Characterization of Phytophthora spp. Isolated from Ornamental Plants in Florida

Abstract

This report investigates population structure and genetic variability of Phytophthora spp. isolated from botanically diverse plants in Florida. Internal transcribed spacer-based molecular phylogenetic analyses indicate that Phytophthora isolates recovered from ornamental plants in Florida represent a genetically diverse population and that a majority of the isolates belong to Phytophthora nicotianae (73.2%), P. palmivora (18.7%), P. tropicalis (4.9%), P. katsurae (2.4%), and P. cinnamomi (0.8%). Mating type analyses revealed that most isolates were heterothallic, consisting of both mating type A1 (25.2%) and mating type A2 (39.0%), and suggesting that they could outcross. Fungicide sensitivity assays determined that several isolates were moderate to completely insensitive to mefenoxam. In addition, several isolates were also moderately insensitive to additional fungicides with different modes of action. However, correlation analyses did not reveal occurrence of fungicide cross-resistance. These studies suggest that a genetically diverse Phytophthora population infects ornamental crops and the occurrence of mefenoxam-insensitive Phytophthora populations raises concerns about disease management in ornamentals. Mitigating fungicide resistance will require prudent management strategies, including tank mixes and rotation of chemicals with different modes of actions.

Full text

Jaimin S. Patel et al, Plant Disease
1
Characterization of Phytophthora spp. isolated from ornamental plants in Florida.
Jaimin S. Patel
1
*, Anne Vitoreli*
2
, Aaron J. Palmateer
2
, Ashraf El-Sayed
1,3
, David J. Norman
1
,
Erica M. Goss
4
, Mary S. Brennan
1
and Gul Shad Ali
1#
1
Mid-Florida Research and Education Center and Department of Plant Pathology, University of
Florida/Institute of Food and Agricultural Sciences, 2725 Binion Rd, Apopka, FL 32703
2
Tropical Research and Education Center, University of Florida/Institute of Food and
Agricultural Sciences, 18905 S.W. 280
th
Street, Homestead, FL 33031
3
Microbiologyand Botany Department, Faculty of Science, Zagazig University, Zagazig, 44519,
Egypt 
4
Department of Plant Pathology, University of Florida, Institute of Food and Agricultural
Sciences, Gainesville, FL 32611-0680.
*
These authors contributed equally to this work.
#
Corresponding author: Gul Shad Ali
Email address: gsali@ufl.edu
Tel:+1-407-410-6933
Fax:+1-407-814-6186
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ABSTRACT
This report investigates population structure and genetic variability of Phytophthora spp isolated
from botanically diverse plants in Florida. ITS-based molecular phylogenetic analyses indicate
that Phytophthora isolates recovered from ornamental plants in Florida represent a genetically
diverse population and that a majority of the isolates belong to P. nicotianae (73.2%), P.
palmivora (18.7%), P. tropicalis (4.9%), P. katsurae (2.4%) and P. cinnamomi (0.8%). Mating
type analyses revealed that most isolates were heterothallic consisting of both mating type A1
(25.2%) and mating type A2 (39.0%) suggesting that they could outcross. Fungicide sensitivity
assays determined that several isolates were moderate to completely insensitive to mefenoxam.
In addition, several isolates were also moderately insensitive to additional fungicides with
different mode of actions. However, correlation analyses did not reveal occurrence of fungicide
cross-resistance. These studies suggest that a genetically diverse Phytophthora population
infects ornamental crops and the occurrence of mefenoxam-insensitive Phytophthora populations
raises concerns about disease management in ornamentals. Mitigating fungicide resistance will
require prudent management strategies including tank mixes and rotation of chemicals with
different modes of actions.
Keywords: Phytophthora, ornamentals, Mefenoxam, fungicide-resistance.
INTRODUCTION
Over a 100 Phytophthora spp. have been described (Kroon et al. 2012), several of which
infect economically important plants including numerous vegetables, fruits, oil crops, ornamental
plants, and landscape and forest trees (Erwin and Ribeiro 1996, Gevens et al. 2007, Grunwald et
al. 2012, Martin et al. 2012). Worldwide Phytophthora diseases are responsible for billions of
dollars in crop losses (Kamoun et al. 2014, Wawra et al. 2012). Phytophthora spp. display
substantial variation in host ranges with some causing disease on a wider variety of plants
whereas others infect only a single crop. They are regularly isolated from diverse ornamental
plants in greenhouses and landscapes throughout the world (Bienapfl and Balci 2014),
(Leonberger et al. 2012, Parke et al. 2014, Prigigallo et al. 2015, Schlenzig et al. 2014). For
example, P. nicotianae infects close to 255 genera in 90 families including many herbaceous and
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Jaimin S. Patel et al, Plant Disease
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woody plants, and is present in nurseries worldwide (Cline et al. 2008). P. nicotianae also
causes root rot in citrus resulting in substantial yield and quality losses where the disease occurs
(Cacciola and Lio 2008). P. ramorum, which is an invasive and quarantined species, causes
sudden oak death disease and infects over 60 ornamental trees and shrubs with devastating
impacts on natural ecosystems (Grunwald et al. 2008, Grunwald et al. 2012). Another species,
P. capsici, which was initially thought to infect peppers only, was also found to infect cucurbits,
tomatoes, eggplants, and beans (Martin et al. 2012, Wang et al. 2013). Common to the
infection process of all Phytophthora spp. is the requirement of moist condition, which favors
dispersal and germination of sporangia and zoospores. Long-distance dispersal of Phytophthora
spp. is facilitated by run-off water from nurseries and by natural streams. The spread of
Phytophthora at a global level is believed to be through commercial trade of ornamentals
(Bienapfl and Balci 2014, Goss et al. 2011a). For example, since the first detection of P.
ramorum in the 1990s, it has spread to several US states and Canada through shipments of
infested ornamental plants (Goss et al. 2011a, Prospero et al. 2009, Rizzo et al. 2002).
Pesticides are the primary means of managing diseases caused by Phytophthora spp.
