Prima segnalazione di <em>Neofusicoccum parvum</em> e <em>Diplodia olivarum</em> quali agenti di cancri e disseccamenti su oleastro in Italia
10.6092/issn.2531-7342/11048
L’oleastro (Olea europaea subsp. europaea var. sylvestris) è una specie legnosa sempreverde ampiamente diffusa nel bacino del Mediterraneo. Questa specie è ritenuta rustica e resistente alle avversità ambientali e parassitarie, ma a partire dal 2017, una grave malattia ad eziologia sconosciuta è stata osservata in varie formazioni della Sardegna nord-orientale. Le piante colpite mostravano sintomi di clorosi fogliare, un progressivo disseccamento della chioma e vistosi cancri su rami, branche e fusto. I cancri spesso trasudavano un fluido di colore bianco-arancione, che asciugandosi formava una massa amorfa e collosa sulla corteccia. Le indagini diagnostiche condotte su campioni legnosi prelevati in corrispondenza dei cancri hanno consentito di isolare 32 colonie fungine appartenenti alle specie: Diplodia olivarum, Dothiorella sarmentorum e Neofusicoccum parvum (Botryosphaeriaceae), Pestalotiopsis sp. (Pestalotiopsidaceae) e Rosellinia corticium (Xylariaceae). Diplodia olivarum è stata isolata con maggiore frequenza, mentre N. parvum nelle prove di patogenicità ha causato le lesioni più estese e riprodotto tutti i sintomi osservati in natura. Le ricerche effettuate hanno permesso di individuare l’esistenza di nuove associazioni ospite-patogeno e di accertare che nell’eziologia della malattia sono coinvolte più specie afferenti alla famiglia delle Botryosphaeriaceae. 
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Benedetto Linaldeddu
Italian Journal of Mycology
Rosellinia
First report of canker and dieback caused by <em>Neofusicoccum parvum</em> and <em>Diplodia olivarum</em> on oleaster in Italy
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2020-05-27
2020-02-10
2020-09-28
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Ital J Mycol
Benedetto Linaldeddu
2020/06/30
10.6092/issn.2531-7342/11048
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White TJ, Bruns T, Lee J, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds). Academic Press, San Diego, California, pp 315-322.
First report of canker and dieback caused by <em>Neofusicoccum parvum</em> and <em>Diplodia olivarum</em> on oleaster in Italy
https://italianmycology.unibo.it/article/download/11048/11246
49
Manca et al.                                                                   Italian Journal of Mycology 49: 85-91 (2020)

DOI: 10.6092/issn.2531-7342/11048

Short note

       First report of canker and dieback caused by Neofusicoccum
            parvum and Diplodia olivarum on oleaster in Italy
                      Daniele Manca1, Carlo Bregant1, Lucia Maddau2, Claudia Pinna2,
                               Lucio Montecchio1, Benedetto T. Linaldeddu1

1 Dipartimento Territorio e Sistemi Agro-Forestali, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD),
Italy
2 Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy

Corresponding author e-mail: benedetto.linaldeddu@unipd.it

Article info
Received 27/05/2020; accepted 17/06/2020
DOI: 10.6092/issn.2531-7342/11048

Abstract
Oleaster (Olea europaea subsp. europaea var. sylvestris) is a sclerophyllous forest tree occurring in the Mediterranean region
including Sardinia (Italy). Oleaster suffers from few major diseases but, since 2017, a new and unusual disease leading to
the death of both young and old trees has been observed in several woodlands of high ecological value in north-eastern
Sardinia. Declining trees showed a variety of symptoms including leaf chlorosis, dieback and sunken canker on trunk
and branches. The bleeding cankers exuded a white to orange sap, which gradually dried to a whitish gluey mass on the
bark. Symptomatic wood samples yielded fungal isolates belonging to five fungal species: Diplodia olivarum, Dothiorella
sarmentorum, Neofusicoccum parvum, Rosellinia corticium and unidentified Pestalotiopsis sp. In pathogenicity trials,
Neofusicoccum parvum proved to be the most aggressive species. Our results provide the first evidence for a combined
involvement of different Botryosphaeriaceae species in the aetiology of a new oleaster disease.

