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2009 Caribbean Division Meeting Abstracts

May 16-19, 2009 - Orlando, Florida
(Joint with The Florida Pathological Society)

Posted online September 29, 2009

Monitoring resistant populations of Xanthomonas citri subsp. citri and epiphytic bacteria on young citrus trees treated with copper or streptomycin
F. BEHLAU (2), J. B. Jones (2), J. H. Graham (1)
(1) Citrus Research and Education Center, University of Florida, Lake Alfred, FL; (2) Plant Pathology Department, University of Florida, Gainesville, FL

Since Florida’s citrus canker (Xanthomonas citri subsp. citri, Xcc) eradication program was halted in 2005 attention has focused on management strategies that include the use of bactericides such as copper and streptomycin for disease control. Widespread use of these chemicals in citrus industries elsewhere in the world has led to development of resistant strains of Xcc. Cu and Sm resistance were monitored in Xcc and epiphytic bacterial populations on citrus trees repeatedly sprayed with these chemicals for control of citrus canker. Copper hydroxide (Cu, Kocide 3000) or streptomycin sulphate (Sm, Firewall) were sprayed on foliage of young ‘Ray Ruby’ grapefruit every 21 days from March to October 2008. Mature canker-symptomatic and non-symptomatic leaves were sampled monthly to assay for resistant Xcc and epiphytic bacteria, respectively. Leaves were washed with MGY broth + 1 mg/L of CuSO(4) for 2 hrs using 10 mL of liquid medium/g of leaf and plated on semi-selective medium MGY-KCC + Cu or Sm for isolation of resistant Xcc or on MGY + Cu or Sm for monitoring resistant population of epiphytic bacteria. No Cu or Sm resistant strains of Xcc were isolated. No major differences in total epiphytic bacterial population were observed among treatments over time in comparison to the check. However, Cu and Sm sprays increased the ratio of epiphytic bacterial population with resistance to these chemicals. Overall, the Sm resistant bacterial populations were proportionally lower than Cu resistant bacterial population.

Survival of Xanthomonas citri subsp. citri on symptomatic fruit under prolonged ambient and cold storage conditions
G. Bonn (1), E. TAYLOR (3), T. Riley (2), T. Gottwald (3), T. Schubert (1)
(1) Florida Department of Agriculture & Consumer Services, Division of Plant Industry, Gainesville, FL; (2) USDA, APHIS, PPQ, Orlando, FL; (3) USDA, ARS, US Horticultural Research Laboratory, Ft. Pierce, FL

Live cells of Xanthomonas citri subsp. citri (Xcc) were detected by excising canker lesions from commercial fresh-packed grapefruit, macerating them in phosphate buffer followed by dilution plating onto a semi-selective agar medium (KCB). After 4–5 days incubation at 28°C, separate colonies were counted at 100X using a dissecting microscope. Confirmation of Xcc was by the use of Agdia’s ImmunoStrip® for suspect plate colonies and a bioassay by needleless infiltration of leaf lamina of young ‘Duncan’ grapefruit seedlings. Xcc was detected from lesions on fruit held at both ambient and 5°C for up to 100 days in storage. There was a general trend for the percentage of Xcc positive lesions and actual bacterial populations to decrease with storage time. Populations of Xcc decreased faster at ambient temperature than at 5°C, possibly due to the higher metabolic activity of cells or microbial competition at the elevated ambient temperature. The low numbers of viable canker bacteria associated with peel lesions in grapefruit, especially over time, suggest that the risk of canker transmission from them is extremely low in comparison to active lesions on the leaves, stems and fruit during the growing season.

Distribution of Candidatus Liberibacter asiaticus in huanglongbing infected citrus trees
R. H. BRLANSKY (1), K. S. Pelz-Stelinski (1)
(1) University of Florida, CREC

The distribution of the huanglongbing (HLB) associated Candidatus Liberibacter asiaticus (Las) bacterium in mature field infected citrus trees was evaluated. The number of tissue samples collected per tree ranged from 16–32 and included: fruit peduncles, bark phloem, and symptomatic leaf midribs and petioles taken from throughout the tree canopy. Overall, the percentage of Las-positive plant tissue samples obtained ranged from 23–44% based on real-time PCR results. Although samples taken from bark phloem varied, phloem from one-year old bark consistently tested positive for Las. Similar variation in the detection of Las occurred in samples obtained from leaf petioles and fruit peduncles. The percentage of positive leaf petioles ranged from 0–100%, while fruit peduncles ranged from 17–56%. Additional replicates continue to be collected in order to firmly establish whether one-year old bark phloem is consistently positive when another part of the tree has tested positive for Las via PCR. These results indicate that distribution of the HLB-associated pathogen varies widely within symptomatic, PCR-positive citrus trees and thus illustrate the importance of obtaining multiple samples from trees where an infection is suspected.

Infectious clones and characterization of a previously unreported bean-infecting begomovirus from Rynchosia minima (L.), an endemic legume species from Puerto Rico
J. K. BROWN (2), M. Rehman (1), A. M. Idris (2)
(1) Department of Microbiology, Hazara University, Mansehra, Pakista; (2) Department of Plant Sciences, The University of Arizona, Tucson, AZ, USA

R. minima
plants exhibiting mild mosaic symptoms that were reminiscent of begomovirus infection were observed in Puerto Rico during the summer, 1997. Total nucleic acids were extracted from symptomatic R. minima leaves using the CTAB method. The viral single-stranded DNA was subjected to rolling circle amplification. The SacI-linearized, multimeric DNA band was ligated into SacI-digested pGEM7Zf+ and cloned. Eight clones bearing a ~2.6 kbp fragment were sequenced using primer walking. Analysis of the resultant sequences indicated that five and three of the clones were DNA-A and DNA-B components, respectively. The genome organization and number of open reading frames (six) was typical of other bipartite begomoviral genomes from the Western Hemisphere. Comparative analysis of the DNA-A (n = 5) and DNA-B (n = 3) component sequences shared 98–99% and 99% nucleotide (nt) identity, respectively. Inspection of the common region (CR) revealed that they were cognate components, and shared an identical iteron. The DNA-A component shared 80% nt identity with its closest relatives, Macroptilium mosaic Puerto Rico virus and Rhynchosia golden mosaic virus. The DNA-B component shared 64% and 62% nt identity with RhGMV and Cabbage leaf curl virus, respectively. This previously undescribed begomovirus species is herein named Rhynchosia mild mosaic virus (RhMMV). Clones of the RhGMV DNA-A and DNA-B component were released with SacI and biolistically inoculated to R. minima and bean (Phaselous vulgaris) seedlings. R. minima seedlings developed mild mosaic symptoms like those observed in field-infected bean plants, thereby fulfilling Koch’s postulates, and the bean seedlings developed severe green-yellow mosaic symptoms, confirming that the virus also infects bean. This previously undescribed virus could pose a serious threat to bean crops in the Caribbean region.

