. Watermelon was more susceptible to infection than balsam apple or smellmelon. However, all weed species were equally susceptible to SqVYV when inoculated by whitefly. For the first 5 h after release, whiteflies had no preference to settle on infected vs. mock-inoculated creeping cucumber leaves. After 24 h, whiteflies preferred to settle on mock-inoculated leaves, and more eggs were laid on mock-inoculated creeping cucumber leaves than on SqVYV-infected leaves. The transmission experiments (source of inoculum and susceptibility) show these weed species as potential inoculum sources of the virus. The changing settling preference of whiteflies from infected to mock-inoculated plants could lead to rapid spread of virus in the agroecosystem.

First Report of Vine Decline of Mature Watermelon Plants Caused by Olpidium bornovanus.

In late May 2013, collapse of mature watermelon plants (Citrullus lanatus L.) at first harvest occurred in several drip-irrigated commercial fields in the Coachella Valley, California. Above-ground symptoms consisted of chlorosis, wilting, and death of leaves starting at the crown and progressing rapidly towards the tip of vines. Structural roots of collapsed plants appeared healthy but feeder roots exhibited a brownish discoloration. Microscopic examination revealed that almost all epidermal cells of feeder roots contained either sporangia or resting spores of a fungus tentatively identified, based upon morphological characteristics, as Olpidium bornovanus (Sahtiy.) Karling. No other fungi or fungal-like organisms were microscopically observed in or isolated from structural roots, feeder roots, or vascular tissue of collapsed plants. Leaf, root, and peduncle samples from collapsed plants were tested for Melon necrotic spot virus (MNSV), a virus known to be transmitted by O. bornovanus, and Squash vein yellowing virus (SqVYV), a whitefly-transmitted ipomovirus known to cause watermelon vine decline (1). No MNSV was detected using previously described methods (3). No SqVYV was detected by testing total RNA from symptomatic plants (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) with reverse transcription-PCR using previously described primers and methods (1,2). Genomic DNA was extracted from zoospores of the fungus which were obtained from a single-sporangial isolate maintained on watermelon seedlings. Analysis of ITS 1 and 2 gene sequences and a subsequent search in NCBI GenBank revealed a 99% identity to nucleotide sequences for O. bornovanus (Accession Nos. AB205215 and AB665758). To confirm Koch's postulates, roots of three 5-day-old watermelon seedlings were inoculated by exposure to zoospores (~1 × 105) in a beaker for 2 min and then transplanted into pots containing vermiculite. Pots were irrigated daily and incubated in a growth chamber (25°C, 12-h photoperiod). Controls consisted of non-inoculated watermelon seedlings. The experiment was repeated twice. Within 15 days of inoculation, all inoculated plants were stunted, and roots of stunted plants were brown and most root epidermal cells were filled with either sporangia or resting spores of O. bornovanus. Within 30 days of inoculation, 40 to 60% of the inoculated plants died in all three experiments. No other microorganisms were microscopically observed in or isolated from necrotic roots. Control plants remained symptomless over the duration of the study. Although O. bornovanus has been reported as a root pathogen of melons in greenhouse conditions (3), this is the first worldwide report of the fungus as a root pathogen of watermelons and its association with a late season vine decline in the field. Near-saturated soil conditions resulting from a daily irrigation regime during the latter part of the growing season apparently favored extensive root colonization by this indigenous and opportunistic zoosporic fungus, suggesting that growers should exercise care regarding the duration and frequency of irrigation events. References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) S. Adkins et al. Plant Dis. 1119, 2008. (3) M. E. Stanghellini et al. Plant Dis. 94:163, 2010.

First Report of Tomato chlorotic spot virus in Lettuce in Puerto Rico.

