Phage therapy for plant disease control.

Bacteria cause a number of economically important plant diseases. Bacterial outbreaks are generally problematic to control due to lack of effective bactericides and to resistance development. Bacteriophages have recently been evaluated for controlling a number of phytobacteria and are now commercially available for some diseases. Major challenges of agricultural use of phages arise from the inherent diversity of target bacteria, high probability of resistance development, and weak phage persistence in the plant environment. Approaches for resistance management--by applying phage mixtures and host-range mutant phages and, for increasing residual activity, by employing protective formulations, avoiding sunlight, and utilizing propagating bacterial strains--resulted in better efficacy and reliability. Deployment of phage therapy as part of an integrated disease management strategy, which includes the use of genetic control, cultural control, biological control, and chemical control, also has been investigated and will likely increase in the future.

Suppression of the bacterial spot pathogen Xanthomonas euvesicatoria on tomato leaves by an attenuated mutant of Xanthomonas perforans.

A bacteriocin-producing strain of the bacterial spot of tomato plant pathogen, Xanthomonas perforans, with attenuated pathogenicity was deployed for biocontrol of a bacteriocin-sensitive strain of the genetically closely related bacterial spot of tomato plant pathogen, X. euvesicatoria. The attenuated mutant (91-118DeltaopgHDeltabcnB) of X. perforans was tested in leaf tissue and shown to significantly inhibit internal populations of the wild-type X. euvesicatoria strain although significantly less than the wild-type 91-118 strain, whereas in a phyllosphere inhibition assay, the mutant strain reduced epiphytic populations comparably to 91-118. Thus, the attenuated mutant limited the sensitive bacterium more efficiently on the leaf surface than inside the leaf. In field experiments, weekly application of 91-118DeltaopgHDeltabcnB significantly reduced X. euvesicatoria populations compared to the growers' standard control (copper hydroxide and mancozeb applied weekly and acibenzolar-S-methyl applied every 2 weeks). The biological control agent, 91-118DeltaopgHDeltabcnB, applied every 2 weeks also significantly reduced X. euvesicatoria populations in one season but was not significantly different from the growers' standard control. Potentially, attenuated pathogenic strains could be deployed as biological control agents in order to improve disease control of foliar plant pathogens.

Bacteriophages for plant disease control.

The use of phages for disease control is a fast expanding area of plant protection with great potential to replace the chemical control measures now prevalent. Phages can be used effectively as part of integrated disease management strategies. The relative ease of preparing phage treatments and low cost of production of these agents make them good candidates for widespread use in developing countries as well. However, the efficacy of phages, as is true of many biological control agents, depends greatly on prevailing environmental factors as well as on susceptibility of the target organism. Great care is necessary during development, production and application of phage treatments. In addition, constant monitoring for the emergence of resistant bacterial strains is essential. Phage-based disease control management is a dynamic process with a need for continuous adjustment of the phage preparation in order to effectively fight potentially adapting pathogenic bacteria.

Factors affecting survival of bacteriophage on tomato leaf surfaces.

The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if applied on the day of phage application but not if applied 4 or 7 days in advance. Sunlight UV was evaluated for detrimental effects on phage survival on tomato foliage in the field. Phage was applied in the early morning, midmorning, early afternoon, and late evening, while UVA plus UVB irradiation and phage populations were monitored. The intensity of UV irradiation positively correlated with phage population decline. The protective formulation reduced the UV effect. In order to demonstrate direct effects of UV, phage suspensions were exposed to UV irradiation and assayed for effectiveness against bacterial spot of tomato. UV significantly reduced phage ability to control bacterial spot. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. The effects of desiccation and fluorescent light illumination on phage were investigated. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation eliminated the reduction caused by both of these factors. Phage persistence was dramatically affected by UV, while the other factors had less pronounced effects. Formulated phage reduced deleterious effects of the studied environmental factors.

Integrated management of TYLCV/TYLCSV on greenhouse hydroponic tomatoes in Southern Italy.

