14th Biennial Meeting of the
Florida Phytopathological Society
June 4-6, 2015
Hosted by:
University of Florida/ IFAS
Dept. of Plant Pathology
Gainesville, FL
Sponsors of the event:
Location for the meeting: University of Florida, Fifield Hall, Conference rooms 1304,
1306 and 1308.
Physical address: 2550 Hull Road, Gainesville, FL 32611
Parking: Link for parking restrictions at University of Florida during the meeting
(http://parking.ufl.edu/subpages/liftedrestrictions.html). Everyone should park on the
west side of Fifield Hall (Green Lot). This is the lot on the opposite side of the
Diagnostic Clinic. State vehicles can park in any lot.
1 May 4th, Welcome Reception: 5.30 - 7.30 p.m, The Lodge at Gainesville (Beverages and snacks will be
provided)
May 5th, Tuesday
8.30 a.m
Introduction: Dr. Rosemary Loria
Dr. Aaron Palmateer
8.45 a.m
Keynote speaker: Dr. Jim Marois “A Career in the Science of Plant Pathology”,
introduced by Mason Newark
Bacteriology session: Moderator: Dr. Frank White
9.30 a.m
Detection of Xanthomonas albilineans in sugarcane stalks using LAMP, PCR and selective
isolation assays. Vanessa Duarte Dias, E. Fernandez, M.G. Da Cunha, P. Roumagnac, J.C.
Comstock, And P. Rott
9.45 p.m
Mechanisms of evolution of plant pathogenic Streptomyces species. Jose C. Huguet-Tapia
10.00 a.m
CsLOB1 homologs can function as disease susceptibility genes in citrus bacterial canker.
Junli Zhang, Jeff Jones, Nian Wang, and Frank White
10.15 a.m
Characterization of Pseudomonas cichorii and P. viridiflava causing bacterial speck-like
symptoms on tomato in Florida. Sujan Timilsina
10.30 a.m
Session break (15 minutes)
Virology session: Moderator: Dr. Svetlana Folimonova
10.45 a.m
Diagnostic tools for Rose rosette virus: TaqMan® qRT-PCR and Recombinase polymerase
assay. Binoy Babu, Jeyaprakash Ayyemperumal, Debra Jones, Timothy S. Schubert, Carlye
Baker, Gary Knox and Mathews L. Paret
11.00 a.m
A nonconserved protein of Citrus tristeza virus shows membrane association and shares
characteristics of viral movement proteins. Sung-Hwan Kang, Aurélie Bak, Ok-Kyung
Kim, and Svetlana Y. Folimonova.
11.15 a.m
Viral diversity in Caladium x hortulanum. Ricardo I. Alcalá-Briseño, Aurora Londoño, and
Jane E. Polston
11.30 a.m
Concave Gum in Citrus: New Approaches to an Old Disease. Mark E. Hilf and Scott J.
Harper.
11.45 a.m
Yellow leaf of sugarcane caused by Sugarcane yellow leaf virus: What do we know and
what do we need to know? Philippe C. Rott.
12.00 p.m
Weeds, Whiteflies, and Squash Vein Yellowing Virus: Taking a Closer Look. Deepak
Shrestha and Susan Webb.
Lunch (Provided)
2 Disease Management session: Moderator: Dr. Phil Harmon
1.00 p.m
Plant Disease Control Solutions from BioSafe Systems: An Overview of Available Products
and Research. Vijay Choppakatia
1.15 p.m
Potential spread of Diaphorina citri and Ca. Liberibacter asiaticus based on species
distribution models. Narouei-Khandan H.A., Halbert, S.E., and van Bruggen, A.H.C.
2.00 p.m
A new, simple simulation model for potato late blight development in relation to oscillations
in temperature and relative humidity. Ariena H.C. van Bruggen, Hossein A. Narouei
Khandan, Shankar K. Shakya, Erica M. Goss, Nicholas S. Dufault, Jorge L. Andrade-Piedra,
Senthold Asseng and Daniel Wallach
2.15 p.m
Two New Active Ingredients From Syngenta Crop Protection. Katherine Buxton
2.30 p.m
An integrated approach to battle an old enemy; Fusarium wilt of watermelon. Tatiana
Sanchez
2.45 p.m
Disease diagnosis, habitat decline and fungal diversity associated with endangered
Gondwanan conifers in New Caledonia. Jason Smith
3.00 p.m
Sensitivity of Phytophthora cactorum from strawberry to azoxystrobin and mefenoxam.
