Australasian Plant Disease Notes (2021) 16: 1
https://doi.org/10.1007/s13314-020-00414-8
First report of Peronospora saturejae-hortensis in Iran
Hadi Khateri1
· Nahid Moarrefzadeh1 · Samad Jamali1
Received: 11 June 2020 / Accepted: 20 December 2020 / Published online: 6 February 2021
© Australasian Plant Pathology Society Inc. 2021
Abstract
In November 2018 severe downy mildew infection was observed on summer savory plants in Kermanshah Province, Iran.
Leaves on infected plants displayed chlorosis-browning on the upper surfaces and abundant dark gray-brown sporulation of
the asexual morph on the lower surfaces. Based on the morphological characteristics and molecular analyses using ITS-1
and ITS-4, the species was identified as Peronospora saturejae-hortensis. The nucleotide sequence of this pathogen had a
very high (99.9%) identity with a previously reported sequence of P. saturejae-hortensis from Germany. The possibility of
infection on other native Satureja species in Iran is discussed.
Keywords Aromatic plants · Lamiaceae · ITS · rDNA · Peronosporales
Summer savory (Satureja hortensis) is an important
aromatic plant in the Lamiaceae family. It is being used
as a fresh vegetable, food additive and in many medicinal
applications (Hassanzadeh et al. 2016; Mozaffarian 2013).
In November 2018 severe downy mildew infection was
observed on approximately 70% of summer savory plants
growing in experimental plots inside a research greenhouse
at the College of Agriculture, Razi University (Kermanshah,
Iran). This greenhouse had high humidity conditions and
the temperatures inside were between 18-25 °C. Leaves on
infected plants were chlorotic-brown on the upper surface
(Fig. 1a) and there was a dark gray-brown sporulation
consisting of conidiophores bearing conidia which covered
the entire lower surface (Fig. 1b). Later, most infected leaves
died. A voucher specimen of diseased material (designated
as Felfelak1) was deposited with the herbarium number
RUHK-2903 in Razi University Herbarium, (Kermanshah,
Iran).
The fungal mass (comprising the asexual morph) was
scraped from the lower leaf surfaces and mounted in water
or lactophenol-cotton blue on microscope slides. Slides
were examined and photographed using an Olympus CH40
microscope equipped with a JVC color video camera (model
TK-C1480E). Measurements of conidia and conidiophores
* Hadi Khateri
hkhateri@razi.ac.ir
1
Department of Plant Protection, College of Agriculture, Razi
University, Kermanshah, Iran
(30 for each) were done by BioloMICS software (Version
1.0.2, BioAware) and are reported as follows, (minimum-)
y-z(-maximum), where y = mean-standard deviation and
z = mean + standard deviation. All the values were rounded
to the nearest whole number. The conidiophores were
(190-)345-585(-610) µm in length and had four to five
branches (Fig. 1c). The lengths of the longer and the shorter
ultimate branchlets (Fig. 1d) were (7-)9-18(-22) µm and
(4-)4-7(-8) µm, respectively. Obovoid, brown-olive conidia
(17-)17-23(-25) µm × (12-)13-18(-21) µm, length/width
ratio 1.1–1.7, developed singly at the tips of the ultimate
branchlets (Fig. 1e). Conidia germinated by producing a
single unbranched germ tube. No oospore was found in the
examined samples.
The species had morphological characteristics very
similar to those of Peronospora saturejae-hortensis (Gabler
et al. 2012). The dimensions of conidia were similar to
those reported by Gabler et al. (2012), i.e. (15-)16-25
(-26) × (9-)10-18(-19), but were larger than the dimensions
published in the original description (Novotelnova and
Pystina 1985), i.e. 14.5-18 × 12-15 µm. The reason for this
difference with the latter could be due to the influence of the
environment, differences in host matrix (Runge et al. 2012)
and that the species author had not examined fresh, fully
developed material (Gabler et al. 2012).
