Specific Detection of Phytophthora Crown Rot and Anthracnose in Strawberry
Frank J. Louws
Dept. Plant Pathology
North Carolina State University
Raleigh NC 27695
BRIEF INTRODUCTION TO THE PROBLEM:
Strawberry growers face numerous production challenges. High capital costs to establish a
strawberry crop make growers keenly aware of the need to adequately manage weeds, diseases,
nematodes, insects and mites that prove to be a constant threat. Disease problems are a primary
focus of pesticide input and crop losses in strawberry production systems. In particular,
anthracnose caused by Colletotrichum species, and Phytophthora crown rot caused primarily by
P. cactorum (also causal agent of leather rot in northern production regions), are serious
concerns in the industry. In our region, we have experienced severe losses in the last three years
in particular, due to either or both of these pathogens and they are typically introduced on
contaminated transplants. There is a real need to develop rapid diagnostic assays to allow rapid
disease identification and to develop plant quality assays to ensure reduced risk of planting
contaminated plants. We have initiated development of standard and real-time PCR assays and
sampling protocols to aid in our goal to ensure the use of disease free plants.
In 2005 we articulated the following objectives: 1) to evaluate the potential of specific detection
and diagnosis of Colletotrichum acutatum, C. gloeosporioides and Phytophthora cactorum to
monitor production of “disease-free” transplants; 2) to develop a real-time PCR assay to rapidly
identify the presence of Colletotrichum (with emphasis on the acutatum strain) or Phytophthora
in diseased plant tissue and 3) to initiate work to develop sampling protocols to detect latent
infections of these pathogens in nursery production systems.
During 2005, we did not focus on our Phytophthora work. The Phytophthora work presented
here was completed during phase 1 of this project. We have done extensive work with
Colletotrichum assays in 2005.
DESCRIPTION OF THE EXPERIMENTAL DESIGN:
Phytophthora Work (2004):
Identifying Phytophthora cactorum morphologically requires familiarity with the shapes and
sizes of oomycete sexual structures. It can take several days and may require multiple steps to
encourage cactorums to reproduce sexually. Even under good conditions some specimens of
cactorum may not form these structures and could require molecular identification. To make
PCR based identification faster and cheaper, we have combined four primers (multiplex-PCR);
CACTF1, CACTR1, DC6 and ITS2 (Fig. 1). Together, they form four bands for “cactorums”,
one band for other oomycetes and no band for all other organisms. Eddie Croom (undergraduate
research assistant); L.M. Ferguson; L. Leandro and F.J. Louws. We have used this multiplex
assay extensively to identify and characterize problems associated with strawberry root and
crown rot problems. An hourly student can competently assay 40 samples in an afternoon.
ladder 1 2 3 4 5 6 7 8 9 10 ladder Lane #
Figure 1:The ladders are the 100bp size standards on either end. For lanes 1 and 8 the samples
do not contain any oomycete DNA. They have no bands. In lane 3, the sample is cactorum and
has four bands. Three of these bands are unique for cactorum. All other lanes have the one band
that is formed from oomycete DNA.
In phase 1 of the project (2004) we compared DNA extraction kits to determine the best kit to
obtain fungal DNA that could be assayed by PCR. Of all the kits, only the MoBio PowerSoil kit
yielded consistent bands. We successfully adopted or adapted primer sets from the literature and
could reliably amplify C. acutatum, C. gloeosporioides and C. fragariae (the later two could not
be differentiated by PCR).
IN 2005, a real-time PCR protocol was initiated using the TaqMan technology. Specific primers
were developed for Colletotrichum (based on “acutatum” sequences although we anticipate, but
have not proven, that the primers will amplify DNA from all three species) as well as a specific
probe. The sensitivity of the PCR assay was determined using purified DNA as well as DNA
spiked into strawberry petiole extract (to determine if strawberry samples will inhibit or affect
the results). Data on these experiments is being compiled.
The assay was then evaluated on inoculated plants in controlled experiments in growth chambers
as well as on field samples during a Colletotrichum epidemic in several production nurseries.