(Foster and Hausbeck, 2010; Hausbeck and Lamour, 2004), which add to the total cost of
production. A number of fungicides with different modes of action such as phenyl amides,
benzamides, phosphonate, dimethomorph, quinone outside inhibitors (QoI), and quinone inside
inhibitors (QiI) have been recommended to control diseases caused by Phytophthora spp. These
fungicides are very effective because most of them inhibit single molecular sites in essential
cellular processes such as mitochondrial function, rRNA synthesis, cell wall biosynthesis and
cell division. Due to frequent use of single-site fungicides, Phytophthora spp. pose a high risk
of developing resistance to fungicides and therefore, potentially disrupting effective disease
management programs (FRAC 2012, Hu et al. 2012). Fungicide-insensitive isolates of various
Phytophthora spp. have been reported throughout the world (Dobrowolski et al. 2008, Gisi and
Cohen 1996, Hu et al. 2012, Hu et al. 2005, Hu et al. 2008, 2010, Hwang and Benson 2005,
Meng et al. 2011, Perez-Sierra et al. 2011, Timmer et al. 1998). Therefore, regular monitoring of
Phytophthora populations is essential for sustainable disease management (Brent 1992,
Grunwald et al. 2006, Hu et al. 2012, Staub and Sozzi 1984)
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Historically, Phytophthora spp. have been characterized through morphological features,
which may not be practical for in-depth analyses of larger populations. More recently several
groups have used DNA-based molecular phylogenetic analyses for characterizing Phytophthora
communities in agricultural lands, nurseries and natural ecosystems (reviewed in Blair et al.
2008, Grunwald et al. 2011, Park et al. 2008). One of the first DNA regions to be used in
phylogenetic analysis of Phytophthora species was the 5.8S ribosomal RNA gene and the
flanking internal transcribed spacers 1 and 2 (Lee and Taylor 1992). These studies have revealed
important information about the genetic makeup, lineages and spread of previously known and
some newly discovered Phytophthora spp. throughout the world.
Several Phytophthora species are heterothallic with two known mating types, type A1
and A2 (Gisi and Cohen, 1996). Hybridization between opposite mating types increases genetic
variability and promotes survival of the pathogen (Lamour and Hausbeck, 2000). Sexual
reproduction may also spread fungicide resistance through genetic recombination and, therefore,
it is important to monitor mating types as well as fungicide sensitivity of Phytophthora spp.
Florida is home to thousands of commercial nurseries that produce a wide variety of
ornamental plants ranging from annual herbaceous plants to tropical foliage and woody trees and
shrubs (DPI 1998). Florida is the major port of entry for ornamental plants arriving in the
United States (Merritt et al. 2012), and subsequently has a high number of pest and disease
introductions. Phytophthora spp. are no exception and frequently isolated from various
ornamental plants in Florida. Based on morphological characteristics and previous pathological
history, these isolates are tentatively assigned to specific species. However, molecular species
identification is not routine. Accurate characterization of the genetic composition of the
Phytophthora spp. infecting ornamentals is necessary for investigating Phytophthora evolution,
spread and adaptation to new hosts and environments. Many commercial nurseries where these
isolates were collected co-cultivate many host plants of different Phytophthora species,
potentially increasing risk of development of hybrid species. Since the ornamental crop industry
in Florida relies on frequent fungicide sprays for controlling diseases, the risks of selecting
fungicide resistant Phytophthora isolates are high. The objectives of this study were (1) To
conduct molecular phylogenetic analyses of a spatio-temporal population of Phytophthora
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Jaimin S. Patel et al, Plant Disease
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recovered from ornamental crops in Florida. (2) To determine mating types of this population.
(3) To investigate the sensitivity of these Phytophthora spp. to commonly used fungicides with
different modes of actions. Findings in this report will help in formulating effective strategies
and policies for preventing the spread of Phytophthora to new niches and mitigate risks of
fungicide resistance development.
MATERIALS AND METHODS
Phytophthora isolates. Between 1993 and 2014, a total of 156 putative Phytophthora isolates
were recovered from the leading edges of lesions of different diseased plants with characteristics
symptoms of Phytophthora at the University of Florida Plant Disease Clinics at the Mid-Florida
Research and Education Center, Apopka, Florida and the Tropical Research and Education
Center, Homestead, Florida. These isolates were initially recovered on water agar and then
stored in sterile water or 10% V8 juice agar slants in glass bottles at room temperature. For this
study, we attempted to recover all these isolates on 10% V8, PDA and water agar. All cultures
that grew were checked for any bacterial or fungal contamination, and contaminated cultures
were excluded from further analysis, resulting in a population of 123 isolates (Table 1). A single
hyphal tip from pure isolate cultures was transferred to and maintained on selective medium V8-
PARP agar (20% clarified V8 juice, 0.4% CaCO
3
, 1.5% Bacto-agar, 25 µg ml
-1
pimaricin, 100
µg ml
-1
ampicillin, 25 µg ml
-1
rifampicin and 25 µg ml
-1
pentachloronitrobenzene). The first two
digits in an isolate designation indicate year of collection. For this study, all isolates were grown
on V8-PARP at 22
o
C for 6-8 days.
PCR and sequencing of ITS. Approximately 100 mg mycelia of each isolate, grown on
clarified 20% V8 PARP agar plates, were harvested by scraping off with a sterile spatula into
sterile 1.5 ml eppendorf tubes. The mycelia were flash frozen with liquid nitrogen and ground
using a pestle and mortar. DNA was isolated from the ground mycelia according to a previously
described method (Aljanabi and Martinez 1997), and DNA was quantified using NanoDrop
2000C Spectrophotometer (Thermo Fisher Scientific, USA). To accurately identify the isolates
to species level, ITS regions were sequenced using the ITS5 forward primer and ITS4 reverse
primers, to amplify the ITS1, 5.8s rRNA and ITS2 regions (White et al. 1990) (Figure 1). PCR
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Jaimin S. Patel et al, Plant Disease
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reactions consisted of 1x Ex Taq
TM
reaction buffer, 0.8 mM all dNTPs, 0.3 µM each primer, 0.1
U/µl TaKaRa Ex Taq
TM
polymerase (TaKaRa, Shiga, Japan)) and 0.8 ng/µl genomic DNA in a
final volume of 25 µl. PCR conditions consisted of an initial denaturation step at 95°C for 5 min,
followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. PCR products were
purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Both strands of the
amplified DNA products were directly sequenced using the ITS4 reverse and ITS5 forward
primers. ITS sequences were searched against both the non-redundant nucleotide sequence
database at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the Phytophthora database (Kang
et al. 2007).
Phylogenetic analysis. Genetic diversity of all isolates was determined using phylogenetic
analyses. To infer genetic relationships of the Phytophthora isolates reported in this study,
phylogenetic analyses were conducted using the ITS sequences. Representative ITS sequences
of Phytophthora spp. were also included in constructing the phylogenetic tree for comparison.
Sequences were aligned using clustalW; alignment with the Muscle algorithm returned similar
results. The ClustalW alignments were examined manually and ambiguous overhanging
sequences from both ends were trimmed resulting in an alignment block consisting of 772 sites,
which spanned the ITS1, 5.8S rRNA and ITS2 regions. For multiple sequence alignment, a
FASTA file containing ITS sequences determined in this study and representative ITS sequences
of Phytophthora spp. were imported into Mega 6.0 software (Larkin et al. 2007, Tamura et al.
