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Identification of Fungal Species Associated with Branch Dieback of Olive and
Resistance of Table Cultivars to Neofusicoccum mediterraneum and
Botryosphaeria dothidea
Juan Moral, Departamento de Agronom´
ıa, ETSIAM, Universidad de C´
ordoba, Campus de Rabanales, Edif. C4, 14071 C ´
ordoba, Spain; and
Kearney Agricultural Research and Extension Center, University of California-Davis, Parlier 93648; Carlos Agust´
ı-Brisach, Mario P´
erez-Rodr´
ıguez,
Carlos Xavi´
er, and M. Carmen Raya, Departamento de Agronom´
ıa, ETSIAM, Universidad de C ´
ordoba, Campus de Rabanales; Ali Rhouma,
Institute de l’Olivier, Mahraj`
ene, BP208, 1082, Tunisia; and Antonio Trapero, Departamento de Agronom´
ıa, ETSIAM, Universidad de
C´
ordoba, Campus de Rabanales
Abstract
Over two consecutive seasons, 16 olive orchards with trees exhibiting die-
back symptoms on branches were surveyed in southern Spain. The six
dominant fungal species recovered were characterized by means of pheno-
typic observations, DNA analysis (by sequencing of the internal transcribed
spacer, b-tubulin, and large subunit nuclear ribosomal DNA regions), and
pathogenicity tests. Additionally, three isolates collected from Tunisian ol-
ive trees showing similar dieback symptoms, one isolate of Colletotrichum
godetiae, and a reference isolates of Neofusicoccum mediterraneum
were included. The resistance of the 11 most important table cultivars to
N. mediterraneum and Botryosphaeria dothidea, the causal agent of
“escudete”(small shield) of fruit, was studied by the inoculation of
branches and immature fruit, respectively. The species Cytospora pruinosa,
N. mediterraneum,Nothophoma quercina,Comoclathris incompta, and
Diaporthe sp. were identified. Only N. mediterraneum and C. incompta
were able to induce the typical dieback symptoms and cankers that af-
fected the development of the plants. The species N. mediterraneum
was the most virulent among the evaluated species, although differences
in virulence among its isolates were observed. The remaining fungal species
were weakly pathogenic to nonpathogenic on plants. According to resistance
tests, ‘Gordal Sevillana’and ‘Manzanilla Cacereña’were the most suscep-
tible to branch dieback caused by N. mediterraneum. Furthermore, the fruit
of ‘AloreñadeAtarfe’and ‘Manzanilla de Sevilla’were the most susceptible
to B. dothidea. Knowledge of the etiology and cultivar resistance of these
diseases will help to establish better control measures.
Cultivated olive (Olea europaea subsp. europaea L) is the most
important perennial crop in Spain. Spain leads the world in olive pro-
duction, generating about 43% of the world’s olive fruit. The Spanish
olive industry accounts for 25% of the global acreage designated
for olive production, occupying 2.5 × 10
6
ha (for both table fruit
and oil). Approximately 65% of this land lies in the Andalusia re-
gion of the southern Iberian Peninsula, in itself producing 85% of
the total production for Spain (Barranco et al. 2008). Global table
fruit production is around 2.3 × 10
6
tons per year, 23% of which
come from Spain (MAGRAMA 2016). The most important table
cultivars are ‘Gordal Sevillana’and ‘Manzanilla de Sevilla’,both
comprising approximately 85% of table fruit production. In addi-
tion, other table cultivars such as ‘Aloreña’and ‘Morona’have a
secondary importance but their fruit are appreciated for their or-
ganoleptic properties. Finally, some cultivars such as ‘Hojiblanca’,
‘Manzanilla Cacereña’,and‘Verdial de Hu´
evar’have a twofold
purpose as both table fruit and as oil, while some other cultivars
such as ‘Picual’are used exclusively for oil production (Rallo
et al. 2005).
At the beginning of the 2000s, a serious disease showing typical
dieback symptoms was observed in olive orchards in the Andalusia
region, where ‘Gordal Sevillana’was the most affected cultivar
(Romero et al. 2005). Similar symptoms were also observed affecting
other cultivars such as ‘Arbequina’,‘Manzanilla de Sevilla’, and
‘Picual’. Differences in susceptibility among cultivars to the disease
are evident in the field but knowledge regarding the resistance of cul-
tivars to twig-branch dieback is unknown. Affected trees showed an
abundance of dead twigs and wilted leaves that remained attached to
blighted branches, which were generally associated with the decline
of entire young stems or older branches (Moral et al. 2010; Romero
et al. 2005; ´
Urbez-Torres et al. 2013). The branches showing cankers
are less tolerant to water stress and had an insufficient water and nutrient
flow through both xylem and phloem vessels. Consequently, char-
acteristic dieback symptoms such as bud mortality, leaf chlorosis,
fruit rot, and twig dieback occur when water and nutrient demand
exceeds the conductive capacity of the vascular tissues ( ´
Urbez-
Torres et al. 2013).
Olive fruit can be affected by numerous species of the family
Botryosphaeriaceae, including species belonging to the genera
Botryosphaeria and Neofusicoccum that are well known to cause
cankers, dieback, and rot of mature and immature fruit (Carlucci
et al. 2013; Lazzizera et al. 2008a, 2008b; Moral et al. 2008a,
2010; ´
Urbez-Torres et al. 2013). However, further studies may
elucidate the etiology of these diseases in Spain, and the relation-
ship between affected host tissue and pathogen species.
The species N. luteum (Pennycook & Samuels) Crous, Slippers &
A. J. L. Phillips was described causing stem cankers and tip dieback
in olive branches in New Zealand (Taylor et al. 2001), and causing
leaf necrosis in Australia (Sergeeva et al. 2009). In Spain, Moral
et al. (2010) observed that N. mediterraneum Crous, M. J. Wingf. &
A. J. L. Phillips was the prevalent species associated with branch die-
back, mainly on ‘Gordal Sevillana’. The Botryosphaeriaceae species
Diplodia mutila (Fr.) Mont.; D. seriata De Not.; Dothiorella iberica
A. J. L. Phillips, J. Luque & A. A lves; Lasiodiplodia theobromae (Pat.)
Griffon & Maubl.; N. luteum;N. parvum;andN. vitifusiforme (Van
Niekerk & Crous) Crous, Slippers & A. J. L. Phillips were also reported
to cause branch dieback and blight, and eventual death of shoots in dif-
ferent olive-growing areas (Carlucci et al. 2013; Moral et al. 2010;
´
Urbez-Torres et al. 2013).
On olive drupes, Diplodia olivarum A. J. L. Phillips, Frisullo &
Lazzizera and D. seriata were reported causing rot on mature fruit
in Italy (Lazzizera et al. 2008a) and Spain (Moral et al. 2008b), re-
spectively. In Australia, N. luteum was also observed causing rot
of mature fruit (Sergeeva et al. 2009). Conversely, because immature
Corresponding author: J. Moral; E-mail: jmoral@ucdavis.edu
J. Moral and C. Agust´
ı-Brisach contributed equally to this article.
Accepted for publication 5 October 2016.
©2017 The American Phytopathological Society
306 Plant Disease / Vol. 101 No. 2
Plant Disease •2017 •101:306-316 •http://dx.doi.org/10.1094/PDIS-06-16-0806-RE
olive fruit are highly resistant to fungi, no significant pathogens have
been observed causing rot on them, with the exception of Botryos-
phaeria dothidea (Moug.) Ces. & De Not. (Moral et al. 2008b,
2010). This pathogen is the causal agent of Dalmatian disease of olive,
which can be found in most of the Mediterranean basin (Iannotta et al.