Viral diseases have not previously been described in lettuce (Lactuca sativa) in Puerto Rico. In April 2013, lettuce samples from a hydroponic greenhouse in Guayanilla were submitted to the Plant Disease Clinic at the University of Puerto Rico's Juana Díaz Experimental Station. Lettuce plants were symptomatic for virus and had thrips. Eight samples reacted with Tomato spotted wilt virus (TSWV) DAS-ELISA (Envirologix, Portland, ME) and lateral flow immunoassay (Envirologix). Further sampling at the hydroponic greenhouse, which had 45,000 lettuce plants in different growth stages, revealed leaf symptoms of necrotic ringspots and browning with an incidence of 38%. Losses were high because plants had to be destroyed, resulting in $160,000 of lost earnings to date. Symptoms appeared in the younger core leaves 5 days after transplanting and consisted of small chlorotic spots that developed into necrotic ringspots. The leaves became pale, then brown and wilted. In 15-day-old plants, lesions coalesced and within 1 to 2 days, leaf tissue appeared burned. Soft rot in the crown was observed in 5% of the affected plants. Stunting was also observed when young plants were affected. Due to recent identification of Tomato chlorotic spot virus (TCSV) in Puerto Rico (4) and known cross reaction of TSWV serological reagents with closely related tospoviruses, plants were tested for TCSV, TSWV, and Groundnut ringspot virus (GRSV) by reverse transcription (RT)-PCR as previously described (4). Total RNA was extracted from representative symptomatic leaves of two lettuce plants using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and tested by RT-PCR with TCSV-specific nucleocapsid (N) or RNA-dependent RNA polymerase (L) gene primers (4) or movement protein (NSm) gene primers (2). Amplicons of the expected sizes were produced with all three TCSV primer sets from both samples, whereas primers specific for the N gene of TSWV (1) or GRSV (3) did not amplify products from either sample. Three TCSV amplicons (N, L, and NSm) from one sample were gel-purified and cloned (pGEM-T, Promega, Madison, WI). Six clones of each amplicon were sequenced in both directions and consensus sequences were deposited in GenBank (KF819827 to 29). All three genes showed greater than 96% nucleotide identity with all TCSV isolates in GenBank, including 99 to 100% nucleotide identity with previously characterized TCSV isolates from tomato, pepper, and jimsonweed in Puerto Rico (4). Consistent with the identification of TCSV, the known TCSV vector Frankliniella schultzei was identified in the lettuce with an adult population of 10 to 21 thrips per plant. Symptomatic lettuce leaves were used to mechanically inoculate 10-day-old lettuce and 56-day-old tobacco (Nicotiana tabacum) plants. Symptoms reminiscent of the original lettuce developed, and the presence of TCSV was confirmed by RT-PCR as described above. This is the first report of TCSV infection of lettuce in Puerto Rico and demonstrates that TCSV can be a limiting factor to lettuce production here and elsewhere in the Caribbean. References: (1) S. Adkins and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) M. S. Silva et al. Arch. Virol. 146:1267, 2001. (3) C. G. Webster et al. Virology 413:216, 2011. (4) C. G. Webster et al. Plant Health Progress doi:10.1094/PHP-2013-0812-01-BR, 2013.

Development and Field Evaluation of Multiple Virus-Resistant Bottle Gourd (Lagenaria siceraria).

In an effort to develop bottle gourd (Lagenaria siceraria) as a widely adapted rootstock for watermelon grafting, we sought to identify lines with broad resistance to several cucurbit viruses that are economically important in the United States. Preliminary analysis under greenhouse conditions indicated that the currently available commercial watermelon rootstocks were either highly susceptible or somewhat tolerant to one or more viruses. However, in greenhouse screening, several breeding lines of bottle gourd displayed broad-spectrum resistance to four viruses tested, including Zucchini yellow mosaic virus, Watermelon mosaic virus (WMV), Papaya ringspot virus watermelon strain (PRSV-W), and Squash vein yellowing virus. Resistance to PRSV-W and WMV was confirmed through field trials in two consecutive years at two different locations in South Carolina. Two breeding lines (USVL#1-8 and USVL#5-5) with broad-spectrum virus resistance could be useful materials for watermelon rootstock development.

First Report of Squash vein yellowing virus Affecting Watermelon and Bitter Gourd in Puerto Rico.

In 2005, symptoms of watermelon vine decline (WVD) were observed on a 200-acre watermelon farm in Santa Isabel, on the south central coast of Puerto Rico. WVD symptoms included leaf curling, mosaics, and internode necrosis. In early growth stages of WVD, reduced vigor and plant stunting occurred. At flowering, symptoms progressed to necrosis and wilting of vines. A 2006 to 2007 survey demonstrated that fungal pathogens were not associated with the presence of WVD symptoms (3,4). By 2006, other watermelon fields were also affected. Field trials in 2007 and 2008 with insect-proof cages and insecticides suggested a role of whiteflies (Bemisia tabaci) in the transmission of a virus (3,4). Here, we report that watermelon and pumpkin plants were successfully infected in Puerto Rico by mechanical inoculation and through B. tabaci transmission assays, similarly to transmissions previously conducted in Florida with Squash vein yellowing virus (SqVYV) (1). In addition, plants of Cucurbita moschata exhibited vein clearing symptoms typical of SqVYV after mechanical inoculation with extracts from watermelon plants with WVD symptoms. In 2011, eight watermelon samples from plants exhibiting WVD syndrome were collected in Guánica, Santa Isabel, Juana Díaz, and Mayagüez, and two Momordica charantia samples were collected from Mayagüez. RNA was extracted from all 10 original samples, as well as from plants that were used in mechanical and vector transmission assays, using RNeasy Plant Mini Kit (Qiagen, Valencia, California), and all samples were found positive for SqVYV by reverse transcription-PCR, using previously described primers and methods (1,2). In all cases, a single ~1-kb PCR fragment was revealed, and PCR fragments from four samples were selected for direct sequencing. All sequences showed high levels (>99%) of nucleotide identity with SqVYV sequences from Florida (JF897989, JF897985, and JF897984). Sequences of the SqVYV CP gene from Puerto Rico were deposited in GenBank under accession numbers KC713961 to KC713964. To our knowledge, this is the first report of SqVYV in Puerto Rico associated with WVD syndrome in cucurbits, and thus has implications for management of viral diseases of watermelon in the Caribbean. This is also the first detection of SqVYV outside of the continental United States in both watermelon and a wild species, M. charantia (bitter gourd). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) S. Adkins et al. Plant Dis. 92:1119, 2008. (3) C. Estévez de Jensen et al. Phytopathology 98:S52, 2008. (4) L. Polanco-Florián. El marchitamiento súbito de la sandía [Citrullus lanatus (Thumb.) Matsum & Nakai]. M.S. thesis, University of Puerto Rico, Mayagüez, PR, 2009.