Elias Zuchelli and Natalia Peres
Session break (15 minutes)
3.15 p.m
Efficacy of drip tape and shank applications of Dominus™ against Macrophomina
phaseolina, the cause of charcoal rot in strawberry. Manuel Chamorro Rodriguez
3.30 p.m
Evaluation of rotation and tank-mixture programs for gray mold management in strawberry.
Adrian Zuniga
3.45 p.m
Assessing the pathogenicity of Raffaelea spp. isolated from Xyleborus glabratus to swamp
bay, Persea palustris. Tyler Dreaden and Jason Smith
4.00 p.m
Screening sugarcane clones for rust resistance. Sushma Sood and Jack Comstock
4.15 p.m
Influence of ontogenic resistance, environment and timing of fungicide applications on
development of powdery mildew on strawberries. Rodrigo Borba Onofre and Natalia Peres
4.30 p.m
Impact network analysis to identify strategies for improving disease management. Karen
Garrett
4.45 p.m
Pomegranate disease in Florida: current status and future possibilities. Archana Nepal
5.00 p.m
Evaluation of redbay (Persea borbonia) germplasm for tolerance to laurel wilt disease. Marc
Hughes and Jason Smith
6.00 p.m
Dinner (Provided) and Felicitation for Dr. Tim Schubert, Division of Plant Industry,
FDACS, introduced by Amanda Strayer
3 May 6th, Wednesday; Graduate Student presentations: Moderator: Dr. Brantlee Spakes Richter
8.00 a.m
Superinfection exclusion by Citrus tristeza virus is a complex phenomenon that requires multiple viral
factors
Osama O. Atallah1 and Svetlana Y. Folimonova1
1
Plant Pathology Department, University of Florida, Gainesville, Florida 32611
Superinfection exclusion (SIE), a phenomenon in which a primary virus infection prevents a secondary infection
by the same or closely related virus, was observed with various viruses. We are examining SIE by Citrus tristeza
virus (CTV), a member of the family Closteroviridae. Previously we demonstrated that isolates of the same
strain can exclude each other, while isolates from different strains do not. Later on, we showed that SIE is a
virus-controlled function that requires viral p33 protein. In this work we found that p33 protein alone is not
sufficient for exclusion of secondary infections. The hybrid virus containing substitution of the p33 gene with a
cognate region from a different strain did not elicit exclusion of the latter donor strain. In order to define the
additional viral components that are involved in this phenomenon and considering our previous observation that
substitutions throughout the 3’ portion of the CTV genome do not affect SIE, we engineered a T36-based hybrid
virus harboring substitution within its leader proteases region substituted with a corresponding region from the
genome of the T68 strain. Unlike wild type CTV T36 virus, the L1-L2 T68/T36 hybrid was unable to exclude
the parental isolate of T36. This result indicated the strict requirement of viral leader proteases beside p33
protein for SIE between CTV isolates. Further analysis seeking for minimal sequences within L1 and L2 that are
required for SIE indicated that the entire region is essential and each of N- and C-terminal domains are
determinants for exclusion of superinfections.
8.15 a.m
Detection and characterization of a new Mastrevirus of sugarcane in Florida
W. Boukari1&5, S. Kraberger2, E. Fernandez3, D. Filloux3, J.C. Comstock4, A. Varsani2, P. Roumagnac3, J. E.
Polston1 and P. Rott5
1
University of Florida, IFAS, Plant pathology Department, Gainesville, FL 32611.
2
University of Canterbury, School of Biological Sciences and Biomolecular Interaction Centre, Christchurch,
New Zealand.
3
CIRAD-INRA-Montpellier SupAgro, UMR BGPI, Campus International de Baillarguet, Montpellier, France.
