A Coordinated Effort to Manage Soybean Rust in North America: A Success Story in Soybean Disease Monitoring.

Existing crop monitoring programs determine the incidence and distribution of plant diseases and pathogens and assess the damage caused within a crop production region. These programs have traditionally used observed or predicted disease and pathogen data and environmental information to prescribe management practices that minimize crop loss. Monitoring programs are especially important for crops with broad geographic distribution or for diseases that can cause rapid and great economic losses. Successful monitoring programs have been developed for several plant diseases, including downy mildew of cucurbits, Fusarium head blight of wheat, potato late blight, and rusts of cereal crops. A recent example of a successful disease-monitoring program for an economically important crop is the soybean rust (SBR) monitoring effort within North America. SBR, caused by the fungus Phakopsora pachyrhizi, was first identified in the continental United States in November 2004. SBR causes moderate to severe yield losses globally. The fungus produces foliar lesions on soybean (Glycine max) and other legume hosts. P. pachyrhizi diverts nutrients from the host to its own growth and reproduction. The lesions also reduce photosynthetic area. Uredinia rupture the host epidermis and diminish stomatal regulation of transpiration to cause tissue desiccation and premature defoliation. Severe soybean yield losses can occur if plants defoliate during the mid-reproductive growth stages. The rapid response to the threat of SBR in North America resulted in an unprecedented amount of information dissemination and the development of a real-time, publicly available monitoring and prediction system known as the Soybean Rust-Pest Information Platform for Extension and Education (SBR-PIPE). The objectives of this article are (i) to highlight the successful response effort to SBR in North America, and (ii) to introduce researchers to the quantity and type of data generated by SBR-PIPE. Data from this system may now be used to answer questions about the biology, ecology, and epidemiology of an important pathogen and disease of soybean.

First Report of Turnip mosaic virus Infecting Brassica carinata (Ethiopian Mustard) in the United States.

Brassica carinata L. Braun (Ethiopian mustard) is an annual oil seed crop currently being evaluated for its potential use as a source of biofuel. Due to its high content of erucic acid, it provides a biodegradable non-fossil fuel feedstock that has many applications ranging from biofuels to other industrial uses such as polymers, waxes, and surfactants. Moreover, high glucosinolate content adds the scope of B. carinata being used as a bio-fumigant. B. carinata is amenable to low input agriculture and has great economic potential to be used as a winter crop, especially in the southeastern United States. Virus-like leaf symptoms including mosaic, ringspot, mottling, and puckering were observed on B. carinata (cvs. 080814 EM and 080880 EM) in field trials at Quincy, FL, during spring 2013, with disease incidence of >80%. A more extensive survey of the same field location indicated that mosaic symptoms were the most common. Viral inclusion assays (1) of leaves with a range of symptoms indicated the presence of potyvirus-like inclusion bodies. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA) from six symptomatic samples and one non-symptomatic B. carinata sample were subjected to reverse transcription (RT)-PCR assays using SuperScript III One-Step RT-PCR System (Invitrogen, Life Technologies, NY), and two sets of potyvirus-specific degenerate primers MJ1-F and MJ2-R (2) and NIb2F and NIb3R (3), targeting the core region of the CP and NIb, respectively. The RT-PCR assays using the CP and NIb specific primers produced amplicons of 327 bp and 350 bp, respectively, only in the symptomatic leaf samples. The obtained amplicons were gel-eluted and sequenced directly (GenBank Accession Nos. KC899803 to KC899808 for CP and KC899809 to KC899813 for NIb). BLAST analysis of these sequences revealed that they came from Turnip mosaic virus (TuMV). Pairwise comparisons of the CP (327 bp) and NIb (350 bp) segments revealed 98 to 99% and 96 to 98% nucleotide identities, respectively, with corresponding sequences of TuMV isolates. These results revealed the association of TuMV with symptomatic B. carinata leaf samples. Although TuMV has been reported from B. carinata in Zambia (4), this is the first report of its occurrence on B. carinata in the United States. Considering the importance of B. carinata as a biofuel source, this report underscores the need for developing effective virus management strategies for the crop. References: (1) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986. (2) M. Grisoni et al. Plant Pathol. 55:523, 2006. (3) L. Zheng et al. Plant Pathol. 59:211, 2009. (4) D. S. Mingochi and A. Jensen. Acta Hortic. 218:289, 1988.