Pathogenicity testing was conducted as described by
Gabler et al. (2012). The inoculum was prepared from the
same infected samples that were used for morphological
and molecular studies by stirring 150 g infected leaves in
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Australasian Plant Dis. Notes (2021) 16: 1
Fig. 1 Symptoms, signs and
microscopic images of downy
mildew caused by Peronospora saturejae-hortensis on
summer savory; a browning of
upper leaf surfaces on infected
plants, b sporulation of the
asexual morph on the lower
leaf surfaces, c conidiophore
(bar = 100 µm), d ultimate
branchlets (bar = 20 µm), e
conidia (bar = 10 µm)
400 ml water with the addition of 30 μl Tween 20. The
suspension was adjusted to approximately 1 × 106 conidia/
ml. Inoculation was done by submerging the eight weeks old
summer savory plants in the aqueous suspension of conidia
for about 30 s. The inoculated plants were then covered for
the entire period of the experiment in a moistened plastic
bag starting immediately after inoculation and incubated in
a greenhouse at 18–24 °C. The first symptoms appeared after
12 days as small yellow spots on the upper leaf surfaces and
after 20 days sporulation consisting of conidiophores and
conidia identical to those of P. saturejae-hortensis occurred
on the abaxial surfaces of the spots. Microscopic examination
of the conidiophores and conidia on the inoculated plants
confirmed that it was consistent with P. saturejae-hortensis.
A spore suspension was collected by spraying the lower
leaf surfaces of a downy mildew infected summer savory
sample by a hand-held water sprayer and after centrifugation
was stored at -20 °C for DNA extaction. DNA was extracted
using a commercial kit (Zagros Bioidea Co., Razi University
Incubator, Kermanshah, Iran). PCR was done by using primers
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ITS1 and ITS4 (White et al. 1990). The PCR product was
sequenced by a commercial service (Pishgam Biotech Co.,
Tehran, Iran) and the sequence was deposited in GenBank
under accession number MT510002. BLAST analysis
revealed a very high (99.9%) nucleotide identity (the only
difference was an ambiguous nucleotide) with the ITS and
ribosomal RNA sequence of P. saturejae-hortensis previously
reported from Germany under the GenBank accession number
JN882274 (Gabler et al. 2012). The phylogenetic tree (not
shown) that was reconstructed by the same method and the
same set of sequences used by Gabler et al. (2012), was
similar to that report and the Iranian isolate was in the same
clade as the the German isolate that was characterised by
Gabler et al. (2012). Except for these two accession numbers
(JN882274 and MT510002), there is currently no other
available sequences from the ITS and ribosomal RNA genes
of this species. The almost identical nucleotide sequences
from these two accession numbers reveal a very high genetic
conservation in this region and a potentially identical source
for both occurrences. Based on both morphological and
Australasian Plant Dis. Notes (2021) 16: 1
molecular characterisation, the Iranian isolate RUHK-2903
can be considered to be P. saturejae-hortensis.
Although many Peronospora species show a high degree
of host specialization (Spring et al. 2019; Thines and Choi
2016), there are also some species including Peronospora
belbahrii (Ben-Naim et al. 2019; Cohen et al. 2017) and
Peronospora tabacina (Hall 1989; Kröber and Massfeller
1961) that infect multiple plant species and genera. More
than 16 species of Satureja such as Satureja kermanshahensis
and Satureja khuzistanica (Jamzad 2010) grow naturally as
annual or perennial plants in Iran (Mozaffarian 2013) and
there is a possibility that P. saturejae-hortensis also causes
a similar disease on one or more of these species which may
act as inoculum sources and overwintering hosts. Another
possible source of inoculum for the infection of summer
savory plants that needs to be studied in future is infected
seed which has been documented in the case of P. belbahrii,
the causal agent of basil downy mildew (Garibaldi et al.
2004; Wyenandt et al. 2015).
P. saturejae-hortensis has been reported as the causal
agent of downy mildew on summer savory from Germany
and Armenia (Gabler et al. 2012; Novotelnova and Pystina
1985), whilst the causal agent of downy mildew on summer
savory in Bulgaria and Lithuania has been recorded as P.
calaminthae (Gabler et al. 2012). The only published report
of Peronospora on summer savory in Iran was that of
Peronospora lamii from Khuzestan Province in the southwest of the country (Ershad 1977). Except for this report and
others on the occurrence of downy mildew on basil in the
south-west (Ebrahimi and Minassian 1973), north (Khateri
et al. 2007) and west (Khateri et al. 2019) of Iran, there was
no information about the downy mildews on plants in the
Lamiaceae in other parts of the country. Previous studies
have identified many Peronospora collections from different
Lamiaceae plants as P. lamii which is due to the application
of a morphologically-based broad species concept (Gabler
et al. 2012). This assumption may have been the reason for
neglecting the possible preventive measures such as seed
tests (Thines and Choi 2016). The molecular analysis by
nucleotide sequences helps to override such problems and
differentiate the nearly related but different species such as
P. lamii, P. belbahrii and P. saturejae-hortensis (Gabler et al.
2012).