Tomato yellow leaf curl (TYLC) caused by Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), vectored by the whitefly Bemisia tabaci, is a major disease of tomato in Sardinia and Sicily, and is becoming a serious threat in Southern Italy too. TYLCSV was first reported in Calabria region in 1991, but apparently it was an occasional outbreak, and B. tabaci was not detected. Later, during the 2003-2004 winter, a serious epidemic was observed in protected tomato crops in Castrovillari, Cosenza province. TYLCV was first described in Sicily in 2003 and during 2004 in continental Italy. Both viruses were detected in winter 2005-2006 on the Basilicata Ionic coast, in the Metapontum area, both in protected and in open field tomato crops. Experiments were conducted in Calabria Region, Southern Italy, under controlled conditions in a group of greenhouses where several tomato crops were grown hydroponically to determine the separate and integrated effects of UV-reflective mulch (UVRM), Acibenzolar-S-methyl (Actigard) and the two insecticides Imidacloprid (ADMIRE 2F) and Thiamethoxam (ACTARA 25WG). Highly UV-reflective mulch covered plots were treated with Actigard and insecticides, both alone or in combination. TYLC disease incidence was determined from late August 2005 to late January 2006. The highly UVRM alone was effective in reducing disease incidence of about 28.6% at the end of October, and of 31.7% at the end of January. However, Actigard with UVRM significantly reduced TYLC disease incidence to 70% and 48.5%, in 2 months and 5 months after the first treatment, respectively. The insecticides with UVRM, resulted in a moderate reduction of disease incidence (22.5%) at the end of October. At the end of January a reduction in disease incidence due to insecticide applications was not significant. The use of Actigard combined with the insecticides on UVRM reduced the disease incidence (63.4% with Admire and 56.1% with Actara) at the end of January. Actigard alone or with insecticides on UVRM was effective in reducing disease incidences. Highly UVRM and Actigard were effective in reducing the primary spread of TYLCV/TYLCSV in greenhouse hydroponic tomatoes. Comparative analysis of their effects at different periods post-treatment suggests that multiple applications of Actigard may be necessary to reduce progress of this disease.

Relative importance of bacteriocin-like genes in antagonism of Xanthomonas perforans tomato race 3 to Xanthomonas euvesicatoria tomato race 1 strains.

In a previous study, tomato race 3 (T3) strains of Xanthomonas perforans became predominant in fields containing both X. euvesicatoria and X. perforans races T1 and T3, respectively. This apparent ability to take over fields led to the discovery that there are three bacteriocin-like compounds associated with T3 strains. T3 strain 91-118 produces at least three different bacteriocin-like compounds (BCN-A, BCN-B, and BCN-C) antagonistic toward T1 strains. We determined the relative importance of the bacteriocin-like compounds by constructing the following mutant forms of a wild-type (WT) T3 strain to evaluate the antagonism to WT T1 strains: Mut-A (BCN-A-), Mut-B (BCN-B-), Mut-C (BCN-C-), Mut-AB, Mut-BC, and Mut-ABC. Although all mutant and WT T3 strains reduced the T1 populations in in planta growth room experiments, Mut-B and WT T3 were significantly more effective. Mutants expressing BCN-B and either BCN-A or BCN-C reduced T1 populations less than mutants expressing only BCN-A or BCN-C. The triple-knockout mutant Mut-ABC also had a significant competitive advantage over the T1 strain. In pairwise-inoculation field experiments where plants were coinoculated with an individual mutant or WT T3 strain and the T1 strain, the mutant strains and the WT T3 strain were reisolated from more than 70% of the lesions. WT T3 and Mut-B were the most frequently reisolated strains. In field experiments where plants were group inoculated with Mut-A, Mut-B, Mut-C, Mut-ABC, and WT T1 and T3 strains, Mut-B populations dominated all three seasons. In greenhouse and field experiments, the WT and mutant T3 strains had a selective advantage over T1 strains. Bacterial strains expressing both BCN-A and BCN-C appeared to have a competitive advantage over all other mutant and WT strains. Furthermore, BCN-B appeared to be a negative factor, with mutant T3 strains lacking BCN-B having a selective advantage in the field.

Application of Acibenzolar-S-Methyl Enhances Host Resistance in Tomato Against Ralstonia solanacearum.