Odontonema cuspidatum and Psychotria punctata, Two New Hosts of Cucumber mosaic virus in the United States.

Cucumber mosaic virus (CMV) has a reported host range of 750 to 1,200 species (2,3) that includes weeds, wild plants, crops, and ornamentals. Two new CMV hosts were recently identified in Florida. In July 2011, leaves of Odontonema cuspidatum (firespike), a member of the Acanthaceae, with virus-like symptoms were sent to FDACS-DPI. Firespike is an ornamental shrub native to Mexico with evergreen ovate leaves tapering to a pointed tip. Leaf symptoms included severe leaf distortion with some subtle yellowing or mosaic on younger leaves. Pink-red crystals were seen in leaf strips stained with the nucleic acid stain Azure A, indicating a viral infection. In January 2012, leaves of Psychotria punctata (dotted wild coffee), a member of the Rubiaceae, with virus-like symptoms were sent to FDACS-DPI. Dotted wild coffee is a small exotic tropical tree found in south Florida with many tiny leaf nodules inhabited by endosymbiotic bacteria. In addition to the nodules, these leaves had many large dark green ringspots surrounded with a yellow halo. Both samples were positive for CMV when tested with ImmunoStrips and/or by conventional ELISA using CMV antiserum (Agdia, Elkhart, IN). To confirm CMV infection, reverse transcriptase (RT)-PCR on total RNA from a leaf sample of each plant species was used with previously published cucumovirus primers (1). An expected ~940 bp product was amplified from each sample and cloned into pGEM-T (Promega, Madison, WI). Ten clones from each plant species were sequenced in both directions. After removal of primer sequences, the 906 bp products were 96.3% identical with each other and showed 96.8 to 98.9% nucleotide identity with CMV sequences from Hungary, the United States, and Austria (GenBank Accession Nos. AF517802, U20668, and HQ916354, respectively). Identification of CMV infection in these two species expands the known host range and therefore the reservoir of this plant virus. This has implications for the ornamental industry in general and Florida farmers in particular. References: (1) S. K. Choi et al. J. Virol. Methods 83:67, 1999. (2) E. J. Sikora. Cucumber Mosaic Virus, Pant Disease Notes, Alabama Cooperative Extensions System, retrieved online at http://www.aces.edu/pubs/docs/A/ANR-0868/ANR-0868.pdf , 2004. (3) T. A. Zitter and J. F. Murphy. The Plant Health Instructor. DOI: 10.1094/PHI-I-2009-0518-01, 2009.

Outbreak of Cucurbit Powdery Mildew on Watermelon Fruit Caused by Podosphaera xanthii in Southwest Florida.

Cucurbit powdery mildew caused by the obligate parasite Podosphaera xanthii occurs commonly on foliage, petioles, and stems of most cucurbit crops grown in the United States. (3). However, in the field, fruit infection on cucurbits including watermelon (Citrullus lanatus), is rarely, if ever, observed (2). Consequently, it was atypical when severe powdery mildew-like symptoms were observed on seedless and seeded watermelon fruit on several commercial farms in southwestern Florida during November and December 2010. Severe powdery mildew was also observed on 'Tri-X 313' and 'Mickey Lee' fruit grown at SWFREC, Immokalee, FL. Infected fruit developed poorly and were not marketable. Powdery mildew symptoms were mainly observed on young immature fruit, but not on mature older fruit. Abundant powdery mildew conidia occurred on fruit surface, but not on the leaves. Conidia were produced in chains and averaged 35 × 21 µm. Observation of conidia in 3% KOH indicated the presence of fibrosin bodies commonly found in the cucurbit powdery mildew genus Podosphaera (3). Orange-to-dark brown chasmothecia (formerly cleisthothecia) containing a single ascus were detected on the surface of some fruit samples. Conidial DNA was subjected to PCR using specific primers designed to amplify the internal transcribed spacer (ITS) region of Podosphaera (4). The resulting amplicons were sequenced and found to be 100% identical to the ITS sequences of P. xanthii in the NCBI database (D84387, EU367960, AY450961, AB040322, AB040315). Sequences from the watermelon fruit isolate were also identical to several P. fusca (synonym P. xanthii), P. phaseoli (GQ927253), and P. balsaminae (AB462803) sequences. On the basis of morphological characteristics and ITS sequence analysis, the pathogen infecting watermelon fruit can be considered as P. xanthii (1,3,4). The powdery mildew isolate from watermelon fruit was maintained on cotyledons of squash (Cucurbita pepo, 'Early Prolific Straight Neck'). Cotyledons and leaves of five plants each of various cucurbits and beans were inoculated with 10 µl of a conidial suspension (105conidia/ml) in water (0.02% Tween 20). Two weeks after inoculation, abundant conidia were observed on cucumber (Cucumis sativus, 'SMR-58') and melon (Cucumis melo) powdery mildew race differentials 'Iran H' and 'Vedrantais'. However, no growth was observed on melon differentials 'PI 414723', 'Edisto 47', 'PMR 5', 'PMR 45', 'MR 1', and 'WMR 29' (2,3). The powdery mildew isolate from watermelon fruit behaved as melon race 1 (3). Mycelium and conidia were also observed on fruit surface of watermelon 'Sugar Baby' and a susceptible U.S. plant introduction (PI 538888) 3 weeks after inoculation. However, the disease was not as severe as what was observed in the fields in fall 2010. The pathogen did not grow on plants of Impatiens balsamina or on select bean (Phaseolus vulgaris) cultivars ('Red Kidney', 'Kentucky Blue', and 'Derby Bush'), but did grow and produce abundant conidia on 'Pinto bush bean'. Powdery mildew on watermelon fruit in production fields can be considered as a potentially new and serious threat requiring further studies to develop management strategies. References: (1) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000. (2) A. R. Davis et al. J. Am. Soc. Hortic. Sci. 132:790, 2007. (3) M. T. McGrath and C. E. Thomas. In: Compendium of Cucurbit Diseases. American Phytopathological Society, St. Paul, MN, 1996. (4) S. Takamatsu and Y. Kano. Mycoscience 42:135, 2001.