2007; Latinovi´
c et al. 2013; Moral et al. 2010; Zachos and Tzavella-
Klonari 1983). B. dothidea produces a sunken, necrotic, and circular
lesion (approximately 8 mm in diameter, never larger than 1 cm) with
a sharp edge delineating infected and healthy fruit tissues; this appear-
ance is called “escudete”(or small shield) in Portuguese and Spanish.
As the disease progresses, the necrotic spots expand and eventually en-
compass the entire fruit. Then, when the fruit matures, it falls to the
ground and is mummified (Moral et al. 2010). Overall, the incidence
of olive fruit showing the Dalmatian symptoms is relatively low but,
frequently, it exceeds the tolerance level for the “Extra”Class standard
for the olive fruit, which is usually at a 2 or 4% level, according to t he
Spanish Royal Decree 1230/2001 or the “Codex Alimentarius”of
the FAO, respectively.
Botryosphaeriaceae asexual stage is predominant in the majority
of its hosts. In olive, the sexual stagehasbeenreportedinonlyafew
cases for B. dothidea and N. mediterraneum in California and Spain,
respectively (Moral et al. 2010, 2015). Recently, Eldesouki (2013)
elucidated the Dalmatian disease cycle, in which the mosquito Prola-
sioptera berlasiana, a predator of olive fly eggs (Bactrocera oleae),
carries the B. dothidea spores in a mycangium (a specialized structure,
like a pocket). The mosquito is attracted to ovipositor punctures on the
olive surface made by the olive fly. When the mosquito deposits its
own egg adjacent to the fly egg, it also inoculates the puncture with
the fungus. Because the mosquito cannot penetrate intact fruit, the
control of olive flies incidentally involves the control of the Dalma-
tian disease (Moral et al. in press). However, there is little informa-
tion on the control of Dalmatian disease by the use of resistant olive
cultivars. In Montenegro, Latinovi´
c et al. (2013) studied the resis-
tance of olive cultivars infected with B. dothidea, demonstrating
that ‘Crnjaka’and ‘Gloginja’(native cultivars) and ‘Pendolino’
and ‘Cassanesse’(Italian cultivars) were highly resistant to the dis-
ease. These authors evaluated the resistance of only one Spanish cul-
tivar, ‘Manzanilla’; however, the identity of this cultivar is unclear
because the name “Manzanilla”includes more than 20 cultivars, such
as ‘Manzanilla de Sevilla,’‘Manzanilla Cacereña,’and so on (Barranco
and Rallo 2000). Even so, the resistance of a broad range of Spanish olive
cultivars to B. dothidea and N. mediterraneum has not been evaluated yet
in Spain.
During the last few decades, other pathogens have also been asso-
ciated with olive twig-branch dieback. For example, the species
Cytospora oleina Berl. and Eutypa lata (Pers.) Tul. & C. Tul. have
been associated with olive cankers and consequent branch dieback
in Greece (Rumbos 1988, 1993). The latter pathogen was recently re-
ported in California and Italy causing dieback of olive trees (Tosi and
Natalini 2009; ´
Urbez-Torres et al. 2013). The species Comoclathris
incompta (Sacc. & Martelli) Ariyaw. & K. D. Hyde has also been de-
scribed causing branch dieback in olive in Croatia, Greece, and Italy
(Ivic et al. 2010; Malathrakis 1979; Tosi and Zazzerini 1994). Re-
cently, Phoma fungicola has been associated with branch dieback
of olive trees in Tunisia (Rhouma et al. 2010; Taieb et al. 2014). In
addition, several fungi, including Diaporthe viticola Nitschke; Diatrype
oregonensis (Wehm.) Rappaz; D. stigma (Hoffm.) Fr.; Phaeoacremonium
aleophilum W. Gams, Crous, M. J. Wingf. & Mugnai; Phaeomoniella
chlamydospora (W. Gams, Crous, M. J. Wingf. & Mugnai) Crous
& W. Gams; Phoma sp.; Phomopsis (Sacc.) Bub´
ak sp. (synonym
Diaporthe Nitschke); Trametes versicolor (L.) Lloyd; and Schizo-
phyllum commune Fr., have also been associated with olive branch
dieback, stem canker, and twig necrosis in California ( ´
Urbez-
Torres et al. 2013). In southern Italy, N. parvum;Pleurostomophora
richardsiae (Nannf.) L. Mostert, W. Gams & Crous; six species be-
longing to genus Phaeoacremonium; and Pseudophaeomoniella
oleicola Nigro, Antelmi & Crous were isolated from the wood of ol-
ive trees displaying wilting and branch dieback (Carlucci et al. 2013,
2015; Crous et al. 2015). Colletotrichum spp., the causal agents of
olive anthracnose, have also been implicated as responsible for leaf
wilting and branch dieback symptoms (Moral et al. 2014). Although
these fungi affect fruit, preliminary studies demonstrated that the
toxic substances produced by Colletotrichum spp. on rotten fruit
are able to incite leaf wilting and branch dieback (Moral et al.
2009). In the latter study, for example, olive plants without fruit were
infected by the pathogen but did not show any disease symptoms.
However, the plant inoculations were conducted by using a conidial
suspension and not using the conventional mycelium plugs of the
pathogen (Moral et al. 2009).
In Spain, little attention has been given to olive branch dieback or
the Dalmatian disease. Nevertheless, both diseases have increas-
ingly become concerns among growers in the main table-olive-
producing areas of the Andalusia region. Determination of the eti-
ology, epidemiology, and control of these diseases are required;
therefore, the objectives of the current study were to (i) identify
on the basis of morphology and molecular phylogeny the different
fungal species associated with the olive twig-branch syndrome, (ii)
evaluate the pathogenicity of the different fungi associated with the
olive twig-branch dieback, and (iii) evaluate the resistance of the
most common table cultivars to N. mediterraneum and B. dothidea,
the causal agents of twig-branch dieback and Dalmatian disease,
respectively.
Material and Methods
Field surveys and fungal isolation. In 2009 and 2010, field sur-
veys were conducted in orchards of ‘Gordal Sevillana’and ‘Picual’
olive located in the Andalusian provinces of Ja´
en and Sevilla
(Table 1). In total, 163 samples were collected from 16 olive or-
chards, each being 25 years old or older. Branches and twigs from
olive trees showing the typical symptoms of branch dieback such
as cankers, internal wood necrosis, black vascular streaking. or dis-
colored tissues were collected. Parafilm (Parafilm, Menasha, WI)
was used to seal both ends of all samples; then, samples were placed
in black plastic bags and kept at 4°C until isolations were made.
Branches and twigs were surface disinfected with 50% ethanol.
The outer bark was removed with a sterile scalpel. Small pieces from
the edge between healthy and affected wood tissue were plated on potato
dextrose agar (PDA; Biokar-Diagnostics, Zac de Ther, France) acidified
with lactic acid (2.5 ml at 25% [vol/vol] per liter of medium). Petri dishes
were incubated at 23 to 27°C under a 12-h photoperiod of cool fluores-
cent light (350 mmol m
−2
s
−1
) until colonies were large enough to be ex-
amined. To obtain pure cultures, mycelial fragments from the margin of
the fungal colonies were transferred to acidified PDA and incubated as
described before. Several isolates collected in affected ‘Meski’orchards
from Tunisia were also included in this study (Table 1). Fungal species
were isolated for further study from 10 olive trees from three different
orchards. The studied isolates are maintained in the collection of the
Departamento de Agronom´
ıa, Universidad de C´
ordoba, Spain.
Morphological characterization. Based on morphological char-
acteristics, fungal species were first identified to genus. Pycnidial and
conidial characteristics such as shape, size, color, and presence or ab-
sence of septa were recorded from the colonies developed in vitro.