Developing an effective international education program for management of Ralstonia solanacearum Race 3 biovar 2
P. G. CHAMPOISEAU (1), J. B. Jones (1), C. Allen (3), T. M. Momol (2)
(1) University of Florida, Department of Plant Pathology, Gainesville, FL, USA; (2) University of Florida, District Directors Office, Gainesville, FL, USA; (3) University of Wisconsin-Madison, Department of Plant Pathology, Madison, WI, USA

Because it threatens both potato and ornamental production, Ralstonia solanacearum Race 3 biovar 2 (R3bv2) is considered a serious quarantine pest in Canada and Europe and is listed as a Select Agent plant pathogen in the United States, where it is subject to the strictest biosecurity regulations. Although this pathogen is not known to be established in the US, import of infected geranium cuttings from off-shore production sites has already proved to be a possible pathway for introduction. Previous accidental introductions resulted in multi-million dollar losses due to quarantine responses. Therefore it is critical to prevent further re-introduction and possible spread of R3bv2 in the US. This involves exclusion, early detection, and unambiguous identification of the pathogen at both national and international levels. This can be achieved by use of reliable diagnostic tools for the pathogen and effective phytosanitary measures; however, this is not enough. It is also essential to ensure preparedness and effective training of official regulators, diagnosticians and other individuals responsible for first detection and response to a possible R3bv2 discovery in the US. We have therefore developed an integrated education and outreach program, as part of a USDA-founded project for a better management of R3bv2. This program involves development of educational and training content by a team of experts, delivery of educational materials to target audiences by diverse means including current web-based technologies, as well as use of various evaluation tools to assess program effectiveness. Monitored access of our Ralstonia/bacterial wilt-dedicated website shows that stakeholders from diverse organizations both within and outside the US regularly use this resource to obtain updated and accurate information on R. solanacearum R3bv2 and bacterial wilt disease management.

Nitrocellulose membranes as a solid matrix for Cucumber mosaic virus immuno-detection and subgroup identification by RT-PCR
P. S. CHANG (1), S. A. Tolin (1)
(1) Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

Cucumber mosaic virus
(CMV) is an important and widespread plant virus. The strains and isolates of CMV are highly diverse and assigned to either subgroup 1A, 1B or 2. Here we report the application of tissue blot immunoassay (TBIA) followed by reverse transcription-polymerase chain reaction (RT-PCR) for the immuno-detection and subgroup identification of CMV from various hosts and locations. Freshly torn leaves were blotted onto nitrocellulose membranes (NCM), which were used as sources of viral RNA after processing by TBIA. CMV positive samples show a purple precipitate at the blot site. A 3 mm disc was removed from the positive sites and cleaned by rinsing with Triton X-100, followed by TE buffer, then dried and added directly to RT reactions that included a reverse primer to the CMV coat protein (CP) gene. The resulting cDNA was added to PCR reactions containing forward and reverse primers for the CMV CP gene. Agarose gel electrophoresis revealed amplicons of the expected size. The subgrouping of CMV samples was predicted from sequences of PCR products and confirmed by monoclonal antibodies specific to CMV subgroups 1 and 2. Successful amplification was possible from NCM blotted and TBIA processed up to 15 months previously, but amplification levels from older blots were lower. The PCR protocol was adjusted by increasing the number of cycles for consistent results from blots older than 8 months. This method eliminates the need for leaf tissue storage or costly RNA extraction when sampling for CMV diversity or monitoring virus prevalence and incidence. NCM are thus a suitable matrix for obtaining viral RNA for RT-PCR and archival storage of viral nucleic acids, similar to Whatman’s FTA® Plant Cards.

Sugarcane orange rust, an emerging disease in the western hemisphere
J. C. COMSTOCK (1), N. C. Glynn (1), L. A. Castlebury (2)
(1) USDA-ARS Sugarcane Field Station, Canal Point, FL; (2) Yystematic Mycology and Microbiology Laboratory, ARS, USDA, Beltsville, MD

Symptoms consistent with sugarcane orange rust were first observed in Florida in June 2007, these were subsequently confirmed morphologically and molecularly as being caused by Puccinia kuehnii, the causal agent of orange rust. This was the first documented occurrence of sugarcane orange rust in the Western Hemisphere. Since then it has been reported in Guatemala, Costa Rica and Nicaragua and has been confirmed in several other Central American and Caribbean Countries. A comparison of brown rust and its causal agent, P. melanocephala and P. kuehnii, will be presented. Orange rust has impacted both the commercial production and the cultivar development program in Florida. One major difference in the epidemiology of the two pathogens is that P. kuehnii tolerates warmer temperatures and orange rust severity continues throughout the summer and early fall lasting much longer than brown rust. This is significant as it means that commercial cultivars susceptible to both pathogens are impacted by either one or both pathogens depending on the month of the growing season. A cultivar that occupies 25% of the acreage in Florida, CP 80-1743, is susceptible to the disease and has had reduced cane yields. It is being withdrawn from production. Results from a comprehensive approach towards developing sugarcane cultivars resistant to orange rust that is being adopted in the Canal Point breeding program will be presented. This involves identifying and discarding susceptible sugarcane clones as early in the breeding program as possible, the development of novel screening methods and the identification of sources of resistance for breeding.

Candidatus Liberibacter solanacearum’ on tomato and potential losses in field production
R. D. FRENCH-MONAR (3), R. W. Wallace (2), J. A. Abad (1), T. A. Wheeler (4)
(1) APHIS-PPQ-PGQP, Beltsville, MD, USA; (2) Dept. of Horticultural Sciences, AgriLife Extension-Texas A&M System, Lubbock, TX, USA; (3) Dept. of Plant Pathology and Microbiology, AgriLife Extension-Texas A&M System, Amarillo, TX, USA; (4) Dept. of Plant Pathology and Microbiology, AgriLife Research-Texas A&M System, Lubbock, TX, USA

In August 2008, tomato (Solanum lycopersicum) plots in Lubbock County, TX that were utilized for a chemical test aimed at management of root knot nematode became infected with ‘Candidatus Liberibacter solanacearum’. Overall symptoms on tomato cv. Spitfire included leaf yellowing, lateral stem dieback, upward leaf curling, enlargement of stems, adventitious roots, and swollen nodes. PCR amplification was done using 16S rDNA OA2 and OI2c primers for ‘Ca. L. solanacearum’ used for potato, tomato, and other solanaceous crops in New Zealand, which amplifies a 1.1 kb fragment of the 16S rRNA gene of this new species. A 1.1 kb fragment was obtained, sequenced, and found to be 99.9% identical in sequence to a ‘Ca. L. solanacearum’ obtained last year from a potato production field in Texas. In the tomato field, a total of 32 plots (one-row wide, 7.7 m long) comprised of 24 plants per plot were evaluated for disease symptoms and galling by root-knot nematode. Foliar disease incidence in plots ranged from one (4.2%) to 19 (79.2%) plants showing symptoms by the last harvest date. Regression analysis was used to determine losses in yield associated with the bacterium and with root-knot nematode. Percent galling by root-knot nematode only explained 14% of the variability in yield, and 100% galling was predicted to cause a 1.5% loss in yield, based on the regression model. In contrast, for each 1% incidence in plants with disease symptoms, there was a 0.9% loss in yield. In essence, there was no yield contribution if a plant developed symptoms (R(^2) = 0.41). The potential exists for ‘Ca. L. solanacearum’ to be a detriment in tomato production and a source for survival of this bacterium that has been found to be associated with the Zebra Chip disease in potato, tomato, pepper (Capsicum annuum), and other solanaceous crops.