Keywords
Sardinia; emerging diseases; Botryosphaeriaceae; Pestalotiopsis; Rosellinia


Introduction

Oleaster (Olea europaea subsp. europaea var. sylvestris) is a long-living tree economically, socially,
and culturally intertwined with the populations of Mediterranean region as well as a key component
of the rural landscape (Marcuzzi, 1996; Gianguzzi and Bazan, 2019). In the Mediterranean region, it
is genetically divided into seven areas that could overlay glacial refuges (Breton et al., 2006).
        Oleaster is commonly considered a tree tolerant to environmental stresses, pathogens and insect
pests (Jiménez-Fernández et al., 2016; Sesli and Yegenoglu, 2017). However, during the last years an
increase in root rot symptoms caused by Phytophthora spp. was reported in southern Spain (González
et al., 2017, 2019).
        In Sardinia oleaster is a typical component of the Mediterranean maquis. It is considered as a
species of high ecological and economic value, given its high adaptability to different environments
and the good quality of its wood (Bacchetta et al., 2003). Since 2017, a new and unusual severe disease
leading to the death of both young and old oleaster trees has been observed in several woodlands in
north-eastern Sardinia. Since there is no information about this new disease, a survey was conducted
to establish the causal agents.


ISSN 2531-7342 Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2020 The Authors. This work is licensed under CC BY 4.0
Manca et al.                                                               Italian Journal of Mycology 49: 85-91 (2020)

DOI: 10.6092/issn.2531-7342/11048



Materials and Methods

Field surveys, sampling procedure and fungal identification
From spring 2017 to winter 2018, the health status of oleaster trees was monitored in fourteen
woodlands located in north-eastern Sardinia. Samples of branches showing whitish exudations, canker
and dieback were collected from 38 symptomatic trees of different age (Table 1). All samples were
taken to the laboratory to be inspected and then immediately processed. The outer bark surface was
removed with a sterile scalpel. Longitudinal and transversal cuts were made to observe any internal
symptoms. Isolations were made from approx. 5 mm2 chips of inner bark and xylem cut aseptically
from the margin of infected tissues.
      All plant samples were placed on petri dishes containing potato dextrose agar (PDA 39 g l-1,
Oxoid Ltd) growing medium. After incubation at 25 ± 1 °C for 5-7 days in the dark, hyphal tips
from the emerging fungal colonies were sub-cultured onto half-strength PDA supplemented with
autoclaved holm oak twigs and incubated at room temperature under natural daylight to enhance
sporulation.
      Fungal isolates were initially grouped in morphotypes on the basis of colony growth
characteristics, including surface and reverse colony appearance, observed after 7 days of incubation
on PDA at 25 °C in the dark and morpho-biometric data of conidia. Measurements of conidia were
taken with the software Motic Images Plus 3.0 paired with a Moticam 10+ camera connected to a
Motic BA410E microscope.

               Table 1 - Study sites information and number of symptomatic branches sampled
                 Study              Coordinates                                         Number of
                                                             Elevation (m a.s.l.)
                  sites               (N, E)                                         branches sampled
                    1         40°57’58”N - 9°33’40”E                         74                        1
                    2         40°58’00”N - 9°33’97”E                         44                        1
                    3         40°58’00”N - 9°33’97”E                         36                        1
                    4         41°00’20”N - 9°35’27”E                         31                        1
                    5         40°59’96”N - 9°32’92”E                         27                        1
                    6         41°00’41”N - 9°30’18”E                           7                       1
                    7         41°03’74”N - 9°28’00”E                         25                        1
                    8         41°06’47”N - 9°27’57”E                         53                        1
                    9         41°07’11”N - 9°26’11”E                         84                        1
                   10         41°09’89”N - 9°24’12”E                         38                        1
                   11         41°10’35”N - 9°17’47”E                         41                        6
                   12         40°58’56”N - 9°31’45”E                        103                       17
                   13         40°28’29”N - 9°08’74”E                        327                        1
                   14         40°26’10”N - 9°11’44”E                        226                        4

DNA extraction, PCR amplification and sequencing
Molecular analysis was used to confirm the identity of all isolates at species level. InstaGene Matrix
(BioRad Laboratories, Hercules) was used to extract genomic DNA from 5-day-old cultures grown
on PDA and incubated at 25 °C in the dark. The primers ITS1 and ITS4 (White et al., 1990) were used