2011) and aligned using the clustalW algorithm (Larkin et al. 2007). Alignments were also
performed using the Muscle algorithm (Edgar 2004) implemented in Mega 6.0. A phylogenetic
tree was constructed using the Maximum Likelihood (ML) method based on the Tamura-Nei
model with 1000 bootstrap replication (Tamura and Nei 1993, Tamura et al. 2011); Neighbor-
joining method was also employed, which yielded results similar to the ML method.
Evolutionary diversity estimates were calculated for the P. nicotianae group. Estimates of
average evolutionary divergence over all isolate pairs were calculated as number of base
substitutions per nucleotide site. Analyses were conducted using the Maximum Composite
Likelihood model (Tamura et al. 2004). All positions containing gaps and missing data were not
included in analyses. There were a total of 772 base positions in the final dataset.
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Mating type analysis. Mating type of isolates were determined using known P. nicotianae
mating type A1(#13-724) and type A2 (#13-723) tester strains in a 24-well culture plates
(Nunc
TM
, Thermo Fisher, USA). Each well of the culture plate contained 0.5 ml V8 juice agar
(20% clarified V8 juice, 0.4% CaCO
3
, 1.5% Bacto-agar). A mycelial plug (2 mm diameter) from
a test isolate was placed at the margin of the well of a culture plate opposite a mycelial plug (2
mm diameter) of either mating type A1 or A2. Each isolate was also tested for homothallism
using two agar plugs from the same isolate. Plates were wrapped with Parafilm and incubated in
a growth chamber at 22-25
0
C for up to 6 weeks under dark. Plates were checked for the presence
of oospores every week for at least 6 weeks.
Fungicide sensitivity tests. Most isolates were tested for sensitivity to Mefenoxam (FRAC
group 4, Subdue Maxx, Syngenta Crop Protection), fluopicolide (FRAC group 43, Adorn, Valent
USA Corporation), aluminum tris (FRAC group 33, Aliette WDG, Bayer CropScience),
azoxystrobin (FRAC group 11, Heritage, Syngenta Crop Protection), cyazofamid (FRAC group
21, Segway, FMC Corporation) and dimethomorph (FRAC group 40, Stature SC, BASF).
Fungicides formulated as commercial products were dissolved in autoclaved clarified V8
medium at the following active ingredient rates: mefenoxam 0.1 mg mL
-1
, fluopicolide 0.1 mg
mL
-1
, aluminum tris 1 mg mL
-1
, azoxystrobin 1 mg mL
-1
, cyzofamid 1 mg mL
-1
and
dimethomorph 1 mg mL
-1
. Fungicide concentrations chosen in this study were based on
previous studies (Jackson et al. 2010, Kousik and Keinath 2008, Kuhajek et al. 2003, Stein and
Kirk 2004, Ziogas et al. 2006). A single 5-mm mycelial plug from the leading edge of a 1-week
old Phytophthora culture, grown in the dark at 22
o
C, was placed on fungicide amended and non-
amended clarified V8 agar plates. Plates were incubated in the dark at 22 to 25
o
C for 7 days.
Colony diameters were measured and percent growth inhibition was calculated using the
following formula:
ℎℎ =
 − 

× 100
where C and F are average colony diameters on non-amended control and fungicide-amended V8
agar plates, respectively. To determine the effect of mefenoxam dosage on resistance, isolate 05-
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Jaimin S. Patel et al, Plant Disease
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297 (resistant with less than 10% growth inhibition) and isolate 05-217 (sensitive with >90%
growth inhibition) were assayed over a range of mefenoxam concentrations. All experiments
were arranged in a completely randomized design with four replications for each fungicide. The
experiment was repeated once. The emergence of fungicide resistant populations has been
reported in several Phytophthora spp. (Dobrowolski et al. 2008, Gisi and Cohen 1996, Hu et al.
2012, Hu et al. 2005, Hu et al. 2008, 2010, Hwang and Benson 2005, Meng et al. 2011, Perez-
Sierra et al. 2011, Timmer et al. 1998) and poses serious threat to effective management of
Phytophthora diseases. Therefore, Phytophthora isolates collected in this study were screened
against commonly used fungicides. Based on the % growth inhibition, isolates were categorized
into sensitive (
≥
80% growth inhibition), moderately sensitive (51-79% growth inhibition) and
resistant (≤50% growth inhibition). Cross-resistance of isolates to different fungicides was
analyzed using the Spearman’s rank correlation coefficients (rho) on ranked percent growth
inhibition data. Spearman’s rho (ρ) values and their associated p-values were calculated using
the open source statistical software R (http://www.r-project.org/).
RESULTS
Molecular identification of Phytophthora isolates.
Consensus ITS contigs for each isolate were constructed from the alignment of forward
and reverse sequences obtained with ITS5 and IT4 primers. The resulting contigs were searched
using BLASTn at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and at the manually curated
Phytophthora database (Park et al. 2008). BLAST search results from NCBI and the
Phytophthora database were identical for each isolate (Table 1). Among the 123 isolates, 90
isolates were identified as P. nicotianae, 23 as P. palmivora, 6 as P. tropicalis, 3 as P. katsurae
and 1 as P. cinnamomi (Table 1). ). Since double peaks were not observed in the DNA sequence
chromatograms of any of the isolates, this suggested that these isolates are homozygous for ITS.
Diversity and genetic composition of Phytophthora isolates. ClustalW alignment showed that
approximately 33% (252/772) of the sites were polymorphic. An ML tree was constructed from
these alignments, which separated all isolates into five major groups, consistent with BLAST
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Jaimin S. Patel et al, Plant Disease
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results such that each group closely clustered with a representative published ITS sequence of P.
parasitica (GU983635), P. palmivora (KF263691), P. tropicalis (KC479199), P. katsurae
(JQ901391) and P. cinnamomi (JX996044) (Fig. 1). Using the Maximum Composite Likelihood
model (Tamura et al. 2004), evolutionary diversity estimate, calculated as mean base substitution
per site over all sequence pairs, was 0.0286 (2.86%).
The P. nicotianae group, which consisted of 90 isolates, displayed substantial variation
and was further analyzed in detail for genetic diversity. These analyses classified the P.
nicotianae isolates roughly into 5 subgroups (Fig. 1). Overall, mean divergence between P.
nicotianae isolates was 0.64 % (range, 0 to 7.5%). In pairwise comparisons, a majority of the P.
nicotianae isolates displayed mean divergence below 2%. These isolates infected plants from
diverse families. Groups PpIV and PpV displayed high within-group and between-group
divergences (Table 2 and 3). Similarly, P. palmivora isolates also displayed considerable
variation in ITS sequence with a 0.44% (range, 0 to 2.3%) overall mean divergence.
