Content uploaded by Juan Moral
Author content
All content in this area was uploaded by Juan Moral on Jul 04, 2018
Content may be subject to copyright.
1
Identification of Fungal Species Associated with Branch Dieback of
1
Olive and Resistance of Table Cultivars to Neofusicoccum
2
mediterraneum and Botryosphaeria dothidea
3
4
Juan Moral
1,2,*
, Carlos Agustí-Brisach
1,*
, Mario Pérez-Rodríguez
1
, Carlos Xaviér
1
, M.
5
Carmen Raya
1
, Ali Rhouma
3
, and Antonio Trapero
1
6
7
1
Departamento de Agronomía, ETSIAM, Universidad de Córdoba, Campus de
8
Rabanales, Edif. C4, 14071 Córdoba, Spain.
9
2
University of California, Davis, Kearney Agricultural Research and Extension Center,
10
Parlier CA 93648
11
3
Institute de l’Olivier, Mahrajène, BP208, 1082, Tunisie.
12
*These authors contributed equally to this article
13
2
email: jmoral@ucdavis.edu
14
ABSTRACT
15
Moral, J., Agustí-Brisach, C., Pérez-Rodríguez, M., Xaviér, C. J., Raya, M.C., Rhouma,
16
A., and Trapero, A. 2016. Identification of fungal species associated with branch
17
dieback of olive and resistance of table cultivars to Neofusicoccum mediterraneum and
18
Botryosphaeria dothidea. Plant Dis. XX:XXX-XXX.
19
Over two consecutive seasons, 16 olive orchards with trees exhibiting dieback
20
symptoms on branches were surveyed in Southern Spain. The six dominant fungal
21
Page 1 of 46
2
species recovered were characterized by means of phenotypic observations, DNA
22
analysis (by sequencing of the ITS, TUB, and LSU regions), and pathogenicity tests.
23
Additionally, three isolates collected from Tunisian olive trees showing similar dieback
24
symptoms, one isolate of Colletotrichum godetiae, and a reference isolates of
25
Neofusicoccum mediterraneum were included. The resistance of the 11 most important
26
table cultivars to N. mediterraneum and Botryosphaeria dothidea, the causal agent of
27
“escudete” (small shield) of fruit, was studied by the inoculation of branches and
28
immature fruits, respectively. The species Cytospora pruinosa, N. mediterraneum,
29
Nothophoma quercina, Comoclathris incompta, and Diaporthe sp. were identified. Only
30
N. mediterraneum and Co. incompta were able to induce the typical dieback symptoms
31
and cankers that affected the development of the plants. The species N. mediterraneum
32
was the most virulent among the evaluated species, although differences in virulence
33
among its isolates were observed. The remaining fungal species were weakly
34
pathogenic to nonpathogenic on plants. According to resistance tests, cvs. Gordal
35
Sevillana and Manzanilla Cacereña were the most susceptible to branch dieback caused
36
by N. mediterraneum. Furthermore, the fruits of cvs. Aloreña de Atarfe and Manzanilla
37
de Sevilla were the most susceptible to B. dothidea. Knowledge of the etiology and
38
cultivar resistance of these diseases will help to establish better control measures.
39
40
KEYWORDS
41
Botryosphaeriaceae, canker, dieback, Olea europaea,
42
43
44
Page 2 of 46
3
INTRODUCTION
45
The cultivated olive (Olea europaea subsp. europaea L) is the most important perennial
46
crop in Spain. It leads the world in olive production, generating about 43% of the
47
world’s olives. The Spanish olive industry accounts for 25% of the global acreage
48
designated for olive production, occupying 2.5 × 10
6
ha (for both table fruit and oil).
49
Approximately 65% of this land lies in the Andalusia region of the southern Iberian
50
Peninsula, in itself producing 85% of the total production for Spain (Barranco et al.,
51
2008). Global table fruit production is around 2.3 × 10
6
tons per year, 23% of which
52
come from Spain (MAGRAMA, 2016). The most important table cultivars are ‘Gordal
53
Sevillana’ and ‘Manzanilla de Sevilla’, both comprising ≈ 85% of table fruit production.