Diverse tomato cropping systems affect arbuscular mycorrhizal fungal community diversity and structure
E. G. JOHNSON (3), D. O. Chellemi (2), T. Wu (1), J. H. Graham (3)
(1) Division of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, USA; (2) USDA-ARS, US Horticulture Research Laboratory, Fort Pierce, FL, USA; (3) University of Florida, Citrus Research and Education Center, Lake Alfred, FL, USA

In conventional agricultural systems, AMF are greatly affected by factors including soil disruption, intermittent lack of host root tissue, and plant inhibition of AMF colonization due to high soil fertilization. To determine the effect of diverse agricultural land management and crop production practices on the AMF community structure and diversity, five tomato crop production systems consisting of bahiagrass pasture cover, conventional, continuous removal of vegetation (disk fallow), organic, and undisturbed (weed fallow) were initiated. The plots were adjusted to the new management regime, except for conventional, for three or four years followed by one or two years of tomato cropping. Soil DNA samples were taken in the off season, at planting, and after harvest. Phylogenetic analysis of AMF 18S rDNA sequence combined with multivariate statistical analysis using PRIMER-E was used to compare community structure and diversity. Initial analysis shows that bahiagrass, weed fallow, and organic land management practices support different, diverse AMF communities, while disk fallow and conventional practices greatly reduced detection of AMF sequences. Tomato cropping caused the emergence of common sequences for the Glomus mosseae group, in all cropping systems. Bahiagrass and weed fallow diversity were unaffected by the emergence of the G. mosseae group, while organic, conventional and disk fallow all converge on a low diversity community dominated by the G. mosseae group. Current analyses will determine if the shift in AMF community caused by tomato cropping in organic and bahiagrass plots is seasonal or persistent, and if other factors such as soil fertility and disease incidence correlate to these community changes.

Identification and characterization of powdery mildew caused by Golovinomyces cichoracearum on sunn hemp
S. A. JORDAN (1), G. S. Maia (1), A. J. Gevens (1)
(1) University of Florida, Gainesville, FL, USA

Crotalaria juncea
, or sunn hemp, is a warm season legume grown in FL as a cover crop. In 2008, powdery mildew was observed on sunn hemp in a research field in Hastings, FL. This disease is important because it has the potential to impact the quality of sunn hemp and this powdery mildew can infect cucurbits which are grown in north FL in late summer. Fungal growth appeared first on lower, more mature leaves as white, powdery mildew colonies initially seen on upper leaf surfaces and later moving to undersides; petioles and floral parts were disease-free. As disease progressed, colonies enlarged, coalesced, and covered entire leaf surfaces; heavily infected leaves senesced and abscised. Mycelia produced white accumulations of conidiophores and conidia. Hyphae were superficial with papillate appressoria and produced conidiopohores with cylindrical foot cells that measured 48.5 × 10.0 µm. Conidia were hyaline, short-cylindrical-ovoid, lacked fibrosin bodies, borne in short chains, had sinuate edge lines with other immature conidia, and measured 22.5-40.0 × 12.5-20.0 µm. The teleomorph was not observed. The nuclear rDNA internal transcribed spacer (ITS) regions were amplified by PCR and sequenced. On the basis of morphological characteristics of the asexual state and ITS sequence data, the pathogen was identified as G. cichoracearum. Pathogenicity was confirmed on healthy plants. This is the first report of G. cichoracearum causing powdery mildew on C. juncea.

Phytophthora cactorum
a serious problem on prefinished Cattleya orchid liners from Thailand
R. T. MCMILLAN (1), A. J. Palmateer (2), R. A. Cating (2)
(1) Kerrys, Homestead, FL; (2) University of Florida, Homestead, FL

The major pathogen on Cattleya orchids in Florida and in the New and Old World countries is Phytophthora cactorum (Lebert & Cohn) J. Schroet., which causes Black Rot during the wet months of the year. All species of Cattleya and their interspecific and intergeneric hybrids are susceptible. Phytophthora cactorum infects leaves, pseudobulbs, rhizomes, and flower buds. Shipments of prefinished Cattleya orchid liners from Thailand during the monsoon season often infected with P. cactorum. Orchid plants with visual symptoms of P. cactorum were removed from the shipment and drenched with fungicides such as Banrot, Natriphene, Shield Brite, Truban, and Phyton 27, Heritage, Shield Brite, Stature, Truban, Pentathlon, Aliette, Subdue Maxx, and Insignia, in an effort to salvage some of the plants. In spite of the effort to save the orchids, the level of Phytophthora over rode the attempt to control the fungus resulting in destroying the shipments. Cattleya liner shipments during the dry season are found to be P. cactorum free.

Controlling angular leaf spot in Florida annual strawberry
J. C. MERTELY (1), N. A. Peres (1)
(1) University of Florida-GCREC, Wimauma, FL, USA

Angular leaf spot (ALS) is a bacterial disease caused by Xanthomonas fragariae that produces unsightly lesions on strawberry leaves and sepals. Leaves with numerous spots and/or vein-following lesions become blighted and die prematurely. During the 2008–2009 growing season, an epidemic of ALS occurred in the principal Florida strawberry production area west of Tampa. During the season, a replicated trial evaluating products for ALS control was conducted at the Gulf Coast Research and Education Center in Wimauma, FL. Treatments included the plant defense activator acibenzolar-s-methyl (Actigard); copper fungicides Badge, Cuprofix, IRF070, Kentan, Kocide, and Quint; hydrogen peroxide (Oxidate); and Streptomyces lydicus (Actinovate) applied weekly throughout the season to the foliage with a CO(2) back pack sprayer. A late-season evaluation of foliar symptoms showed that Badge, IRF070, Kentan, Kocide, and Actigard significantly reduced the proportion of leaves killed and partially blighted by X. fragariae. Alternating applications of Actinovate and Cuprofix also showed this effect. However, only Badge and the low rate of Actigard significantly increased marketable yield during a season with markedly high disease pressure. Future experiments may target applications to periods favorable to infection and disease spread, such as rain events associated with approaching cold fronts, and periods of prolonged overhead irrigation for freeze protection.