Cucurbit yellow stunting disorder virus Detected in Pigweed in Florida.

Pigweeds (genus Amaranthus) are problematic weeds in crop production throughout the world and are responsible for significant yield losses in many crops (2). Members of this genus can produce hundreds of thousands of seeds per plant and are also capable of supporting populations of important crop pathogens including viruses, nematodes, fungi, and oomycetes. Thirty-one pigweed samples (tentatively identified as Amaranthus lividus L. based on leaf notch and growth habit) were collected in November and December of 2009 from a watermelon field near Immokalee, FL, previously found to contain watermelon plants infected with three whitefly-transmitted viruses: Cucurbit yellow stunting disorder virus (CYSDV), Cucurbit leaf crumple virus (CuLCrV), and Squash vein yellowing virus (SqVYV). Although no obvious virus symptoms were observed on any of the pigweed plants, whiteflies (Bemisia tabaci), a known vector of CYSDV, CuLCrV, and SqVYV, were observed on leaves. Consequently, replica tissue blots were made from all pigweed samples and tested independently by tissue blot nucleic acid hybridization assay for CYSDV, CuLCrV, or SqVYV (3). Tissue blots indicated CYSDV infection in six pigweed samples. Neither CuLCrV nor SqVYV was detected. Three of the tissue blot-positive pigweed samples were further tested by reverse transcription (RT)-PCR amplification from total RNA (extracted from leaf tissue with TRIzol Reagent [Invitrogen, Carlsbad, CA]) with HSP70 and coat protein (CP) gene primers (1). HSP70 and CP gene RT-PCR products of the expected sizes (175 and 707 nt, respectively) were amplified, sequenced, and found to be 100% identical for all three pigweed samples. The partial HSP70 gene sequence from pigweed shared 98.3 to 100% nucleotide identity with CYSDV isolates from Arizona, California, and Spain (GenBank Accession Nos. FJ492808, EU596530, and NC_004810, respectively). The partial CP gene sequence from pigweed shared 88.8 to 100% nucleotide identity with CYSDV isolates from Arizona, Saudi Arabia, Texas, and Spain (GenBank Accession Nos. EF210558, AF312811, AF312806, and AF312808, respectively). To our knowledge, this is the first report of CYSDV infection of pigweed in Florida. Infection of redroot pigweed (A. retroflexus) was recently reported in California (4). These results collectively indicate that control of noncucurbit weeds may be important for effective management of CYSDV in cucurbit crops. References: (1) S. Adkins et al. Online publication. doi:10.1094/PHP-2009-1118-01-BR. Plant Health Progress, 2009. (2) L. Holm et al. World's Weeds: Natural Histories and Distributions. John Wiley and Sons, Inc. New York, NY, 1997. (3) W. W. Turechek et al. Phytopathology 100:1194, 2010. (4) W. M. Wintermantel et al. Plant Dis. 93:685, 2009.

First Report of Tomato chlorosis virus Infecting Tomato in Georgia.