54
In addition, other table cultivars such as ‘Aloreña’ and ‘Morona’ have a secondary
55
importance, but their fruits are appreciated for their organoleptic properties. Finally,
56
some cultivars such as ‘Hojiblanca’, ‘Manzanilla Cacereña’, and ‘Verdial de Huévar’
57
have a twofold purpose, both as table fruit and as oil, while some other cultivars as
58
‘Picual’ are used exclusively for oil production (Rallo et al., 2005).
59
At the beginning of the 2000s, a serious disease showing typical dieback symptoms was
60
observed in olive orchards in the Andalusia region, where ‘Gordal Sevillana’ was the
61
most affected cultivar (Romero et al., 2005). Similar symptoms were also observed
62
affecting other cultivars such as ‘Arbequina’, ‘Manzanilla de Sevilla’, and ‘Picual’.
63
Differences in susceptibility among cultivars to the disease are evident in the field, but
64
knowledge regarding the resistance of cultivars to twig-branch dieback is unknown.
65
Affected trees showed an abundance of dead twigs and wilted leaves that remained
66
attached to blighted branches which were generally associated with the decline of entire
67
young stems and/or older branches (Moral et al., 2010; Úrbez-Torres et al., 2013;
68
Page 3 of 46
4
Romero et al., 2005). The branches showing cankers are less tolerant to water stress and
69
had an insufficient water and nutrient flow through both xylem and phloem vessels.
70
Consequently, characteristic dieback symptoms such as bud mortality, leaf chlorosis,
71
fruit rot, and twig dieback occur when water and nutrient demand exceeds the
72
conductive capacity of the vascular tissues (Úrbez-Torres et al., 2013).
73
Olives can be affected by numerous species of Botryosphaeriaceae, including species
74
belonging to Botryosphaeria and Neofusicoccum that are well known to cause cankers,
75
dieback, and rot of mature and immature fruit (Carlucci et al., 2013; Lazzizera et al.,
76
2008a, 2008b; Moral et al., 2008a, 2010; Úrbez-Torres et al., 2013). However, further
77
studies may elucidate the etiology of these diseases in Spain, and the relationship
78
between affected host tissue and pathogen species.
79
The species N. luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was
80
described causing stem cankers and tip dieback in olive branches in New Zealand
81
(Taylor, 2001), and causing leaf necrosis in Australia (Sergeeva et al., 2009). In Spain,
82
Moral et al. (2010) observed that N. mediterraneum Crous, M.J. Wingf. & A.J.L.
83
Phillips was the prevalent species associated with branch dieback mainly on the cv.
84
Gordal Sevillana. The Botryosphaeriaceae species Diplodia mutila (Fr. :Fr.) Fr., D.
85
seriata De Not., Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves, Lasiodiplodia
86
theobromae (Pat.) Griffon & Maubl, N. luteum, N. parvum, and N. vitifusiforme (Van
87
Niekerk & Crous) Crous, Slippers & A.J.L. Phillips were also reported to cause branch
88
dieback and blight, and eventual death of shoots in different olive-growing areas (Moral
89
et al., 2010; Carlucci et al., 2013; Úrbez-Torres et al., 2013).
90
On olive drupes, D. olivarum A.J.L. Phillips, Frisullo & Lazzizera and D. seriata were
91
reported causing rot on mature fruits in Italy (Lazzizera et al., 2008a) and in Spain
92
Page 4 of 46
5
(Moral et al., 2008b), respectively. In Australia, N. luteum was also observed causing
93
rot of mature fruits (Sergeeva et al., 2009). Conversely, because immature olive fruits
94
are highly resistant to fungi, no significant pathogens have been observed causing rot on
95
them with the exception of B. dothidea (Moug.) Ces. & De Not. (Moral et al., 2008b;
96
2010). This pathogen is the causal agent of Dalmatian disease of olive, which can be
97
found in most of the Mediterranean basin (Iannotta et al., 2007; Latinovic et al., 2013;
98
Moral et al., 2010; Zachos et al., 1983). Botryosphaeria dothidea produces a sunken,
99
necrotic, and circular lesion (~8mm in diameter, never larger than 1 cm) with a sharp
100
edge delineating infected and healthy fruit tissues; this appearance is called “Escudete”
101
(or small shield) in Portuguese and Spanish. As the disease progresses, the necrotic
102
spots expand and eventually encompass the entire fruit. Then when the fruit matures, it
103
falls to the ground and is mummified (Moral et al., 2010). Overall, the incidence of
104
olive fruit showing the Dalmatian symptoms is relatively low, but frequently, it exceeds
105
the tolerance level for the 'Extra' Class standard for the olive fruit, which is usually at a
106
2 or 4% level, according to the Spanish Royal Decree 1230/2001 or the “Codex
107
Alimentarius” of the FAO, respectively.