Cultivar susceptibility, temperature and leaf wetness durations required for lesion production by Alternaria alternata on tangerine and tangerine hybrids
S. N. Mondal (1), L. Timmer (1), M. M. DEWDNEY (1)
(1) University of Florida

Alternaria brown spot, caused by A. alternata, is an important disease of tangerines and their hybrids in many citrus-producing regions. A prediction model for fungicide applications, the Alter-Rater, was developed previously, but it was unknown whether the relationship between temperature and leaf wetness duration (LWD) would be consistent across all tangerine hybrids. We tested the LWD and temperature relationships on 5 tangerine and tangerine hybrids: Dancy, Minneola, Murcott, Nova and Sunburst. The LWDs were 2, 4, 8, 16, 24 and 30h at temperatures of 20, 24, 28 and 32°C. The rating scale for the number of lesions/leaf was: 0 = 0; 1 = 1-2; 2 = 3-5; 3 = 6-10; 4 = 11-15; 5 => 15 lesions/leaf and the data were taken from 15 leaves. The experiment was an incomplete block design. Cultivar differences were observed; lesions formed on Minneola and Dancy with as little as 2h of leaf wetness at all temperatures. Lesions were observed on Murcott, Nova and Sunburst with 4h of leaf wetness. The optimal temperature range for lesion production was 24 and 28°C for all LWDs. On the more susceptible Minneola and Dancy, 24h LWD was required to reach the max. lesion rating, but that level was never reached on Murcott, Nova and Sunburst even with 30h of leaf wetness. The results should be incorporated into the Alter-Rater model so that unnecessary sprays are not applied to less susceptible tangerine and tangerine hybrids.

Identification of the Florida torreya canker pathogen
L. L. MOUNT (1), J. A. Smith (1)
(1) University of Florida

The Florida torreya (Torreya taxifolia Arn.) is a conifer of the Taxaceae. The native population is restricted to within 20 miles of the Apalachicola River in northern Florida and southern Georgia. Torreya wood is resistant to decomposition. For this reason, it was lumbered for railroad ties in the 19th century. Surveys conducted prior to 1970 detail a dramatic reduction of population numbers, size and health. Historically, trees reached 18 m at maturity. Of the 58 trees surveyed in November 2008, no individual surpassed a height of one meter. Disease symptoms associated with this decline are leaf spot, shoot tip dieback, and cankers. Fungal cultures were isolated from symptomatic tissue and from the initial 58 trees tested, cultures of Fusarium spp. and Botryosphaeria spp. were each isolated 20 times. The same tree sample often produced both genera. Products were amplified by polymerase chain reaction using internal transcribed spacer region rDNA (ITS-rDNA) specific primers and the sequences obtained were compared to those deposited in the GenBank database. Four unique sequences of Fusarium spp. and Botryosphaeria obtusa were identified. Fusarium solani and F. lateritium matched GenBank sequences to the species level. Fusarium lateritium was previously identified as the causal agent of the leaf spot, but was cultured during this study directly from cankered tissue. Two species of Phomopsis, as well as Diaporthe, Lasiodiplodia, and Hypoxylon were also infrequently isolated and identified in the same manner from dying shoots and cankers sampled in November 2008 and January 2009. Inoculations of as many of these genera as possible were conducted on torreyas grown using sterile tissue culture at Atlanta Botanical Gardens in April 2009.

The impact of silicon soil amendments on cucumber anthracnose in the greenhouse
J. PALENCHAR (1), S. Taber (1), L. E. Datnoff (2), and A. J. Gevens (1)
(1) Department of Plant Pathology, University of Florida, Institute of Food and Agricultural Sciences, Gainesville, FL; (2) Current address: Dept. of Plant Pathology and Crop Physiology, Louisiana State University

Cucumber is an economically important crop in Florida and in other parts of the U.S. In Florida, one of the most common diseases on cucumber is anthracnose, caused by the ascomycetous fungus Colletotrichum orbiculare. Anthracnose can cause serious yield and quality losses, and produces symptoms on all aboveground plant parts at any stage of growth. Organic producers have limited options for anthracnose control. The use of silicon (Si) as a tool for disease control has been established in other crop systems. In the greenhouse, the control of anthracnose was demonstrated on 2-week-old ‘Straight Eight’ cucumber seedlings by amending soil (organic Fafard FOF 30) with a high rate of Si (Vansil W50). Treatments included cucumber seeds planted into 1) Si-amended soil (600 kg Si/ha) + no C. orbiculare inoculation 2) Si-amended soil (600 kg Si/ha) with C. orbiculare inoculation, 3) non-amended soil with no C. orbiculare inoculation, and 4) non-amended soil with C. orbiculare inoculation. Each treatment included 5 replications and the experiment was repeated 4 times. Disease evaluations (Horsfall-Barrett scale) were recorded for leaves at 3, 7, and 14 days post inoculation (dpi) and Si levels in plant tissues were determined. Significant differences between treatments were observed at 7 and 14 dpi. Si treatment reduced disease severity on leaves by 20–60% when compared to the inoculated control. This is the first study demonstrating the efficacy of soil-applied Si for the control of cucumber anthracnose.

Diagnostics and emerging plant pathogens
(1) USDA/APHIS/PPQ, Molecular Diagnostic Laboratory, Beltsville, MD, USA

Accurate identification of plant pathogens is essential for making management decisions and determining appropriate regulatory actions. An accurate identification is facilitated when a group is well understood taxonomically based on robust systematics studies. Such studies provide information on which morphological and molecular characters are taxonomically useful and tools can then be developed to use in detection and identification. Identification of emerging plant pathogens poses a particular challenge in that they are often understudied and poorly characterized. Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, exemplifies how regulatory diagnostics evolve as scientific knowledge about the disease and its causal pathogen is gathered and evaluated. The discovery of new and related taxa, including P. foliorum that cross-reacted in the P. ramorum nested assay, led to the development of additional assays. The use of new markers to compare large numbers of P. ramorum isolates has provided a clearer picture of the genetic diversity in the pathogen and a means of tracing origins of newly found isolates. This case study is one of many examples of the importance of a strong understanding of the systematics of a group as the basis for accurate identification and the development of diagnostic tools.

Basil downy mildew in Florida: A disease of new importance
R. N. RAID (1), P. D. Roberts (4), P. F. Harmon (2), A. J. Palmateer (3), S. A. Jordan (2)
(1) University of Florida, Belle Glade, FL, USA; (2) University of Florida, Gainesville, FL, USA; (3) University of Florida, Homestead, FL, USA; (4) University of Florida, Immokalee, FL, USA

Sweet basil (Ocimum basilicum L.) is one of the most important herbs currently grown in Florida, with both commercial field and greenhouse production. In addition, it is one of the most commonly propagated herbs in home gardens. Fortunately, it has had very few foliar disease problems and has, for that reason, required little or no disease management. During fall 2007, a new disease was first reported on field-grown basil in south Florida. Symptoms initially appeared as a yellowing of the lower canopy, with chlorotic areas frequently delineated by leaf veins. Gray, fuzzy fungal growth was apparent on the abaxial leaf surface. The disease was subsequently reported to be incited by a species of Peronospora. Yield losses during this initial outbreak were near total, since preventative control measures were formerly unnecessary, and therefore, non-existent. Since the initial outbreak, basil downy mildew has become firmly established in Florida. It has been observed from all regions within the state, as well as in numerous other states. Although there is ample evidence that the disease may have been introduced on infested seed, alternative sources (i.e. from closely related hosts) have not been totally ruled out. With widely-scattered, year-round greenhouse and/or field production providing a host continuum, it is very likely that basil downy mildew will be a disease to contend with on a permanent basis. Nearly all basil varieties or types appear susceptible at this time. Management programs are currently under development. Aside from cultural practices to limit leaf wetness and hence fungal infection, preventive foliar applications of phosphonates and strobilurin fungicides have proven useful. Used together in a program, these have provided economic but not total control.