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to amplify and sequence the internal transcribed spacer (ITS) regions, including the complete 5.8S
gene. Polymerase chain reaction (PCR) mixtures and amplification conditions were as described by
Linaldeddu et al. (2016a). The PCR products were purified using a EUROGOLD gel extraction kit
(EuroClone S.p.A.) following the manufacturer’s instructions.
      The ITS regions were sequenced by the BMR Genomics DNA sequencing service (www.bmr-
genomics.it), in both directions, with the primers used for amplification. The nucleotide sequences
were read and edited with FinchTV 1.4.0 (Geospiza, Inc. http://www.geospiza.com/finchtv) and
then compared with reference sequences (type material) retrieved from GenBank using the BLASTn
algorithm. ITS sequences from representative isolates obtained in this study were deposited in
GenBank (www.ncbi.nlm.nih.gov/genbank).

Pathogenicity test
To verify the pathogenicity of the species isolated, a field inoculation trial was conducted in June-
August 2018 on asymptomatic branches of oleaster growing in a natural stand in Sardinia (40°26’10”N
- 9°11’44”E). During the experimental period, the daily mean air temperature was 13–36 °C. Six
branches (6-9 cm in diameter) were inoculated with a representative isolate of each fungal species,
and six uninoculated branches were used as controls. The inoculated region of the branch was surface-
disinfected with 70% ethanol and a piece (0.7 mm in diameter) of outer and inner bark was removed
with a flamed cork borer and replaced with an agar-mycelium plug of the same size taken from the
margin of an actively growing colony on PDA. The inoculation point was covered with cotton wool
soaked in sterile water and wrapped in a piece of aluminium foil secured with masking tape. Controls
were inoculated with a sterile PDA plug applied as described above. After 3 months, the outer bark
was carefully removed with a scalpel and the size of necrotic lesion surrounding each inoculation
point was measured.
      Re-isolation of inoculated species was attempted by transferring 10 pieces of inner bark and
wood taken around the margin of each lesion onto PDA. Cultures were grown in daylight and room
temperature and then identified by analysis of ITS sequences.

Statistical analyses
Pathogenicity assay data were checked for normality and then subjected to analysis of variance
(ANOVA). Significant differences among mean values were determined using Fisher’s least significant
differences multiple range test (P ≤ 0.05) after one-way ANOVA using XLSTAT software (Addinsoft).

Results

Symptomatology
Field surveys have shown that symptomatic oleaster trees are widespread along the north-eastern coast
of Sardinia. Sampled trees displayed a variety of symptoms including leaf chlorosis, a progressive
dieback of twigs and sunken canker on trunk and branches. These cankers often exuded a white to
orange sap, giving them the appearance of bleeding, which gradually dried to a whitish gluey mass
on the bark. After removing the outer and inner bark from cankers, dark brown necrotic lesions of
variable size were visible on the xylem tissue (Figs. 1a-h). In cross-section, necrotic lesions appeared
with the characteristic wedge-shaped aspect typical of Botryosphaeriaceae infections (Figs. 1i-k).

Aetiology
Isolations performed from 38 cankered branch samples yielded a total of 32 fungal colonies. On the
basis of morphological features and DNA sequence data (ITS region), five distinct species namely
Diplodia olivarum A.J.L. Phillips, Frisullo & Lazzizera (16 isolates from 5 sites), Neofusicoccum

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parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (11 isolates from 4 sites),
Pestalotiopsis sp. (2 isolates from 2 sites), Rosellinia corticium (Schwein.) Sacc. (2 isolates from
2 sites) and Dothiorella sarmentorum (Fr.) A.J.L. Phillips, A. Alves & J. Luque (1 isolate) were
identified (Figs. 1l-p).




Fig. 1 - Main disease symptoms detected on oleaster trees: branch dieback and leaf chlorosis (a); active sunken cankers
on the trunk and branches wet due to the emission of a whitish gluey mass and extensive necrotic lesion of xylem tissues
in correspondence of the cankers (b-h); cross-section of cankers on branches and main stem with wedge-shaped necrotic
sectors (i-k). Colony morphology of Diplodia olivarum (l), Dothiorella sarmentorum (m), Neofusicoccum parvum (n),
Pestalotiopsis sp. (o) and Rosellinia corticium (p) after 7 days growth at 25 °C on PDA in the dark. Symptoms observed
on oleaster branches 90 days after inoculation with: Diplodia olivarum (q), Dothiorella sarmentorum (r), Neofusicoccum
parvum (s), Pestalotiopsis sp. (t), Rosellinia corticium (u). Asymptomatic control branch (v)

      For each species BLAST searches against GenBank showed 100% identity to reference sequences
of ex-type isolates and/or representative strains. The ITS sequence of a representative isolate of each
species was deposited in GenBank (D. olivarum MT509988, D. sarmentorum MT509989, N. parvum
MT509990, Pestalotiopsis sp. MT509991 and R. corticium MT509992).