The chemical elicitor acibenzolar-S-methyl (ASM; Actigard 50 WG), which induces systemic acquired resistance (SAR), was investigated to determine the effect on bacterial wilt of tomato caused by Ralstonia solanacearum on moderately resistant cultivars under greenhouse and field conditions. In greenhouse experiments, ASM was applied as foliar spray and/or soil drench (3 µg/ml) before and as foliar spray (30 µg/ml) after transplanting. The chemical elicitor was ineffective in reducing bacterial wilt incidence on susceptible tomato cultivars Equinox and FL 47 when plants were inoculated with R. solanacearum. However, greenhouse studies indicated that ASM significantly enhanced resistance in cultivars with moderate resistance to bacterial wilt such as Neptune and BHN 466. It appeared that ASM-mediated resistance was partially due to prevention of internal spread of R. solanacearum toward upper stem tissues of tomato plants. The effect of ASM on moderately resistant cultivars was consistent in field experiments conducted in 2002 and 2003 in Quincy, FL, where bacterial wilt incidence was significantly reduced in ASM-treated BHN 466 (in 2002), FL 7514 (in 2003), and Neptune (both years) plants. ASM-treated BHN 466 and FL 7514 produced significantly higher tomato yield than the untreated controls. This is the first report of ASM-mediated control of bacterial wilt under field conditions, which suggests that use of this treatment for moderately resistant genotypes may be effective for control of bacterial wilt of tomato.

Integration of Biological Control Agents and Systemic Acquired Resistance Inducers Against Bacterial Spot on Tomato.

Two strains of plant growth-promoting rhizobacteria, two systemic acquired resistance inducers (harpin and acibenzolar-S-methyl), host-specific unformulated bacteriophages, and two antagonistic bacteria were evaluated for control of tomato bacterial spot incited by Xanthomonas campestris pv. vesicatoria in greenhouse experiments. Untreated plants and plants treated with copper hydroxide were used as controls. The plant growth-promoting rhizobacteria or a tap water control were applied as a drench to the potting mix containing the seedlings, while the other treatments were applied to the foliage using a handheld sprayer. The plant growth-promoting rhizobacteria strains, when applied alone or in combination with other treatments, had no significant effect on bacterial spot intensity. Messenger and the antagonistic bacterial strains, when applied alone, had negligible effects on disease intensity. Unformulated phage or copper bactericide applications were inconsistent in performance under greenhouse conditions against bacterial spot. Although acibenzolar-S-methyl completely prevented occurrence of typical symptoms of the disease, necrotic spots typical of a hypersensitive reaction (HR) were observed on plants treated with acibenzolar-S-methyl alone. Electrolyte leakage and population dynamics experiments confirmed that acibenzolar-S-methyl-treated plants responded to inoculation by eliciting an HR. Application of bacteriophages in combination with acibenzolar-S-methyl suppressed a visible HR and provided excellent disease control. Although we were unable to quantify populations of the bacterium on the leaf surface, indirectly we determined that bacteriophages specific to the target bacterium reduced populations of a tomato race 3 strain of the pathogen on the leaf surface of acibenzolar-S-methyl-treated plants to levels that did not induce a visible HR. Integrated use of acibenzolar-S-methyl and phages may complement each other as an alternative management strategy against bacterial spot on tomato.

Evaluation of Thymol as Biofumigant for Control of Bacterial Wilt of Tomato Under Field Conditions.