First Report of Phakopsora pachyrhizi Adapting to Soybean Genotypes with Rpp1 or Rpp6 Rust Resistance Genes in Field Plots in the United States.

During the years following the first detection of soybean rust, caused by Phakopsora pachyrhizi Syd., in the continental United States in November, 2004, soybean (Glycine max [L.] Merr.) genotypes with the Rpp1 or Rpp6 resistance genes exhibited high levels of resistance there (1,2,3). When challenged with 72 different American isolates collected between 2006 and 2009, PI 200492 (source of Rpp1) produced no sporulating lesions (2). In 2011 and 2012, however, field populations of P. pachyrhizi from Gadsden County, FL, caused higher rust severity on plants with Rpp1 or Rpp6 than in previous years. To assess aggressiveness, sporulation ratings were made using a 1 to 5 scale (no sporulation to profuse sporulation) on leaflets collected from field plants at or near the R6 (full seed) stage of development. A dissecting microscope was used to examine 3 replications of 5 leaflets each in 2009 or 2 replications of 10 leaflets each in 2012. The sporulation ratings increased on PI 200492 (from 1.1 ± 0.1 in 2009 to 4.1 ± 0.4 in 2012), PI 567102B (Rpp6; from 1.1 ± 0.1 in 2009 to 2.4 ± 0.2 in 2012), and L85-2378, a 'Williams 82' isoline carrying the Rpp1 gene (from 1.0 ± 0 in 2009 to 4.0 ± 0.3 in 2012). The mean 2009 and 2012 sporulation ratings for susceptible control Williams 82 were 5.0 ± 0 and 4.2 ± 0.1, respectively. Single-uredinium-derived isolates were purified from bulk isolates collected from field plots in 2009 (FL-Q09-1), 2011 (FL-Q11-1), and 2012 (FL-Q12-1). Greenhouse and detached leaflet assays were then used to test the virulence of these isolates under controlled conditions. Detached leaflets from 3-week-old seedlings of Williams 82, PI 200492, PI 567102B, and L85-2378 were inoculated by pipetting 15-µl drops of a 30 to 40 urediniospore µl-1 suspension onto the abaxial side of 3 to 4 leaflets per genotype, which were then sealed in Petri plates and incubated in a growth chamber at 20 to 22°C. Plates were kept in the dark for 12 h following inoculation. For the greenhouse assay, the first trifoliolate leaves of at least 3 seedlings were each sprayed with 1.5 ml of a 40 urediniospore µl-1 suspension and incubated 24 h at 22 to 24°C in a dark mist chamber. The plants were then maintained at 22 to 24°C and 76 to 86% relative humidity in a greenhouse with 10 h of daylight on average. Two weeks after inoculation with FL-Q11-1 or FL-Q12-1, all of the genotypes had developed TAN lesions with abundant sporulation, indicating susceptibility. On leaves inoculated with FL-Q09-1, however, no visible reaction was observed on PI 200492, and PI 567102B developed reddish-brown (RB) lesions associated with incomplete resistance. Although the lesions on Rpp1 and Rpp6 greenhouse seedlings inoculated with the FL-Q11-1 and FL-Q12-1 isolates were slightly darker than those that developed on Williams 82 plants or on detached leaflets, the profuse sporulation that is characteristic of the TAN infection type was observed. The higher virulence of the 2011 and 2012 Florida isolates on two soybean genotypes with Rpp1 and one with Rpp6 confirmed the presence of a P. pachyrhizi pathotype in north-central Florida that is more virulent against these genes than earlier populations from the southeastern United States. References: (1) S. Li. Crop Sci. 49:887, 2009. (2) Twizeyimana and Hartman. Plant Dis. 96:75, 2012. (3) Walker et al. Crop Sci. 51:678, 2011.