Phytophthora isolates are a mixture of A1 and A2 mating types.
Mating types of all isolates were identified using known A1 and A2 mating type tester isolates.
A summary of this analysis is presented in Table 4. Overall, 25.2% of all Phytophthora isolates
belonged to A1 mating type and 39.0% belonged to A2 mating types. The three isolates
identified as P. katsurae were all homothallic and produced oospores. The remaining 33.3% did
not develop any oospores, antheridia or oogonia up to 6 weeks of culture suggesting that they are
sterile.
Several Phytophthora isolates displayed mefenoxam-insensitivity. A majority of isolates
were either sensitive (53%) or moderately sensitive (37%) to mefenoxam, suggesting that they
can be controlled using mefenoxam. There was substantial variation in mefenoxam sensitivities
of these isolates (Figure S1). One P. tropicalis and three P. nicotianae isolates were found to be
resistant to mefenoxam. Among these, isolate 05-297 (P. nicotianae) was highly resistant to
mefenoxam with only 9.4% growth inhibition (Figure 2). The other three isolates, 05-215 (P.
nicotianae), 11-38 (P. nicotianae) and 14-138 (P. tropicalis) had 43.7%, 47.7% and 23.9%
growth reduction, respectively (Figure S1). Isolate 05-297 displayed high level of resistance
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Jaimin S. Patel et al, Plant Disease
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over all tested concentrations. Interestingly, at sub-lethal concentrations (0.1 to 2 µg mL
-1
), the
growth of this isolate was enhanced by approximately 20%, which was observed consistently in
several independent experiments (Figure 2A). Similar results were observed with the other three
resistant isolates 05-215, 11-38 and 14-138 (data not shown). Since mefenoxam is used
extensively in nurseries, the resistant strains will likely be selected thus increasing the probability
of mefenoxam-resistance build-up in future Phytophthora populations.
Phytophthora isolates did not display any resistance to different fungicides. A majority of
the tested isolates were sensitive to fluopicolide (Adorn), aluminum tris (Aliette), azoxystrobin
(Heritage), cyzofamid (Segway) and dimethomorph (Stature) (Figure S1. Frequency distribution
of sensitive, moderately resistant and resistant isolates are shown in Fig. 3. More than 80% of
isolates were sensitive to Mefenoxam, Adorn and Stature (Fig. 3). Although a majority of
isolates were inhibited by each of these fungicides, several isolates displayed variable resistance
levels to at least one of these fungicides. Less than 50% growth inhibition was displayed by 1
(Adorn), 9 (Aliette), 10 (Heritage) and 11 (Segway) isolates (Fig. 3). The remaining isolates
displayed either moderate resistance or were sensitive to these fungicides. Ornamental facilities,
where the isolates reported in this study originated, often apply fungicides with different modes
of action on the same crop multiple times. This led us to speculate that some isolates might
display resistance to multiple fungicides. With the exception of a very low-level and statistically
insignificant co-resistance between mefenoxam and Adorn (ρ=0.31, p=0.09), and between
mefenoxam and Aliette (ρ=0.30, p=0.09), none of the other fungicides showed resistance to
multiple isolates. All isolates, which were resistant to one fungicide, were inhibited by at least
one other fungicide with a different mode of action. For example, isolates 05-215, 05-217 and
05-222 showed moderate resistance to Adorn, but were sensitive to Aliette, Heritage and Stature.
No correlation was found between genetic diversity of isolates and their sensitivity to
fungicides (Table 2 and Fig. 2 and 3, Spearmans’s rank correlation p>0.1). For example, isolates
05-297 (resistant to mefenoxam) and 12-5 (sensitive to mefenoxam), belong to the same group
PpV in the phylogenetic tree (Fig.1). Isolates 05-297, 05-215 and 11-38, which were
mefenoxam-resistant displayed single nucleotide polymorphism (SNPs) at different sites and
belonged to different phylogenetic groups (Fig. 1), suggesting that mefenoxam-insensitivity is
not necessarily associated with overall genetic variation.
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Jaimin S. Patel et al, Plant Disease
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DISCUSSION
Florida is renowned for its ornamental plant industry and it ranks only second to
California in total trade. Altogether, thousands of ornamental plant species are grown
throughout Florida in greenhouses and nurseries. Ornamental plant producers in Florida often
face heavy pressure from pathogens and insects, which due to a hot and humid climate can be a
year round challenge. In addition, every year millions of plants are traded through Florida,
which can serve as an unintentional conduit for the spread of pathogens to new areas, where they
can infect resident plants. Conglomeration of so many diverse plants and their associated
pathogens in confined greenhouses can also serve as a potential breeding ground for the
evolution of more aggressive pathogens through intra- and inter-specific cross hybridizations.
Therefore, it is important to investigate population structure and genetic diversity of plant
pathogens on ornamentals in Florida. Phytophthora spp. are one of the most frequently isolated
pathogens from symptomatic ornamental crops in Florida (Merritt et al. 2012). Based on
symptoms, the type of host species infected and previous etiology, these isolates were tentatively
assigned to a previously described Phytophthora species. Beyond these initial assessments, little
was known about the genetic variation and population structure of this Phytophthora collection.
Cook et al (2000) conducted a phylogenetic study of Phytophthora spp. using ITS
sequences. Moreover, a large number of ITS sequences of Phytophthora species are available to
compare in GenBank. Phylogenetic analysis was based on the ITS sequences, a commonly used
molecular marker and a barcode for oomycetes, which provided sufficient resolution to interpret
phylogenetic trees and to distinguish various Phytophthora spp (reviewed in Kroon et al. 2012,
Martin et al. 2012, Robideau et al. 2011). Based on these analyses, this collection was separated
into five groups (Figure 1). Group 1 consisted of P. nicotianae, with the majority of isolates
displaying very low single nucleotide polymorphisms. Based on SNP data, the P. nicotianae
group can be further divided into additional subgroups. These groups were not associated with a
particular group of hosts ruling out any strong host specificity among the isolates. Moreover, the
tree branches defining these groups were not supported by strong bootstrap values (< 50%) and
therefore each ITS sequence type has a broad host range. Our analyses, however, focused on
species identification and it is likely that variation at other loci important for pathogenicity and
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Jaimin S. Patel et al, Plant Disease
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aggressiveness, such as effector genes, could correlate with host range in P. nicotianae With
the ever increasing ease and feasibility of sequencing entire genomes, this issue can be addressed
by sequencing the entire genomes of select sets of isolates in this collection in the future.