Volatile plant essential oils thymol and palmarosa oil, used at a concentration of 0.7%, were evaluated under field conditions for control of bacterial wilt of tomato caused by Ralstonia solanacearum. The experimental fields were artificially infested with the bacterial pathogen. Two hours after infestation, the plant essential oils were applied, then the plots were sealed with plastic mulch for 3 or 6 days. Tomato seedlings were transplanted into the field 7 days later. In fall of 2002, 92.5% of tomato plants (cv. Equinox) wilted in the untreated control plots. Both thymol and palmarosa oil soil treatments reduced bacterial wilt incidence significantly. Thymol was more effective than palmarosa oil based on the final assessment, when 33.1 and 48.1% of the plants had wilted in plots treated with thymol and palmarosa oil, respectively. Soil treatment with either thymol or palmarosa oil produced significantly higher yield of tomato than the untreated control. In 2003, only thymol was evaluated. Thymol application significantly reduced bacterial wilt incidence on the susceptible cultivar Solar Set. Disease incidence in untreated plots reached 65.5%, while in thymol treated plots only 12% of plants wilted. Thymol treatment also increased yield of Solar Set significantly compared with the untreated control. This is the first report on the use of thymol for controlling a plant disease under field conditions, which indicated that this compound provided effective control of bacterial wilt on susceptible tomato cultivars when used as preplant treatment of soils. Because of its volatile property and broad-spectrum functions, thymol shows potential to be used as a soil biofumigant for the management of various plant pathogens.

Management of Tomato Bacterial Spot in the Field by Foliar Applications of Bacteriophages and SAR Inducers.

Various combinations of the harpin protein, acibenzolar-S-methyl, and bacteriophages were compared for controlling tomato bacterial spot in field experiments. Harpin protein and aciben-zolar-S-methyl were applied every 14 days beginning twice before transplanting and then an additional four applications throughout the season. Formulated bacteriophages were applied prior to inoculation followed by twice a week at dusk. A standard bactericide treatment, consisting of copper hydroxide plus mancozeb, was applied once prior to inoculation and then every 7 days, while untreated plants served as an untreated control. Experiments were conducted in north and central Florida fields during fall 2001, spring 2002, and fall 2002. In three consecutive seasons, acibenzolar-S-methyl applied in combination with bacteriophage or bacteriophage and harpin significantly reduced bacterial spot compared with the other treatments. However, it did not significantly affect the total yield compared with the standard or untreated control. Application of host-specific bacteriophages was effective against the bacterial spot pathogen in all three experiments, providing better disease control than copper-mancozeb or untreated control. When results of the disease severity assessments or harvested yield from the bacteriophage-treated plots were grouped and compared with the results of the corresponding nonbacteriophage group, the former provided significantly better disease control and yield of total marketable fruit.

Integrated Management of Tomato Spotted Wilt on Field-Grown Tomatoes.

Epidemics of spotted wilt caused by Tomato spotted wilt virus (TSWV) vectored by Frankliniella occidentalis and possibly other thrips species occur regularly in tomato in the southeastern United States. Field experiments were conducted to determine the effects of UV-reflective mulch, acibenzolar-S-methyl (plant activator), and insecticides on progress of tomato spotted wilt incidence and population dynamics of flower thrips (including F. occidentalis, F. tritici, and F. bispinosa). Whole plots of tomatoes grown on UV-reflective and black polyethylene mulch were divided into subplots of acibenzolar-S-methyl and no acibenzolar-S-methyl, and sub-subplots of insecticide and no insecticide for thrips control. The UV-reflective mulch was more effective than black polyethylene mulch each year in reducing colonization of thrips in May and the consequent primary infections of tomato spotted wilt. Application of acibenzolar-S-methyl further reduced tomato spotted wilt incidence in 2000 and 2002, when disease pressure was great. Reproduction of thrips on tomato was poor in these experiments, but their control in the insecticide-treated sub-subplots prevented secondary spread in both years. The combination of UV-reflective mulch, acibenzolar-S-methyl, and insecticides was very effective in reducing tomato spotted wilt incidence in tomato.

Efficacy of Plant Growth-Promoting Rhizobacteria, Acibenzolar-S-Methyl, and Soil Amendment for Integrated Management of Bacterial Wilt on Tomato.