First Report of Sclerotinia Stem Rot Caused by Sclerotinia sclerotiorum on Brassica carinata in Florida.

Brassica carinata A. Braun, commonly referred to as Ethiopian rapeseed, a near relative of collards and mustard, has become the object of increasing interest as an oil crop. It has been reported that B. carinata adapts better and is more productive than B. napus (canola) in adverse conditions, such as clay and sandy soils and under low management cropping systems (1). In late February 2012, symptoms typical of sclerotinia stem rot were observed in B. carinata trials (cultivars 090867 EM and 080814 EM) at the University of Florida, North Florida Research and Education Center located in Quincy, FL. Approximately 20 to 30% of the B. carinata cultivar 090867 EM were observed to have symptoms and approximately 5% of cultivar 080814 EM displayed symptoms. Stems had white mycelia growing on the outside, plants were lodging and spherical to cylindrical, 3 to 8 mm, and black sclerotia were found outside and inside bleached stems. Sclerotia from diseased stems were surface sterilized and placed in 9-cm diameter petri plates on quarter strength potato dextrose agar (PDA) amended with 25% lactic acid. Fungal cultures consisting of white mycelia and medium-sized (mean 4 mm), black, irregular sclerotia were consistently recovered and identified as Sclerotinia sclerotiorum (Lib.) de Bary based on morphological characteristics (3). Sequence analyses were conducted on mycelium by extracting fungal DNA with the Qiagen DNeasy Plant Mini Kit (Valencia, CA). PCR amplification was performed using primers ITS1 and ITS4. The BLAST search revealed that the sequence (GenBank Accession No. JX307092) had 99 and 100% sequence identity with S. sclerotiorum GenBank accessions JN013184.1 and JN012606.1. Pathogenicity was determined by inoculating six 1-month-old B. carinata plants (cultivars 090867 EM and 080814 EM) that were grown in greenhouse pots (20 cm in diameter). Mycelia plugs (8 mm in diameter) were excised from the colony margin after 6 days of incubation at room temperature (approximately 25°C), and placed on stems, at the soil line, of B. carinata plants. Six control plants were inoculated with noncolonized PDA plugs. All plants were enclosed in plastic bags that had been sprayed with water on the inside to maintain high humidity and kept in the laboratory at room temperature (approximately 25°C). Symptoms similar to those observed in the field were evident after 3 days on inoculated plants and S. sclerotiorum was reisolated. In the controls, no symptoms developed and the fungus could not be isolated. The experiment was repeated with similar results. The majority of rapeseed production is in North Dakota, where sclerotinia stem rot caused by S. sclerotiorum is a major fungal disease affecting production (2). Currently, there is no significant B. carinata production in Florida; however, interest in biofuels could lead to an increase in planted acreage and sclerotinia stem rot could become a significant disease problem in areas of Florida were B. carinata is planted. To our knowledge, this is the first report of sclerotinia stem rot of B. carinata caused by S. sclerotiorum in Florida. References: (1) M. Cardone et al. Biomass and Bioenergy. 25:623, 2003. (2) L. E. del Río et al. Plant Dis. 91:191, 2007. (3) L. M. Kohn. Phytopathology 69:881, 1979.

First Report of Downy Mildew (Hyaloperonospora camelinae) on Camelina sativa in Florida.