4
USDA-ARS, Sugarcane Field Station, Canal Point, FL 33438.
5
University of Florida, IFAS, Everglades Research & Education Center, Belle Glade, FL 33430.
A metagenomics study of the viruses associated with 180 plants of Saccharum species collected from
commercial fields and germplasm collections in Florida was conducted in 2013/2014. The objective of that
study was to identify the occurrence of known and potentially new sugarcane viruses. All four previously
reported sugarcane viruses known to occur in Florida were detected: Sugarcane yellow leaf virus, Sugarcane
mosaic virus, Sugarcane mild mosaic virus, and Sugarcane bacilliform virus. Additionally, potential new
viruses of sugarcane that appear to be species in the genera Chrysovirus (family Chrysoviridae), Mastrevirus
(family Geminiviridae), and Umbravirus (family Unassigned) were identified. We further investigated the
mastreviruses because mastreviruses such as Sugarcane streak virus and Maize streak virus are known to cause
diseases in sugarcane in several locations in the world where this plant is grown. Two genome sequences of
about 2.8 Kb, each closely related to sequences of known mastreviruses, were recovered, cloned and sequenced
from infected Saccharum spp. Comparison of these two genomes with sequences available in GenBank revealed
that the two genomes from Florida represent a new species of Mastrevirus. This new species was found in four
plants of a germplasm collection that was introduced to Florida several decades ago. It was not found by
metagenomics in commercial fields, suggesting that no insect vector of this novel virus is currently present in
Florida.
4 8.30 a.m
Low-volume spray technology for control of major diseases of strawberry in Florida.
Cordova, L. G., Peres, N. A.
IFAS, Gulf Coast Research and Education Center, University of Florida.
The two main diseases that affect strawberry production in Florida are Botrytis and anthracnose fruit rots,
caused by Botrytis cinerea and Colletotrichum acutatum, respectively. Fungicide applications are the most
effective method for control of both diseases. Traditionally, Florida strawberry growers use sprayers equipped
with hydraulic nozzles with a flow rate of about 935 l/ha. The objectives of this study were to investigate the
effects of reduced flow rates on the control of both diseases and to compare the efficiency of the standard
hydraulic nozzle sprayer with a low-volume atomizer. Moreover, the reduction of fungicide rate by 25% was
also evaluated. The experiment consisted of four treatments: 1) and 2) hydraulic nozzle technology with a flow
rate of 935 and 561 l/ha, respectively, and 3) and 4) atomizer with a flow rate of 234 l/ha, but in treatment 4, the
fungicide rate was reduced by 25%. Fungicides were sprayed 10 times over the season (November to March)
when weather conditions were favorable for disease. Water sensitive papers were placed during spray operations
to evaluate penetration and coverage, and plots were harvested twice weekly to evaluate yield and disease
incidence. No significant differences in either yield or disease incidence was observed among the treatments.
Botrytis isolates have been collected to evaluate the effect of reduced rate on fungicide resistance development.
8.45 a.m
Resistance in strawberry isolates of Colletotrichum acutatum from Florida to quinone-outside inhibitor
fungicides
Bruna B. Forcelini, Achour Amiri, Teresa E. Seijo, and Natalia A. Peres. IFAS, Gulf Coast Research and
Education Center, University of Florida.
Anthracnose Fruit Rot (AFR) of strawberry, caused by Colletotrichum acutatum, is a major disease in Florida
and frequent fungicide applications are needed for disease control. Quinone outside inhibitor (QoI) fungicides
such as azoxystrobin and pyraclostrobin are commonly used to control AFR. From 1994 to 2014, 177 C.
acutatum isolates were collected from multiple strawberry fields in Florida with or without a QoI spray history.