Tomato yellow leaf curl virus (TYLCV) and Tomato spotted wilt virus (TSWV) are prevalent in field-grown tomato (Solanum lycopersicum) production in Georgia. Typical TYLCV symptoms were observed during varietal trials in fall 2009 and 2010 to screen genotypes against TYLCV at the Coastal Plain Experiment Station, Tifton, GA. However, foliar symptoms atypical of TYLCV including interveinal chlorosis, purpling, brittleness, and mottling on upper and middle leaves and bronzing and intense interveinal chlorosis on lower leaves were also observed. Heavy whitefly (Bemisia tabaci (Gennadius), B biotype) infestation was also observed on all tomato genotypes. Preliminary tests (PCR and nucleic acid hybridization) in fall 2009 indicated the presence of TYLCV, TSWV, Cucumber mosaic virus, and Tomato chlorosis virus (ToCV); all with the exception of ToCV have been reported in Georgia. Sixteen additional symptomatic leaf samples were randomly collected in fall 2010 and the preliminary results from 2009 were used to guide testing. DNA and RNA were individually extracted using commercially available kits and used for PCR testing for ToCV, TYLCV, and TSWV. Reverse transcription (RT)-PCR with ToCV CP gene specific primers (4) produced approximately 750-bp amplicons from nine of the 16 leaf samples. Four of the nine CP gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 100.0% identical with each other (GenBank Accession Nos. HQ879840 to HQ879843). They were 99.3 to 99.5%, 97.2 to 97.5%, and 98.6 to 98.9% identical to ToCV CP sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. The presence of ToCV was confirmed by amplifying a portion of the HSP70h gene using the primers HSP-1F and HSP-1R (1). RT-PCR produced approximately 900-bp amplicons in the same nine samples. Four HSP70h gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 99.7% identical to each other (Accession Nos. HQ879844 to HQ879847). They were 99.2 to 99.5%, 98.0 to 98.4%, and 98.9 to 99.3% identical to HSP70h sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. TYLCV was also detected in all 16 samples by PCR using degenerate begomovirus primers PAL1v 1978 and PARIc 496 (3) followed by sequencing. TSWV was also detected in two of the ToCVinfected samples by RT-PCR with TSWV N gene specific primers (2) followed by sequencing. To our knowledge, this is the first report of the natural occurrence of ToCV in Georgia. Further studies are required to quantify the yield losses from ToCV alone and synergistic interactions between ToCV in combination with TSWV and/or TYLCV in tomato production in Georgia. References: (1) T. Hirota et al. J. Gen. Plant Pathol. 76:168, 2010. (2) R. K. Jain et al. Plant Dis. 82:900, 1998. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) L. Segev et al. Plant Dis. 88:1160, 2004.

Cucurbit leaf crumple virus Identified in Common Bean in Florida.

Virus-like symptoms of leaf deformation and rugosity, especially of younger leaves, and a mild mosaic were observed on fresh market common (green) bean (Phaseolus vulgaris L.) plants in Hendry County in southwest Florida in December of 2007 and again in February of 2008. All bean fields were adjacent to watermelon fields in which Cucurbit leaf crumple virus (CuLCrV), Squash vein yellowing virus (SqVYV), and Papaya ringspot virus type W (PRSV-W) infections had previously been confirmed (fall of 2007) by PCR, reverse transcription (RT)-PCR, and/or ELISA. Whiteflies, Bemisia tabaci, were observed on both bean and watermelon plants in December and February. Fifteen samples (eleven with symptoms) were collected in December and two (both with symptoms) in February. Initial ELISA assays using commercially available antisera for potyviruses or Cucumber mosaic virus (Agdia, Elkhart, IN) were negative. Total nucleic acids were extracted and used for PCR testing. All samples tested negative by RT-PCR using specific primers for SqVYV, PRSV-W, and Cucurbit yellow stunting disorder virus, and degenerate primers for potyviruses. Ten of fifteen December samples (ten of eleven symptomatic samples) and both February samples yielded PCR products of the expected size with the degenerate begomovirus primers, PAR1c496/PAL1v1978, which amplify a portion of the begomovirus A component (3). PCR products from three December and both February samples were cloned and sequenced. The 1,159-nt PCR products shared 99% identity with each other and 96% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF256200 and AF224760, respectively). Additional degenerate begomovirus primers PBL1v2040/PCRc154, which amplify a 381-nt portion of the hypervariable region of the begomovirus B component (3), and AC1048/AV494, which amplify a 533-nt portion of a conserved region of the coat protein gene (4), were used to confirm the identity of CuLCrV in the three December samples. The PBL1v2040/PCRc154 PCR products shared 98 to 99% identity with each other and 94 to 95% identity with the corresponding region of B component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF327559 and AF224761, respectively), whereas the AC1048/AV494 PCR products shared 99% identity with each other and 97% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates. Nucleic acid dot-blot hybridization assays of sap from homogenized leaves of the three December samples (from which the PCR product clones were obtained) with a digoxigenin-labeled CuLCrV cDNA probe also confirmed the presence of CuLCrV. Although CuLCrV has been reported to experimentally infect common bean and tobacco (2), to our knowledge, this is the first report of CuLCrV infecting any noncucurbit host in Florida. This finding suggests that CuLCrV may be more widely distributed than previously known in Florida (1) and that common bean (and potentially other legumes) are potential reservoirs for CuLCrV. References: (1) F. Akad et al. Plant Dis. 92:648, 2008. (2) J. K. Brown et al. Phytopathology 92:734, 2002. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Mimicking a semi-arid tropical environment achieves dormancy alleviation for seeds of Australian native Goodeniaceae and Asteraceae.