Camelina sativa (L.) Crantz, Brassicaceae, whose common name is Crantz-large-seeded false flax, is an annual oilseed species. It is grown as a forage and biofuel crop in Europe and North America. False flax is an ideal low-input crop for biodiesel production, because of its low requirements for nitrogen fertilizer and pesticides. Production costs of this crop are substantially lower than those of many other oilseed crops such as rapeseed, corn, and soybean. It is an excellent rotation crop and can reduce disease and insect and weed pressure in wheat fields. During the spring of 2011, commercial and research plantings of C. sativa cultivar Calena in Liberty and Gadsden counties in north Florida developed symptoms typical of downy mildew. In spring of 2012, the same symptoms were observed in experimental plantings of false flax. A white downy mold was observed on the upper third portion of the plants, on the upper stem internodes, and on the developing seed. The affected stems exhibited a twisted growth. Conidiophores had main trunks with dichotomous branches terminating in slender curved tips. Conidia were ovoid and 28 to 45 (mean 36) µm long and 22 to 38 (mean 30) µm wide. Conidiophores were branched (three to four branches, each with six to eight curved tips) and ranged from 107 to 236 µm long and 5 to 14 µm wide. Mycelium was obtained directly from diseased plants for DNA extraction (Qiagen DNeasy Plant Mini Kit, Valencia, CA). Primers ITS1 and ITS4 were used for PCR amplification (4). The PCR product was sequenced bidirectionally with the PCR primers. A consensus nucleotide sequence (Accession JQ997103) was compared to those in the NCBI GenBank database using a BLAST search. The sequence was 99% similar to sequence from Hyaloperonospora camelinae (Gäum.) Göker, Voglmayr, Riethm, M. Weiss & Oberw. (Accession AY198249.1) with a 95% query coverage (1). Pathogenicity was established by applying white conidial masses of downy mildew from field samples to stems of 4-week-old plants grown in pots in a greenhouse maintained at 25 ± 2°C. Noninoculated plants maintained under the similar conditions served as control. Symptoms and signs of downy mildew developed after 14 days only on inoculated plants. Downy mildew constitutes a serious threat to the cultivation of C. sativa in Florida because of the humid climate favoring disease development. Diseased plants may reduce yield and disease management would increase production costs. H. camelinae was previously reported on C. sativa in Oregon, Minnesota, Montana (3), and Nebraska (2). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Florida. References: (1) M. Göker et al. Canad. J. Bot. 81:672, 2003. (2) R. M. Harveson et al. Plant Health Progress. 2011. doi: 10.1094/PHP-2011-1014-01-BR. (3) M. L. Putnam et al. Plant Health Progress. 2009. doi: 10.1094/PHP-2009-0910-01-BR. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Effects of Silicon Applications on Soybean Rust Development Under Greenhouse and Field Conditions.

Soybean rust (SBR), caused by Phakopsora pachyrhizi, is one of the most destructive fungal diseases affecting soybean production. Silicon (Si) amendments were studied as an alternative strategy to control SBR because this element was reported to suppress a number of plant diseases in other host-pathogen systems. In greenhouse experiments, soybean cultivars inoculated with P. pachyrhizi received soil applications of wollastonite (CaSiO3) (Si at 0, 0.96, and 1.92 t ha-1) or foliar applications of potassium silicate (K2SiO3) (Si at 0, 500, 1,000, or 2,000 mg kg-1). Greenhouse experiment results demonstrated that Si treatments delayed disease onset by approximately 3 days. The area under disease progress curve (AUDPC) of plants receiving Si treatments also was significantly lower than the AUDPC of non-Si-treated plants. For field experiments, an average 3-day delay in disease onset was observed only for soil Si treatments. Reductions in AUDPC of up to 43 and 36% were also observed for soil and foliar Si treatments, respectively. Considering the natural delayed disease onset due to the inability of the pathogen to overwinter in the major soybean production areas of the United States, the delay in disease onset and the final reduction in AUDPC observed by the soil Si treatments used may lead to the development of SBR control practices that can benefit organic and conventional soybean production systems.

Fusarium verticillioides (Saccardo) Nirenberg associated with hardlock of cotton.