Analyses based on entire genome sequences will also allow full-genome concatenated SNP-
based analysis, which provides in-depth resolution in differentiating even closely related
pathogen strains (Bart et al. 2012).
Evolutionary diversity estimates, which were as high as 7.5%, indicate that this P.
nicotianae population is highly diverse. The high divergence suggests that most of the isolates
are not a single clonal lineage and that they were probably introduced along with their hosts into
Florida. One of the most divergent isolates 08-168 and several less divergent isolates were
isolated from the same host (Spathiphyllum). This kind of host overlap can potentially provide a
favorable breeding ground for hybridization and the emergence of new isolates containing novel
complements of virulence factors, which can further expand their already wide host range. It is
interesting to note that the first natural hybrid between two Phytophthora spp. was also isolated
from a Spathiphyllum sp. (Man In 't Veld et al. 1998), suggesting that this plant and probably
other tropical foliage plants are susceptible to multiple species of Phytophthora. Similarly,
occurrence of several different Phytophthora spp. with so much variation within a small
geographic location can also potentially lead to the emergence of novel hybrid species with
adaptability to new environments and hosts (Leonberger et al. 2012). Several emerging
pathogenic Phytophthora spp. such as P. andina and P. alni have been shown to be hybrids of
different Phythophthora spp., which most likely co-inhabited the same environment or even the
same plant species (Brasier et al. 1999, Ersek and Nagy 2008, Goss et al. 2011b, Ioos et al.
2006). Similarly, hybrids between P. nicotianae (nicotianae) and P. cactorum, which possess an
expanded host range likely due to a combination of novel virulent factors (Bonants et al. 2000,
Hurtado-Gonzales et al. 2009, Man In 't Veld et al. 1998, Man 2001, Nirenberg et al. 2009). All
these examples suggest that if brought together, Phytophthora spp. can potentially hybridize
resulting in development of aggressive strains. This possibility is supported by the fact that both
mating types A1 and A2 were represented in the isolates analyzed in this study. In our
Phytophthora collection, we did not see any evidence of natural intra- or inter-specific
Phytophthora hybrids despite the fact that many isolates were recovered from Spathiphyllum. A
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Jaimin S. Patel et al, Plant Disease
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possible explanation could be incompatibility among Phytophthora spp. co-inhabiting with the
same individuals of a host plant species. For example, we recovered P. nicotianae and P.
palmivora from Spathiphyllum sp., and assuming that these two Phytophthora species are
sympatric, their chances of hybridization might have been obscured by their genetic divergence.
This is because P. nicotianae belongs to clade 1 and P. palmivora belongs to clade 4 according
to recent molecular classification (Blair et al. 2008, Kroon et al. 2012). Similarly P. tropicalis
and P. palmivora, both infect Hedera sp. and thus can potentially occur concurrently on the same
individual plant, belong to distinct clades, clade 2 (P. tropicalis) and clade 4 (P. palmivora)
(Blair et al. 2008, Kroon et al. 2012), suggesting that they may be also genetically diverse to
cross-hybridize. Evolutionary diversity estimates, which were as high as 7.5%, indicate that this
P. nicotianae population is highly divergent. The high divergence suggests that most of the
isolates are not a single clonal lineage and that they were probably introduced along with their
hosts into Florida. In our study, most isolates collected from 1993-2012 belong to A2 mating
type; whereas most isolates collected in 2013 belong to A1 mating type. Further, most isolates
collected in 2014 belong to A2 mating type. Such a change in mating type year over year was
evident in Europe for A2 mating type (Gisi and Cohen, 1996). This pattern might involve
epidemiological properties of the mating type, which is less competitive than other mating type.
Assessing fungicides sensitivity of Phytophthora spp. is an essential step in planning and
employing proper disease management strategies. Occurrence of fungicide resistance is a
recurring problem with most single-site fungicides including mefenoxam-resistance in
Phytophthora spp. (Ferrin and Kabashima 1991, Hu et al. 2005, Hu et al. 2008, 2010, Hwang
and Benson 2005, Jeffers et al. 2004, Timmer et al. 1998), which can severely impact disease
management programs. In several cases, mefenoxam-resistance has been suggested to be
associated with repeated use of mefenoxam (Hu et al. 2008). The isolates reported in this study
were collected over a longer time from nurseries that rely on chemicals for disease control, thus
raising the probability of fungicide resistance development. In our analyses, a majority of
isolates were sensitive to all fungicides including mefenoxam. This may be due to growers
practicing fungicide rotation and tank mixes, which is widely publicized and emphasized through
extension and outreach efforts in Florida. Moreover, only few isolates (1-7) were collected each
year starting from 1993 with exceptions of year 2013 and 2014. Such a few number of isolates
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Jaimin S. Patel et al, Plant Disease
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collected each year could be a reason for the narrow range of species. For example, four species
were recovered from a total of 45 isolates in year 1993 to 2012, whereas four species were
recovered from a total of 78 isolates only from two years (2013-2014). However, there were
notable exceptions of two isolates displaying insensitivity to mefenoxam. One isolate was
recovered in 2005 and another isolate was recovered in 2014. In 2014, a total of 33 isolates were
collected which increased a chance of capturing resistant isolates. This was expected since
mefenoxam has been used extensively in nurseries in Florida. The substantial variation in
mefenoxam sensitivities in tested isolates suggests that they are still evolving. It is likely that
these isolates could further evolve and become more resistant to mefenoxam, especially if
mefenoxam is used exclusively. These results agree with those reported by Ferrin and
Kabashima (1991) where 7.7% of the P. nicotianae isolates collected from nurseries in Southern
California were mefenoxam-resistant (Ferrin and Kabashima 1991). Our results differed
substantially from those reported from P. nicotianae isolates collected from nurseries elsewhere,
which reported 21% (North Carolina) and 26% (Virginia) mefenoxam-resistant isolates (Hu et al.
2008, Hwang and Benson 2005). Hu et al (2008) collected 95 isolates of P. nicotianae from
diverse nursery crops and irrigation water from Virginia, whereas Hwang and Benson (2005)
recovered 483 isolates from seven different plant species from North Carolina. Based on our
data, if we assume that ~8.8% P. nicotianae isolates present in Florida are resistant to
mefenoxam and that mefenoxam is used frequently, we should have seen an increase of
mefenoxam-resistant isolates over time. However, this is not what was observed in the analyses.
