Phomopsis juniperivora identified in Greece causing dieback on Juniperus macrocarpa
Panaghiotis Tsopelas1, Stavros Palavouzis2, Aliki K. Tzima2, Nikoleta Soulioti1 and Epaminondas J. Paplomatas2
1Hellenic Agricultural Organization “Demeter”-Institute of Mediterranean Forest Ecosystems, Forest Pathology Laboratory, Terma Alkmanos, 115 28 Athens, Greece.
2Agricultural University of Athens, Laboratory of Plant Pathology, 75 Iera Odos, 118 55 Athens, Greece.
Juniperus macrocarpa Sibth. & Sm. is a large spreading shrub 2-5 m high, often taking the form
of a tree up to 10-15 m tall. It is a native species in Greece, forming natural woodlands in the
coastal dunes on many of the Aegean islands. These coastal woodlands are very important from
an ecological point of view, since they stabilize sand and provide habitat for rare animals and
On the island of Paros, Cyclades group of central Aegean Sea, symptoms of branch dieback
were observed during the summer of 2014, on several naturally growing shrubs of J. macrocarpa
in different localities [Fig. 1, 2]. According to local people, these symptoms on the junipers were
present for many years.
The objective of this study was to determine the cause of the disease and characterize the
pathogen using morphological and molecular methods.
In 2014 and 2017, plants of J. macrocarpa with intense symptoms of branch necrosis were observed in different
sites of the Santa Maria locality of Paros Island. Some of the plants that had symptoms of dieback in 2014 were
completely dead in 2017 [Fig. 2]. However, the disease was not widespread to all individual plants in each site;
certain neighboring plants to heavily diseased ones were showing slight symptoms of dieback or were without
symptoms. Also, no symptoms have been seen on Juniperus phoenicea L. growing in the same sites.
A fungus of the genus Phomopsis was consistently isolated from infected branches collected from different
areas of the island. Colonies on PDA were white to creamy in colour [Fig. 7], developing pycnidia with exuding
creamy spore droplets after 2-3 weeks [Fig. 8]. Characteristic alpha (α) and beta (β) spores [Fig. 9] were
observed from conidia formed in culture. Conidia of similar shape and size were also observed from pycnidia on
juniper seed cones, however, β conidia from pycnidia on seed-cones were very rare. Alpha conidia were
hyaline, fusoid, with two oil globules, 8-11 x 2.5-3 μm. Beta conidia were hyaline, filiform curved, 33-46 × 1–1.5
μm. Phylogenetic analysis of the nucleotide sequence of the ITS region of 3 isolates, compared with sequences
available in the GenBank database was performed by applying Neighbor-Join and BioNJ algorithms to a matrix
of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach . All isolates from
J. macrocarpa clustered together with Phomopsis juniperivora G. Hahn (≡P. juniperovora) (about 1% difference)
[Fig. 6]. Spore morphology and size were also very similar with this fungal pathogen (Hahn 1943).
All three plant species (J. macrocarpa, J. oxycedrus and C. sempervirens) were infected by the Phomopsis
isolate, forming elongated cankers on the stems of the inoculated plants, while no symptoms developed on
control plants where sterile agar plugs were used instead of inoculum. In the majority of the inoculated J.
macrocarpa plants the distal part of the stem above the developed canker was dead six months after
inoculation, because the canker had girdled the thin stems (diameter 5-7 mm). When infected parts of stems
were incubated in a moist chamber, pycnidia were developed on the cankered area with spores extruding in the
form of tendrils [Fig. 4]. The same fungus was consistently isolated from the cankers of all three inoculated
MATERIALS & METHODS
Sampling. Plants of Juniperus macrocarpa growing in natural woodlands in the Santa Maria
locality on Paros Island were examined for the presence of disease symptoms. Samples of
branches with symptoms were collected from different sites in September and October 2014.
Observations and sample collection were also performed in April 2017. From trees with
symptoms, they were also collected ripe seed-cones (strobili) with pycnidia on the outer surface
Isolation. Seed-cones were incubated in moist chambers at room temperatures (20-23 oC) for a
week and examined under the dissecting microscope for the presence of pycnidia. Spore masses
were mounted on microscope slides and microscopic observations were performed under the oil
immersion lens. Branch samples were surface-disinfected with 70% ethanol, the outer bark was
removed with a sterile scalpel and small pieces of affected wood and bark tissues from the
margins of cankers were aseptically transferred onto Petri dishes with potato dextrose agar
(PDA) and incubated at 25 oC. Spores from pycnidia formed on fungal cultures were also
examined under the microscope.