Isolate sensitivity to both fungicides was tested based upon mycelial growth, germ tube elongation and fruit
inoculation assays. Mean EC50 values for isolates collected prior to 2013 based upon mycelial growth and germ
tube elongation were 0.22 and 0.013 µg/ml for azoxystrobin and pyraclostrobin, respectively. Mycelial growth
and germ tube elongation of 43 isolates collected from 2013 to 2014 were not inhibited at the highest
concentrations of azoxystrobin (3 µg/ml) and pyraclostrobin (0.110 µg/ml). Azoxystrobin and pyraclostrobin
sprayed preventively on detached strawberry fruit inoculated with C. acutatum failed to control resistant
isolates. Isolates were characterized by sequencing of the cytochrome b gene and resistant isolates had the
G143A amino acid substitution at the cytb locus. To our knowledge this is the first report of resistance to QoI
fungicides in C. acutatum. Our findings indicate that development of AFR management practices is essential to
limit selection and spread of QoI resistant populations of C. acutatum.
9.00 a.m
Use of quantitative real-time PCR for quantifying spores of Phyllosticta citricarpa, causal agent of citrus
black spot
Mareli Kellerman1,2, Megan M. Dewdney3, Adéle McLeod1 and Paul H. Fourie1,2
1
Department of Plant Pathology, Stellenbosch University, Private bag X1, Stellenbosch 7602, South Africa
2
Citrus Research International, PO Box 28, Nelspruit, 1200, South Africa
3
Citrus Research and Education Centre, University of Florida, Lake Alfred, FL 33850, USA
Phyllosticta citricarpa is classified as an A1 quarantine pathogen, and major restrictions have been placed on
citrus exports from South Africa (SA) to European Union (EU) countries. Since ascospores of P. citricarpa, the
causal agent of citrus black spot (CBS), cannot be morphologically distinguished from the harmless endophyte
P. capitalensis, only generic epidemiological models of Phyllosticta spp. are available. This complicates the pest
risk assessment that CBS will establish in the EU territory, as well as timing of fungicide spray applications to
5 effectively control CBS in SA. A previously published qPCR assay was adapted for quantification of P.
citricarpa spores in CBS infested citrus orchards. In spiked samples, the limit of detection was 20,000
pycnidiospores. This method will allow for the quantification of the relative proportions of P. citricarpa and the
harmless endophyte (P. capitalensis) on spore trap discs, which was to date impossible based on spore
morphology alone. Preliminary screening of spore trap samples from groves showed that P. citricarpa DNA was
detected successfully by the qPCR, but comparisons of qPCR results with microscope spore counts showed
differences, which indicate that ascospores of P. capitalensis may be present. This assay will enable the
validation of existing Phyllosticta epidemiological models, with regards to P. citricarpa specifically. This will
assist in more accurate pest risk analyses and timing of fungicide sprays to control CBS.
9.15 a.m: Break - 15 min
9.30 a.m
Preliminary examination of potential fitness cost associated with maintaining fungicide resistance in
absence of selection for Didymella bryoniae epidemics in Florida watermelon production
M. J. Newark, N.S. Dufault, M.L. Paret
University of Florida, Department of Plant Pathology
Gummy stem blight of watermelon, caused by Didymella bryoniae, is primarily managed using fungicidal
products despite the identification of recent fungicide resistant isolates. The purpose of this study was to assess
the shifts of D. bryoniae populations in-vivo under known fungicide selection pressures. Two populations of D.
bryoniae, one sensitive and one resistant to the active ingredient boscalid were inoculated onto watermelons at
equal proportions. Treatments were applied to either select for the resistant isolate or to control both pathogen
populations. Three samples were collected throughout the duration of the trial. Sampling was done to assess
initial population levels and to examine population ratios based on fungicide treatments. 237 Isolates were
obtained from symptomatic leaf or stem tissue, cultured on quarter strength potato dextrose agar (QPDA) and
pure cultures were replated onto QPDA amended with two rates of boscalid (0.0068 and 0.017mg/l). Radial
growth was measured at 48, 72, and 96 hours post-culturing and compared to the unamended control to calculate
percent inhibition. Phenotypes were characterized as either sensitive or resistant based on inhibition. After
analyzing frequencies using a chi-square test, it was observed that after 3 continuous sprays of boscalid that
resistant isolates became significantly more prevalent within the field plots (p<0.01 and p=0.07 for the Citra and
Quincy locations respectively). Differences in disease severity were consistent with treatments when based on
phenotype frequencies. These results indicate the importance of fungicide rotations in adequately managing
gummy stem blight.