For morphological identification, single conidial cultures were de-
rived using the serial dilution method (Dhingra and Sinclair 1995)
and grown on PDA for up to 2 weeks at 25 ± 2°C with a 12-h pho-
toperiod of cool fluorescent light (350 mmol m
−2
s
−1
). To induce pyc-
nidia development, the same isolates were cultured on sterile ‘Gordal
Sevillana’leaves, which were previously autoclaved at 120°C for
20 min, and placed into petri dishes containing 5 ml of molten
PDA (Moral et al. 2009). Petri dishes were incubated as described
above for 10 days. Three petri dishes per isolate were used. The ex-
periment was performed twice.
Fungal structures were measured using a Nikon Eclipse 80i micro-
scope and images were captured using the NIS-Element software
(Nikon Corp., Tokyo). Conidial masses were observed from pycnidia
mounted in water. Color (using a color scale) (Kornerup and Wanscher
1963), morphology, and diameter of pycnidia from these isolates were
recorded. For conidial measurements, mycelial plugs were removed
from the petri dishes, placed on slides with a drop of 0.005% acid
fuchsine in lactoglycerol (1:1:1 lactic acid, glycerol, and water),
Plant Disease / Februar y 2017 307
and covered. For each isolate, 30 measurements were obtained for each
structure. The averages of length and width and the length/width rela-
tion were calculated. Characteristics of conidia (color, shape, and sep-
tation) and mycelia (texture, density, color, and zonation) were also
recorded after 14 days of incubation at 23 to 27°C in the dark (Barnett
and Hunter 1998; Sutton 1980).
DNA extraction, amplification, and phylogenetic analysis. One
to three isolates were selected based on their morphology as repre-
sentative of each of three fungal taxa, and their identification was
confirmed by using molecular techniques (Table 1). Fungal isolates
were grown on PDA for 14 days. FastDNA Kit (BIO 101, Inc., Vista,
CA) was used to extract total genomic DNA. The nuclear ribosomal
DNA (nrDNA) repeats, including the internal transcribed spacer
(ITS) 1, 5.8S ribosomal RNA (rRNA), ITS5, and portions of the
genes encoding both small and large subunit rRNA, were amplified
using primers ITS1 and ITS4 (White et al. 1990). The partial large
subunit nrDNA (LSU) was amplified with the primer pair LR0R
and LR7 (Rehner and Samuels 1994; Vilgalys and Hester 1990). Ol-
igonucleotide primers Bt2a and Bt2b were used to amplify a portion
of the b-tubulin (BT) gene (Glass and Donaldson 1995). Finally, am-
plification of part of the translation elongation factor 1-a(EF) gene
was done with the primers EF1-728F and EF1-986R (Carbone
et al. 1999). The polymerase chain reaction (PCR) of BT, EF, ITS,
and LSU was conducted according to previous studies (Chen et al.
2015; Lazzizera et al. 2008b; Moral et al. 2010). The PCR products
were purified with an Ultra Clean PCR Clean-Up Kit (MO BIO Lab-
oratories, Inc., Solana Beach, CA). The resulting amplicons were se-
quenced in both directions using an automated sequencer by the
University of C´
ordoba sequencing facility (ABI Prism 3130XL Genetic
Analyzer; Applied Biosystems, Foster City, CA). The nucleotide se-
quences were read and edited with FinchTV 1·4·0. Sequences were as-
sembled and edited to resolve ambiguities using the program SeqMan
(DNASTAR Lasergene, Madison, WI), and consensus sequences for
all isolates were compiled into a single file (Fasta format).
Phylogenetic analysis with all isolates was first conducted with the
BT, EF, ITS, and LSU data sets individually, and the four topologies
were compared. A partition homogeneity test was performed to de-
termine whether BT, EF, ITS, and LSU data sets could be combined.
The combined alignment of the two loci BT and ITS was first analyzed
for inferring the phylogeny of the isolates. Phylogeneticanalysis on the
LSU single-locus alignment was used to elucidate the organismal
phylogeny. GenBank sequences (Table 1) from different species of
Neofusicoccum,Colletotrichum,Cytospora,Phoma-like, or Diaporthe
were selected based on their high similarity with our query sequences
using MegaBLAST. Ganoderma resinaceum (GR145) and Coltriccia
cinnamomea (Dai 2464) were used as the outgroups of the combined
alignment of the two loci BT and ITS, and the LSU single-locus align-
ment. These were added to the sequences obtained and aligned using
CLUSTALW v. 2.0.11 (Larkin et al. 2007). The genetic distances were
calculated using the Kimura two-parameter model. For phylogenetic
inference, the neighbor-joining (NJ) method was used (Saitou and
Nei 1987). The NJ tree and the statistical confidence of a particular
group of sequences in the tree, evaluated by bootstrap (BS) test
(2,000 pseudoreplicates), were performed using the computer program
MEGA 6 (Tamura et al. 2013). Sequences derived in this study were
lodged at GenBank. GenBank accession numbers of the strains col-
lected during this study are listed in Table 1.
Pathogenicity tests. One representative isolate of each identified
species was inoculated toward to determine their pathogenicity capacity.
In addition, one Colletotrichum godetiae isolate (CH-21), which be-
longs to the C. acutatum species complex and is the dominant species
in the Andalusian population of the pathogen (Moral et al. 2014), was
selected to determine pathogenicity on olive detached branches or
potted plants (Table 1).
Pathogenicity on detached branches. In March 2010, branch seg-
ments (14 to 18 cm long and 1.0 to 1.5 cm in diameter) were collected
from ‘Gordal Sevillana’trees from The Olive Cultivars Garden or-
chard that belongs to the Andalusian Institute for Research and For-
mation in Agriculture and Fishery (IFAPA in Spanish) and is located
in Meng´
ıbar (Ja´
en province, Andalusia region). The branch segments
were sealed at both ends with Parafilm to reduce desiccation, and
bark surfaces were sterilized with 70% ethanol at the point of inocu-
lation, situated in the middle of each branch. In order to remove the
outer bark yet leave the inner bark intact, 10-mm-diameter holes were
made in the bark using a cork borer. In the cavities, a 10-mm-
diameter mycelium plug of each isolate was inserted such that the
inoculum was placed in direct contact with vascular tissues, as de-
scribed by Moral et al. (2010); then, the inoculated area was wrapped
with Parafilm. In total, 11 representative isolates belonging to six
species were individually tested using five replicate branches per iso-
late. The experiment was repeated three times and five branches were
treated with acidified PDA as control. Inoculated and control branches
were then incubated in humid chambers (plastic containers, 30 by 23 by
6 cm, with 100% relative humidity [RH] obtained by adding 300 ml
of water) at room temperature (26 to 33°C) under continuous cool
fluorescent lights (350 mmol m
−2
s
−1
) for 30 days. Humid chambers
were placed inside at room temperature in a completely randomized
design. For each branch, evaluation of the lesion length and the
length of the colonized bark surface bearing pycnidia, and calculation
of the relative (percent) affected length of each branch were obtained
according to Moral et al. (2010). Small wood fragments that form the
margin of the affected area of each inoculated branch were plated on
acidified PDA to isolate the pathogens.
Pathogenicity on potted plants. Five-year-old potted ‘Gordal Sev-
illana’plants were obtained from a commercial nursery. Each plant
was inoculated with mycelium plugs as described above for detached
branches. The same 11 isolates were inoculated using three replicate
plants per isolate and four branches per plant. After inoculation,
plants were placed in a greenhouse at 25 to 30°C in a completely ran-
domized design and were irrigated as needed. The area around the ex-
periment was abundantly irrigated to increase the RH. Lesion length
and percentage of dead branches were assessed 90 days after inocu-
lation. The pathogens were isolated from the olive branches as de-
scribed above. The experiment was repeated three times and five
branches from the same plant were treated with acidified PDA as
control in each replicate of the experiment.