The most resistant isolate (05-297) was isolated in 2005, where isolates collected after 2005 were
more sensitive to mefenoxam. An explanation for this is that the plants could have been shipped
elsewhere before Phytophthora could become established in the nursery, or they could have been
phased out from natural populations due to use of fungicides with modes of actions that are
different from mefenoxam. In addition, mefenoxam has been in the market since 1996 for use on
various crops but as years have passed more fungicides are available which belong to different
modes of action. Commercialization of new products may have reduced the exposure of
Phytophthora spp. to mefenoxam. More intensive and extensive sampling might be needed to
gain a true assessment of the prevalence of mefenoxam resistant isolates in Florida.
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Jaimin S. Patel et al, Plant Disease
15
Our analysis finds resistance in Phytophthora spp. to Adorn (fluopicolide), Aliette
(fosetyl-Al), Heritage (azoxystrobin) and Segway (cyazofamid). To our knowledge this is the
first report of resistance in an isolate of Phytophthora sp. to fluopicolide. The finding of an
isolate resistant to Adorn is not surprising as fluopicolide has a single site mode of action for
efficacy against Oomycetes. In fact, the product manufacture recommends that Adorn is not to
be applied alone. There are reports of reduced sensitivity in Phytophthora spp. to Aliette. For
example, Phytophthora cinnamomi obtained from avocado trees continuously treated with
phosphonates were less inhibited by Aliette (Duvenhage, 1994). Similarly, all Phytophthora
capsici isolates collected from diverse vegetable crops found either resistant or intermediate
sensitivity to Segway (Jackson et al. 2012).
Several groups have reported increased fitness of Metalaxyl-resistant isolates as increased
vegetative growth, oospores formation and lesion sizes (Hu et al. 2008, Kadish and Cohen
1988a, b, Kadish et al. 1990, Porter et al. 2007). Interestingly, in our study we found that in the
presence of lower mefenoxam doses, mefenoxam-resistant isolates displayed increased mycelial
growth, suggesting that these isolates have adapted to exploit mefenoxam to their advantage
through an unknown mechanism. Since mefenoxam is suggested to affect the activity of RNA
polymerases (Davidse et al. 1983, Davidse et al. 1988, Fisher and Hayes 1984), it is possible that
the resistant isolates have accumulated mutations that instead of inhibition, enhances the activity
of RNA polymerase for favorable mycelial growth. Mefenoxam has been popular for controlling
Phytophthora diseases in ornamental nurseries. The frequencies of resistant P. nicotianae
isolates may increase in the future if these isolates have greater fitness compared to sensitive
isolates. Moreover, by cross hybridizing with other Phytophthora isolates or species,
mefenoxam resistance can be transferred into hybrids with better fitness in natural ecosystems.
Therefore, caution is advised in using mefenoxam where it has been found to be ineffective,
under such situations it is advisable to discontinue using mefenoxam altogether as this can lead
to the selection and reappearance of mefenoxam-resistant populations as was suggested for P.
infestans (Hu et al. 2012). Resistance can also be managed by rotating fungicides with different
modes of action such as fosetyl-Al and dimethomorph, which provided effective control against
those isolates, which were resistant to mefenoxam. In addition, all other possible integrated
disease management approaches should be practiced such as using clean potting mixes, clean
water, and including biological and bio-rational pesticides with broad-spectrum activity. This
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Jaimin S. Patel et al, Plant Disease
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work provides important information about the genetic structure of Phytophthora population in
Florida. These findings will help in formulating effective strategies and policies for preventing
the spread of Phytophthora to new niches and mitigate risks of fungicide resistance development.
ACKNOWLEDGEMENTS
This work was supported by funds from the Institute of Food and Agricultural Sciences at the
University of Florida to GSA. We are thankful to Robina Ali for her help with mating typing
analyses and fungicide resistance assays.
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Jaimin S. Patel et al, Plant Disease
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Figure legends.
Fig. 1. Molecular Phylogenetic analysis of Phytophthora isolates. A, Upper panel shows
schematics of the ITS region of Phytophthora. Locations of ITS4 and ITS5 primers are
indicated. Lower panel shows Phylogenetic tree, which was constructed from the ITS sequences
using the Maximum Likelihood method. Analysis consisted of 128 sequences including 5
reported representative ITS sequence of P. nicotianae (GU983635), P. palmivora (KF263691),
P. tropicalis (KC479199), P. katsurae (JQ901391) and P. cinnamomi (JX996044).
Fig. 2. Sensitivity of Phytophthora isolates to mefenoxam.
A, Dose-response curves of P. nicotianae isolates 05-297 (resistant) and 05-217 (sensitive) to
mefenoxam on V8 juice media. Each data point is a mean of 4 replications; Error bars are SE. B,
Representative photographs of isolates 05-297 and 05-217 on mefenoxam -containing and
control plates 5 days after the start of experiment.
Fig. 3. Frequency distribution of resistant, moderately resistant and sensitive Phytophthora
isolates to different fungicides.
Resistant, ≤50% growth inhibition; Moderate, moderately resistant with 51-79% growth
inhibition; Sensitive, ≥80% growth inhibition.
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Jaimin S. Patel et al, Plant Disease
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Tables
Table 1: Isolates of Phytophthora spp. collected from various ornamental hosts in Florida
Species
a
Isolates ITS
Identity
(%)
b
Host Matin
g
types
c
Mefenoxam
sensitivity
d
cinnamomi
07-385 99.9
Rhododendron sp.
A2 S
katsurae
14-374B 99.0
Podocarpus sp.
A1A2 A1A2 S
katsurae
14-374C 99.0
Podocarpus sp.
A1A2 A1A2 S
katsurae
14-761 99.0
Podocarpus sp.
A1A2 A1A2 S
palmivora
01-118 100.0
Hedera sp.
A2 S
palmivora
03-550 99.4
Chamaedorea elegans
A1 S
palmivora
11-65 99.9
Spathiphyllum sp.
A2 S
palmivora
10-1733 99.0
Liriope muscari
– – S
palmivora
13-1483A 100.0
Chrysothemis sp.
– – S
palmivora
13-1483B 100.0
Chrysothemis sp.
– – S
palmivora
13-1726 100.0
Mandevilla sp.
– – S
palmivora
13-1734 98.0
Pheonix dactylifera
– –
palmivora
13-1735 97.0
Catharanthus sp.
– –
palmivora
14-130 100.0
Cordyline sp.
A2 S
palmivora
14-32 100.0
Latania sp.
– – S
palmivora
14-33 100.0
Latania sp.
– – S
palmivora
14-34 99.0
Latania sp.
– – S
palmivora
14-35 100.0
Latania sp.
