First report of Pythium schmitthenneri on olive trees and in Morocco

Abstract

In a grove in north-eastern Morocco in 2020, severe root rot and crown rot symptoms were noticed on olive trees (Olea europaea). Pythium schmitthenneri was identified as the pathogen based on conidia morphology and cytochrome oxidase subunit II (COXII) gene sequence analysis. Koch's postulates were verified by re-isolation of the fungus P. schmitthenneri from artificially inoculated olive seedlings after a pathogenicity test. This is the first time P. schmitthenneri has been identified as the cause of olive tree root and crown rot in Morocco.

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Australasian Plant Disease Notes (2022) 17:3
https://doi.org/10.1007/s13314-022-00449-z
First report ofPythium schmitthenneri onolive trees andinMorocco
IkramLegrifi1,2· JamilaAlFiguigui2· NabilRadouane1· SaidEzrari1· ZinebBelabess3· AbdessalemTahiri1·
SaidAmiri1· RachidLahlali1
Received: 22 October 2021 / Accepted: 26 January 2022
© The Author(s) under exclusive licence to Australasian Plant Pathology Society Inc. 2022
Abstract
In a grove in north-eastern Morocco in 2020, severe root rot and crown rot symptoms were noticed on olive trees (Olea
europaea). Pythium schmitthenneri was identified as the pathogen based on conidia morphology and cytochrome oxidase
subunit II (COXII) gene sequence analysis. Koch's postulates were verified by re-isolation of the fungus P. schmitthenneri
from artificially inoculated olive seedlings after a pathogenicity test. This is the first time P. schmitthenneri has been identi-
fied as the cause of olive tree root and crown rot in Morocco.
Keyword Olea europaea· Pythium schmitthenneri· Sequence analysis· Pathogenicity
The olive tree (Olea europaea) is one of the Mediterranean
Basin's oldest and most traditional crops, and it was one of
the first plants to adapt to the Mediterranean environment
(Fraga etal. 2021). Olive cultivation supports the economies
of Mediterranean countries (Caselli and Petacchi 2021),
including Morocco, which is among the top countries in
terms of olive output and area (Anonyme 2019; Harbouze
etal.2019, 2021).
Olive trees, like all crop plants, are susceptible to a
variety of pest and disease problems that can result in
major consequences and financial losses by causing tree
death (Chliyeh etal. 2017). Severe root rot and crown
rot symptoms (Fig.1) were noticed on olive trees inside
a grove in Ain Blouz, Fes Prefecture (North-eastern,
Morocco), in February 2020. The disease's causative
agent was effectively identified from affected roots using
cornmeal agar (CMA). Small sections of root tissues
were disinfested in 2% sodium hypochlorite for 2min,
then rinsed three times in distilled sterile water (DSW)
for 30s, plated on CMA, and incubated for four days
at 25°C. Single spore cultures produced from the iso-
lates were sub-cultured on potato dextrose agar (PDA) to
identify the pathogen. White colonies with non-septate
hyphae grew from these cultures (Fig.1). Sporangia were
globular or sub-globular with/without papilla and meas-
ured 15.13 × 33.44µm on V8 medium after 7days of
incubation (Fig.1). The shape of the hyphae and conidia,
as well as the size of the spores, were similar to Pythium
sp. (Ellis etal. 2012).
Two isolated fungus was identified at the species level
using sequencing analysis of the cytochrome oxidase subu-
nit II (COXII) gene. Total DNA was isolated from each
fungal isolate's mycelium using the Doyle and Doyle (1990)
protocol. Total DNA was used in the polymerase chain reac-
tion (PCR), which amplifies 563bp of the highly conserved
COXII gene using the primer pair FM66/FM58 (Martin
2000). Sanger sequencing was used to determine the
nucleotide sequences of two PCR amplicons. MZ466379
and MZ466380 are the accession numbers for these ampli-
cons in GenBank. Both isolates clustered with Pythium
schmitthenneri (GenBank Accession Number JM895530)
according to phylogenetic analysis, with a 100% bootstrap
value, thery were also clustred with Pythium hypogynum
and Pythium acrogynum, as these species are closely related
to eachother (Ellis etal. 2012; Senda etal. 2009) (Fig.2),
moreover, temperature growth experiment have also
* Rachid Lahlali
rlahlali@enameknes.ac.ma
1 Department ofPlant Protection, Phytopathology Unit,
Ecole Nationale d’Agriculture de Meknès, Km10, Rte Haj
Kaddour, BP S/40, 50001Meknès, Morocco
2 Laboratory ofFunctional Ecology andEnvironmental
Engineering, Sidi Mohamed Ben Abdellah University, PO
Box2202, Route d’Imouzzer, Fez, Morocco
3 Plant Protection Laboratory, Regional Center ofAgricultural
Research ofOujda, National Institute ofAgricultural
Research, Avenue Mohamed VI, BP428 Oujda, 60000Oujda,
Morocco