Evaluation of cultivar resistance. To evaluate the resistance of
different table cultivars to N. mediterraneum, which was the most
virulent species of the previous experiments, branches and 5-year-
old plants of 10 table cultivars were inoculated using the reference
isolate N. mediterraneum BOO071. In the same way, the resistance
of cultivars to B. dothidea was evaluated using the reference isolate
B. dothidea BOO046 (Moral et al. 2010).
Branch inoculation with N. mediterraneum.In March 2010,
branch segments (14 to 18 cm long and 1.0 to 1.5 cm in diameter)
were cut from 10 table olive cultivars growing at The Cultivar
Garden orchard (Table 2). Sampling and inoculation were conducted
as described above. Ten branches per cultivar were inoculated and
the experiment was conducted three times. Likewise, another 10
branches per replicate were treated with acidified PDA as control.
In total, 270 branches were inoculated with N. mediterraneum and
90 branches were used as control. All of the branches were incubated
in humid chambers as described above. Evaluation of lesion length
and the length of the colonized bark surface bearing pycnidia, and
calculation of the relative (percent) affected length of each branch
were obtained according to Moral et al. (2010). The pathogen was
reisolated from the branches as described above.
Plant inoculation with N. mediterraneum.Five-year-old potted
plants of seven table cultivars were obtained from a commercial
nursery in Cordoba province from Andalusia (Table 2). Each plant
was inoculated with N. mediterraneum using mycelial plugs as
described above. Three replicate plants and four branches of each
one per cultivar were inoculated. Five branches from the same plant
were treated with acidified PDA as control in each replicate (plant)
of the experiment. Inoculated plants were then incubated in a green-
house from 25 to 30°C in a completely randomized design, and irrigated
abundantly to increase the RH. The percentage of dead branches and the
lesion length of inoculated branches were assessed 90 days after inoc-
ulation. The experiment was repeated four times. The pathogen was iso-
lated from the branches as described above.
308 Plant Disease / Vol. 101 No. 2
Fruit inoculation with B. dothidea.In September 2010, immature
fruit of 10 table cultivars were collected from The Cultivar Garden
orchard (Table 2). Fruit were washed, disinfested, and inoculated
according to Moral et al. (2010). Inoculated and control fruit (PDA
plugs without mycelia) were incubated in humid chambers as
described above. Disease severity was assessed every 2 weeks for
2 months using a 0-to-5 rating scale previously described by Moral
et al. (2008a). Disease severity index (DSI) was calculated according
to the formula DSI = (Sni ×i)/N, where irepresents severity (0 to 5),
ni is the number of fruit with the severity of i, and Nis the total num-
ber of fruit (Moral et al. 2008a). The area under the disease progress
curve (AUDPC) was calculated by trapezoidal integration of DSI val-
ues over time. There were three replicates (humid chambers) per
treatment and 30 fruit per replicate. Treatments were arranged in a
completely randomized design.
Statistical analysis. Analysis of variance (ANOVA) and means
comparison tests were conducted to determine the differences in viru-
lence among isolates and in resistance among cultivars. For the differ-
ent analyses conducted, dependent variables were lesion length to
evaluate differences in virulence among isolates; lesion length and pyc-
nidia colonization or dead branches (percent) to evaluate differences
in resistance among cultivars by using detached branches or potted
plants, respectively; and AUDPC to evaluate differences in resis-
tance among cultivars by using detached fruit. When ANOVA
showed significant differences, the treatment means were compared
according to Tukey’s honestly significant difference test at P=0.05.
The nonpathogenic isolates were excluded from the statistical anal-
ysis. The data were logarithmically transformed to meet the homoge-
neity of the variances or normality. Because the data of cultivar
resistance to N. mediterraneum did not show homogeneity of vari-
ances, even with the logarithmic transformation, they were analyzed
using the Kruskal-Wallis nonparametric test and the cultivars were
compared according to Dunn’stestatP= 0.05. Linear regression
was used to study the relationship between dependent variables of this
study. In addition, we studied the relation between fruit resistance to
B. dothidea and the susceptibility to fruit rot caused by Colletotrichum
spp. (Moral et al. 2014). All the data were analyzed by using Statistix
10 (Analytical software 2013).
Results
Collection of fungal isolates. In total, 120 fungal isolates from
affected olive trees showing twig-branch dieback were recovered
in Spanish olive orchards. Overall, olive twig death was usually as-
sociated with cankered stems. Among the collected isolates, five
fungal groups were clearly differentiated, the most frequent being
Botryosphaeriaceae species (mainly N. mediterraneum), followed
by Cytospora spp., a Phoma-like species (two subgroups), and
Diaporthe spp. Representative isolates from each of the five fungal
groups were selected for further studies. In addition, a fungal group
of isolates that were frequently isolated from affected orchards in
Tunisia was included.
Morphological characterization. All studied isolates showed
mycelial growth and sporulation within 14 days of incubation on ol-
ive leaves, except one Phoma-like isolate, CH-12, which showed
very poor mycelial growth but developed some pycnidia around
the original mycelium plug. Based on their appearance in culture,
the isolates were again assigned to five main fungal groups (Table 3).
The first group comprised a Botryosphaeriaceae species with a
Neofusicoccum anamorph (isolates BOO071 and CH-06); this spe-
cies was characterized by floccose to felted aerial mycelium show-
ing colors that varied from light to olive green on acidified PDA.
Conidia were hyaline, aseptate, and fusiform. Pycnidia were dark,
ostiolate, globose, and erumpent. Two groups (group 2 and 3) were
clearly differentiated, belonging to Phoma-like isolates. Group 2
(isolates CH-04 and CH-16) was characterized by brown to yellow-
ish mycelium with a regular margin. Conidia were dark, ellipsoid or
ovoid, slightly flattened, and thin-walled. Pycnidia were irregular,
branched, wooded, velvety, and sprawled. Group 3 (isolates CH-14
and CH-15) showed flat, fine, medium density to very dense myce-
lium; the color varied from white to pink, or green to yellow, with a
regular colony margin. Conidia were hyaline, aseptate, and oblong.
Pycnidia were dark, ostiolate, and globose. A fourth group included
aDiaporthe sp. that was characterized by having white, cottony,
slow-growing, raised mycelium with a regular colony margin. In
this fourth group, two types of hyaline conidia were observed: ob-
long, slightly fusiform, and ligulated conidia (a); and filiform and
curved conidia (b). Pycnidia were dark, ostiolate, globose, erumpent,
and velvety. Finally, Cytospora isolates formed a fifth group charac-
terized by white, flat, felty, texture uniform mycelium with an irreg-
ular colony margin. Conidia were hyaline, aseptate, and with an
alantoide form. Pycnidia were dark, erumpent, ostiolate, tuberculate,
globous, and velvet. (Tables 3 and 4; Fig. 1).
Molecular characterization. To confirm the identification based
on morphology, BT, EF, ITS, and LSU regions were obtained for all
isolates studied. The combined alignment of BT and ITS included 18
taxa (including the outgroup G. resinaceum) (Fig. 2). In this phylo-
genetic tree, combined sequences of the genera Colletotrichum (iso-
late CH-21), Cytospora (isolate CH-13), Diaporthe (isolates CH-01
and CH-03), Neofusicoccum (isolates CH-06 and BOO071), and
Nothophoma (isolates CH-4, CH-14, and CH-15) formed five well-
supported clades, in which BS support ranged from 94 to 100%. Each
clade incorporated one representative isolate of C. godetiae (CBS
127561), Cytospora pruinosa (Fr.) Sacc. (CBS 118555), Diaporthe
sp. (UCR1395), N. mediterraneum (UCD720SJ) and Nothophoma
quercina (Syd. & P. Syd.) Q. Chen & L. Cai (CBS 633.92) for
Colletotrichum,Cytospora,Diaporthe,Neofusicoccum, and Notho-
phoma clades, respectively. However, the isolates CH-12 and
CH-16 clustered in a subclade close to Leptosphaeria biglobosa
Shoemaker & H. Brun (CBS 532.66) with a low BS value (BS =
74%) and were hypothesized as a Phoma-like species. Subsequently,
phylogenetic analysis on the LSU single-locus alignment was analyzed
for the elucidation of the organismal phylogeny of the doubtful isolates
(Fig. 3). This alignment was successful in identifying allof the isolates.