108
Botryosphaeriaceae asexual stage is predominant in the majority of its hosts. In olive,
109
the sexual stage has been only reported in few cases for B. dothidea and N.
110
mediterraneum in California and Spain, respectively (Moral et al., 2010; 2015).
111
Recently, Eldesouki (2013) elucidated the Dalmatian disease cycle, in which the
112
mosquito Prolasioptera berlasiana, a predator of olive fly eggs (Bactrocera oleae),
113
carries the B. dothidea spores in a mycangium (a specialized structure, like a pocket).
114
The mosquito is attracted to ovipositor punctures on the olive surface made by the olive
115
fly. When the mosquito deposits its own egg adjacent to the fly egg, it also inoculates
116
the puncture with the fungus. Because the mosquito cannot penetrate intact fruits, the
117
Page 5 of 46
6
control of olive flies incidentally involves the control of the Dalmatian disease (Moral et
118
al., 2016). There is, however, little information on the control of Dalmatian disease by
119
the use of resistant olive cultivars. In Montenegro, Latinovic et al. (2013) studied the
120
resistance of olive cultivars infected with B. dothidea, demonstrating that the cultivars
121
‘Crnjaka’ and ‘Gloginja’ (native cultivars), and ‘Pendolino’ and ‘Cassanesse’ (Italian
122
cultivars) were highly resistant to the disease. These authors evaluated the resistance of
123
only one Spanish cultivar, ‘Manzanilla,’ but the identity of this cultivar is unclear as the
124
name “Manzanilla” includes more than 20 cultivars, such as ‘Manzanilla de Sevilla’,
125
‘Manzanilla Cacereña’, etcetera (Barranco and Rallo, 2000). Even so, the resistance of a
126
broad range of Spanish olive cultivars to B. dothidea and N. mediterraneum has not
127
been evaluated yet in Spain.
128
During the last few decades, other pathogens have also been associated with olive twig-
129
branch dieback. For example, the species Cytospora oleina Berl. and Eutypa lata (Pers.)
130
Tul. & C. Tul. have been associated with olive cankers and consequent branch dieback
131
in Greece (Rumbos, 1988; 1993). The latter pathogen was recently reported in
132
California and Italy causing dieback of olive trees (Tosi and Natalini, 2009; Úrbez-
133
Torres et al., 2013). The species Comoclathris incompta (Sacc. & Martelli) Ariyaw. &
134
K.D. Hyde has also been described causing branch dieback in olives in Croatia, Greece,
135
and Italy (Ivic et al., 2010; Malathrakis, 1979; Tosi and Zazzerini, 1994). Recently, P.
136
fungicola has been associated with branch dieback of olive trees in Tunisia (Rhouma et
137
al., 2010; Taieb et al., 2014). In addition, several fungi, including Diaporthe viticola
138
Nitschke, Diatrype oregonensis (Wehm.) Rappaz, Diat. stigma (Hoffm.) Fr.,
139
Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai,
140
Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. & Mugnai) Crous & W.
141
Gams, Phoma sp., Phomopsis (Sacc.) Bubák sp. (synonym Diaporthe Nitschke),
142
Page 6 of 46
7
Trametes versicolor (L.) Lloyd and Schizophyllum commune Fr., have also been
143
associated with olive branch dieback, stem canker, and twig necrosis in California
144
(Úrbez-Torres et al., 2013). In southern Italy, N. parvum, Pleurostomophora
145
richardsiae (Nannf.) L. Mostert, W. Gams & Crous, six species belonging to
146
Phaeoacremonium, and Pseudophaeomoniella oleicola Nigro, Antelmi & Crous and Ps.