9.45 a.m
Distribution of azoxystrobin resistance in nonpathogenic Alternaria alternata isolates of citrus
Katrina Nicoletta, Byron Vega, and Megan M. Dewdney
First and third author: Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA;
second author: DuPont Crop Protection-Discovery, Stine-Haskell Research Center, Newark, DE, USA
Currently, the greatest proportion of tangerine-infecting A. alternata isolates in Florida are QoI-resistant
populations; however the selection pressure of QoI fungicides has not been measured for nonpathogenic A.
alternata isolates. Pathogenic and nonpathogenic isolates of A. alternata are found on tangerine cultivars, and
126 isolates of the 1,258 total collected were confirmed nonpathogenic to cultivars Dancy, Minneola, Murcott,
and Sunburst through phenotypic approaches. Also, the absence of the ACT-toxin production was molecularly
confirmed in 37 isolates by amplifying the ACTTR locus. Isolates were tested for azoxystrobin (AZ) sensitivity
using the resazurin-based microtiter assay. Isolates with an effective concentration to reduce growth by 50%
(EC50) values greater than 5.0 µg/ml were categorized resistant. Of the isolates, 75% were resistant to AZ. The
mean EC50 value for the 32 sensitive isolates was 0.044 µg/ml. Twenty of the sensitive isolates came from
Murcott and 12 from Sunburst cultivars. A χ2 comparison of the populations of pathogenic and nonpathogenic
resistant isolates was not significantly different (χ2 = 0.699, P = 0.403) among cultivars. The presence of the
G143A mutation in the cytochrome b gene was determined in a subset of 37 isolates using PCR-restriction
6 fragment length polymorphism with the developed primers CytF1/CytR1 and restriction enzyme Fnu4HI. All
nonpathogenic QoI-resistant isolates carry the G143A mutation.
10.00 a.m
Optimization of a resazurin-based assay testing sensitivity of Botrytis cinerea to respiration-inhibitor
fungicides
Oliveira, M.S., Cordova, L.G., Peres, N.A.
Resistance of Botrytis cinerea to fungicides is rapidly developed due to its abundant sporulation and high
genetic variability. Conidial germination test is the common assay to determine resistance to respiration
inhibitor fungicides; however, this test requires time, space, and its measurements are subjective. Our objective
was to adapt a resazurin-based assay to evaluate B. cinerea sensitivity to boscalid and pyraclostrobin and
determine the effective dose to provide 50% control of the fungus (EC50) in both fungicides. Eleven isolates
were tested in three liquid media (Complete Medium, Minimal Medium and HA Medium with pH 6.5), four
conidial concentrations (from 102 to 105 conidia/ml), eight fungicide concentrations (0, 0.001, 0.01, 0.1, 0.5, 1,
5, and 10 µg/ml for boscalid and 0, 0.001, 0.01, 0.1, 0.5, 1, 10, and 50 µg/ml for pyraclostrobin), and evaluated
from 16 to 32h after plating. HA at 24h and 104 conidia/ml were the optimal conditions for evaluation of
resazurin reduction. The EC50 calculated for the sensitive isolates (n=5) varied between 0.39 and 3.25 ppm in the
conidial germination test, and between 0.13 and 0.20 ppm in the resazurin-based assay. For the resistant isolates
(n=6), the EC50 was higher than 10 ppm on boscalid and higher than 50 ppm on pyraclostrobin. Our findings
suggest that this rapid and reliable technique can be used on a large scale to evaluate the sensitivity of B. cinerea
to respiration inhibitor fungicides.
10.15 a.m
Managing impatiens downy mildew in the nursery and landscape.