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DOI: 10.6092/issn.2531-7342/11048



Pathogenicity
All five species proved to be pathogenic on oleaster. At the end of the experimental period, all branches
inoculated with D. olivarum, D. sarmentorum, N. parvum, Pestalotiopsis sp. and R. corticium
displayed dark brown bark lesions that spread up and down from the inoculation site (Figs. 1q-v). The
average lesion length differed significantly among species (F4,25 = 105.332, P < 0.001; Tab. 2). The
lesions caused by N. parvum were significantly larger than those caused by other species. In addition,
branches inoculated with N. parvum displayed wilting symptoms and a wedge-shaped necrotic sector
in cross-section, congruent with field observations.
       Control branches inoculated with sterile PDA plugs remained symptomless. All five fungal
species were successfully re-isolated from symptomatic wood and inner bark tissues from inoculated
branches, thus fulfilling Koch’s postulates.

       Table 2 - Mean lesion length ± standard deviation caused by fungal species on branches of oleaster
                                        Identification Mean lesion length                Re-isolation
        Study sites                                    (cm)               Leaf chlorosis
                                        code                                             frequency (%)
        Diplodia olivarum                 DM1                   3.2 ± 0.41b    no                       100
        Dothiorella sarmentorum           DM2                   1.3 ± 0.17d    no                       100
        Neofusicoccum parvum              DM3                   8.1 ± 1.47a    yes                      100
        Pestalotiopsis sp.                DM4                   2.2 ± 0.18c    no                       100
        Rosellinia corticium              DM5                   2.1 ± 0.52c    no                       83.3
        Control                                                        0.00
        LSD critical value                                             2.06

Discussion and Conclusion

The results obtained have allowed us to clarify the aetiology of a new disease of oleaster in different
natural ecosystems in Sardinia. In particular, two Botryosphaeriaceae species, D. olivarum and N.
parvum were isolated and identified as the main causal agents of this emerging disease.
       Diplodia olivarum was originally described from rotting olive drupes in Apulia (Italy)
(Lazzizera et al., 2008) and it was subsequently reported associated with cankered branches of carob
and declining lentisk shrubs in Italy (Granata et al., 2011; Linaldeddu et al., 2016b) and declining
almond trees in Spain (Gramaje et al., 2012).
       Neofusicoccum parvum is a plurivorous pathogen reported on more than 100 plant hosts
worldwide (Sakalidis et al., 2013; Dissanayake et al., 2016). In Italy N. parvum has previously been
reported as a pathogen on several hosts such as cannabis, cork oak, grapevine and pomegranate
(Linaldeddu et al., 2007, Carlucci et al., 2015; Riccioni et al., 2017; Alberti et al., 2018). It has also been
reported associated with a decline of olives in southern Italy (Carlucci et al., 2013). Interestingly, on
four branches D. olivarum and N. parvum were isolated from the same canker, suggesting a potential
synergistic interaction in the pathogenesis process.
       Given the low frequency of isolation of D. sarmentorum, Pestalotiopsis sp. and R. corticium,
it is not possible to establish the exact role of these species in the aetiology of the disease. All three
species have proven to be weak pathogens on oleaster. It remains to establish a possible synergic
interaction with D. olivarum and N. parvum. All five species are reported here for the first time as
oleaster pathogens.
       Members of the Botryosphaeriaceae family represent a growing threat to agricultural crops,
urban and forest ecosystems in the Mediterranean region (Phillips et al., 2005; Alves et al., 2013;

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Dissanayake et al., 2017). In particular, over the last few years there has been an exponential increase
in the occurrence of diseases caused by species of this family in natural ecosystems (Piškur et al.,
2011; Bragança et al., 2016; Linaldeddu et al., 2017; Shami et al., 2017).
      The site factors that may have contributed to the onset of this new oleaster disease remain
unknown. However, given the high susceptibility of oleaster to N. parvum, great caution should be
taken in the use of oleaster as a source of genetic resistance against fungal pathogens in olive trees
breeding programmes.