Boll rots of cotton (Gossypium hirsutum L.) are common in the humid areas of the Southeastern US. One type of boll damage that may be differentiated from others is hardlock, with symptoms that include compression of the fibers within individual locules of mature, open cotton bolls without further obvious disintegration of the lint or damage to the carpel wall. The principal economic effect is that the boll's lint is unharvestable by mechanical cotton pickers. This disease is endemic to the Southeast and can cause severe yield losses up to 70% in some fields. Scanning electron microscopy images of fibers from hardlocked bolls showed flattened and twisted tissue compared to fibers from healthy bolls. Fusarium verticillioides (Saccardo) Nirenberg was the fungus most commonly isolated from seeds of developing cotton bolls. Flowers inoculated with F. verticillioides on the day of bloom by spraying a spore suspension onto the flowers developed significantly (P < 0.05) more hardlock symptoms compared to untreated controls. The infection process was analyzed using a F. verticillioides isolate tagged with green fluorescent protein (GFP). When it was applied to cotton flowers on the day of bloom, the GFP-tagged F. verticillioides strain was detected in the stigma and style by 2 days after bloom (DAB) and in developing seeds at 4, 6, 8, 10, 16, 20, 40, and 60 (open bolls) DAB. By 8 DAB, the GFP F. verticillioides was isolated from over 80% of developing seeds.

Removal of Wet Deposited Phakopsora pachyrhizi Urediniospores from Soybean Leaves by Subsequent Rainfall.

Urediniospores of Phakopsora pachyrhizi, the soybean rust fungus, have a high probability of being removed from a soybean leaf by water runoff associated with subsequent rainfall after wet deposition. The effects of rainfall intensity, subsequent spore-free rainfall duration, and soybean leaf sample height on uredinia density were used to evaluate the retention of urediniospores on soybean leaf tissue. Rainfall simulations of 45 and 85 mm/h were conducted on potted soybean plants that were inoculated with 2 min of urediniospore-injected simulated rainfall and exposed to 0, 1, and 30 min of subsequent spore-free rainfall. Urediniospore retention was estimated using uredinia density values obtained from a detached leaf bioassay for the sample heights of soil level, mid-canopy, and upper-canopy. Soil level leaflets inoculated with the 45 mm/h rainfall intensity treatment had a higher (P < 0.01) mean number of uredinia/cm2 than the 85 mm/h treatment, even though they were inoculated with approximately 40% fewer urediniospores. Subsequent spore-free rainfall reduced (P < 0.01) uredinia density by as much as 38 and 91% for the 1- and 30-min durations, respectively. The relationship between uredinia density proportion and depth of rainfall was best fit using an inverse power empirical model. Our results indicate that a majority of the wet deposited P. pachyrhizi urediniospores would be removed from soybean leaf surfaces by subsequent rainfall, but sufficient percentages of spores (10 to 25%) will likely remain on the leaf tissue long enough to germinate and infect during heavy summer rains lasting ≥30 min.

Effect of Fungicide and Timing of Application on Soybean Rust Severity and Yield.

Soybean rust, caused by Phakopsora pachyrhizi, is a devastating foliar disease of soybean that may cause significant yield losses if not managed by well-timed fungicide applications. To determine the effect of fungicide timing on soybean rust severity and soybean yield, field trials were completed in Paraguay (four locations), the United States (two locations), and Zimbabwe (one location) from 2005 to 2006. Treatments at each location included applications of tebuconazole, pyraclostrobin, or a combination of azoxystrobin + propiconazole, and in some locations pyraclostrobin + tebuconazole at the following soybean growth stages (GS): (i) GS R1 (beginning flowering), (ii) GS R3 (beginning pod), (iii) GS R5 (beginning seed), (iv) GS R1 + R3, (v) GS R3 + R5, and (vi) GS R1 + R3 + R5. Soybean yields from plots treated with fungicides were 16 to 114% greater than yields from no fungicide control plots in four locations in Paraguay, 12 to 55% greater in two locations in the United States, and 31% greater in Zimbabwe. In all locations, rust severity measured over time as area under the disease progress curve (AUDPC) was negatively correlated (r = -0.3, P < 0.0001) to yield. The effectiveness of any given treatment (timing of application and product applied) was often dependent on when rust was first detected and the intensity of its development. For example, when soybean rust was first observed before GS R3 (two locations in Paraguay), the plants in plots treated with a fungicide at GS R1 had the lowest AUPDC values and highest yields. When soybean rust was first observed after GS R3, plants treated with a fungicide at GS R3 and/or GS R5 had the lowest AUDPC values and highest yields with a few exceptions.