S. N. Suarez and A. J. Palmateer
Since the 2012 outbreak, Florida growers and landscapers have experienced the devastating effects of downy
mildew on impatiens caused by Plasmopara obducens. An overview of the disease history will be provided as
well as results of effective fungicide chemistries and nutritional amendments for containerized grown impatiens
and landscape beds. Managing this disease for nursery and greenhouse production is simpler as fungicides for
use in these sites are readily available for commercial growers. Fungicides for landscape use are limited in a
legal sense and are not particularly cost effective. Due to these restrictions, residual efficacy of fungicides
applied in the nursery or greenhouse have been tested in order to prolong protection before plants are
transplanted into the landscape. The disease seems to remain active year-round. Thus, gardening impatiens has
been replaced by other bedding plant alternatives due to the persistence of the disease. Collaborative research
efforts are currently focusing on short-term management strategies that include fungicide efficacy trials to
reduce environmental impact and cost as well as long-term strategies, such as identifying host resistance in
Impatiens walleriana.
10.30 a.m
Population genetic structure of the late blight pathogen, Phytophthora infestans, in Mexico
Jianan Wang, Department of Plant Pathology, University of Florida
Meg Larsen, Horticultural Crops Research Laboratory, USDA ARS, Corvallis, Oregon
Rosario Gregorio-Cipriano and Gerardo Rodríguez-Alvarado, Laboratorio de Patología Vegetal, Universidad
Michoacana de San Nicolás de Hidalgo, IIAF, Morelia, Michoacán, México
Niklaus J. Grünwald, Horticultural Crops Research Laboratory, USDA ARS, Corvallis, Oregon
Sylvia P. Fernandez-Pavia, Laboratorio de Patología Vegetal, Universidad Michoacana de San Nicolás de
Hidalgo, IIAF, Morelia, Michoacán, México
Erica M. Goss, Department of Plant Pathology and Emerging Pathogen Institute, University of Florida.
7 Phytophthora infestans, the causal agent of the late blight in potato and tomato, causes annual losses of more
than $6 billion worldwide. In the central highlands of Mexico, P. infestans undergoes regular sexual
reproduction in contrast to the asexual reproduction that characterizes this pathogen in the United States. The
biology of these populations together with multilocus genetic data points to Central Mexico as its center of
origin. The diversity and population structure of P. infestans in Mexico has not been characterized using the
simple sequence repeat (SSR) markers currently being used to monitor P. infestans in the United States, Europe,
and elsewhere. We genotyped P. infestans isolates sampled from several states in Mexico at 12 microsatellite
loci to examine genetic diversity within and among populations using analysis of molecular variance, Bayesian
clustering, and discriminant analysis of principal components. We found significant genetic variation both
within and between states. The Michoacán population is distinct from the Toluca population and is also
characterized by an asexual population on tomato. Understanding the genetic structure and diversity of P.
infestans in Mexico, using the same set of hypervariable markers as used to study P. infestans in other countries,
will contribute to our understanding of the contemporary global diversity and migration of this problematic
pathogen.
10.45 a.m
Break - 15 min
11.00 a.m
The effect of relative humidity on colonization of citrus twigs by Phyllosticta citricarpa, the citrus black
spot pathogen
Nan-Yi Wang, Citrus Research and Education Center, University of Florida, Lake Alfred
Megan M. Dewdney, Citrus Research and Education Center, University of Florida, Lake Alfred
Citrus black spot, caused by P. citricarpa, in Florida has led to substantial economic loss to local growers since
March 2010. In the subtropical conditions of Brazil, conidia produced on dead twigs were demonstrated to be an
important inoculum source of P. citricarpa. While alternative wetting and drying cycles are critical for
pseudothecia and ascospore formation of P. citricarpa on leaf litter, conditions needed for pycnidia and conidia
production on twigs remain unclear. To determine the conditions for pycnidia and conidia formation of P.
citricarpa, inoculated twigs were incubated at various relative humidities (RH; 43, 72, 82, and 100%) at 24°C
under a 12-h light cycle. The formation of pycnidia and conidia was observed at 3, 5, and 7 weeks postinoculation at 82 and 100% RH but not at 43 and 72% RH, indicating that RH from 82 to 100% would be
suitable for pycnidia and conidia production of P. citricarpa. While no pycnidia or conidia were observed on
twigs at 43 and 72% RH, approximately 10 pg of P. citricarpa DNA per twig was detected using qPCR,
implying that P. citricarpa may survive under such conditions. Given that frequent summer rains and high %
RH also occur in Florida, the importance of conidia produced on dead twigs should not be underestimated. The
information obtained could improve our understanding of conditions required for inoculum production of P.
citricarpa on twigs, ultimately leading to the development of better control techniques.