BACKGROUND AND AIMS: Seed physiological dormancy (PD) limits the use and conservation of some of Queensland's (Qld) native forb species. It was hypothesised that optimum dormancy-alleviating treatments would reflect environmental conditions that seeds experience in situ, and this premise was tested for PD seeds of four species native to south-west Qld. METHODS: High temperatures and increased rainfall during summer are characteristic of this semi-arid tropical environment. Ex situ treatments were designed to mimic conditions that seeds dispersed in spring experience during the summer months before germinating in cooler autumn temperatures. Seeds received between 4 and 20 weeks of a dry after-ripening (DAR), warm stratification or dry/wet cycling treatment (DAR interspersed with short periods of warm stratification), in darkness, before being transferred to germination test conditions. In addition, natural dormancy alleviation of one of the Goodeniaceae species was investigated in situ. KEY RESULTS: Dry/wet cycling resulted in higher levels of germination of Actinobole uliginosum (Asteraceae), Goodenia cycloptera and Velleia glabrata (Goodeniaceae) when compared with constant DAR or stratification, while Goodenia fascicularis (Goodeniaceae) responded better to short durations of warm stratification. Long durations of DAR partially alleviated PD of A. uliginosum; however, stratification induced and maintained dormancy of this species. Modifications to the dry/wet cycling treatment and germination test conditions based on data collected in situ enabled germination of G. cycloptera and V. glabrata to be further improved. CONCLUSIONS: Treatments designed using temperature, relative humidity and rainfall data for the period between natural seed dispersal and germination can successfully alleviate PD. Differences between the four species in conditions that resulted in maximum germination indicate that, in addition to responding to broad-scale climate patterns, species may be adapted to particular microsites and/or seasonal conditions.

Detection of Cucurbit leaf crumple virus in Florida Cucurbits.

In October of 2006, yellow straightneck and zucchini squash plants (Cucurbita pepo L.) with crumpled, curled, thickened leaves were found in St. Johns and Marion counties in central Florida, respectively. Both locations had high populations of the whitefly, Bemisia tabaci. Incidences of symptomatic plants were greater than 95% in three squash fields (33 ha total) in St. Johns County and 35% in an experimental plot in Marion County. Twenty-three samples were collected from symptomatic plants (two from St. Johns County and 21 from Marion County). DNA was extracted for PCR and tested for the presence of begomoviruses using the following pairs of degenerate primers: AC1048/AV494, which amplifies a conserved region of the coat protein gene (2), PAR1c496/PAL1v1978, which amplifies a region of the begomovirus A component, and PBL1v2040/PCRc154, which amplifies a hypervariable region of the begomovirus B component (1). All squash samples yielded amplicons of sizes expected for a bipartite begomovirus: 1,159 nt with PAR1c496/PAL1v1978, 550 nt with AC1048/AV494, and 493 nt with PBL1v2040/PCRc154. The 1,159- and 493-nt amplicons obtained from two squash plants were cloned and sequenced. The 1,159 nt sequences from both plants shared 98% sequence identity with each other and 97% identity with equivalent regions of the A component of Cucurbit leaf crumple virus (CuLCrV) from Arizona and California (GenBank Accession Nos. AF256200 and AF224760, respectively). The 493-nt sequences amplified with PBL1v2040/PCRc154 were identical and shared a 96% identity with CuLCrV sequence (GenBank Accession No. AF327559) from Arizona and 97% identity with CuLCrV B component sequence (GenBank Accession No. AF224761) from California. Leaves were collected from eight symptomatic squash plants from Citra, FL and used for whitefly transmission assays. Approximately 100 adults of Bemisia tabaci biotype B were released onto each caged leaf and given a 24-h acquisition access period, after which a healthy squash seedling was introduced. Symptoms developed within 10 days on all test plants, and the presence of CuLCrV was confirmed by PCR assays, (primer pairs PAR1c496/PAL1v1978 and PBL1v2040/PCRc154) followed by sequencing. In 2007, similar symptoms were seen in several locations around the state. The same assays confirmed the presence of CuLCrV in watermelon (Citrullus lanatus L.) and squash in the following counties: Collier and Hendry in southwest Florida and Hillsborough, Manatee, and Sarasota in west-central Florida. To our knowledge, this is the first report of CuLCrV, and the first report of any begomovirus in cucurbits in Florida. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Bidens mottle virus and Apium virus Y Identified in Ammi majus in Florida.