The use of magnetic field effects on photosensitizer luminescence as a novel probe for optical monitoring of oxygen in photodynamic therapy.

The effect of a magnetic field on the steady-state and time-resolved optical emission of a custom fullerene-linked photosensitizer (PS) in liposome cell phantoms was studied at various oxygen concentrations (0.19-190 microM). Zeeman splitting of the triplet state and hyperfine coupling, which control intersystem crossing between singlet and triplet states, are altered in the presence of low magnetic fields (B < 320 mT), perturbing the luminescence intensity and lifetime as compared to the triplet state at B = 0. Measurements of the luminescence intensity and lifetime were performed using a time-domain apparatus integrated with a magnet. We propose that by probing magnet-affected optical emissions, one can monitor the state of oxygenation throughout the course of photodynamic therapy. Since the magnetic field effect (MFE) operates primarily by affecting the radical ion pairs related to type I photodynamic action, the enhancement or suppression of the MFE can be used as a measure of the dynamic equilibrium between the type I and II photodynamic pathways. The unique photo-initiated charge-transfer properties of the PS used in this study allow it to serve as both cytotoxic agent and oxygen probe that can provide in situ dosimetric information at close to real time.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Erythrina herbacea (Coral Bean).

Soybean rust (SBR), caused by the obligate fungus Phakopsora pachyrhizi Syd. & P. Syd., was initially reported on soybean (Glycine max L.) in Louisiana in 2004 and has since been reported on soybean and/or kudzu (Pueraria lobata (Willd.) Ohwi) in 9 states in 2005, 15 states in 2006, and 19 states in 2007 (1). The host range of P. pachyrhizi includes plants that are all in the Fabaceae or legume family. Six plant species in the United States have been reported as hosts of P. pachyrhizi: soybean, kudzu, Florida beggarweed (Desmodium tortuosum (Sw) DC.), dry bean (Phaseolus vulgaris L.), lima bean (P. lunatus L.), and scarlet runner bean (P. coccineus L.) (4). On 17 April 2008, a rust disease was observed on a weedy legume host with red showy flowers that was growing with kudzu in an overgrown vacant lot in the understory of live oak trees (Quercus virginiana Mill.) in Citra, FL. The discovery was made during routine scouting of this Integrated Pest Management Pest Information Platform for Extension and Education (IPM PIPE) mobile sentinel plot (3). The plant was confirmed by University of Florida botanists to be Erythrina herbaceae L., commonly known as coral bean. Coral bean is native to the southeastern United States and also is planted as a perennial ornamental. A sample of leaves exhibiting rust pustules characteristic of P. pachyrhizi uredinia was collected and examined with a microscope. Brown-to-brick red, angular lesions that were 3 to 11 mm in diameter (average 6.75 mm) were observed on the undersides of the leaves of two trifoliates. Within these lesions, there were several uredinia, some exuding hyaline, echinulate urediniospores (20 × 25 µm). The visual diagnosis and the species of the rust fungus were confirmed to be P. pachyrizi by a real-time PCR protocol (2). The diagnosis on this new host was verified by a USDA, APHIS National Mycologist in Beltsville, MD. Coral bean may serve as an additional overwintering host for P. pachyrhizi in the southeast. To our knowledge, this is the first report of soybean rust caused by P. pachyrhizi on E. herbaceae. References: (1) R. S. C. Christiano and H. Scherm, Phytopathology 97:1428, 2007. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) S. A. Isard et al. Online publication. doi:10.1094/PHP-2006-0915-01-RV. Plant Health Progress, 2006. (4) T. L. Slaminko et al. Plant Dis. 92:767, 2008.