References
Ben-Naim Y, Falach-Block L, Ben-Daniel B-H, Cohen Y (2019) Host
range of Peronospora belbahrii, causal agent of basil downy
mildew, in Israel. Eur J Plant Pathol 155:789–799. https://doi.
org/10.1007/s10658-019-01809-9
Page 3 of 3 1
Cohen Y, Ben-Naim Y, Falach L, Rubin AE (2017) Epidemiology of
basil downy mildew. Phytopathology 107:1149–1160. https://doi.
org/10.1094/PHYTO-01-17-0017-FI
Ebrahimi A, Minassian V (1973) An index of cultivated and wild
plant diseases in Khuzestan. Jundi-shapour University, College
of Agriculture, Ahvaz, Iran
Ershad D (1977) Fungi of Iran. Plant Pests and Diseases Research
Institute (Iran), Department of Botany, Publication No. 10,
Tehran, Iran
Gabler J, Hagedorn G, Braun U (2012) Taxonomy and phylogenetic
placement of the downy mildew Peronospora saturejae-hortensis.
Mycotaxon 121:455–463. https://doi.org/10.5248/121.455
Garibaldi A, Minuto A, Minuto G, Bertetti D, Gullino M (2004) The
downy mildew of sweet basil: seed transmission and disease
control. J Plant Pathol 86:320–321
Hall G (1989) Peronospora hyoscyami f. sp. tabacina, CMI
Descriptions of Pathogenic Fungi and Bacteria No. 975.
Mycopathologia 106:191–193
Hassanzadeh MK, Tayarani Najaran Z, Nasery M, Emami SA (2016)
Summer Savory (Satureja hortensis L.) Oils. In: Preedy VR (ed)
Essential Oils in Food Preservation, Flavor and Safety. Academic
Press, San Diego, pp 757–764. https://doi.org/10.1016/B978-012-416641-7.00086-9
Jamzad Z (2010) A new species of Satureja (Lamiaceae) from Iran.
Iran J Bot 16:213–217
Khateri H, Calmin G, Moarrefzadeh N, Belbahri L, Lefort F (2007)
First report of downy mildew caused by Peronospora sp. on basil
(Ocimum basilicum) in northern Iran. J Plant Pathol 89:S70
Khateri H, Moarrefzadeh N, Jamali S (2019) The first report of
Peronospora belbahrii the causal agent of downy mildew on
sweet basil in the west of Iran. Paper presented at the First Iranian
Phytopathology Congress, Karaj, Iran, 31 Aug-1 Sep 2019
Kröber H, Massfeller D (1961) Untersuchungen über die
blauschimmelkrankheit des tabaks in Deutschland, IV. Das
wirtsspektrum von Peronospora tabacina Adam. Nachrichtenblatt
des Deutschen Pflanzenschutzdienstes 13:81–85
Mozaffarian V (2013) Identification of Medicinal and Aromatic Plants
of Iran. Farhange-Moaser Publishing, Tehran, Iran
Novotelnova N, Pystina K (1985) Flora Plantarum Cryptogamarum
URSS, Vol XI Fungi (3) Ordo Peronosporales (Fam. Pythiaceae,
Phytophthoraceae, Peronosporaceae, Cystopaceae). Nauka Press,
Leningrad (In Russian)
Runge F, Ndambi B, Thines M (2012) Which morphological
characteristics are most influenced by the host matrix in downy
mildews? A case study in Pseudoperonospora cubensis. PLoS
ONE 7:e44863. https://doi.org/10.1371/journal.pone.0044863
Spring O, Gomez-Zeledon J, Hadziabdic D, Trigiano RN, Thines M,
Lebeda A (2019) Biological characteristics and assessment of
virulence diversity in pathosystems of economically important
biotrophic oomycetes. Crit Rev Plant Sci 37:439–495. https://doi.
org/10.1080/07352689.2018.1530848
Thines M, Choi YJ (2016) Evolution, diversity, and taxonomy of
the Peronosporaceae, with focus on the genus Peronospora.
Phytopathology 106:6–18. https ://doi.org/10.1094/PHYTO 05-15-0127-RVW
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.
In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR
Protocols: A Guide to Methods and Applications. Academic Press,
San Diego, CA, pp 315–322
Wyenandt CA et al (2015) Basil downy mildew (Peronospora
belbahrii): discoveries and challenges relative to its control.
Phytopathology 105:885–894. https://doi.org/10.1094/PHYTO02-15-0032-FI
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