– – S
palmivora
14-37 99.0
Latania sp.
– – S
palmivora
14-684A 100.0
Podocarpus sp.
– – S
palmivora
14-684B 99.0
Sabal palmetto
– – S
palmivora
14-704 97.0
Pothos sp.
– – S
palmivora
14-705 97.0
Pothos sp.
– – S
palmivora
14-708 99.0
Palm
A2 S
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Jaimin S. Patel et al, Plant Disease
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palmivora
14-709 99.0
palm
A2 S
palmivora
14-710 98.0
Pachira sp.
– – S
palmivora
14-711 98.0
Pachira sp.
– – S
nicotianae
93-653 99.9
Vinca sp.
A2 S
nicotianae
99-191 99.9
Spathiphyllum sp.
A2 S
nicotianae
00-334 100.0
Vinca sp.
A1 S
nicotianae
01-138 98.4
Spathiphyllum sp.
A2 S
nicotianae
01-280 99.7
Anthurium sp.
A2 S
nicotianae
01-302 99.3
Viola sp.
– – S
nicotianae
02-29 99.1
Calceolaria sp.
A2 S
nicotianae
03-131 99.9
Spathiphyllum sp.
A2 S
nicotianae
03-132 100.0
Spathiphyllum sp.
– – S
nicotianae
03-361 100.0
Dieffenbachia sp.
A1 – S
nicotianae
03-407 99.3
Pothos sp.
A1 – S
nicotianae
04-130 99.9
Spathiphyllum sp.
A1 S
nicotianae
04-325 100.0
Spathiphyllum sp.
A2 S
nicotianae
04-410 99.8
Spathiphyllum sp.
A2 S
nicotianae
05-215 99.6
Alocasia sp.
A2 S
nicotianae
05-217 100.0
Coleus sp.
– – S
nicotianae
05-222 99.6
Gerbera sp.
A2 S
nicotianae
05-287 97.2
Pothos sp.
A2 S
nicotianae
05-297 98.5
Poinsettia
A2 R
nicotianae
06-141 99.9
Spathiphyllum sp.
A2 S
nicotianae
06-174 100.0
Cattleya sp.
A2 S
nicotianae
07-57 99.8
Alocasia sp.
A1 S
nicotianae
08-168 99.7
Spathiphyllum sp.
– – S
nicotianae
10-43 99.9
Spathiphyllum sp.
A1 S
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Jaimin S. Patel et al, Plant Disease
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nicotianae
11-1 99.9
Spathiphyllum sp.
A2 S
nicotianae
11-3 100.0
Spathiphyllum sp.
– – S
nicotianae
11-21 99.0
Rhoeo discolor
A2 S
nicotianae
11-38 100.0
Impatiens sp.
A2 S
nicotianae
12-1 98.6
Spathiphyllum sp.
A2 S
nicotianae
12-5 98.2
Hibiscus sp.
A2 S
nicotianae
10-524A 99.0
Iris sp.
A2 S
nicotianae
10-524B 99.0
Iris sp.
A2 S
nicotianae
10-534B 100.0
Spathiphyllum sp.
– – S
nicotianae
10-616A 100.0
Spathiphyllum sp.
– – S
nicotianae
10-616B 99.0
Spathiphyllum sp.
– – S
nicotianae
10-616C 100.0 Unknown – – S
nicotianae
13-1133A 100.0
Mandevilla sp.
– – S
nicotianae
13-1134B 100.0
Rhododendron sp.
A1 S
nicotianae
13-1667 100.0
Mandevilla sp.
– – S
nicotianae
13-1727 100.0
Mandevilla sp.
– – S
nicotianae
13-1728 99.0
Mandevilla sp.
– – S
nicotianae
13-1729 100.0
Allamanda sp.
– – S
nicotianae
13-1730 100.0
Spathiphyllum sp.
– – S
nicotianae
13-1731 100.0
Spathiphyllum sp.
– – S
nicotianae
13-1732 100.0
Spathiphyllum sp.
A1 S
nicotianae
13-1815 100.0
Kalanchoe sp.
A2 S
nicotianae
13-1816 100.0
ND
A2 S
nicotianae
13-1817 100.0
ND
A1 S
nicotianae
13-1818 99.0
Swietenia sp.
– – S
nicotianae
13-1819 100.0
Strelitzia nicolai
A2 S
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Jaimin S. Patel et al, Plant Disease
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nicotianae
13-1820 100.0
Bursera sp.
A1 S
nicotianae
13-1821 99.0
Syagrus romanzoffiana
A2 S
nicotianae
13-1822 100.0
Casia sp.
A1 S
nicotianae
13-1823 100.0
Casia sp.
A1 S
nicotianae
13-1824 99.0
Impatiens sp.
walleriana
A2 S
nicotianae
13-1825 100.0
Impatiens sp.
walleriana
A1 S
nicotianae
13-1826 99.0
Croton sp.
A2 S
nicotianae
13-1827 100.0
Oncidium sp.
A1 S
nicotianae
13-1828 100.0
Catharanthus sp.
A1 S
nicotianae
13-1829 100.0
Catharanthus sp.
A1 S
nicotianae
13-1830 100.0
Catharanthus sp.
A1 S
nicotianae
13-1831 100.0
Tilandsia sp.
A1 S
nicotianae
13-1832 100.0
Dracaena sp.
A1 S
nicotianae
13-1833 100.0
Dracaena sp.
A1 S
nicotianae
13-1834 100.0
Schefflera sp.
A1 S
nicotianae
13-1835 99.0
Schefflera sp.
A1 S
nicotianae
13-1836 100.0
Dracaena sp.
A1 S
nicotianae
13-1837 100.0
Anthurium sp.
– – S
nicotianae
13-1838 99.0
Sansevieria sp.
A1 S
nicotianae
13-1839 100.0
Epipremnum sp.
A1 S
nicotianae
13-1840 99.0
Mandevilla sp.
A2 S
nicotianae
13-1841 100.0
Philodendrun
A2 S
nicotianae
13-1842 99.0
Epipremnum sp.
A2 S
nicotianae
13-1843 99.0
Adenium sp.
A2 S
nicotianae
13-1844 100.0
Mandevilla sp.
A2 S
nicotianae
13-1845 100.0
Geranium sp.
A2 S
nicotianae
14-1030 99.0
Anthurium sp.