Orange rust of sugarcane: Prospects for fungicidal control
R. N. RAID (2), J. C. Comstock (1), N. C. Glynn (1)
(1) USDA Sugar Cane Field Station, Canal Point, FL, USA; (2) University of Florida, Belle Glade, FL, USA

Orange rust of sugarcane, incited by Puccinia kuehnii, was first observed in Florida during June 2007 on one of the industry’s most important commercial cultivars, CP80-1743. This was the first report of this disease in the Western Hemisphere. It has since been reported in several other Central American and Caribbean Countries. With host-plant resistance being a worthy long-term goal, studies were initiated to investigate the feasibility of fungicides serving as an interim or supplementary management strategy. Thirteen different fungicide treatments were examined for their efficacy in controlling orange rust during the 2008/2009 growing season. Experimental units consisted of two rows of cane 15m in length replicated four times in a randomized complete block design. Fungicide treatments consisted of select candidates from two major classes of fungicides, the strobilurins (FRAC group 11) and triazoles (FRAC group 3), alone, and in combination or alternation. Fungicide applications were made using a CO(2) backpack sprayer and were initiated following canopy closure (approx. 1.5-m ht) at 21 day intervals. Rust severity in the trial area was moderately severe, with severities in excess of 30% on the distal third of the fourth leaf beneath the top-visible-dewlap leaf in the untreated check. Results indicate that the strobilurin fungicides provided the highest level of control, followed by strobilurin/triazole combinations, and finally, the triazole fungicides alone. In separate trials using the strobilurin fungicide pyraclostrobin, fungicide treatments were demonstrated capable of reducing orange rust to levels sufficient to significantly reduce yield losses by as much as 40%. While economic factors will ultimately be an important consideration, levels of orange rust control obtained in these studies show promise regarding prospects for fungicides as a potential management tool.

Fungicidal control of basil downy mildew
R. N. RAID (1)
(1) University of Florida, Belle Glade, FL, USA

Sweet basil (Ocimum basilicum L.) is one of the most common herbs grown by home gardeners in Florida. Commercially, basil ranks as Florida’s most important potted herb and the state ranks second nationally in field production, shipping to the entire eastern seaboard. Since 2007, basil downy mildew, incited by a species of Peronospora, has caused considerable losses for commercial basil growers in the U.S. In the absence of control, total crop failure is common. Two fungicide field trials were conducted to determine the efficacy of various foliar applications for the control of this disease. The tests included both registered and non-registered compounds. Experimental units consisted of four rows 2m in length separated on the ends by alleyways and replicated four times in a randomized complete block design. All experimental compounds were topically applied on approximately a weekly basis using a CO(2) backpack sprayer equipped with 3 flat-fan nozzles mounted on a hand-held boom. Treatment commenced at the 4-6 leaf stage, with mildew present in the area at time of initial application. Disease severity was considered extreme. Products tested in these trials included: acibenzolar, azoxystrobin, Bacillus subtilis, chlorothalonil, copper hydroxide, cyazofamid, cymoxanil/famoxadone, dimethomorph, fenamidone, mandipropamid, mefenoxam, potassium phosphanates, propamocarb, pyraclostrobin/boscalid, and Streptomyces lydicus. All products provided for significant suppression of downy mildew early in the trials, but only a few provided for significant control by the end of the tests. Dimethomorph, mandipropamid, cyazofamid, fenamidone, and mefenoxam provided the highest levels of control, but are not currently labeled for use on basil. Of currently registered products, only azoxystrobin and the potassium phosphonates provided control levels that could be considered acceptable from a marketing perspective. No compounds licensed for use in organic herb production provided acceptable levels of mildew control when sprayed on a weekly basis in these trials.

Current distribution of Texas Phoenix palm decline in Florida
D. A. RESTOM GASKILL (2), N. A. Harrison (3), M. L. Elliot (3), T. R. Smith (1)
(1) Florida Dept. of Agriculture and Consumer Services, Gainesville, FL; (2) USDA-APHIS-PPQ, Sarasota, FL; (3) University of Florida IFAS, Fort Lauderdale, FL

Texas Phoenix palm decline (TPPD) is a fatal disease of date (Phoenix dactylifera, P. sylvestris, P. canariensis, P. reclinata), queen (Syagrus romanzoffiana) and cabbage (Sabal palmetto) palms caused by a ‘Candidatus Phytoplasma palmae’-related strain belonging to subgroup 16SrIV-D. In addition to the economic costs of disease management in nurseries and landscapes, the potential ecological impact due to reduction in S. palmetto populations is incalculable. TPPD was first reported and characterized in 2002 from samples obtained in Corpus Christi, Texas, and was detected in the Tampa Bay area of Florida in 2006. Surveys were conducted by the Cooperative Agricultural Pest Survey (CAPS) in 2008 and 2009 to determine the current distribution in Florida. Palms displaying characteristic symptoms of TPPD were sampled and analyzed by polymerase chain reaction assay. Phytoplasma positive samples from new locations were sequenced. Samples were also submitted by personnel from the University of Florida, Institute of Food and Agricultural Sciences (UF-IFAS) and from private landscape companies. TPPD was determined to occur at a high incidence in a three county area (Hillsborough, Manatee, Sarasota), while it was found to be of limited distribution in seven additional counties (Pinellas, Polk, Hardee, Desoto, Highlands, Lake, Duval). Spread of TPPD is likely occurring locally through an unknown insect vector and over long distances through the transportation of infected palms.

Biosafety regulation and biotechnology: How it affects public research in Latin America and the Caribbean
M. M. ROCA (1)
(1) Zamorano University, Tegucigalpa, Honduras

While poverty in developing countries is usually linked with low agricultural output, pest and plant diseases are major factors that contribute significantly to this low productivity. Genetic engineering and transgenic’s have great potential to improve crop production. However, the application and use of this biotechnology has not materialized for the public sector because of the politics associated with the regulatory process. As a result, the time, effort and expense required for commercialization of transgenic crops are way beyond what public-sector investigators can muster leaving only the private sector to accomplish this task. Restrictive regulations were established when the commercial use of transgenic crops was just beginning, and have not taken into account the more than 12 years of extensive experience gained on crops tested on more than 100 million hectares in 23 countries. This information has scientifically demonstrated that crops obtained through biotechnologies do not have risk profiles that are any different from those developed through more traditional plant breeding methods. The potential health and environmental risks originally foreseen have not materialized. Furthermore, it has been demonstrated that this biotechnology provides environmental and economic benefits. As it now stands, the current biosafety regulatory standards in place continue to delay the development and use of transgenic technology. The time has come where there is a great need to consider both the benefits and the risks of this technology, and analyze them relative to those of the present agricultural production systems especially in Latin America and the Caribbean.