Ammi majus (bishop's weed), a member of the Apiaceae, is grown from seed for cut flowers in South Florida. In March 2005, plants were found to be showing virus-like symptoms including mosaic, vein clearing, and leaf rugosity (3) that rendered their flowers unmarketable. Inclusion morphology in epidermal strips from these infected plants indicated the presence of one or more potyviruses. This was confirmed by ELISA with commercially available antiserum for potyvirus identification (Agdia, Elkhart, IN). Clover yellow vein virus (ClYVV) was identified by sequencing and confirmed with specific antiserum (4). However, ClYVV was not identified in all potyvirus-infected samples from 2005, indicating the presence of one or more additional potyviruses. Bidens mottle virus (BiMoV) was subsequently identified in one of three potyvirus-infected samples by immunodiffusion tests using specific antiserum for BiMoV (Department of Plant Pathology, University of Florida), cylindrical inclusion morphology in epidermal strips, host range data, and sequencing of cloned reverse transcription (RT)-PCR products from degenerate potyvirus primers (2). Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence (GenBank Accession No. EU255631) were 95 and 98% identical, respectively, to a Florida isolate of BiMoV recently reported from tropical soda apple (1). Similar virus-like symptoms were again observed in A. majus in January 2007 and persisted through March. ELISA testing again indicated the presence of a potyvirus. However, neither ClYVV nor BiMoV were identified in the initial 2007 samples. Instead, sequence analysis of the cloned RT-PCR products amplified with degenerate potyvirus primers (2) from seven potyvirus-infected samples collected on two dates in January and one each in February and March revealed the presence of Apium virus Y (ApVY). The 3' terminal portion of the genome (GenBank Accession No. EU255632) was found to be 90 to 91% identical to ApVY sequences in GenBank at the nucleotide level. Deduced amino acid sequences of the NIb and CP regions of these RT-PCR products were 96 and 95% identical, respectively, to ApVY sequences in GenBank. One of these seven ApVY-infected samples (collected in March 2007) was determined to be coinfected with BiMoV by sequence analysis of the cloned RT-PCR products. Six clones were sequenced. Three were determined to be ApVY as indicated above. Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence from the other three clones were 95 and 97% identical, respectively, to the 2005 A. majus BiMoV isolate. Although ClYVV and BiMoV have previously been reported in other hosts in Florida, to the best of our knowledge, this is the first report of BiMoV and ApVY in A. majus anywhere and the first report of ApVY in North America. References: (1.) C. A. Baker et al. Plant Dis. 91:905, 2007. (2.) A. Gibbs and A. J. Mackenzie. J. Virol. Methods 63:9, 1997. (3.) M. S. Irey et al. (Abstr.) Phytopathology (suppl.)95:S46, 2005. (4.) M. S. Irey et al. Plant Dis. 90:380, 2006.

Bidens mottle virus Identified in Tropical Soda Apple in Florida.

Tropical soda apple (TSA) (Solanum viarum Dunal), a plant native to South America, was first identified in Florida in 1988 (4). It rapidly became a noxious weed in pastures throughout the state and it is known to be a reservoir for Cucumber mosaic virus, Potato leafroll virus, Potato virus Y (PVY), Tobacco etch virus (TEV), Tomato mosaic virus, and Tomato mottle virus, viruses that infect important vegetable crops in Florida (3). During a routine survey of Florida weeds during May of 2004, a TSA plant with chlorotic, young leaves found near Okeechobee, FL was determined to be infected with a potyvirus by using a commercially available enzyme linked immunosorbent assay kit (Agdia, Elkhart, IN). The results of a host range study indicated this potyvirus was neither PVY nor TEV. The virus caused local lesions in Chenopodium amaranticolor and systemic symptoms in C quinoa, Coreopsis sp. (C. A. Baker, unpublished), Helianthus annus, Nicotiana benthamiana, Petunia × hybrida, Verbena hybrida, and Zinnia elegans. It did not infect Gomphrena globosa, N. glutinosa, Pisum sativum, or Phaseolus vulgaris (1). Cylindrical inclusions consistent with those observed in plants infected with Bidens mottle virus (BiMoV) were observed in Z. elegans. Immunodiffusion tests with antiserum to BiMoV (Department of Plant Pathology, University of Florida) gave a reaction of identity with leaf extracts of the symptomatic zinnia, a known sample of BiMoV originally isolated from Bidens pilosa and a recent isolate of BiMoV from lettuce in Belle Glade, FL (C. A. Baker and R. Raid, unpublished). A partial polyprotein gene fragment (GenBank Accession No. EF467235) was amplified from total RNA of an inoculated C. quinoa plant by reverse transcription (RT)-PCR with previously described degenerate potyvirus primers (2). Analysis of the RT-PCR product sequence confirmed the host range results and indicated that the potyvirus infecting TSA was neither PVY nor TEV. However, the nucleotide and deduced amino acid sequences of a 247-bp portion of the RT-PCR product were 94 and 98% identical, respectively, with the coat protein sequence (GenBank Accession No. AF538686) of Sunflower chlorotic spot virus (SCSV). SCSV is a tentative potyvirus species described from Taiwan that is not yet recognized as an accepted species by the International Committee on Taxonomy of Viruses. On the basis of our concurrent host range, inclusion body, and serological data, it is likely that SCSV is in actuality the previously described and currently accepted potyvirus species BiMoV, for which no previous sequence data existed. As part of a comprehensive viral disease management plan, it is recommended that TSA plants growing in and around lettuce-production areas be controlled along with other weed hosts of this virus. References: (1) A. A. Brunt et al., eds. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20 at http://biology.anu.edu.au/Groups/MES/vide/ , 1996. (2) A. Gibbs and A. J. Mackenzie. Virol. Methods 63:9, 1997. (3) R. J. McGovern et al. Int. J. Pest Manag. 40:270, 1994. (4) J. J. Mullahey et al. Weed Technol. 7:783, 1993.

Squash vein yellowing virus Identified in Watermelon (Citrullus lanatus) in Indiana.