The isolates CH-01 and CH-03 were grouped together in a clade (BS =
99%) with a representative isolate of Diaporthe sp. (PHAg); the iso-
late CH-13 clustered (BS = 100%) with a representative isolate of
C. pruinosa (CBS119207); and the isolates CH-12 and CH-16 clus-
tered (BS = 93 and 100%, respectively) with a representative isolate
of Comoclathris incompta (CBS 467.76). The rest of the isolates
formed the same clades as those observed in the combined alignment
of BT and ITS. Finally, sequences of EF and its single-locus alignment
were not helpful in identifying our Phoma-like isolates.
Pathogenicity tests. Pathogenicity on detached branches. Ne-
crotic lesions on ‘Gordal Sevillana’branches were observed 2 weeks
after inoculation. In general, vascular tissues showed sunken and sec-
torial necrotic lesions in the affected wood. Eventually, pathogens
belonging to the Cytospora,Phoma-like, or Diaporthe genera pro-
duced cankers on the bark of the inoculated branches. Only the
two N. mediterraneum isolates (BOO071 and CH-06), a Cytospora
sp. isolate (CH-13), and a C. incompta isolate (CH-16) caused visible
lesions on detached branches. Both N. mediterraneum isolates were
the most virulent (P< 0.05), causing an average length of necrosis of
15.93 ± 3.04 and 13.51 ± 5.96 cm, respectively (Fig. 4). The remain-
ing fungal species (Colletotrichum godetiae,Phoma-like sp., and
Diaporthe sp.) did not cause lesions on inoculated branches. Further,
no lesions were observed in control branches. The species N. mediter-
raneum,Cytospora pruinosa,andComoclathris incompta were iso-
lated from affected tissues, and Colletotrichum godetiae,Diaporthe
sp., and Nothophoma quercina were isolated from asymptomatic
branches. A significant and positive linear correlation (P< 0.001,
R
2
= 0.724) was observed between the length of necrosis and pycnidia
production, the bark surface inoculated with N. mediterraneum isolate
BOO071 being that producing the highest number of pycnidia of the
pathogen (data not shown).
Pathogenicity on potted plants. The first symptoms were ob-
served 30 days after inoculation with mycelium plugs on the xylem
tissues of Gordal Sevillana. Symptoms—mainly vascular necrotic
lesions—were similar to those observed in detached branches.
Only the N. mediterraneum isolates BOO071 and CH-06 and the
C. incompta isolate CH-06 were able to induce the typical dieback
Plant Disease / Februar y 2017 309
symptoms and cankers that affected the development of the plants,
where the BOO071 isolate was the most virulent (Fig. 4). The
remaining fungal species (C. godetiae,C. pruinosa,Diaporthe sp.,
and N. quercina) did not cause any lesions on inoculated plants as
with the control branches. The species N. mediterraneum and
C. incompta were consistently isolated from affected tissues while
C. godetiae,C. pruinosa,Diaporthe sp., and N. quercina were iso-
lated distantly from points away from the inoculation point on
asymptomatic branches.
Evaluation of cultivar resistance. Branch inoculation with
N. mediterraneum.Olive branches of the nine inoculated cultivars
showed vascular necrotic tissues after 2 weeks of inoculation. Symp-
toms observed were the same as those described above. According to
the lesion on detached and inoculated branches, ‘Gordal Sevillana’
was significantly (P< 0.05) the most susceptible to the pathogen (ne-
crosis length 15.72 ± 4.62 cm), followed by ‘Santa Caterina’and
‘San Agostino’, which did not show significant differences (P>
0.05) among them. Conversely, ‘Manzanilla Cacereña’was the least
susceptible cultivar (necrosis length 6.19 ± 2.11 cm), followed by
‘Verdial de Hu´
evar’and ‘Morona’, which did not show significant
(P> 0.05) differences among them (Table 2). For all of the cultivars,
a weak but significant negative correlation (R
2
= 0.190, P= 0.0001)
between branch diameter and length of necrosis was observed, show-
ing that branch susceptibility increased when the diameter of branches
decreased. Finally, a significant positive linear correlation (R
2
=
0.1359, P< 0.001) was observed between the length of necrosis
and pycnidia production (data not shown), the bark surface of ‘San
Agostino’being the most occupied by pycnidia.
Plant inoculation with N. mediterraneum.Olive plants showed the
first symptoms at 14 days after inoculation, while the first dead
branches were observed at 8 weeks. In the potted plant trials, ‘Man-
zanilla Cacereña’and ‘Gordal Sevillana’showed a high percentage
of dead branches, 83.33 and 62.50%, respectively, whereas ‘Aloreña
de Atarfe’,‘Hojiblanca’,and‘Verdial de Hu´
evar’did not produce any
dead branches (Table 2). According to necrosis length, ‘Manzanilla
Cacereña’and ‘Gordal Sevillana’were also the most susceptible to
the pathogen (necrosis lengths of 10.6 ± 3.27 and 10.38 ± 2.52 cm, re-
spectively) without significant differences between them. The remain-
ing cultivars (‘Aloreña de Atarfe’,‘Hojiblanca’,‘Manzanilla de
Sevilla’,‘Morona’,and‘Verdial de Hu´
evar’) were significantly less
susceptible (necrotic lesions of 3.06 to 5.25 cm) than the previous
two cultivars and formed a homogeneous group (P> 0.05). When
we studied the relationship between the percentage of dead branches
of the different cultivars and their canker lengths (from detached
branches and branches in vivo), the percentage of dead branches was
observed to be significantly (R
2
= 0.965, P< 0.001) related to the can-
ker lengths of inoculated potted plants, whereas the relationship was not
significant (P= 0.8740) when detached branches were used.
Fig. 1. Two-week-old colonies on potato dextrose agar of the different fungal species isolated from olive trees in Spain and Tunisia. A, Colletotrichum godetiae isolate CH-21; Band
C, Comoclathris incompta isolates CH-12 and CH-16, respectively; D, Cytospora pruinosa isolate CH-13; Eand F, Diaporthe sp. isolates CH-01 and CH-03, respectively; G,
Neofusicoccum mediterraneum isolate CH-06; Hto J, Nothophoma quercina isolates CH-04, CH-14, and CH-15, respectively; K, Pycnidia of N. mediterraneum isolate CH-06
embedded in the bark of a young olive stem; L, Canker lesion caused by N. mediterraneum isolate CH-06 on olive branch from San Agostino olive.
310 Plant Disease / Vol. 101 No. 2
Fruit inoculation with B. dothidea.When immature olive fruit were
inoculated with B. dothidea, the first symptoms were observed at 21 days
after inoculation. These were small, depressed, necrotic lesions surround-
ing the inoculation point that advanced until they covered the entire sur-
face of the fruit. ‘Hojiblanca’,‘Manzanilla de Sevilla’,and‘Morona’
olive fruit were the most resistant to B. dothidea, although there was
an extensive overlap for the AUDPC showed by the fruit of the different
cultivars (Table 2). The resistance to B. dothidea was not correlated (P=
0.6968) with the resistance to fruit rot caused by C. godetiae.