Greenhouse experiments were conducted to study the effect of plant growth promoting rhizobacteria (PGPR; Bacillus pumilus SE 34, Pseudomonas putida 89B61, BioYield, and Equity), acibenzolar-S-methyl (Actigard), and a soil amendment with S-H mixture (contains agricultural and industrial wastes such as bagasse, rice husk, oyster shell powder, urea, potassium nitrate, calcium super phosphate, and mineral ash) on bacterial wilt incidence caused by Ralstonia solanacearum (race 1, biovar 1) in susceptible tomato (Lycopersicon esculentum cv. Solar Set). In experiments with PGPR, Pseudomonas putida 89B61 significantly reduced bacterial wilt incidence when applied to the transplants at the time of seeding and 1 week prior to inoculation with Ralstonia solanacearum. BioYield, a formulated PGPR that contained two Bacillus strains, decreased disease significantly in three experiments. Equity, a formulation containing more than 40 different microbial strains, did not reduced wilt incidence compared with the untreated control. With inoculum at low pathogen densities of 1 × 105 and 1 × 106 CFU/ml, disease incidence of Actigard-treated plants was significantly less than with nontreated plants. This is the first report of Actigard-mediated reduction of bacterial wilt incidence in a susceptible tomato cultivar. When PGPR and Actigard applications were combined, Actigard plus P. putida 89B61 or BioYield reduced bacterial wilt incidence compared with the untreated control. Incorporation of S-H mixture into infested soil 2 weeks before transplanting reduced bacterial wilt incidence in one experiment. Combination of Actigard with the S-H mixture significantly reduced bacterial wilt incidence in tomato in two experiments.

Improved Efficacy of Newly Formulated Bacteriophages for Management of Bacterial Spot on Tomato.

Bacteriophages are currently used as an alternative method for controlling bacterial spot disease on tomato incited by Xanthomonas campestris pv. vesicatoria. However, the efficacy of phage is greatly reduced due to its short residual activity on plant foliage. Three formulations that significantly increased phage longevity on the plant surface were tested in field and greenhouse trials: (i) PCF, 0.5% pregelatinized corn flour (PCF) + 0.5% sucrose; (ii) Casecrete, 0.5% Casecrete NH-400 + 0.5% sucrose + 0.25% PCF; and (iii) skim milk, 0.75% powdered skim milk + 0.5% sucrose. In greenhouse experiments, the nonformulated, PCF-, Casecrete-, and skim milk-formulated phage mixtures reduced disease severity on plants compared with the control by 1, 30, 51, and 62%, respectively. In three consecutive field trials, nonformulated phage caused 15, 20, and 9% reduction in disease on treated plants compared with untreated control plants, whereas plants treated with PCF- and Casecrete-formulated phage had 27, 32, and 12% and 30, 43, and 24% disease reduction, respectively. Plants receiving copper-mancozeb treatments were included in two field trials and had a 20% decrease in disease in the first trial and a 13% increase in the second one. Skim milk-formulated phage was tested only once and caused an 18% disease reduction. PCF-formulated phage was more effective when applied in the evening than in the morning, reducing disease on plants by 27 and 13%, respectively. The Casecrete-formulated phage populations were over 1,000-fold higher than the nonformulated phage populations 36 h after phage application.

Effects of Plant Essential Oils on Ralstonia solanacearum Population Density and Bacterial Wilt Incidence in Tomato.

Greenhouse experiments were conducted to determine the effectiveness of plant essential oils as soil fumigants to manage bacterial wilt (caused by Ralstonia solanacearum) in tomato. Potting mixture ("soil") infested with R. solanacearum was treated with the essential oils at 400 mg or µl and 700 mg or µl per liter of soil in greenhouse experiments. R. solanacearum population densities were determined just before and 7 days after treatment. Populations declined to undetectable levels in thymol, palmarosa oil, and lemongrass oil treatments at both concentrations, whereas tea tree oil had no effect. Tomato seedlings transplanted in soil treated with 700 mg/liter of thymol, 700 ml/liter of palmarosa oil, and 700 ml/liter of lemongrass oil were free from bacterial wilt and 100% of plants in thymol treatments were free of R. solanacearum. Soil amendment with fresh leaves of essential oil-producing plants did not reduce bacterial wilt incidence compare to untreated inoculated control. Some thyme oil-producing plants such as thyme (Thymus vulgaris) cv. German winter, Creeping thyme (Thymus serpyllum), and Greek oregano (Origanum vulgare subsp. hirtum), while remaining symptomless, became systemically infected by R. solanacearum and were therefore identified as hosts of R. solanacearum.