Strawberry plants were obtained from the NCSU Micropropagation Unit and grown in the NCSU
Phytotron growth chambers. Two sets of 48 plants were sampled before inoculation and 5, 12,
and 19 days after inoculation. The infected petioles had an average detection rate of 4.3% by the
Paraquat assay (standard method of detecting latent infections of the pathogen) and 79.9% by the
realtime PCR assay. The uninfected petioles, however, showed a 0% detection rate by the
Paraquat and 4% by the realtime assay in the first run of the experiment. A second run is
currently under analysis. The realtime assay was also tested within a field setting where there
was a natural epidemic of C. gloeosporioides; 60 petioles were collected from each of three
cultivars in the field and tested by the Paraquat assay. Thirty petioles of each were also tested by
the realtime assay. The Paraquat and realtime assays yielded 6.6% and 0% for ‘Bish,’ 10% and
13% for ‘Sweet Charlie,’ and 100% and 70% for ‘Chandler,’ respectively. In the ‘Bish’ assay,
25/30 petioles showed late amplification, indicating that there were fewer than 10 DNA copies
per reaction. The lower detection rate seen in the ‘Chandler’ petioles may be due to high levels
of wound phenolics, which inhibit PCR, suggesting additional work is needed to manage
inhibitors in the assays. Detection of the pathogen in planting stock by realtime-PCR has the
potential to become an important technique for management of strawberry anthracnose.
SUMMARY OF RESULTS AND CONCLUSIONS:
Primers have been adapted or developed for the specific detection of Phytophthora cactorum and
Colletotrichum species. We have used these protocols for rapid diagnosis and identification of
these serious pathogens in commercial samples and controlled experiments. A real-time PCR
protocol has also been developed and further work is underway. We plan to adapt these protocols
for rapid detection and identification of plant material. We have met some challenges in
extracting pathogen DNA from some of the tissues in attempts to develop protocols that would
be useful in “clean plant” programs. Work will continue. Good progress has been achieved. We
have utilized $5000.00 of NASGA research funds to accomplish an estimated $25000 to $30000
worth of research (in direct costs) in 2005.
Evaluation of strobilurin fungicides (Abound and Cabrio),
Potassium phosphite and Ridomil Gold for control of strawberry leather rot and Vascular
collapse, caused by Phytophthora cactorum.
Michael A. Ellis and Angel Rebollar-Alviter
Department of Plant Pathology, The Ohio State University/OARDC
1680 Madison Avenue, Wooster, OH 44691
Phone: 330-263-3849, Fax: 330-263-3841, e-mail: firstname.lastname@example.org
Studies were conducted to determine the post-infection or curative activity of Abound, Cabrio,
Potassium Phosphite and Ridomil Gold EC for control leather rot. Commercial formulations of
mefenoxam as Ridomil Gold 2E, potassium phosphite as Agri-Fos, azoxystrobin as Abound, and
pyraclostrobin as Cabrio were used in greenhouse experiments. The concentration of each
fungicide used in the experiments was the label recommended rate in 50 gallons of water/A. To
evaluate post-infection (curative) activity of the fungicides, inoculated plants with fruits were
incubated in the dark in a mist chamber for 12 hours at room temperature to insure infection.
After this time, plants with fruits were sprayed to runoff with each fungicide at 12, 24, 36 and 48
hours after inoculation. Pre-infection (protectant) activity was evaluated by applying the
fungicides at 2, 4 and 7 days before inoculation. Diseased and healthy fruits were recorded 5
days after inoculation.
Results and Conclusions:
1.) All fungicides provided excellent protectant activity for at least 7 days with, no
significant differences between fungicides.
2.) Significant differences in leather rot control were observed when the fungicides were
applied after inoculation.
3.) The systemic fungicides mefenoxam and potassium phosphite provided excellent post-
infection activity for up to 36 h. Mefenoxam provided a moderate level of disease control
at 48 hours after inoculation.
4.) Strobilurin fungicides did not provide satisfactory curative activity. Although a
certain level of control was observed at 12 hours after inoculation, no control was observed
when these fungicides were applied after this time.