Discussion
Six fungal species belonging to six different genera were isolated
from olive plants showing branch dieback symptoms in Spain and
Tunisia. Affected trees showed defoliation or leaf wilting in young
twigs and darker, sunken areas along affected branches, which
revealed perennial cankers under the bark. Cankered zones presented
a well-defined dark line of demarcation between infected and healthy
tissues. Dieback diseases characterized by perennial cankers af-
fecting branches and trunks have been studied in many different
perennial hosts worldwide, including olive (Moral et al. 2010;
´
Urbez-Torres et al. 2013). The current work elucidates the etiology
of olive branch dieback in Spain, with a special emphasis on the path-
ogenicity of associated fungi. Moreover, the resistance of the main ta-
ble cultivars against the main causal agents of the disease has also been
evaluated.
Morphological characteristics (conidial and mycelial) were useful
to separate the isolates into four different groups (Botryosphaeria-
ceae, Cytospora,Phoma-like, and Phomopsis), in agreement with
those described for each genus (Chen et al. 2015; Liu et al. 2015; Phillips
et al. 2013; van Niekerk et al. 2004). Nevertheless, taxa of Phoma-like
species are morphologically difficult to distinguish. This is also true for
Ascochyta spp. Both of these genera have, in the past, been linked
to Didymella sexual morphs. Recently, Chen et al. (2015) clarified
the generic delimitation in the family Didymellaceae by combining
multilocus phylogenetic analyses using ITS, LSU, the RNA poly-
merase II second largest subunit (rpb2) gene, partial gene regions
of BT (tub2), and morphological observations. LSU, in particular,
has been described as one of the most helpful regions to identify
Table 1. Fungal isolates from olive trees located in Spain and Tunisia, and sequences from GenBank used in this study
GenBank Accession number
y
Species Isolate
z
Host, cultivar Symptoms Collector Origin BT ITS LSU
Botryosphaeria
dothidea
BOO046 Olea europaea
Santa Caterina
Dalmatian
disease
J. Moral Mengibar, Jaen,
Andalusia, Spain
GU292738 GU292626 n/a
Colletotrichum
godetiae
CH-21 O. europaea
Gordal Sevillana
Soapy fruit A. Trapero Palomera,
Cordoba,
Andalusia, Spain
KU973701 KU973719 KU973721
CBS 127561 Ugni molinae Twig, tip
necrosis
U. Damm Chile, South
America
JQ950093 JQ948442 n/a
Coltriccia
cinnamomea
Dai 2464 ……T. Wagner and
M. Fischer
Finland n/a -n/a AF311003
Comoclathris
incompta
CH-12 O. europaea
Picual
Branch canker J. Moral Jaen, Jaen,
Andalusia, Spain
KU973707 KU973715 KU973728
CH-16 O. europaea
Meski
Branch canker A. Rhouma Tunisia KU973708 KU973716 KU973729
CBS 467.76 O. europaea …M. M. Averskamp Greece n/a n/a GU238087
Cytospora
pruinosa
CH-13 O. europaea
Gordal Sevillana
Branch canker J. Moral Mengibar, Jaen,
Andalusia, Spain
KU973702 KU973711 KU973722
CBS 118555 O. europaea
africana
Branch canker Y. Li Wang South Africa KM034893 DQ243790 n/a
CBS 119207 ……J. Z. Groenewald …n/a n/a EU552121
Diaporthe sp. CH-01 O. europaea
Gordal Sevillana
Branch canker A. Trapero Sevilla, Sevilla,
Andalusia, Spain
KU973709 KU973717 KU973730
CH-03 O. europaea
Gordal Sevillana
Branch canker M. P ´
erez-
Rodr´
ıguez
Sevilla, Sevilla,
Andalusia, Spain
KU973710 KU973718 KU973731
UCR1395 Persea americana
Hass
Fruit M. Twizeyimana San Diego, CA JX898989 JX869042 n/a
PHAg …Branch canker M. Pilloti Rome n/a AY620999 AY621002
Ganoderma
resinaceum
GR145 ……C. L. Su Shanghai, China DQ288101 KC311374 n/a
Leptosphaeria
biglobosa
CBS 532.66 Brassica sp. ……The Netherlands KT389840 KT389541 KT389759
Neofusicoccum
mediterraneum
CH-06 O. europaea
Gordal Sevillana
Branch canker J. Moral Sevilla, Sevilla,
Andalusia, Spain
KU973703 KU973712 KU973723
BOO071 O. europaea
Gordal Sevillana
Branch canker A. Trapero Arahal, Sevilla,
Andalusia, Spain
GU292757 GU292645 KU973724
UCD720SJ Vitis vinifera Branch canker J. R. ´
Urbez-Torres California GU799475 GU799452 n/a
CBS 268.80 ……… …n/a n/a AY004336
Nothophoma
quercina
CH-04 O. europaea
Gordal Sevillana
Branch canker M. P ´
erez-
Rodr´
ıguez
Sevilla, Sevilla,
Andalusia, Spain
KU973704 KU973713 KU973725
CH-14 O. europaea
Meski
Branch canker A. Rhouma Tunisia KU973705 KU973714 KU973726
CH-15 O. europaea
Meski
Branch canker A. Rhouma Tunisia KU973706 KU973720 KU973727
CBS 633.92 Microsphaera
alphitoides,
Quercus sp.
……Ukraine GU237609 GU237900 EU754127
y
BT = b-tubulin, ITS = internal transcribed spacer, LSU = large subunit ribosomal RNA, and n/a = not available at the time of this publication.
z
CBS = Centraalbureau voor Schimmecultures, Utrech, The Netherlands; UCD = University California-Davis; and UCR = University California-Riverside. Se-
quences from GenBank used in the phylogenetic analysis are indicated in bold.
Plant Disease / Februar y 2017 311
species belonging to the Didymellaceae family. Here, the BT, ITS,
and LSU regions were needed to identify the species of each genus,
LSU being especially helpful to identify Phoma-like spp. Con-
versely, sequences of EF were unhelpful for identification. The
ITS and EF combined phylogenetic analysis was useful in describing
the diversity of species that affect olive trees in Spain and Tunisia,
confirming the five described groups based on morphology.
Concerning the Botryosphaeriaceae group, only one species,
N. mediterraneum, was recovered, the prevalent pathogen in this
work. The current study corroborates the presence of this species
in lignified tissues of olive trees in Spain, suggested as the main
causal agent of the disease (Moral et al. 2010; Romero et al. 2005).
In addition, N. mediterraneum was the most virulent species from
this study, although differences in virulence between tested isolates
were observed. These differences were only observed when pathoge-
nicity was evaluated on potted plants; they were not observed on
detached branches. This result suggests that there is a possibility of
finding genetic diversity within N. mediterraneum populations
with geographic and host preferences. In fact, the teleomorph of
N. mediterraneum was reported for the first time in the world in
the Andalusian region (Moral et al. 2015), which invites us to further
research its genetic diversity. In addition, studies conducted in Spain
by Romero (2012), in which olive plants were inoculated with
B. dothidea,Diplodia corticola,Dothiorella iberica, and N.
mediterraneum, showed that only N. mediterraneum was able to in-
duce cankers on plants with a high level of virulence. In other olive-
growing areas, a broad range of species belonging to the Botryosphaer-
iaceae family (D. mutila,D. seriata,D. iberica,L. theobromae,N.
luteum,andN. parvum) has been associated with olive trees showing
branch dieback (Sergeeva et al. 2009; Taylor et al. 2001; ´
Urbez-Torres
et al. 2013).