147
oleae Nigro, Antelmi & Crous were isolated from the wood of olives displaying wilting
148
and branch dieback (Carlucci et al., 2013, 2015; Crous et al., 2015). Colletotrichum
149
species, the causal agents of olive anthracnose, have also been implicated as responsible
150
for leaf wilting and branch dieback symptoms (Moral et al., 2014). Although these fungi
151
affect fruits, preliminary studies demonstrated that the toxic substance produced by
152
Colletotrichum spp. on rotten fruit are able to incite leaf wilting and branch dieback
153
(Moral et al., 2009). In the latter study, for example, olive plants without fruits were
154
infected by the pathogen but they did not show any disease symptoms. However, the
155
plant inoculations were conducted by using a conidial suspension and not using the
156
conventional mycelium plugs of the pathogen (Moral et al., 2009).
157
In Spain, little attention has been given to olive branch dieback, nor the Dalmatian
158
disease. Nevertheless, both diseases have increasingly become concerns among growers
159
in the main table olive-producing areas of the Andalusia region. As the determination of
160
the etiology, epidemiology, and control of these diseases are required, the objectives of
161
the current study were to (i) identify on the basis of morphology and molecular
162
phylogeny the different fungal species associated with the olive twig-branch syndrome;
163
(ii) evaluate the pathogenicity of the different fungi associated with the olive twig-
164
branch dieback; and (iii) evaluate the resistance of the most common table cultivars to
165
N. mediterraneum and B. dothidea, the causal agents of twig-branch dieback and
166
Dalmatian disease, respectively.
167
Page 7 of 46
8
MATERIAL AND METHODS
168
Field surveys and fungal isolation
169
In 2009 and 2010, field surveys were conducted in orchards of cvs. ‘Gordal Sevillana’
170
and ‘Picual’ located in the Andalusian provinces of Jaén and Sevilla (Table 1). One
171
hundred sixty-three samples in total were collected from 16 olive orchards, each being
172
25 years or older. Branches and twigs from olive trees showing the typical symptoms of
173
branch dieback such as cankers, internal wood necrosis, black vascular streaking or
174
discolored tissues were collected. Parafilm (Parafilm, Menasha, WI) was used to seal
175
both ends of all samples, then samples were placed in black plastic bags, and kept at 4ºC
176
until isolations were made.
177
Branches and twigs were surface disinfected with 50 % ethanol. The outer bark was
178
removed with a sterile scalpel. Small pieces from the edge between healthy and affected
179
wood tissue were plated on Potato Dextrose Agar (PDA) (Biokar-Diagnostics, Zac de
180
Ther, France) acidified with lactic acid (2.5 ml of 25% [vol/vol] per liter of medium).
181
Petri dishes were incubated at 23-27°C under a 12-h photoperiod of cool fluorescent
182
light (350 µmol m
-2
s
-1
) until colonies were large enough to be examined. To obtain pure
183
cultures, mycelial fragments from the margin of the fungal colonies were transferred to
184
acidified PDA and incubated as described before. Several isolates collected in affected
185
‘Meski’ orchards from Tunisia were also included in this study (Table 1). Fungal
186
species were isolated for further study from 10 olive trees from three different orchards.
187
The studied isolates are maintained in the collection of the Departamento de
188
Agronomía, Universidad de Córdoba, Spain.
189
190
191
Page 8 of 46
9
Morphological characterization
192
Based on morphological characters, fungal species were first identified to genus.
193
Pycnidial and conidial characteristics such as shape, size, color, presence or absence of
194
septa were recorded from the colonies developed in vitro. For morphological
195
identification, single conidial cultures were derived using the serial dilution method
196
(Dhingra and Sinclair, 1995) and grown on PDA for up to two weeks at 25±2°C with a
197
12-h photoperiod of cool fluorescent light (350 µmol m
-2
s
-1
). To induce pycnidia
198
development, the same isolates were cultured on sterile cv. Gordal Sevillana leaves,
199
which were previously autoclaved at 120°C for 20 min and placed into Petri dishes
200
containing 5 ml of molten PDA (Moral et al., 2009). Petri dishes were incubated as
201
described above for 10 days. Three Petri dishes per isolate were used. The experiment
202
was performed twice.