Gene expression analysis in soybean in response to the causal agent of Asian soybean rust (Phakopsora pachyrhizi Sydow) in an early growth stage.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi Sydow is a potentially devastating disease posing a serious threat to the soybean industry. Understanding plant host response at the molecular level is certainly important for control of the disease. The main objective of this study was to perform a transcriptome profiling of P. pachyrhizi-exposed young soybean plants (V2 growth stage) using whole genome Affymetrix microarrays of soybean. Three-week-old soybean cv. 5601 T plants at the V2 growth stage were inoculated with P. pachyrhizi, and leaf samples were collected 72 h post inoculation with subsequent microarray analysis performed. A total of 112 genes were found to be differentially expressed from P. pachyrhizi exposure, of which 46 were upregulated, and 66 were downregulated. Most of the differentially expressed genes were general defense and stress-related genes, and 34 of these were unknown. Confirmational real-time reverse transcription-polymerase chain reaction was performed on a subset of 5 out of 112 differentially expressed genes. These results were congruent with the microarray analysis. Our results indicated that low and nonspecific innate response to the pathogen may account for the failure to develop rust resistance in the soybean variety studied. To our knowledge, this is the first microarray analysis of soybean in response to ASR.

Influence of Flower Thrips on Fusarium Hardlock Severity.

Cotton (Gossypium hirsutum) fiber is sometimes affected by hardlock, which is characterized by a failure of the fiber to expand outward from the boll at maturity. Because affected fiber is inaccessible to mechanical harvesters, yield loss can be considerable. Hardlock has been linked to infection by Fusarium verticillioides. The involvement of flower thrips (Frankliniella spp.), which are commonly found in cotton flowers, was explored. At 1100 h, approximately 10% of cotton flowers contained thrips that were carrying F. verticillioides. The effect of thrips and/or Fusarium in flowers and bolls was explored under greenhouse conditions. Exposing flowers to Fusarium and thrips resulted in bolls with the most severe symptoms. Exposure to either Fusarium or thrips alone resulted in more hardlock than was noted in the control group. The impact of thrips was also evaluated under field conditions. Field plots were treated with insecticides, a fungicide, both, or left untreated. Insecticides reduced thrips numbers and reduced hardlock severity. The fungicide had no impact on thrips numbers and was less effective at reducing hardlock. Combining insecticide and fungicide applications was no more effective than using insecticides alone, although it more frequently increased yield. The untreated control plots generally had the most severe hardlock and lowest yields. Reducing hardlock severity resulted in higher yields, although not consistently. These studies suggest that thrips increase the severity of hardlock, and reducing their numbers may diminish hardlock severity.

First Report of Soybean Rust Caused by Phakopsora pachyrhizi on Phaseolus spp. in the United States.

Phakopsora pachyrhizi Syd. & P. Syd., the cause of soybean rust, was first observed in the continental United States in November 2004 (2). During the growing season of 2005, P. pachyrhizi was confirmed on soybean (Glycine max) and/or kudzu (Pueraria montana) in nine states in the southern United States. It is known that P. pachyrhizi has a much larger host range within the Fabaceae family. On 29 September 2005 in Quincy, FL, 45 entries of mostly large-seeded legumes were planted next to soybeans that were infected with P. pachyrhizi. Several seeds of each entry were planted on one hill. Soybean plants growing adjacent to these potential hosts had 15 to 25% of the leaf area affected, 95% incidence, and 73% defoliation on 16 November. On 7 December 2005, all the plants of Phaseolus coccineus L. (scarlet runner bean, PI311827), Phaseolus lunatus L. (lima bean, PI583558), and two Phaseolus vulgaris L. (kidney bean) cvs. Red Hawk and California Early Light Red Kidney (CELRK) were found to have leaves with suspected rust lesions. These plants were at physiological maturity but had not senesced. None of the hosts had been inoculated other than from spores produced by the adjacent rust-infected soybean plants or from unknown locations. On the basis of microscopic examination, suspected infected leaves from plants of the Phaseolus spp. had rust pustules characteristic of P. pachyrhizi uredinia. Uredinia were counted within a randomly selected 2-cm2 area of one leaf of each sample. The mean and range of uredinia per lesion for Phaseolus coccineus was 29 uredinia with a range of 0 to 3 uredinia per lesion, Phaseolus lunatus had 2 uredinia with 0 to 1 uredinium per lesion, Phaseolus vulgaris cv. Red Hawk had 22 uredinia with 0 to 5 uredinia per lesion, and Phaseolus vulgaris cv. CELRK had 43 uredinia with 0 to 4 uredinia per lesion. Polymerase chain reactions using two sets of primers (Ppa1/Ppa2 and Pme1/Pme2) were performed on DNA extracted from leaves of the three species with sporulating rust pustules (1). The results of these tests and further tests conducted by the USDA/APHIS confirmed that P. pachyrhizi was the causal organism for the observed rust. References: (1) P. F. Harmon et al. On-line publication. doi:10.1094/PHP-2005-0613-01-RS. Plant Health Progress, 2005. (2) R. W. Schneider et al. Plant Dis. 89:774, 2005.