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confirmed the identity of our isolates as P. schmitthenneri,
which is the only one species able to grow at temperature
below 4°C as previously reported by Ellis etal. (2012).
Both PH1 and PH2 isolates have been deposited in the
PhytoENA-Meknès-Microorganisms Collection (CMENA)
under the accession number CMENA01 and CMENA02
respectively. As a result of morphological traits, tempera-
ture experimpent and sequence analysis of one gene, the
COXII, the pathogen was identified as P. schmitthenneri.
The above two isolates were utilized individually to arti-
ficially inoculate healthy one-year-old olive seedlings for
pathogenicity assays (Santilli etal. 2020). One-year-old
olive seedlings were transplanted into free-draining pots
with sterile soil and inoculum. The latter involved a 21-day
culture of the isolates in darkness at 25°C in pots contain-
ing a sterilized medium composed of 50g wheat seeds and
50mL V8 juice. Plants in the healthy controls were trans-
planted into pots with sterile soil but no inoculum. After
transplantation, all of the plants were kept in wet soil for
48h before getting moved to the greenhouse room and kept
at 25°C. This experiment was carried out twice using 5 olive
saplings per treatment.
All inoculated plants exhibited significant root rot, leaf
chlorosis, defoliation, wilting, and eventual mortality six
weeks following inoculation (Fig.3), whereas controls
remained symptomless. The pathogen was re-isolated and
morphologically characterized from infected roots. Koch's
postulates were met by the technique outlined above.
Pythium sp. is a fungus that affects a wide range of plants
all over the world. Several Pythium species have been
associated with olive tree root rot in previous research
(El-Morsi etal. 2009; Hernàndez etal. 1998; Hiar etal.
2017; Shaima and Elkanzi 2021). Chliyeh etal. (2014)
described the symptoms of root rot and wilt diseases on
olive plants and concluded that they are caused by Fusar-
ium species. This is the first report of P. schmitthenneri
as the cause of root rot and crown rot on olive trees in
Morocco that we are aware of its impact. To ensure proper
management of fungal diseases widespread in Moroccan
olive orchards, greater efforts are undoubtedly required
to study species diversity, community composition, and
disease incidence caused by P. schmitthenneri and other
similar species.
Fig. 1 Pythium schmittenneri
causing root rot on olive trees
(Olea europaea L.), A. Decline
symptom observed on an olive
tree in a grove in north-eastern
Morocco, B. Morphology
of 6-days-old colonies of P.
schmittenneri grown on potato
dextrose agar at 25 ± 1°C in
the dark, C, D. sporangia and
hyphal structure, Bars = 10µm

Australasian Plant Dis. Notes (2022) 17:3
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Fig. 2 Phylogenetic analysis
of 8 Pythium (two of this study
GenBank under the Acces-
sion Numbers MZ466379 and
MZ466380), 7 Phytopithium,
and 3 Phytophthora isolates
based on cytochrome oxidase
subunit II sequences using
MEGA X. The tree was con-
structed using the maximum
likelihood method. Numbers on
the branches represent bootstrap
values
AB690665 Phytopythium megacarpum
AB690677 Phytopythium boreale
AB920504 Pythium mercuriale
KJ595436 Phytopythium sindhum
AB690672 Pythium delawarense
AB690679 Phytopythium citrinum
AB690667 Phytopythium montanum
AB690678 Phytopythium carbonicum
AB690674 Pythium chamaihyphon
AF196623 Pythium sylvaticum
KJ595412 Pythium segnitium
MZ466379 Pythium schmitthenneri
MZ466380 Pythium schmitthenneri
JF895530 Pythium schmitthenneri
AB362325 Pythium hypogynum
AB362324 Pythium acrogynum
DQ469734 Phytophthora capsici
AY129207 Phytophthora lateralis
AY129220 Phytophthora palmivora
KY473921 Phytopythium vexans
100
98
54
24
80
78
100
98
100
88
74
44
44
88
0.0000.0500.1000.1500.2000.2500.3000.350

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Acknowledgements The authors wish to acknowledge the olive farm-
ers of Meknes-Fes for providing them with diseased plants.
Funding This study was supported by the Phytopathology Unit of
the Department of Plant Pathology- Ecole Nationale d’Agriculture de
Meknès.This Research was financially supported by MESRSI under
PRIMA Project Section2, SIRAM.
Data availability statement The data that support the findings of this
study are available from the corresponding author upon reasonable
request.
Declarations
Conflict of interest The authors of this work declare that no conflict
of interest.
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Pythium schmitthenneri Ph1/
Ph2 through the soil, 6weeks
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soil (Ph1 and Ph2), and an
uninoculated olive seedling (on
the right)

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