During September 2006, moderate vine decline symptoms including vine collapse and wilt and root rot were observed on numerous watermelon plants growing in a commercial field in Sullivan County, Indiana. No symptoms were observed on the fruit. Six plants displaying typical vine decline symptoms were collected and assayed for potyvirus infection and subsequently for Squash vein yellowing virus (SqVYV) and Papaya ringspot virus type W (PRSV-W). SqVYV is a whitefly-transmitted member of the Potyviridae, recently shown to cause watermelon vine decline in Florida (1,4). Plants infected with SqVYV in Florida are also frequently infected with PRSV-W, although SqVYV is sufficient for watermelon vine decline. The six field samples harbored one or more potyviruses as determined by ELISA (Agdia, Elkhart, IN). Mechanical inoculation of squash (Cucurbita pepo) and watermelon with sap from three of the field samples induced mosaic symptoms in both that are typical of potyviruses. Vein yellowing in squash and plant death in watermelon typical of SqVYV (1) later developed in plants inoculated with one field sample. A coat protein gene fragment was amplified by reverse transcription (RT)-PCR with SqVYV primers (1) from total RNA of five of the six field samples and also from the symptomatic, inoculated plants. Nucleotide and deduced amino acid sequences of a 957-bp region of the RT-PCR product (primer sequences deleted prior to analysis) were 100% identical to SqVYV (GenBank accession No. DQ812125). PRSV-W also was identified in two of the five SqVYV-infected field samples by ELISA (Agdia) and by sequence analysis of a 3' genome fragment amplified by RT-PCR with previously described degenerate potyvirus primers (3). No evidence for infection by other potyviruses was obtained. To our knowledge, this is the first report of SqVYV in Indiana and the first report of the virus anywhere outside of Florida. The whitefly (Bemisia tabaci, B strain) vector of SqVYV is relatively uncommon in Indiana and the cold winter temperatures make it unlikely that any SqVYV-infected watermelon vines or whiteflies will overseason, necessitating reintroductions of virus and vector each season. We feel that the moderate and restricted occurrence of SqVYV in Indiana observed in September 2006 should pose little or no threat to commercial watermelon production in Indiana and should not cause growers to alter their growing practices. The occurrence of SqVYV in Indiana does not appear to explain the similar symptoms of mature watermelon vine decline (MWVD) that has been observed in Indiana since the 1980s. In contrast with the insect vectored SqVYV, MWVD seems to be caused by a soilborne biological agent (2). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) D. S. Egel et al. Online publication. doi:10.1094/PHP-2000-1227-01-HN. Plant Health Progress, 2000. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) P. Roberts et al. Citrus Veg. Mag. December 12, 2004.

Tropical soda apple mosaic virus Identified in Solanum capsicoides in Florida.

Red soda apple (Solanum capsicoides All.), a member of the Solanaceae, is a weed originally from Brazil (3). It is a perennial in southern Florida and is characterized by abundant prickles on stems, petioles, and leaves. Prickles on stems are more dense than those on its larger, noxious weed relative, tropical soda apple (Solanum viarum Dunal), and the mature red soda apple fruits are bright red in contrast to the yellow fruits of tropical soda apple (2). Virus-like foliar symptoms of light and dark green mosaic were observed on the leaves of a red soda apple in a Lee County cow pasture during a tropical soda apple survey during the fall of 2004. The appearance of necrotic local lesions following inoculation of Nicotiana tabacum cv. Xanthi nc with sap from the symptomatic red soda apple leaves suggested the presence of a tobamovirus. Tropical soda apple mosaic virus (TSAMV), a recently described tobamovirus isolated from tropical soda apple in Florida, was specifically identified by a double-antibody sandwich-ELISA (1). An additional six similarly symptomatic red soda apple plants were later collected in the Devils Garden area of Hendry County. Inoculation of N. tabacum cv. Xanthi nc with sap from each of these symptomatic plants also resulted in necrotic local lesions. Sequence analysis of the TSAMV coat protein (CP) gene amplified from total RNA by reverse transcription (RT)-PCR with a mixture of upstream (SolA5'CPv = 5'-GAACTTWCAGAAGMAGTYGTTGATGAGTT-3'; SolB5'CPv = 5'-GAACTCACTGARRMRGTTGTTGAKGAGTT-3') and downstream (SolA3'CPvc = 5'-CCCTTCGATTTAAGTGGAGGGAAAAAC-3'; SolB3'CPvc = 5'-CGTTTMKATTYAAGTGGASGRAHAAMCACT-3') degenerate primers flanking the CP gene of Solanaceae-infecting tobamoviruses confirmed the presence of TSAMV in all plants from both locations. Nucleotide and deduced amino acid sequences of the 483-bp CP gene were both 98 to 99% identical to the original TSAMV CP gene sequences in GenBank (Accession No. AY956381). TSAMV was previously identified in tropical soda apple in these two locations in Lee and Hendry counties and three other areas in Florida (1). Sequence analysis of the RT-PCR products also revealed the presence of Tomato mosaic virus in the plant from Lee County. To our knowledge, this represents the first report of natural TSAMV infection of any host other than tropical soda apple and suggests that TSAMV may be more widely distributed in solanaceous weeds than initially reported. References: (1) S. Adkins et al. Plant Dis. 91:287, 2007. (2) N. Coile. Fla. Dep. Agric. Consum. Serv. Div. Plant Ind. Bot. Circ. 27, 1993. (3) U.S. Dep. Agric., NRCS. The PLANTS Database. National Plant Data Center. Baton Rouge, LA. Published online, 2006.