Based on the results from greenhouse studies, a field trial was conducted to evaluate the
post-infection activity of the previously mentioned fungicides under commercial production
conditions. Fungicides were applied on a calendar based program on a 7-day schedule or as
curative treatments following flooding events. Fungicides were applied on the calendar based
program on the following dates: May 19 ( late bloom , green fruit present ) ; May 26 ( fruit set,
no bloom and green fruit present ); June 6 ( fruit starting to ripen ) and June 10 ( mature fruit
present, first harvest ). AGRI FOS was applied alone on all dates of the 7-day program in
treatment one. In treatment three, Abound was applied on the first two dates and AGRI FOS was
applied on the second two dates in an alternating program.
Ridomil Gold and AGRI FOS were applied alone as curative treatments after flooding
events. Ridomil Gold was applied on May 22 and June 12 in response to flooding events
(infection periods) on May 20 and June 10. AGRI FOS was applied on May 22, June 5, and
June12 in response to flooding events on May 20, June 3, and June 10. All ripe and diseased fruit
were harvested from each plot on 10, 14 and 17 June. The total number of leather rot-infected
and marketable fruit, and total yield (weight) were recorded for each replication and harvest date.
Results of 2005 field trial:
Removing straw from between the rows and repeatedly flooding test plots resulted in a
mean disease incidence of 66% for all three harvest dates (table1). All fungicide treatments had
significantly less leather rot than the untreated control, and there were no significant differences
in leather rot incidence between any fungicide treatment. Ridomil Gold and AGRI FOS appeared
to provide good control of leather rot when applied as curative treatments. Ridomil and AGRI
FOS in curative treatments provided leather rot control equal to that of the calendar based
treatments with two and one fewer applications, respectively.
Anthracnose fruit rot developed in the trial, and some interesting results were observed. The
untreated control had lower levels of anthracnose than Ridomil Gold. We believe this is due to
the fact that the control had very high levels of leather rot infected fruit, and fruits infected with
leather rot could not be infected by anthracnose. Ridomil Gold had a significantly higher
incidence of anthracnose than most other treatments. We believe this occurred because Ridomil
Gold provided excellent control of leather rot, but has no efficacy against anthracnose. This
suggests that Ridomil may need to be applied in combination with other fungicides that have
activity against anthracnose during wet years when both diseases are present. The alternating
program of Abound and AGRI FOS provided excellent control of both leather rot and
anthracnose. Our data also suggest that AGRI FOS ( potassium phosphite ) may have some
activity against anthracnose fruit rot.
Table 1. Efficacy of fungicides applied in a calendar-based protectant program or a post-infection curative
program for control of leather rot of strawberry.
Leather Rot Anthracnose Botrytis Total Number Marketable
Treatment and (Timing) %* % % of fruit %
1 AGRI FOS (calendar) 4.3 a ** 9.3 ab 3.0 a 702 a 79 ab
2 Abound 2 sprays 1.7 a 2.0 a 1.4 bc 614 a 92 a
AGRI FOS 2 sprays (calendar)
3 Ridomil Gold (curative) 2.0 a 21.0 b 2.8 ab 831 a 71 b
4 AGRI FOS (curative) 1.6 a 7.7 a 2.3 ab 620 a 83 ab
5 Nontreated Control 66.0 b 1.3 a 0.5 c 826 a 33 c
* Mean percentage of fruit infected for three harvest dates with leather rot, (caused by Phytophthora cactorum),
anthracnose (caused by Colletotrichum acutatum) and botrytis gray mold (caused by Botrytis cinerea).
** Numbers followed by the same letters within columns are not significantly different (P=0.05).
Results and Conclusions:
1.) Mefenoxam (Ridomil Gold) and AGRI FOS ( potassium phosphite) both provided good
to excellent control of leather rot when applied in a post-infection ,curative program.
2.) Both of these fungicides have potential for use in a disease forecasting system for
3.) Azoxystrobin (Abound) in an alternating 7-day calendar based program with potassium
phosphite (AGRI FOS) provided excellent control of both leather rot and anthracnose
fruit rot. Potassium phosphite applied on a 7-day protectant program provide good
control of leather rot and a fair level of control of anthracnose fruit rot.
Crown rot or Vascular Collapse
We have developed a technique for inoculating strawberry crowns and roots with Phytophthora
catorum that results in the reliable development of crown rot symptoms. We are currently
conducting studies to determine the efficacy of these fungicides for control of Phytophthora