References

Alberti I, Prodi A, Nipoti P, Grassi G (2018) First report of Neofusicoccum parvum causing stem and
     branch canker on Cannabis sativa in Italy. Journal of Plant Diseases and Protection 125:511–
     513. https://doi.org/10.1007/s41348-018-0174-4
Alves A, Barradas C, Phillips AJL, Correia A (2013) Diversity of Botryosphaeriaceae species
     associated with conifers in Portugal. European Journal Plant Pathology 135:791–804. https://
     doi.org/10.1007/s10658-012-0122-2
Bacchetta G, Bagella S, Biondi E, Farris E, Filigheddu R, Mossa L (2003) Su alcune formazioni a
     Olea europaea L. var. sylvestris Brot. della Sardegna. Fitosociologia 40(1):49–53.
Bragança H, Neno J, Henriques J, Diogo E, Alves A (2016) First report of Diplodia quercivora causing
     dieback on Quercus suber and in Europe. Plant Disease 100:2166. https://doi.org/10.1094/
     PDIS-03-16-0380-PDN
Breton C, Tersac M, Bervillé A (2006) Genetic diversity and gene flow between the wild olive (oleaster,
     Olea europaea L.) and the olive: several Plio-Pleistocene refuge zones in the Mediterranean
     basin suggested by simple sequence repeats analysis. Journal of Biogeography 33:1916–1928.
     https://doi.org/10.1111/j.1365-2699.2006.01544.x
Carlucci A, Cibelli F, Lops F, Luisa M, Raimondo D (2015b) Characterization of Botryosphaeriaceae
     species as causal agents of trunk diseases on grapevines. Plant Disease 99:1678–1688. https://
     doi.org/10.1094/PDIS-03-15-0286-RE
Carlucci A, Raimondo ML, Cibelli F, Phillips AJL, Lops F (2013) Pleurostomophora richardsiae,
     Neofusicoccum parvum and Phaeoacremonium aleophilum associated with a decline of
     olives in southern Italy. Phytopathologia Mediterranea 52:517–527. https://doi.org/10.14601/
     Phytopathol_Mediterr-13526
Dissanayake AJ, Camporesi E, Hyde KD, Yan JY, Li XH (2017) Saprobic Botryosphaeriaceae,
     including Dothiorella italica sp. nov., associated with urban and forest trees in Italy. Mycosphere
     8:1157–1176.
Dissanayake AJ, Phillips AJL, Li XH, Hyde KD (2016) Botryosphaeriaceae: current status of genera
     and species. Mycosphere 7:1001–1073. https://doi.org/10.1094/PDIS-03-15-0286-RE
Gianguzzi L, Bazan G (2019) A phytosociological analysis of the Olea europaea L. var. sylvestris
     (Mill.) Lehr. forests in Sicily. Plant Biosystems (in press). https://doi.org/10.1080/11263504.2
     019.1681532
González M, Pérez-Sierra A, Sánchez ME (2019) Phytophthora oleae, a new root pathogen of wild
     olives. Plant Pathology 68:901–907. https://doi.org/10.1111/ppa.13024
González M, Pérez-Sierra A, Serrano MS, Sánchez ME (2017) Two Phytophthora species causing
     decline of wild olive (Olea europaea subsp. europaea var. sylvestris). Plant Pathology 66:941–
     948. https://doi.org/10.1111/ppa.12649
Gramaje D, Agustí-Brisach C, Pérez-Sierra A, Moralejo E, Olmo L, Mostert L, Damm U, Armengol
     J (2012) Fungal trunk pathogens associated with wood decay of almond trees on Mallorca