Low molecular variability of Potato yellow vein virus (PYVV) isolates of Solanum phureja and Solanum tuberosum from Colombia
P. Rodriguez-Burgos (2), G. Chaves (2), L. Franco-Lara (1), M. GUZMAN-BARNEY (2)
(1) Militar University Nueva Granada, Bogotá-Colombia; (2) National University of Colombia, Bogotá-Colombia

PYVV Closteroviridae-Crinivirus, is a quarentine, phloem-limited potato virus, with a tripartite ssRNA(+). It causes yellowing of foliage with reduction of yield. It is found in Colombia, Peru, Venezuela and Ecuador and is transmitted by white fly Trialeurodes vaporariorum and tubers. To study variability, CPg of 75 isolates of PYVV from Solanum phureja and 50 from S. tuberosum, from 5 Colombian regions, was amplified by RT-PCR. Amplicons were analyzed by SSCP and 32 were sequenced directly. Ten SSCP patterns were observed (P1 to P10); P1 represented 78% of the isolates, P9 9.6% and P6 4.8%. Phylogenetic analysis of 70% of CPg produced two groups: Group I (29 isolates) and Group II (3 isolates). In group I, isolates 1084 of S. phureja and 1114 of S. tuberosum, showed evidence of possible recombination within CPg. No direct correspondence between the number of SSCP patterns and the sequence clusters was found, but P1 was present in Group 1 and P6 was found in all 3 isolates of Group II. The aa relationship dN/dS = 0.214 indicated negative selection, suggesting that PYVV has a tendency for low mutation fixation. This might be related to a selection pressure coming from the insect vector. According to variability of the CPg there are at least 3 virus variants circulating in the Country, although variability among them is low.

Asparagus as host of Phythophtora species prevalent in Michigan and its importance as rotational crop
L. RODRIGUEZ-SALAMANCA (1), M. Hausbeck (1)
(1) Michigan State University, East Lansing, MI, USA

Michigan ranks third in U.S. asparagus production, after Washington and California, with 11.200 acres that produced 12 million kg of asparagus spears in 2007. Phytophthora spear and crown rot has been recently identified in Michigan fields as a major limiting disease of asparagus. Although different species of Phytophthora have been reported in other production areas as causing disease in asparagus, only one species has been found in Michigan and has identified as P. asparagi. Phytophthora sp. isolated from vegetables and ornamentals in Michigan were tested for their ability to infect asparagus spears and cause lesions. A series of growth chambers studies were conducted to determine; (i) an optimum inoculation point for detached asparagus spears using three P. asparagi isolates when incubated at 15, 20 and 25°C, (ii) the ability of different Phythophthora sp. present in Michigan agriculture to infect asparagus spears. All the studies were conducted three times. When detached spears were wounded and inoculated at 2, 9 or 16 cm from asparagus tip and incubated at 20°C, all the inoculation points developed similar-sized lesions. However, when inoculated spears were incubated at either 15 or 25°C, the size of the resulting lesion differed significantly among the inoculation points. Among the select Phythophthora sp. used to inoculate asparagus spears, P. capsici was able to cause small lesions. Further studies that investigate pathogenicity of commonly encountered Phythophthora sp. is important in determining appropriate crop rotation strategies.

Fungal diversity associated with rambutan (Nephelium lappaceum L.) in Puerto Rico
L. M. SERRATO (2), L. I. Rivera (2), R. J. Goenaga (1)
(1) USDA TARS, Mayaguez, Puerto Rico; (2) University of Puerto Rico-Mayaguez Campus, Mayaguez, Puerto Rico

Rambutan (Nephelium lappaceum L.) is an exotic tropical fruit of increasing importance in international markets, and that has awakened great interest from farmers in Puerto Rico. During 2008 and 2009, fruit rot and lesions on leaves, branches, and flowers were observed in rambutan orchards through the island. To examine fungal diversity associated with rambutan, samples from different organs were collected in symptomatic and asymptomatic trees. Plant tissue was superficially disinfected and transferred to acidified potato dextrose agar to promote the development of fungi. A total of 311 fungal isolates were obtained, which include 19 genera. Based on morphology, the following species have been identified: Beltrania rhombica, Botryodiplodia theobromae, Botryosphaeria spp., Colletotrichum gloeosporioides, Colletotrichum spp., Curvularia spp., Cylindrocladium spp., Dolabra nepheliae, Fusarium spp., Gliocephalotrichum bulbilium, Lasmenia spp., Phomopsis spp., and Septoria spp. Pathogenicity tests are in progress under laboratory and greenhouse conditions, using seedlings and detached fruit. PCR amplification of the rDNA ITS region and the beta-tubulin gene will complement morphological identification of fungi.

Use of bio-enhanced organic mulches for integrated management of nutsedge in tomato
Y. SHABANA (2), E. Rosskopf (5), R. Charudattan (2), A. Abou Tabl (1), W. Klassen (3), J. Morales-Payan (4)
(1) Mansoura University, El-Mansoura, Egypt; (2) University of Florida, Gainesville, FL, USA; (3) University of Florida, Homestead, FL; (4) University of Puerto Rico-Mayaguez, Mayaguez, PR; (5) USDA, ARS, USHRL, Fort Pierce, FL, USA

Purple and yellow nutsedges (Cyperus rotundus and C. esculentus, respectively) are among the world’s most problematic weeds that impact virtually every horticultural crop grown in Florida and the Caribbean. As an alternative to conventional methods of control that includes the use of soil fumigation with methyl bromide, we tested nine hay mulches (shoot straw of bahiagrass, cogongrass, cowpea, millet, yellow nutsedge, sorghum Sudangrass, sunnhemp, and rye) and three green mulches (shoot biomass of cowpea, millet, and sorghum Sudangrass) as a means to suppress nutsedge growth in a raised-bed tomato (cv. Tygress) field. In addition, two fungus-infested cogongrass hays (infested with the nutsedge pathogen Dactylaria higginsii [Dh] or the saprophytic fungus Trichoderma sp. [Tri]), and two plastic mulches (black and infra-red transmissible [IRT]) were tested. The black plastic mulch and the Dh-infested cogongrass mulch consistently reduced nutsedge emergence and growth more than the other organic mulches and the IRT plastic mulch. Among the organic mulches, cogongrass infested with Dh or Tri and cowpea, sunnhemp, Bahiagrass, and cogongrass provided the highest levels of nutsedge suppression. No disease symptoms developed on nutsedge plants when Dh- or Tri-infested cogongrass was used as the mulch. Both plastic mulches (black and IRT) and Tri-infested cogongrass enhanced tomato yield and the proportion of larger fruits. The highest yield of extra large tomatoes per plant was obtained when these mulches were applied.