Among all of the fungal taxa isolated from the symptomatic wood
of olive in this study, Phoma-like species were the most diverse. All
of the Tunisian isolates in this study were identified as Phoma-like
species. In concordance with this result, today, only Phoma sp. and
Phoma fungicola have been reported as causing branch dieback of
olive trees in this country (Rhouma et al. 2010; Taieb et al. 2014).
Our Tunisian isolates were identified as N. quercina and C. incompta
but the latter was pathogenic to both detached branches and potted
plants. In agreement with this result, C. incompta has also been de-
scribed as causing branch dieback on olive in Croatia (Ivic et al.
2010), Greece (Malathrakis 1979), and Italy (Tosi and Natalini
2009; Tosi and Zazzerini 1994). Concerning negative pathogenicity
results obtained for the N. quercina isolates (both Spanish and Tunisian),
previous surveys on olive orchards in California revealed that the oc-
currence of Phoma spp. in olive is very low ( ´
Urbez-Torres et al.
2013), leading us to hypothesize that it has limited pathogenicity in
olive. It is well known that the Phoma genus contains plant-
Table 2. Susceptibility of olive cultivar inoculated with Neofusicoccum mediterraneum on detached branches and potted plants and Botryosphaeria dothidea on
detached fruit
w
Neofusicoccum mediterraneum Botryosphaeria dothidea
Detached branches Potted plants Fruit
Cultivar Canker (cm) Pycnidia (cm)
x
Canker (cm) Dead (%)
y
AUDPC
z
Aloreña de Atarfe −−4.11 b 0.00 1.70 a
Ascolana Tenera 9.52 cd 3.56 bc n/e n/e n/e
Gordal Sevillana 15.72 a 3.03 bcde 10.38 a 62.50 1.12 c
Hojiblanca 11.22 bc 3.19 bcde 3.43 b 0.00 0.59 de
Manzanilla Cacereña 6.19 e 2.74 cde 10.60 a 83.33 0.82 cd
Manzanilla de Sevilla 12.38 b 3.26 bcd 4.80 b 16.67 1.54 ab
Morona 7.64 de 2.23 e 5.25 b 16.67 0.82 cd
Ocal n/e n/e n/e n/e 1.19 bc
San Agostino 13.21 ab 5.38 a n/e n/e 0.29 e
Santa Caterina 14.03 ab 3.63 b n/e n/e 1.01 c
Verdial de Hu´
evar 6.32 e 2.24 de 3.06 b 0.00 1.04 c
w
Mean values with the same letter in a row are not significantly different according to Tukey’s honestly significant difference test (P< 0.05); −indicates that no
symptoms were observed and n/e = cultivars not evaluated in each experiment.
x
Pycnidia colonization.
y
Dead branches.
z
Area under the disease progress curve.
Table 3. Morphological characteristics and mycelial growth of Colletotrichum,Cytospora, Diaporthe, Neofusicoccum, and Phoma-like isolates obtained from
olive
Mycelia
Obverse Reverse
Species Isolate Color Zonation Margin Color Zonation Growth
y
Colletotrichum godetiae CH-21 Gray No Regular Dark gray No 6.20
Cytospora sp. CH-13 White No Irregular White No 10.69
Diaporthe sp. CH-01 White Yes Regular White green Yes 12.86
CH-03 White Yes Regular White gray Yes 14.91
Neofusicoccum mediterraneum CH-06 Light green No Regular Gray Green No 17.84
BOO071 Olive green No Irregular Olive green No 16.37
Phoma-like CH-04 Brown yellow Yes Regular Brown red Yes 3.30
CH-12
z
−−−− −−
CH-14 White pink No Regular Orange salmon No 7.95
CH-15 White pink No Regular Orange salmon No 9.28
CH-16 Green yellow No Regular Dark brown No 2.27
y
Growth (mm/day). Single conidial cultures were grown on potato dextrose agar for up to 2 weeks at 25 ± 2°C with a 12-h diurnal photoperiod of cool fluorescent
light (350 mmol m
–2
s
–1
).
z
Mycelia of this isolate was not optimum for development for its characterization.
312 Plant Disease / Vol. 101 No. 2
pathogenic species as well as numerous saprobic and endophytic spe-
cies associated with a wide range of hosts (Chen et al. 2015).
Species in the genus Diaporthe were the third most prevalent fungi
isolated in our study, with two isolates identified as Diaporthe spp.
To date, species of this genus have only been associated with olive
branch dieback in California ( ´
Urbez-Torres et al. 2013). In our study,
pathogenicity tests revealed that all isolates of Diaporthe were not
pathogenic on olive branches. However, these results contrast with
those obtained by ´
Urbez-Torres et al. (2013), who demonstrated
the pathogenicity of some Diaporthe isolates on olive branches. Be-
cause species of Diaporthe are known to be cosmopolitan and found
primarily as endophytes, parasites, and saprotrophs in a wide range of
hosts (Udayanga et al. 2011), this difference in pathogenicity in olive
between both countries could be due to variability in isolate virulence.
Many other possible factors such as the age of the host or differences in
cultivar susceptibility could also be explanatory. Thus, a broader path-
ogenicity study using a higher number of isolates of this genus and dif-
ferent plant cultivars will be required to further clarify the pathogenic
role of Diaporthe spp. as causal agents of branch dieback on olive.
Cytospora pruinosa was isolated with the least prevalence. In ad-
dition, this was the least virulent species to detached branches among
all the pathogenic fungi tested in this study, and was found to not be
pathogenic to the potted plants. To our knowledge, this is the first re-
port of C. pruinosa causing olive branch dieback in Spain. To date,
only the species C. oleina has been associated with olive branch die-
back in Greece (Rumbos 1988). Our results suggest that, although
many fungi such as Diaporthe are pathogenic to a wide range of
crops, they would only be able to induce cankers on hosts such as
weakened or stressed olive trees. Notably, no relationship was found
between the dead branches (percentage) of inoculated plants and can-
ker length on detached branches. This reinforces the idea that inocu-
lation in vivo is essential to the characterization of fungal pathogens
or even to identify differences in virulence among isolates belonging
to the same species. In addition, the nonpathogenic isolates were re-
covered from asymptomatic tissues, which indicates that they are
common endophytic fungi in olive.
Pathogenicity of Colletotrichum godetiae, the main causal agent of
olive anthracnose in Andalusia (Moral et al. 2014), was also evalu-
ated on lignified tissues. This isolate was included to test the hypoth-
esis that the syndrome of dead branches is caused by phytotoxins
produced by this pathogen and often present in rotten fruit (Moral
et al. 2009). The pathogenicity test showed that C. godetiae was
not pathogenic to olive branches. The results support the idea that
the isolates of the complex species C. acutatum do not cause the typ-
ical dieback symptoms through direct infection on lignified tissues,
because the toxin produced by the pathogen is the main cause in in-
ducing twig-branch dieback (Moral et al. 2009). However, a wide
collection of Colletotrichum spp. should be tested for pathogenicity
to elucidate the role of their toxins in branch dieback of olive.
