GENERAL TECHNICAL REPORT PSW-GTR-196
Inoculation Trials With Phytophthora ramorum
on Moorland Species1
Stefan Wagner2, Katrin Kaminski2, and Sabine Werres2
Abstract
Moorland plants are important for landscape and garden culture in many European countries. Studies on
the host range of Phytophthora ramorum, in other words first infection trials with the moorland genera
Calluna and Erica, should give a first impression on the susceptibility of these plants. Two cultivars
each of Calluna vulgaris and Erica gracilis were infected with four different P. ramorum isolates.
Wounded and non-wounded twigs were inoculated with zoospores or mycelium. First results indicate
that P. ramorum can infect C. vulgaris and E. gracilis. E. gracilis seemed to be more susceptible than
C. vulgaris. Both inoculation with zoospores and with mycelium resulted in infection. The experiments
with unwounded plant material indicate that P. ramorum can infect the healthy (twig) tissue. There was
a difference in the aggressiveness of the tested isolates; isolates from the United States exhibited the
highest aggressiveness. There were also several interactions between inoculation method, inoculum,
cultivar and isolate. These must be verified in further experiments.
Key words: Phytophthora ramorum, Calluna vulgaris, Erica gracilis, infection.
Introduction
In many European countries the Moorland genera Calluna and Erica, which are produced in specialized
nurseries, are important for landscape and garden culture. Until now, only a single plant of Calluna
vulgaris, infected with Phytophthora ramorum, has been detected in a Polish nursery. Thus, the aim of
our studies was to test whether P. ramorum can infect cultivars of C. vulgaris and E. gracilis and
therefore pose a potential hazard for these European plants. Furthermore, the best inoculum and
inoculation method should be evaluated for further tests. The present study is part of the research
project RAPRA (“Risk analysis for Phytophthora ramorum”, a recently recognized pathogen threat to
Europe and cause of the sudden oak death in the United States), which is funded by the European Union
and is important for the estimation of the potential host range of P. ramorum in Europe.
1
An abstract of a poster presented at the Sudden Oak Death Second Science Symposium: The State of Our
Knowledge, January 18 to 21, 2005, Monterey, California.
2
Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Horticulture,
Germany; S.Werres@bba.de
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Proceedings of the sudden oak death second science symposium: the state of our knowledge
Material and Methods
Detached twigs of C. vulgaris ('Amethyst', 'Long White') and E. gracilis ('Weißes Schloß', 'Glasers
Rote') were tested. Three European isolates (1376, from Viburnum tinus “Eve Price”, UK; 1577 from
Rhododendron catawbiense, Germany; 1578 isolated from Rhododendron grandiflora, U.K.) and one
American isolate (from Vaccinium ovatum) of P. ramorum were tested. Isolates were cultivated on
carrot piece agar (CPA five percent) at 20o C with 16 hour white light for 14 days. Induction of
zoospore release was achieved by flooding the colonies with five ml of sterile distilled water, then
incubated at 4 °C for one hour, followed by 60 minutes at room temperature. Twigs and sterile distilled
water were used for the negative control. Mycelium plugs, eight mm in diameter, were taken from the
edge of an actively growing colony. Sterile CPA was used for the negative control. Wounded and
unwounded twigs were dipped with the apical end into zoospore suspension for 5 seconds. Wounded
twigs were pressed into a mycelium plug and placed on a wet sterile swab. Twigs were incubated for 6
days in a wet chamber at 20° C with 16 hours light. Seven twigs were used for each trial.
Verification of the infection rate was calculated by the length of discoloration in mm followed by PCR
amplification
using
specific
ITS-Primer
for
P.
ramorum
(http://www.bba.de/inst
/g/pramorumneu/pramorum start.pdf). Kruskal-Wallis ANOVA was used on ranked means with P ≤
0,05.
Results
Both C. vulgaris and E. gracilis could be infected with P. ramorum. 96 percent of the Calluna twigs
and 99 percent of the Erica twigs showed discoloration. The length of discoloration was significantly
longer on the Erica twigs (fig.1). The susceptibility of the two C. vulgaris cultivars differed
significantly, in contrast to the two Erica cultivars (fig. 2). The U.S. isolate 1403 caused longer
discoloration than the three European isolates (fig. 3). Both zoospores and mycelium caused
discoloration on the twigs but the damage was significantly higher with zoospores. In comparison to
non-wounded plants, wounding did not increase the length of necrosis after inoculation with zoospores
(fig. 4).
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GENERAL TECHNICAL REPORT PSW-GTR-196
1 0 ,0
a
b
Length of dicoloration (mm)
Length of dicoloration (mm)
6 ,0
4 ,0
2 ,0
0 ,0
Erica gracilis
Calluna vulgaris
10,0
8 ,0
a
a
8,0
a
b
6,0
4,0
2,0
0,0
C a llu n a v u lg a r is
E r ic a g r a c ilis
n = 1 6 8 , 9 6 % tw ig s
w it h d is c o lo r a t io n
n = 1 6 8 , 9 6 % t w ig s
w it h d is c o lo r a tio n
Glasers Rote`
Long white´
`W eißes Schloß´
`Am ethyst´
n = 84, 96% twigs n = 84, 96% twigs n = 84, 99% twigs n = 84, 100% twigs
with discolouration with discolouration with discolouration with discolouration
Figure 1: Disease rate by C. vulgaris and
E. gracilis.
Figure 2: Susceptibility of different C. vulgaris and
E. gracilis cultivars.
10,0
b
8,0
a
6,0
a
a
8,0
a
Length of discoloration (mm)
Length of discoloration (mm)
10,0
a
4,0
2,0
0,0
6,0
b
4,0
2,0
0,0
Isolate 1376
Isolate 1577
Isolate 1578
Isolate 1403 US
n = 82, 99% twigs
with discoloration
n = 82, 98% twigs
with discoloration
n = 82, 96% twigs
with discoloration
n = 82, 99% twigs
with discoloration
Figure 3: Disease rate in dependence of the
P. ramorum isolate.
522
inoculated with zoospores
twigs wounded
n = 112, 100% twigs
with discoloration
inoculated with zoospores
twigs unwounded
n = 112, 97% twigs
with discoloration
inoculated with mycelium
plugs, twigs wounded
n = 112, 96% twigs
with discoloration
Figure 4: Influence of the inoculum and inoculation
method.