Laurel wilt of avocado: Management and mitigation research in Florida
J. A. SMITH (1), R. C. Ploetz (2), T. J. Dreaden (1)
(1) School of Forest Resources and Conservation, University of Florida, Gainesville, FL, USA; (2) Tropical Research and Education Center, University of Florida, Homestead, FL, USA

Laurel wilt, caused by the fungus Raffaelea lauricola and transmitted by the exotic redbay ambrosia beetle, Xyleborus glabratus, threatens the U.S. avocado industry with elimination if drastic measures are not taken in the near future. Since its introduction in Georgia approximately 6 years ago, the disease has spread on several hosts in the Lauraceae on the southeastern coastal plain and now looms only 100 miles from commercial avocado groves (7,500 acres worth $34 million/yr) in Miami-Dade County, FL. Within the last 2 years, door-yard avocadoes have been rapidly killed and serve as a source of inoculum for the epidemic. Current research efforts include: examining extant avocado germplasm for resistance; using a taxon-specific real-time PCR technique to diagnose the pathogen and identify it in screening and epidemiology studies; and fungicide efficacy trials. The results from these studies will be presented and future work will be discussed. Laurel wilt threatens avocado production worldwide. Thus, we will address its potential impact and preventing its movement to new areas.

Combating the loss of red bay and other native species to laurel wilt
J. A. SMITH (3), M. A. Hughes (1), C. Anderson (3), R. C. Ploetz (4), A. E. Mayfield (2)
(1) Department of Plant Pathology, University of Florida, Gainesville, FL, USA; (2) Florida DACS Division of Forestry, Gainesville, FL, USA; (3) School of Forest Resources and Conservation, University of Florida, Gainesville, FL, USA; (4) Tropical Research and Education Center, University of Florida, Gainesville, FL, USA

Laurel wilt, caused by Raffaelea lauricola, currently threatens all native and some exotic species in the Lauraceae in the United States. Since 2003, the disease has devastated native stands of redbay, Persea borbonia, and threatens several other taxa in the family, including avocado. Two natives, pondspice (Litsea aestivalis) and pondberry (Lindera melissifolia), which are on state endangered and federal critically endangered lists, respectively, face extinction. Despite sanitation and other efforts to slow the movement of laurel wilt, it continues to move to new areas every year, largely due to the efficiency of the disease vector, the exotic redbay ambrosia beetle (Xyleborus glabratus). There are many gaps in our current state of knowledge about the biology of the disease, and several studies are underway. Current research has focused on: protecting existing trees via fungicides; identifying and utilizing putative resistance in redbay and avocado; elucidating the disease’s epidemiology and host range; and determining to what extent genetic and pathogenic variation exist in the R. lauricola population. An update on the results from these studies will be given and future research needs will be discussed.

Effect of acibenzolar-S-methyl on bacterial leaf spot of shrub roses caused by a Xanthomonas sp.
G. E. VALLAD (1)
(1) Gulf Coast REC, University of Florida, Wimauma, FL, USA

A bacterial leaf spot was recently identified on shrub rose varieties ‘Knockout’ and ‘Double Knockout’ caused by a Xanthomonas sp. and can be problematic during vegetative propagation and nursery production. Acibenzolar-S-methyl, the active ingredient of Actigard (Syngenta, Greensboro, NC), is an elicitor of plant defenses that has demonstrated efficacy in the control of several bacterial diseases of vegetable crops. Greenhouse and nursery trials were established to test the effect of Actigard on the severity of bacterial leaf spot on ‘Knockout’ and ‘Double knockout’ roses. While lower rates of 0.25 to 0.5 oz of Actigard per 100 gallons were effective at reducing disease severity on potted roses and liners, higher rates of 0.75 to 1.0 oz gave the best results. Multiple applications of Actigard (1.0 oz/100 gal) prior to disease development improved bacterial leaf spot control over single applications. Results demonstrate the potential to use Actigard for disease management on ornamental and nursery species.

Effect of acibenzolar-S-methyl on the management of early blight and target spot of tomato
G. E. VALLAD (1)
(1) Gulf Coast REC, University of Florida, Wimauma, FL, USA

Acibenzolar-S-methyl, the active ingredient of Actigard (Syngenta, Greensboro, NC), is an elicitor of plant defenses. While labeled for tomato, usage is currently limited to the control of bacterial leaf spot (Xanthomonas spp.) and bacterial speck (Pseudomonas syringae pv. tomato). In 2008, two field trials assessed the performance of Actigard (8 weekly applications at 0.75 oz per acre) when integrated into a standard spray program that included weekly applications of copper sulfate (2.1 lbs a.i. per acre) mixed with either mancozeb (1.5 lbs a.i. per acre) or chlorothalonil (1.5 lbs a.i. per acre). The addition of Actigard reduced the severity of early blight (Alternaria solani) and target spot (Corynespora cassiicola) by 22 to 44% over the standard spray program alone, and by 31 to 60% compared to the non-treated plots. In the spring trial, plots treated with Actigard yielded 336 more cartons (25 lbs) of marketable tomatoes per an acre than those receiving the standard alone, and 1,179 cartons more per an acre than the non-treated plots. No yield improvement was observed in the fall trial, due to the development of disease in the late season. Results demonstrate the benefit of including Actigard as part of an overall spray program to manage common foliar diseases caused by bacterial and fungal pathogens of tomato.

outbreaks associated with vegetables: How high is the risk?
(1) Emerging Pathogens Institute and Plant Pathology Department, IFAS, University of Florida, Gainesville, FL, USA

Gastro-enteritis outbreaks increased in the 1990s and have remained steady since then. Most outbreaks have been associated with seafood, but most individual cases with vegetables and fruits (38% of all cases). Salmonella enterica is the most common pathogen involved in outbreaks associated with vegetables. Several Salmonella outbreaks were traced back to contaminated tomatoes. Salmonella enterica is very versatile: there are more than 2500 serovars, which occur in various environments, including many plant and animal species. The main reservoirs are the intestines of birds, pigs, cattle, wild mammals and reptiles, but they are also harbored by protozoa, earthworms, nematodes and snails. They can multiply in the rhizosphere of various plants and occur on plant surfaces as well as in the endosphere. Because of the human as well as economic costs associated with Salmonella outbreaks, it is important to study the risk of an outbreak to occur. However, there are different kinds of risk: calculated probabilities as well as perceived risks. These last risks are concerns voiced by consumers on a comparative scale. Among various safety concerns, microbiological risks are ranked high, due to some knowledge and experience and the feeling of not being able to control exposure. Perceived risks do not necessarily coincide with calculated probabilities, but may be more influential in terms of the response to an outbreak. Quantitative microbial risk assessment consists of several steps: hazard identification and characterization, exposure assessment and risk characterization. In a project on risk assessment of enteric pathogens in the vegetable production chain, we limited ourselves to exposure assessment through lettuce contaminated from manure and soil. The occurrence and survival of enteropathogens in cattle manure were primarily determined by the feed given to the cattle: low-fiber feed resulted in more shedding and longer survival in the low-fiber and low-pH manure. Other risk factors were low numbers of nonpathogenic coliform bacteria and high dissolved organic carbon contents. Constant temperatures and low oxygen levels also contributed to long survival times in manure. Survival times in soil were negatively correlated to microbial diversity and positively to dissolved organic carbon contents. A probabilistic exposure model for E. coli O157:H7 resulted in a relatively low probability of about 1 contaminated head in 10,000 lettuce heads. The risk can best be reduced at the beginning of the production chain, the cattle farm. There are several quantitative risk assessment models for Salmonella, but most of them are for animal products, except for one model for almond contamination and on for vegetable contamination from irrigation water. No risk model was found for tomato production and processing. In a tomato safety research workshop, research needs were identified, but control at the source of the chain was not mentioned while this was the crucial factor in our lettuce risk model. Risk models based on calculated probabilities could be used to influence perceived risks by the general public.