– –
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Jaimin S. Patel et al, Plant Disease
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a
Phytophthora species were identified based on BLAST match in the NCBI database and phytophthora databse.
b
Percent identity to ITS sequences in the Phytophthora database
c
A1 and A2 mating types were determined using know tester mating type A1 (P. nicotianae #13-724) and A2 (P.
nicotianae #13-723); each isolate was also tested with itself for homothallism; (–) Sterile were defined as those that
did not display any evidence of oospores, anthridia or oogonia; A1A2 represent homothallic isolates.
d
S and R are sensitive and resistant isolates, respectively, which were defined as those that displayed
≥
80% (S) or
≤
50% (R) growth inhibition on V8 agar plates containing 100 µg mL
-1
Mefenoxam.
nicotianae
14-1585B 99.0
Gardenia sp.
– – S
nicotianae
14-1585C 99.0
Gardenia sp.
– – S
nicotianae
14-214A 100.0
Phoenix sp.
A2 S
nicotianae
14-214B 100.0
Gardenia sp.
A2 S
nicotianae
14-215 100.0
Gardenia sp.
A2 S
nicotianae
14-271A 98.0
Podocarpus sp.
A2 S
nicotianae
14-707 99.0
Pothos sp.
A2 S
nicotianae
14-712 99.0
Spathiphyllum sp.
A1 S
nicotianae
14-754A 99.0
Spathiphyllum sp.
A1 S
nicotianae
14-754B 99.0
Spathiphyllum sp.
– – S
nicotianae
14-754C 99.0
Spathiphyllum sp.
A1 S
nicotianae
14-773A 99.0
Strelitzia reginae
A2 S
nicotianae
14-820 99.0
Petunia sp.
A2 S
tropicalis
96-83 100.0
Hedera sp.
A1 S
tropicalis
03-387 100.0
Hedera sp.
A2 S
tropicalis
03-452 100.0
Hedera sp.
A1 – S
tropicalis
08-228 99.8
Hedera sp.
A2 S
tropicalis
14-138 99.0
Pepper
– – R
tropicalis
14-822A 99.0
Rosmarinus officinalis
– – S
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Jaimin S. Patel et al, Plant Disease
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Table 2. Estimates of average evolutionary divergence over sequence pairs between groups
Pp II
Pp III
Pp IV
Pp V
P.
palmivora
P.
tropicalis
P.
katsurae
P.
cinnamomi
Pp I 0.0002 0.0009 0.0081 0.0016 0.0629 0.0503 0.0534 0.0611
Pp II 0.0009 0.0079 0.0015 0.0627 0.0503 0.0533 0.0611
Pp III 0.0088 0.0024 0.0638 0.0492 0.0522 0.0600
Pp IV 0.0094 0.0712 0.0591 0.0627 0.0703
Pp V 0.0636 0.0513 0.0550 0.0625
P.palmivora 0.0434 0.0484 0.0583
P.tropicalis 0.0429 0.0510
P.katsurae 0.0511
PpI, PpII, PpIII, PpIV and PpV are subgroups of P. nicotianae. The number of base substitutions per site from
averaging over all sequence pairs within each group are shown. Analysis involved 128 nucleotide sequences. All
positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6
Table 3. Estimates of average evolutionary
divergence over sequence pairs between groups
Group Within group
distance
Pp I 0.00017
Pp II 0.00010
Pp III 0.00009
Pp IV 0.00216
Pp V 0.01073
P.palmivora 0.02053
P.tropicalis 0.00433
P.katsurae 0.00197
PpI, PpII, PpIII, PpIV and PpV are subgroups of
P. nicotianae. The number of base substitutions
per site from averaging over all sequence pairs
within each group are shown. Analysis involved
128 nucleotide sequences.
Table 4. Results of mating type testing.
Mating
Type A1
a
Mating
Type A2
a
Sterile
b
A1A2
c
P. nicotianae 28 40 22 0
P. palmivora 1 5 17 0
P. tropicalis 2 2 2 0
P. katsurae 0 0 0 3
P. cinnamomi 0 1 0 0
Total 31
(25.2%)
48
(39.0%)
41
(33.3%)
3
(2.4%)
a
Mating types were determined using know tester mating type A1 and
A2; each isolate was also tested with itself for homothallism.
b
Sterile are defined as those that did not display any evidence of
oospores, anthridia or oogonia.
c
A1A2 represent homothallic isolates.
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Jaimin S. Patel et al, Plant Disease
31
Supplementary information.
Fig. S1. Sensitivity of Phytophthora isolates to different fungicides.
Fungicides mefenoxam (Subdue MAX), fluopicolide (Adorn), aluminum tris (Aliette),
azoxystrobin (Heritage), cyzofamid (Segway) and dimethomorph (Stature) were tested in vitro
on V8 media plates. Percent growth inhibition is expressed as the amount of growth inhibited on
fungicide-containing V8 juice media compared to no-fungicide control V8 media. Columns and
error bars represent means (n=4) and SE, respectively.
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Fig. 1. Molecular Phylogenetic analysis of Phytophthora isolates.
A, Upper panel shows schematics of
the ITS region of Phytophthora. Locations of ITS4 and ITS5 primers are indicated. Lower panel shows
Phylogenetic tree, which was constructed from the ITS sequences using the Maximum Likelihood
method. Analysis consisted of 128 sequences including 5 reported representative sequences of ITS
sequence of P. parasitica (GU983635), P. palmivora (KF263691), P. tropicalis (KC479199), P. katsurae
(JQ901391) and P. cinnamomi (JX996044).
136x136mm (300 x 300 DPI)
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Fig. 2. Sensitivity of Phytophthora isolates to mefenoxam.
A, Dose-response curves of P. parasitica isolates 05-297 (resistant) and 05-217 (sensitive) to mefenoxam
on V8 media. Each data point is a mean of 4 replications; Error bars are SE. B, Representative pictures of
isolates 05-297 and 05-217 on mefenoxam and no-mefenoxam control plates 5 days after the start of
experiment.
169x73mm (300 x 300 DPI)
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Fig. 3. Frequency distribution of resistant, moderately resistant and sensitive Phytophthora
isolates to different fungicides.
Resistant, ≤50% growth inhibition; Moderate, moderately resistant with 51-79% growth inhibition;
Sensitive, ≥80% growth inhibition.
98x83mm (300 x 300 DPI)
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Fig. S1. Sensitivity of Phytophthora isolates to different fungicides.
Fungicides mefenoxam (Subdue MAX), fluopicolide (Adorn), aluminum tris (Aliette), azoxystrobin (Heritage),
cyzofamid (Segway) and dimethomorph (Stature) were tested in vitro on V8 media plates. Percent growth
inhibition is expressed as the amount of growth inhibited on fungicide-containing V8 juice media compared
to no-fungicide control V8 media. Columns and error bars represent means (n=4) and SE, respectively.
160x250mm (300 x 300 DPI)
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