First Report of Phakopsora pachyrhizi Telia on Kudzu in the United States.

Soybean rust caused by Phakopsora pachyrhizi H. Sydow & Sydow was first reported in the continental United States during 2004 (2). By 10 November 2005, the disease was confirmed in eight southern states (Florida, Georgia, Alabama, Mississippi, South Carolina, North Carolina, Louisiana, and Texas). Diagnoses have been based on visual observation of uredinia and urediniospores of the pathogen followed by polymerase chain reaction confirmation. On 10 November 2005, uredinia and telia were identified on leaves of kudzu (Pueraria lobata) in central Florida. Telia first were noted as dark brown-to-black flecks on the abaxial leaf surface intermingled with abundant tan-to-light brown uredinia. Of 200 leaves examined, 143 (72%) had telia. The number of telia ranged from a few (1/cm2) that were scattered to many (73/cm2). Telia were approximately the same diameter as uredinia, but were appressed to the leaf surface and pigmented. Twenty telia were excised from host tissue with the aid of a dissecting microscope and a 20 gauge hypodermic needle. Telia averaged 89 × 100 µm (n = 20, σ = 17 and 16 µm, respectively). Four telia were crushed and five teliospores from each averaged 4.3 × 8.3 µm (n = 20, σ = 0.5 and 0.9 µm, respectively). Pale yellowish brown-to-hyaline teliospores were similar in color to urediniospores. Observations matched descriptions by Ono et al. (1). To our knowledge, this is the first report of the telial stage of P. pachyrhizi in the United States. References: (1) Y. Ono et al. Mycol. Res. 96:825, 1992. (2) R. W. Schneider et al. Plant Dis. 89:774, 2005.

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.

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.

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe.

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.

3'-Azido-3'-deoxythymidine (AZT) mediates cross-resistance to nucleoside analogs in the case of AZT-resistant human immunodeficiency virus type 1 variants.

Difficulties in deciphering the mechanisms of 3'-azido-3'-deoxythymidine (AZT)-resistance by human immunodeficiency virus type 1 (HIV-1) variants are due in part to an inability to reconstitute resistance in vitro using AZT-resistant reverse transcriptases. We decided to characterize mechanisms of AZT resistance in tissue culture infections by studying the ability of drug-resistant viruses to synthesize viral DNA in the presence or absence of drug. Through use of PCR amplifications, we discovered an AZT-mediated stimulation of reverse transcription by AZT-resistant viruses carrying the M41L and T215Y mutations that can apparently override the inhibitory effects of AZT-5'-triphosphate. In addition, the presence of AZT also causes viruses containing the M41L and T215Y substitutions to have diminished sensitivity to other nucleoside analogs (i.e., ddC, ddI, and d4T). This AZT-mediated cross-resistance may help to explain the virological failure of treatment regimens that included ddI plus AZT or ddC plus AZT in situations in which the T215Y and/or M41L mutations were present (F. Brun-Vézinet, C. Boucher, C. Loveday, D. Descamps, V. Fauveau, J. Izopet, D. Jeffries, S. Kaye, C. Krzyanowski, A. Nunn, R. Schuurman, J. M. Seigneurin, C. Tamalet, R. Tedder, J. Weber, and G. J. Weverling, Lancet 350:983-990, 1997). Our results suggest that the use of AZT may be contraindicated in those patients for whom resistance to this compound (M41L and/or T215Y) has been demonstrated.