Disease resistance of olive cultivars offer an economically sound
alternative to chemical control, with minimal environmental impact,
which can be integrated in pest management strategies. Here, we con-
ducted the first comparison of cultivar resistance to N. mediterraneum
causing cankers on olive worldwide, and to B. dothidea causing
fruit rot of olive in Spain. Trials conducted on detached branches
showed that ‘Gordal Sevillana’was the cultivar most susceptible to
Table 4. Morphological characteristics of conidia and pycnidia of Colletotrichum,Cytospora, Diaporthe, Neofusicoccum and Phoma-like isolates obtained
from olive
Conidia Pycnidia
Species Isolate L 3W(mm)
v
L/W Morphology Color Morphology Color Diameter (mm)
Colletotrichum
godetiae
CH-21
w
(13.05–) 14.35 (–15.65) ×
(4.03–) 4.59 (–5.15)
3.17 ± 0.48 Aseptate, oblong-
fusiform and ligulated
Hyaline −−−
Cytospora sp. CH-13 (4.92–) 5.47 (–6.01) ×
(1.30–) 1.61 (–1.92)
3.54 ± 0.32 Aseptate, alantoide form Hyaline Erumpent, ostiolate,
tuberculate,
globous and
velvety
Dark 278.5–745.2
Diaporthe sp. CH-01
x
(6.65–) 7.68 (–8.71) ×
(1.78–) 2.21 (–2.64)
3.60 ± 0.83 Oblong, slightly
fusiform and ligulated
Hyaline Ostiolate, globose,
erumpent and
velvety
Dark 819.5–1241.1
(19.12–) 22.50 (–25.88) −Filiform and curved Hyaline −−−
CH-03
x
(5.27–) 6.26 (–7.25) ×
(1.91–) 2.40 (–2.89)
2.73 ± 0.80 Oblong, slightly
fusiform and ligulated
Hyaline Ostiolate, globose,
erumpent and
velvety
Dark 654.5–1778.9
(9.30–) 16.80 (–24.30) −Filiform and curved Hyaline −−−
Neofusicoccum
mediterraneum
CH-06 (16.30–) 20.18 (–24.06) ×
(4.93–) 5.86 (–6.79)
3.54 ± 0.93 Fusiform to oblong-
fusiform, 0–2 septa,
usual truncated base
and thin walled
Hyaline Ostiolate, globose,
erumpent and
velvety.
Dark 401.9–1685.1
BOO071 (23.27–) 25.77 (–28.27) ×
(6.37–) 7.35 (–8.33)
3.57 ± 0.60 Aseptate and fusiform Hyaline Ostiolate, globose,
erumpent, with
conidia in cirrus
Dark 431.2–1465.3
Phoma-like CH-04
y
(4.22–) 4.93 (–5.64) ×
(3.17–) 2.70 (–3.17)
1.87 ± 0.47 Soft texture ellipsoid or
ovoid, slightly
flattened and thin-
walled
Dark Branched, wooded,
velvety, sprawled
Dark −
CH-12 (2.65–) 3.0 (–3.35) ×
(0.92–) 1.17 (–1.42)
2.66 ± 0.56 Aseptate, oblong to
oblong-fusiform
Hyaline Ostiolate, globose Dark 60.7–168.5
CH-14 (4.23–) 4.69 (–5.15) ×
(1.15–) 1.78 (–2.06)
2.69 ± 0.48 Aseptate, oblong and
thin-walled
Hyaline Ostiolate, globose Dark 26.1–142.1
CH-15
z
−−−−Ostiolate, globose Dark 22.8–73.8
CH-16 (2.73–) 3.20 (–3.67) ×
(0.71–) 0.83 (–0.95)
2.01 ± 0.25 Aseptate and oblong Hyaline Ostiolate, globose Dark 27.7–83.8
v
Mean and range values: length (L) by width (W) (mm). Extremes of the conidial measurements are shown inside parenthesis.
w
This isolate produces conidia in acervuli instead of in pycnidia.
x
Isolates with both conidia types (aand b).
y
Isolate with irregular pycnidia.
z
Conidia were not observed for this isolate.
Plant Disease / Februar y 2017 313
N. mediterraneum, followed by ‘Santa Caterina’and ‘San Agostino’,
whereas ‘Manzanilla Cacereña’was the most resistant, followed by
‘Verdial de Hu´
evar’and ‘Morona’. Detached branches could be under
great stress and does not behave physiologically in the same way as
a branch attached to a tree. Thus, results obtained from detached
branches could not be absolutely representative of cultivar resistance.
In this way, cultivar resistance was also evaluated by using potted
plants. In this case, inverted results than those obtained by using
Fig. 3. Phylogenetic analysis of taxa for large subunit nuclear ribosomal DNA sequences. The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei
1987). The optimal tree with the sum of branch length =0.82306726 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test
(2,000 replicates) are shown next to the branches (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used
to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura two-parameter method (Kimura 1980) and are in the units of the number of base
substitutions per site. The rate variation among sites was modeled with a gdistribution (shape parameter =1). The analysis involved 17 nucleotide sequences. All positions
containing gaps and missing data were eliminated. There were a total of 782 positions in the final data set. Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013).
Fig. 2. Phylogenetic analysis of taxa for the combined alignment of internal transcribed spacer and b-tubulin sequences. The evolutionary history was inferredusing the neighbor-joining
method (Saitou and Nei1987). The optimal tree with the sum of branchlength =2.64571884 is shown. The percentage of replicate trees in which the associated taxa clustered together
in the bootstrap test (2,000 replicates) are shown next to the branches (Felsenstein1985). The tree is drawn to scale, with branch lengths in the same units as those ofthe evolutionary
distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura two-parameter method(Kimura 1980) and are in the units of the number of
base substitutions per site. The rate variation among sites was modeled with a gdistribution (shape parameter =1). The analysis involved 18 nucleotide sequences. All positions
containing gaps and missing data were eliminated. There were a total of 667 positions in the final data set. Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013).
314 Plant Disease / Vol. 101 No. 2
detached branches were observed on potted plants, where ‘Manzanilla
Cacereña’was the most susceptible cultivar to N. mediterraneum,fol-
lowed by ‘Gordal Sevillana’.‘Verdial de Hu´
evar’was the most resis-
tant cultivar. The last results are in concordance with those obtained
by Romero (2012), who indicated that ‘Gordal Sevillana’was more
susceptible than ‘Picual’when live plants were inoculated with
N. mediterraneum. Concerning B. dothidea, results revealed that
‘Aloreña de Atarfe’was the most susceptible cultivar, whereas ‘Hoji-
blanca’and ‘San Agostino’were the most resistant. These results
complement those obtained by Latinovi´
c et al. (2013), who described
highly resistant native ‘Crnjaka’and ‘Gloginja’olive. It is interesting to
note that the resistance of green olive fruit to B. dothidea is not correlated
with the resistance of ripe fruit to C. godetiae, showing that different re-
sistance mechanisms are at work in both types of fruit (Moral et al. 2014).
Cultivar resistance to Botryosphaeriaceae species affecting lig-
nified tissues of perennial crops has also been studied in mango
(Mangifera indica L) and pistachio (Pistachio vera L.)(Parfittetal.
2003). According to our results, ‘Spanish Hojiblanca’,‘Manzanilla
Cacereña’,and‘Verdial de Hu ´
evar’olive showed a good level of resis-
tance to branch dieback, making cultivar resistance a potential alternative
tool against both branch dieback types.
This study has resulted in significant information regarding the
etiology of branch dieback of olive as well as cultivar resistance to
N. mediterraneum and B. dothidea. The variation found among fungal
species recovered from affected olive trees with cankers and branch die-
back symptoms should be taken into account to develop optimum con-
trol strategies. Therefore, further research is needed to elucidate the role
of many fungal species associated with branch dieback of olive, as well
as the environmental and cultural practices that influence the disease.
Acknowledgments
This research was funded by the Spanish Ministry of Education and Science
(project AGL2004-7495) and by the Andalusian Regional Government (Project
P08-AGR-03635). Both projects were co-financed by the European Union FEDER
Funds. J. Moral holds a Marie Skłodowska Curie fellowship launched by the Eu-
ropean Union’s H2020 (contract number 658579). C. Agust´
ı-Brisach is the holder
of a “Juan de la Cierva-Formaci ´
on”fellowship from MINECO. We thank J. A.
Layosa and F. Luque for skillful technical assistance; and C. Cunningham,
K. Tomari, and T. J. Michailides for critical review of the manuscript.
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