11.15 a.m
Mating-type distribution and population structure of Phyllosticta citricarpa in Florida
Ke Zhang1, Nan-Yi Wang2, Megan Dewdney2, Jeffrey Rollins1
1
Plant Pathology Department, University of Florida, Gainesville, Florida, US
2
CREC, University of Florida, Lake Alfred, Florida, US
Phyllosticta citricarpa, the causal agent of citrus black spot (CBS), is a quarantine fungal pathogen discovered
in Southwest Florida in 2010. Although new to Florida, CBS was first reported in Australia in 1895. As it
continues to spread in Florida, comparisons of pathogen biology and disease epidemiology between Florida and
Australia may inform the potential for and pattern of spread here in Florida. From our previous work we
determined that P. citricarpa has a heterothallic mating system i.e., requiring two compatible mating-types to
generate airborne ascospores. Each mating-type contains either the MAT1-1 or the MAT1-2 idiomorph. In this
8 project, locus specific primers for MAT1-1-1 and MAT1-2-1 were designed to screen the mating-type of 113
isolates from Florida and 26 from Australia. The Australian isolates showed a 1:1 ratio of both mating-types,
while only MAT1-2-1 has been found from Floridian isolates. Based on this result, we hypothesized that the
Floridian population is clonal. Seven polymorphic microsatellite/simple sequence repeat (SSR) markers were
designed to screen multi-locus genotypes (MLGs) in Floridian and Australian populations. In 24 Australian
isolates, eleven MLGs have been found, but in 72 Floridian isolates collected from multiple groves from 2010 to
2013, only one MLG has been found. From this results, we infer that the P. citricarpa population in Florida is a
clonal asexually reproducing population derived from a limited introduction. From these findings, the control of
the production and movement of conidia-bearing materials is deemed critical to CBS management in Florida.
11.30 a.m
Baseline sensitivity of Guignardia citricarpa, causal agent of citrus black spot, to demethylation inhibitor
fungicides.
Naweena Thapa1, Megan M. Dewdney1. Natalia A. Peres2, Philip F. Harmon3
1
CREC, University of Florida, 2GCREC, University of Florida, 3Department of Plant Pathology, Gainesville
Florida.
Citrus black spot (CBS) is a new fungal disease in Florida, caused by Guignardia citricarpa. Heavy yield losses
due to premature fruit drop and unsightly lesions, that affect the fresh market value, made CBS economically
important disease. Currently, copper and QoIs (quinine outside inhibitors) fungicides are recommended to
manage CBS. Demethylation inhibitor (DMI) fungicides could be a potential alternative mode of action for
fungicide rotation. The project objectives were to determine the in vitro fungal population response and the
baseline sensitivity to the DMI fungicides and characterize the CYP51 gene. Evaluation of difenoconazole,
fenbuconazole, propiconazole, tetraconazole, and imazalil in a mycelial growth inhibition assay resulted in the
mean EC50 values of 0.0018, 0.00713, 0.0089, 0.0317, and 0.117 microg/ml, respectively. The EC50 values were
significantly different for each fungicide (P<0.001). Guignardia citricarpa isolates were highly sensitive to
difenoconazole among the tested DMIs, followed by fenbuconazole, propiconazole and tetraconazole. Imazalil
was the least effective DMI with a comparatively high EC50 value. The CYP51 gene encodes for the CYP51
enzyme, necessary for sterol-biosynthesis. DMIs target and bind CYP51 and inhibit the ergosterol synthesis.
Molecular characterization of G. citricarpa CYP51 generated 1,723 bp gene with 3 introns. Sequence
comparison of twenty isolates used in the baseline study with the contig Gc 12 revealed no polymorphisms
and/or mutations, supporting the results of baseline sensitivity study that all the isolates are DMI-sensitive.
Understanding the genetic basis for and baseline sensitivity to DMI fungicides will aid the long-term efforts
toward managing CBS.
11.45 a.m
Impact of acibenzolar-S-methyl on bacterial wilt disease incidence and marketable yield of grafted
tomatoes.