STAR-D: The NPDN accreditation program for diagnostic laboratories
A. B. VITORELI (1), C. L. Harmon (1)
(1) University of Florida, Gainesville, FL, USA

The Food and Agriculture Defense Initiative was established in 2002 to enable the United States Department of Agriculture to develop a network linking plant and animal disease diagnostic facilities across the USA. The National Plant Diagnostic Network (NPDN) is the plant disease component of this network. The mission of the NPDN requires quick and accurate diagnosis of high consequence plant pathogens, weeds and insect pests that threaten national security; communication of such information response authorities; the ability to scale up and manage sample surge as needed; and diagnostic data security. To accomplish these objectives, the NPDN relies on diagnostic data generated by laboratories in Land-Grant Universities, State Departments of Agriculture, and USDA-APHIS. Traditionally, these laboratories have provided diagnostic services at the State or regional levels at a high level of competence. However, to accomplish the national objectives listed above, a standardized approach to diagnosis is required, particularly if the diagnosis has regulatory implications. The NPDN System for True, Accurate, and Reliable Diagnostics (STAR-D) has been developed to enable participating laboratories to meet standards of quality for laboratory management, facilities, equipment, and trained personnel. The ISO 17025 Quality Standard can be adapted to testing done by NPDN diagnostic laboratories by providing a basis for a quality system to meet the needs of the NPDN STAR-D.

Levels of P in Areca catechu leaves following phosphorous acid application through adventitious roots
G. C. WALL (1)
(1) University of Guam, Mangilao, GU 96923, USA

Bud rot disease of betel nut (Areca catechu L) has been shown to be caused by Phytophthora palmivora. Fosphite, or phosphorous acid, is recommended for the control of Phytophthora, applied by injection to the trunk. After finding evidence of damage associated with injection sites in the trunks of betel nut trees, a decision was made to look for other ways of applying the fungicide in order to avoid damaging the trees. A paired t test was devised to study the effect of applying phosphorous acid solution via adventitious roots of betel nut trees. A group of mature trees was sampled pre- and post-application. Levels of P were determined from leaf samples collected from each frond per tree. There were 12 pairs of trees in the study; one set of trees was treated with the recommended rate (applied by absorption through an adventitious root) and half were controls, treated only with sterile distilled water also via one adventitious root. After the appropriate statistical analysis (NCSS, Kaysville, UT), differences were found in the level of P in the leaf samples according to treatment. Control trees had higher levels of P in their leaf tissue compared to trees given phosphorous acid. The underlying hypothesis was that application of the fungicide via adventitious roots of trees would result in a systemic distribution of the fungicide throughout the tree. It was expected that all leaf samples from treated trees would show higher levels of P compared to untreated controls. Surprisingly, P levels were lower in treated trees, yet there was no difference between fronds, suggesting an even effect throughout treated trees. No explanation is known at this time for the reduced P levels observed after treatment; however, results were consistent enough to yield highly significant differences statistically.

Epidemiology of soybean rust (Phakopsora pachyrhizi) in soybean (Glycine max) sentinel plots in Florida
H. M. YOUNG (2), J. J. Marois (2), D. L. Wright (2), D. F. Narvaez (1), G. K. O’Brien (2)
(1) Monsanto, St. Louis, MO, USA; (2) University of Florida, NFREC, Quincy, FL, USA

The overwintering of soybean rust (SBR) in the Southeastern United States has been variable due to weather conditions which may influence disease incidence and severity in the major soybean producing regions of the Midwest, making it important to understand the epidemiology of the pathogen in Florida. This study examined the incidence and severity of SBR in relation to prevailing weather data, growth stage, and maturity group (MGIII, MGV, MGVII) in 15 m square soybean plots across the Panhandle of Florida from 2005 through 2008. Of the three maturity groups, the MGIII soybean became infected first the least often. Plots became infected first at growth stage R4 (full pod) or later. On average, plots became infected 40 days earlier in 2008 than 2005. Precipitation was the principle factor affecting disease progress, where disease increased rapidly after rain events and was suppressed during dry periods. The area under the disease progress curves (AUDPC) for incidence and severity was the lowest in 2007, most likely due to dry conditions. In 2008, there was a significant increase in disease incidence and severity as reflected in the AUDPC. This can be attributed in part to the occurrence of Tropical Storm Fay, which deposited up to 290 mm of water in the plot locations during the third week of August. Results from this study may lead to a better understanding of the impact of weather on the epidemiology of this pathogen.

Effect of rhizobacteria, acibenzolar and silicon on bacterial spot of tomato
S. ZHANG (1), T. L. White (1), W. Klassen (1), M. C. Martinez (1)
(1) Tropical REC, University of Florida, IFAS, Homestead, FL, USA

Bacterial spot, caused by Xanthomonas perforans, is one of the most economically important diseases of tomato in Florida and other tomato grown areas worldwide. Chemical controls have been only partially effective due to the wet and warm climate in Florida and the development of resistance in populations of this bacterial pathogen. It is imperative that practical alternative strategies be developed to sustain the production of tomatoes. Greenhouse and field trials have been conducted to investigate the effect of plant growth-promoting rhizobacteria (PGPR), acibenzolar-S-methyl (ASM) and silicon nutrient on bacterial spot of tomato. In the greenhouse, eight bacilli PGPR strains were evaluated on two cultivars of tomato (FL47R and Tygress). Tomato seeds were sown into pro-mix in 128-cell Styrofoam flats and grown for 1–2 weeks when solutions of PGPR, Actigard 50WG (ASM) and silicic acid were applied weekly as soil drenches. Tomato seedlings were transplanted into 4-inch pots containing potting mix after 3–4 soil drenches, and inoculated by foliar spray with suspensions of X. perforans at 1 × 10(^8) CFU/ml. Results indicated that PGPR strain SE76 and INR7 significantly (P < 0.05) reduced disease severity of bacterial spot on both tomato cultivars compared to the nontreated control. SE52 on cv. FL47R and SE34, IN937a and IN937b on cv. Tygress each had a significant effect on disease reduction. In the first field trial on tomato cv. FL47R, Actigard 50 WG at 30 mg/l significantly suppressed bacterial spot rated at 8, 9, 10 and 11 weeks after transplanting, whereas silicic acid at 0.15 and 1.5 mM did so only at 8 weeks. In another field experiment, the same eight PGPR strains and Actigard 50WG at 30 and 3 mg/l were tested on tomato cv. Tygress. Significant disease reduction was observed on tomato plants treated with PGPR strains IN937a and IN937b 3 weeks after transplanting.