                                                  90
Manca et al.                                                    Italian Journal of Mycology 49: 85-91 (2020)

DOI: 10.6092/issn.2531-7342/11048



      (Spain). Persoonia 28:1–13. http://dx.doi.org/10.3767/003158512X626155
Granata G, Faedda R, Sidoti A (2011) First report of canker caused by Diplodia olivarum on carob
      tree in Italy. Plant Disease 100:2483–2491. https://doi.org/10.1094/PDIS-12-10-0870
Jiménez-Fernández D, Trapero-Casas JL, Landa BB, Navas-Cortés JA, Bubici G, Cirulli M, Jiménez-
      Díaz RM (2016) Characterization of resistance against the olive-defoliating Verticillium
      dahliae pathotype in selected clones of wild olive. Plant Pathology 65:1279–1291. https://doi.
      org/10.1111/ppa.12516
Lazzizera C, Frisullo S, Alves A, Lopes J, Phillips AJL (2008) Phylogeny and morphology of
      Diplodia species on olives in southern Italy and description of Diplodia olivarum sp. nov.
      Fungal Diversity 31:63–71.
Linaldeddu BT, Alves A, Phillips AJL (2016a) Sardiniella urbana gen. et sp. nov., a new member of
      the Botryosphaeriaceae isolated from declining Celtis australis trees in Sardinian streetscapes.
      Mycosphere 7:893–905. http://dx.doi.org/10.5943/mycosphere/si/1b/5
Linaldeddu BT, Franceschini A, Luque J, Phillips AJL (2007) First report of canker disease caused
      by Botryosphaeria parva on cork oak trees in Italy. Plant Disease 91(3):324. https://doi.
      org/10.1094/PDIS-91-3-0324A
Linaldeddu BT, Maddau L, Franceschini A (2017) First report of Diplodia corticola causing canker
      and dieback of Quercus ilex, Q. petraea and Q. suber in Corsica (France). Plant Disease 101:256.
      https://doi.org/10.1094/PDIS-07-16-1076-PDN
Linaldeddu BT, Maddau L, Franceschini A, Alves A, Phillips AJL (2016b) Botryosphaeriaceae
      species associated with lentisk dieback in Italy and description of Diplodia insularis sp. nov.
      Mycosphere 7:962–977. http://dx.doi.org/10.5943/mycosphere/si/1b/8
Marcuzzi G (1996) Il rapporto uomo-olivo: un problema di ecologia umana. Annuali Museo Civico
      Rovereto 12:15–84.
Phillips AJL, Rumbos IC, Alves A, Correia A (2005) Morphology and phylogeny of Botryosphaeria
      dothidea causing fruit rot of olives. Mycopathologia 159:433–439. https://doi.org/10.1007/
      s11046-005-0256-2
Piškur B, Pavlic D, Slippers B, Ogris N, Maresi G, Wingfield MJ, Jurc D (2011) Diversity and
      pathogenicity of Botryosphaeriaceae on declining Ostrya carpinifolia in Slovenia and Italy
      following extreme weather conditions. European Journal of Forest Research 130:235–249.
      https://doi.org/10.1007/s10342-010-0424-x
Riccioni L, Valente MT, Di Gianbattista G (2017) First report of Neofusicoccum parvum causing
      shoot blight and plant decay on pomegranate in Tarquinia, Italy. Journal of Plant Pathology
      99(1):294. http://dx.doi.org/10.4454/jpp.v99i1.3805
Sakalidis ML, Slippers B, Wingfield BD, Hardy GEStJ, Burgess TI (2013) The challenge of
      understanding the origin, pathways and extent of fungal invasions: global populations of the
      Neofusicoccum parvum – N. ribis species complex. Diversity and Distributions 19:873–883.
      https://doi.org/10.1111/ddi.12030
Sesli M, Yegenoglu ED (2017) Genetic relationships in wild olives (Olea europaea ssp. oleaster) by
      ISSR and RAPD markers. Biotechnology & Biotechnological Equipment 31:897-904. https://
      doi.org/10.1080/13102818.2017.1344107
Smahi H, Belhoucine-Guezouli L, Berraf-Tebbal A, Chouih S, Arkam M, Franceschini A, Linaldeddu
      BT, Phillips AJL (2017) Molecular characterization and pathogenicity of Diplodia corticola
      and other Botryosphaeriaceae species associated with canker and dieback of Quercus suber in
      Algeria. Mycosphere 8(2):1261–1272. http://dx.doi.org/10.5943/mycosphere/8/2/10
White TJ, Bruns T, Lee J, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal
      RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications (Innis MA,
      Gelfand DH, Sninsky JJ, White TJ, eds). Academic Press, San Diego, California, pp 315-322.

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