First Report of Peanut stunt virus in Perennial Peanut in North Florida and Southern Georgia.

In spring 2001, symptoms similar to aphid-vectored peanut stunt disease caused by Peanut stunt virus (PSV) were observed on perennial peanut (Arachis glabrata) cv. Floragraze in Jackson and Gulf counties, FL and Lowndes County, GA. Symptoms observed in commercial hay fields and at the North Florida Research and Education Center in Marianna and Quincy included malformed leaves, plant chlorosis, leaf mottling, and stunted plants, which resulted in reduced foliage yield. Leaf symptoms were visible throughout the growing season. Stunting was more common in spring and early summer. No symptoms were seen on rhizomes. Diagnosis of PSV (genus Cucumovirus) from symptomatic leaves and rhizome materials of 12 plants was confirmed by a direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). ELISA tests were repeated three times. Antibodies specific to the Clemson isolate, serotype E, were obtained from Clemson University, Clemson, SC. DAC-ELISA (1) values of 0.1 A405 above the healthy control for perennial peanut were considered positive for foliage and rhizome material tested. ELISA values ranged from 0.4 to 2.1. The mean ELISA value of the positive controls was 2.65. Symptomatic plants were also tested with ELISA using available antibodies from Agdia Inc., Elkart, IN, for Tomato spotted wilt virus, from ATCC for Peanut stripe virus, and from Clemson University (Cowpea isolate) for Cucumber mosaic virus, but all results were negative. To our knowledge, this is the first report of PSV on perennial peanut in Florida and southern Georgia. At this time, it is not known what role perennial peanut may play as a reservoir of the virus in the vicinity of peanut fields. Little is known about the potential for forage production loss and stand longevity. Next season, molecular detection techniques and epidemiological studies on peanut and perennial peanut will be conducted to ascertain the incidence and possible impact of PSV in Georgia and Florida. Reference: (1) A. G. Gillaspie, Jr. et al. Plant Dis. 79:388, 1995.

Characterization of Agrobacterium vitis Strains Isolated from Turkish Grape Cultivars in the Central Anatolia Region.

Crown gall was detected in several vineyards in the Central Anatolia region of Turkey. Vineyards were planted to cultivars of grape that originated in Turkey and that were not grafted. The predominant species isolated from galls consisted of tumorigenic strains of Agrobacterium vitis. They were identified based on reactions to standard biochemical and physiological tests, by polymerase chain reaction amplification of specific Ti plasmid and chromosomal sequences, and by reaction to a species-specific monoclonal antibody. All strains utilized octopine, suggesting that they may carry similar types of Ti plasmids. Some of the strains exhibited a differential host range compared with others and were less virulent based on the numbers of galls that they induced on grape. When grapevines were treated with nontumorigenic A. vitis strain F2/5 prior to inoculation with the Turkish A. vitis strains, crown gall was effectively controlled. The genetic diversity of strains was evaluated by comparing DNA fingerprints that were generated by restriction enzyme digestion of the intergenic spacer region that lies between 16S and 23S rRNA genes. They segregated into two main groups, one that is similar to previously identified A. vitis strains carrying octopine type Ti plasmids and one that was more similar to strains carrying nopaline and vitopine Ti plasmids. The strains of A. vitis from Turkey may represent ancestral forms of the pathogen that will provide insight into the evolution of the bacterium.

First Report of a Furovirus Infecting Field-Grown Rye in North America.