Sanju Kunwar, Mathews L. Paret, Joshua H. Freeman, Laura Ritchie and Stephen M. Olson, North Florida
Research and Education Center, University of Florida, Quincy, FL
Jeffrey B. Jones, Department of Plant Pathology, University of Florida, Gainesville, FL 32611
Greenhouse and field studies were conducted in Florida from 2012-2014 to assess the impact of Acibenzolar-SMethyl (ASM), a systemic acquired resistance inducer, applied as a drench/drip or foliar, on bacterial wilt
incidence and marketable yield of grafted tomatoes in a field infected with Ralstonia solanacearum. A
susceptible variety BHN 602 and BHN 602 grafted onto resistant rootstock BHN 998 were used in the study. In
greenhouse experiments, drench applications of ASM (50 mg/l) to BHN 602 significantly reduced bacterial wilt
disease severity compared to foliar ASM treatment or non-treated control (P ≤ 0.05). In the field experiments,
grafting alone or in combination with drip ASM treatment (0.5 oz/A) significantly reduced disease incidence
and increased total marketable yield relative to all non-grafted treatments (P ≤ 0.05). There was no statistical
difference in bacterial wilt incidence and total marketable yield between grafted plants and grafted plants treated
with ASM through drip. However, foliar applications of ASM showed a negative effect on grafted plants by
9 significantly reducing total marketable yield compared to drip applications of ASM on grafted plants or nontreated grafted control (P ≤ 0.05).
12.00 p.m
Diverse Pseudomonas syringae associated with foliar diseases of watermelon, cantaloupe, and squash
E. A. Newberry (1), M. L. Paret (1), C. T. Bull (2), Isael Rubio (3), J. B. Jones (5), B. Babu (1), Teresa Jardini
(4), E. M. Goss (5), P. D. Roberts (6)
(1) North Florida Research & Education Center, University of Florida, Quincy, FL;
(2) USDA–ARS, Salinas, CA, U.S.A.;
(3) California State University, Monterey Bay;
(4) UC Davis;
(5) Department of Plant Pathology, University of Florida, Gainesville, FL;
(6) South West Florida Research and Education Center, University of Florida, Immokalee, FL
Pseudomonas syringae pv. lachrymans is widely reported as the causal agent of angular leaf spot of cucurbits.
Reports of P. syringae pathovars lachrymans, aptata, syringae, savastanoi, and tabaci as cucurbit pathogens
indicates that a broader diversity of organisms within P. syringae may be responsible for these diseases. In this
study we have collected 54 isolates recovered in various years from watermelon, cantaloupe, and squash hosts in
Florida, Georgia, and California. Isolates were fluorescent on King’s medium B agar and identified
as Pseudomonas syringae through biochemical and 16S rRNA analysis. Variability was observed in level of
fluorescence, levan production, and ice nucleation ability. Phylogenetic analysis using four housekeeping genes
(gyrB, rpoD, gltA, gap1) including sequences publicly available from the Plant Associated Microbes Database
(PAMDB) indicates that all isolates are distributed among three clades within phylogroup 2, are members of
genomospecies 1, and genetically diverged from P. s. pv. lachrymans. Six isolates formed a sub group with P.
syringae pathotypes aptata and atrofacians, while all others were genetically distinct from any pathotype
sequences. The identification of clonal strains recovered in Florida, Georgia, and California over multiple years
indicates potential dispersion of the pathogen through seed. Pathogenicity and host range tests show that
watermelon, squash, and cantaloupe are susceptible to wide range of genetic diversity among P. syringae.
12.15 p.m: Concluding remarks, Dr. Aaron Palmateer
12.25 p.m: Results, graduate student competition
12.30 p.m: Lunch (provided)
2015 FPS Meeting Organizing Committee:
Aaron Palmateer, President FPS ([email protected])
Mathews Paret, Vice President FPS ([email protected])
Nicholas Dufault, Secretary FPS ([email protected])
Student coordinator: Mason Newark, President, Graduate Student Organization
Staff coordinator: Lauretta Rahmes, Office Manager
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