Molecular characterization and identification. DNA was extracted from freeze dried
mycelium of 3 isolates (‘Jm1,’ ‘Jm2’ and ‘Jm3’) applying the LETS buffer protocol . The ITS
regions of ribosomal DNA was amplified by PCR using ITS4 and ITS5 primers and sequenced.
To perform phylogenetic analysis, ITS sequences from isolates ‘Jm1’, ‘Jm2’, ‘Jm3’ and other
Phomopsis species downloaded from NCBI GenBank database were aligned using Clustal X.
Phylogenetic analysis was performed using MEGA 7 . The Maximum Composite Likelihood
approach was used and the best substitution model was chosen. Finally, the phylogenetic tree
was calculated using K2+G+I model with 500 bootstraps and was rooted to a Valsa ceratosperma
Pathogenicity tests. Inoculation tests were performed on 3-4 year-old potted plants of
Juniperus macrocarpa, J. oxycedrus L. and Cupressus sempervirens L. (Mediterranean cypress)
in October 2014. The trees were wound-inoculated on the main stem with mycelial plugs from
two-week old cultures, using one isolate of the fungus. The inoculated plants were examined
periodically for the development of symptoms and were harvested six months after inoculation
DISCUSSION & CONCLUSIONS
Phomopsis juniperivora was first described in 1920 as P. juniperovora in USA, but it was known to cause twig
blight of junipers since the late 19th century. It is widespread in the United States and Canada and there are
also some reports from UK . The pathogen affects different species of junipers and has also been found to
cause disease on other genera of Cupressaceae (Chamaecyparis, Cupressus and Thuja) , . However,
other Phomopsis spp. have also been reported to infect junipers and other plant species of Cupressaceae .
To the best of our knowledge, only one ITS sequence of P. juniperivora with Accession Number AF462436.1 is
available in the GenBank database. More isolates of P. juniperivora, especially from North America should be
analyzed and compared with isolates from Greece using multi–gene sequence analyses to verify the identity of
the fungus causing disease in junipers on the Paros Island.
Peterson and Hodges  reported P. juniperivora to affect mainly nursery plants, causing significant damage by
killing the shoots and small branches of junipers and in some cases leading to total loss of first year seedlings if
control measures are not applied. The disease in the United States is not very severe to large plants in
landscape plantings and to junipers in natural woodlands. However, in Greece this pathogen was found to kill
even large plants of J. macrocarpa.
No previous reports on juniper dieback from Phomopsis spp. exist in Greece. All available evidence suggests
that P. juniperivora is an invasive pathogen in Greece and the native J. macrocarpa is probably a very
susceptible host. Further research is needed to investigate the epidemiology of this fungus in natural
woodlands of J. macrocarpa and the susceptibility of other native junipers in Greece. The disease has been
detected only in the island of Paros but it is possible to be in other areas of Greece and not reported yet.
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 Peterson G. W. and Hodges C. S. Jr., 1975. Phomopsis blight of junipers. U. S. For. Serv. Pest Leaf. No 154.
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Figure 1. Heavily infected Juniperus
macrocarpa plants by Phomopsis juniperivora.
Figure 5. Infected branches of Juniperus
macrocarpa with characteristic canker
symptoms and wood discoloration.
Figure 2. One infected plant (left) and two
dead plants of Juniperus macrocarpa (right).
Figure 7. Phomopsis
juniperivora in culture with
Figure 8. Creamy spore
masses exuding from
Figure 9. Alpha (α) and
beta (β) spores of P.
Figure 4. Tendrils of conidia exuding from
pycnidia of inoculated plants (left main stem,
Figure 3. Pycnidia of P. juniperivora on seed-
cones of J. macrocarpa; tendrils of conidia are
jm1, jm3 and jm5 differ approx. 1%
from P. juniperivora
Figure 6. Maximum composite likelihood
tree obtained from ITS1, 5.8S and ITS2
rDNA sequence data originated from fungal
isolates of related genera. Nucleotide
sequences acquired in this study are
depicted by a blue box, while all sequences
retrieved from GenBank are shown by
names of fungal species and isolate code,
where applicable. The GenBank accession
number is indicated with a vertical bar.
Bootstrap values (500 replicates) are
indicated below the nodes, when larger than
50%. The tree is rooted to the outgroup