Viral symptoms were present in a dwarf recurrent population (99RP17) of rye (Secale cereale) at the North Florida Research and Education Center in Quincy, Gadsden County, FL, during the winter and spring of 2000. Symptoms and distribution of the infected plants in the field were similar to those caused by Soilborne wheat mosaic virus (SBWMV; acronym WSBMV), which was first recognized in North America in 1919 (4) and found in Florida in wheat in 1970 (3). SBWMV has been observed based on symptoms in rye in North America (4). Interveinal, non-continuous, chlorotic areas of leaves and stunting of plants in patchy patterns occurred in four locations (0.8 to 1.6 km between locations). Incidences of the disease ranged from 3 to 15%. Leaves and roots of more than 25 plants were assessed. Using light microscopy, after staining with Calcomine Orange 2RS/Luxol Brilliant Green BL (1), amorphous, vacuolate inclusions were observed in all assayed leaves. With electron microscopy, rigid rods were present with a bimodal distribution of particle lengths that conformed to size distributions found originally in wheat in 1970 in Florida. Leaves with symptoms were sent to Agdia Inc. (Elkhart, IN) and samples were strongly positive for SBWMV using enzyme-linked immunosorbent assay. Cystosori of Polymyxa graminis were detected from a few roots from symptomatic plants. While these associations are suggestive of SBWMV, and rye is a reported host of SBWMV, the possibility of this virus being soilborne rye mosaic virus exists (2). Such a differentiation will require nucleotide sequence analysis. To our knowledge, this is the first report of a furovirus infecting field-grown rye in Florida and in North America. References: (1) R. G. Christie and J. R. Edwardson. 1994. Light and Electron Microscopy of Plant Virus Inclusions Monogr. 9. University of Florida, Quincy. (2) R. Koenig et al. 1999. Arch. Virol. 144:2125-2140. (3) T. A. Kucharek and J. H. Walker. Plant Dis. Rep. 58:763-765, 1974. (4) H. H. McKinney. J. Agric. Res. 23:771-800, 1923.

First Report of Powdery Mildew on Greenhouse Tomatoes Caused by Oidium neolycopersici in Florida.

In December 1999, typical signs of powdery mildew-dense white mycelium in irregular patterns often covering almost the entire upper surface of leaves-were observed in production greenhouses on tomato cv. Tradiro. Microscopic observations revealed mycelium with lobed appressoria and large, approximately cylindrical conidia that measured 38 to 45 × 16 to18 µm. Short germ tubes were at one end of the conidium and ended in a lobed appressorium. Conidiophores were straight with cylindrical foot-cells (≈40 to 42 µm), followed by two short cells (14 to18 µm). Based on these characteristics the fungus was identified as 0idium neolycopersici Kiss et al. (2) (formerly O. lycopersicum Braun [1]). Disease-free tomato cv. FL47 plants were inoculated at the fourth true-leaf stage with conidia by transferring fungal colonies collected from plants in production greenhouses with a single-edged razor blade to the adaxial surface of the test plants (six plants and three leaves per plant). Plants were grown in the greenhouse at 20 to 25°C. Powdery mildew, exhibiting the same morphological features, was observed 12 days later on inoculated tomato leaves. Powdery mildew on tomatoes in Suwannee Valley area greenhouses in Florida was quite common and severe in 1999 to 2000. Secondary cycles of the disease were observed, resulting in disease incidence up to 50 to 60% in some greenhouses, requiring repeated applications of sulfur for its management. This disease is expected to become a significant problem in greenhouse tomatoes, requiring regular disease control measures. This powdery mildew has not yet been observed in field-grown tomatoes in Florida. The pathogen has been reported in Connecticut on tomatoes grown under greenhouse and field conditions (3). To our knowledge, this is the first report of O. neolycopersici on greenhouse-grown tomatoes in Florida. References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fisher Verlag, New York, 1995. (2) L. Kiss et al. Mycol. Res. 105:684, 2001. (3) J. A. LaMondia et al. Plant Dis. 83:341, 1999.

A Severe Outbreak of Fire Blight in Woody Ornamental Rosaceae Plants in North Florida and South Georgia.

Woody ornamentals including 'Bradford' and 'Aristocrat' pears (Pyrus calleryana), Indian hawthorn (Rhaphiolepis sp.), and cotoneaster (Cotoneaster spp.) developed blossom and shoot blight symptoms suggestive of fire blight caused by Erwinia amylovora. Disease was observed in several counties in north Florida and south Georgia and samples were sent to Florida Extension Plant Diagnostic Clinic-Quincy (FEPDC-Q). The incidences in individual 'Bradford' pear were as high as 30 to 40 strikes per tree. This level of severe E. amylovora infections on 'Bradford' pear has not been seen in Florida for the last 10 years based on FEPDC-Q data. Symptomatic plants first appeared in the landscape and nurseries around the first week of April 2000 in the following counties: Bay, Holmes, Washington, Gadsden, Leon, Jefferson (north Florida), and Decatur (south Georgia). Severely infected 'Bradford' pear trees were also observed in Georgia cities of Cairo, Thomasville, Moultrie, and Tifton. The presence of E. amylovora in samples was verified by pEA29-polymerase chain reaction (PCR) (2,4), fatty acid methyl ester analysis (MIDI, Newark, DE), and colony type on semi-selective modified Miller-Schroth (MMS) medium (3). Of the 16 samples tested by PCR, E. amylovora was present in all. Actively growing shoots of Cotoneaster salicifolius (a susceptible host) were inoculated with a bacterium isolated from 'Bradford' pears that was identified as E. amylovora by PCR. Dark brown necrotic tissues with typical fire blight appearance developed within 10 days. E. amylovora was reisolated on MMS medium from inoculated shoot tissues of C. salicifolius and confirmed by PCR. The presence of E. amylovora from ornamental pears, Indian hawthorn, and cotoneasters was previously reported in Florida (1). Cool spring temperatures in the Florida panhandle and several rain events during the bloom period may have stimulated the severe outbreak of fire blight. This severe outbreak highlights the importance of using a forecasting model such as Maryblyt 4.3 to predict unexpected infection periods so that preventive control measures can be taken. References: (1) S. A. Alfieri, Jr. et al. Florida Department of Agriculture and Consumer Services, Bulletin No. 14, 1994. (2) S. Bereswill et al. Appl. Environ. Microbiol. 58:3522, 1992. (3) W. Brulez and W. Zeller. Acta Hortic. 117:37, 1981. (4) M. T. Momol et al. Plant Dis. 82:646, 1998.

First Report of Tomato spotted wilt virus in Habanero and Tabasco Peppers in Florida.

In spring 2000, symptoms similar to thrips-vectored spotted wilt disease caused by Tomato spotted wilt virus (TSWV) were observed on habanero (Capsicum chinense) and tabasco (Capsicum frutescens) peppers in north Florida. Habanero peppers were from commercial fields grown for specialty markets and tabasco peppers were from research plots. Symptoms observed were leaf necrosis, fruit drop, necrotic stem lesions, and stunting. Fruit symptoms included chlorotic and necrotic spotting and distinct ring pattern and distortion. The incidence of symptomatic habanero peppers was 7 to 8% in one of the three production fields visited, and a lower incidence in two other fields (all in Jackson County). In tabasco pepper, TSWV was detected in spring and fall 1999, and spring 2000 seasons in 10 to 15% of the plants (Gadsden County). Adjacent tomato fields contained scattered plants exhibiting symptoms of TSWV. Diagnosis of TSWV from symptomatic stems, leaves, and fruit of habanero and tabasco peppers was confirmed by a double antibody sandwich enzyme linked immunosorbent assay (ELISA) using a commercially available kit (Agdia Inc., Elkhart, IN). ELISA values ranged from 1.57 to 1.95 for habanero pepper and 0.80 to 0.95 for tabasco pepper. The mean ELISA value of the negative controls was 0.001. To further verify TSWV infection, immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) was performed (1). The primer pair 5'-ATGTCTAAGGTTAAGCTC-3' and 5'-TTAAGCAAGTTCTGTGAG-3' represented the first and last 18 bases of the coding region of the nucleocapsid gene of TSWV, respectively, and produces approximately 800 bp PCR product (1). IC-RT-PCR gave a single DNA band of expected size in both habanero and tabasco samples, while no amplification was found in an uninfected pepper sample. This is the first report of TSWV on habanero and tabasco peppers in Florida. TSWV continues to be an economically important disease constraint to the production of tomato, pepper (C. annuum), peanut, and tobacco in the southeastern United States (observations from Georgia and Florida). Meanwhile, the known host range is expanding to include new species of cultivated vegetables. References: (1) R. K. Jain et al.. Plant Dis. 82:900, 1998.