203
Fungal structures were measured using a Nikon Eclipse 80i microscope and images
204
were captured using the NIS-Element software (Nikon Corp., Tokyo, Japan). Conidial
205
masses were observed from pycnidia mounted in water. Color (using a color scale)
206
(Kornerup and Wanscher, 1963), morphology and diameter of pycnidia from these
207
isolates were recorded. For conidial measurements, mycelial plugs were removed from
208
the Petri dishes, placed on slides with a drop of 0.005% acid fuchsine in lactoglycerol
209
(1:1:1 lactic acid, glycerol, and water) and covered. For each isolate, 30 measurements
210
were obtained for each structure. The averages of length and width and the length/width
211
relation were calculated. Characteristics of conidia (color, shape, and septation) and
212
mycelia (texture, density, color, and zonation) were also recorded after 14 days of
213
incubation at 23–27°C in the dark (Barnett and Hunter, 1998; Sutton, 1980).
214
215
Page 9 of 46
10
DNA extraction, amplification, and phylogenetic analysis
216
One to three isolates were selected based on their morphology, as representative of each
217
of three fungal taxa, and their identification was confirmed by using molecular
218
techniques (Table 1). Fungal isolates were grown on PDA for 14 days. FastDNA Kit
219
(BIO 101, Inc., Vista, CA) was used to extract total genomic DNA. The nuclear
220
ribosomal DNA repeats, including the internal transcribed spacer (ITS) 1, 5.8S rRNA,
221
ITS5, and portions of the genes encoding both small and large subunit rRNAs, were
222
amplified using primers ITS1 and ITS4 (White et al., 1990). The partial large subunit
223
nrDNA (LSU) was amplified with the primer pair LR0R and LR7 (Rehner & Samuels
224
1994; Vilgalys and Hester 1990). Oligonucleotide primers Bt2a and Bt2b were used to
225
amplify a portion of the β-tubulin (BT) gene (Glass and Donaldson, 1995). Finally,
226
amplification of part of the translation Elongation Factor 1-α (EF) gene was done with
227
the primers EF1-728F and EF1-986R (Carbone et al., 1999). The polymerase chain
228
reaction (PCR) of BT, EF ITS, and LSU was conducted according to previous studies
229
(Chen et al., 2015; Lazzizera et al., 2008b; Moral et al., 2010). The PCR products were
230
purified with an Ultra Clean PCR Clean-Up Kit (MO BIO Laboratories, Inc., Solana
231
Beach, CA). The resulting amplicons were sequenced in both directions using an
232
automated sequencer by the University of Córdoba sequencing facility (ABI Prism
233
3130XL Genetic Analyzer; Applied Biosystems). The nucleotide sequences were read
234
and edited with FinchTV 1·4·0. Sequences were assembled and edited to resolve
235
ambiguities using the program SeqMan (DNASTAR Lasergene, Madison, WI, USA),
236
and consensus sequences for all isolates were compiled into a single file (Fasta format).
237
Phylogenetic analysis with all isolates was first conducted with the BT, EF, ITS, and
238
LSU datasets individually, and the four topologies were compared. A partition
239
Page 10 of 46
11
homogeneity test was performed to determine whether BT, EF, ITS, and LSU data sets
240
could be combined. The combined alignment of the two loci BT and ITS was first
241
analyzed for inferring the phylogeny of the isolates. Phylogenetic analysis on the LSU
242
single-locus alignment was used to elucidate the organismal phylogeny. GenBank
243
sequences (Table 1) from different species of Neofusicoccum, Colletotrichum,
244
Cytospora, Phoma-like and/or Diaporthe were selected based on their high similarity
245
with our query sequences using MegaBLAST. Ganoderma resinaceum (GR145) and
246
Coltriccia cinnamomea (Dai 2464) were used as the outgroups of the combined
247
alignment of the two loci BT and ITS, and the LSU single-locus alignment. These were
248
added to the sequences obtained and aligned using CLUSTALW v. 2.0.11 (Larkin et al.
249
2007). The genetic distances were calculated using the Kimura 2-parameter model. For
250
phylogenetic inference, the neighbour-joining (NJ) method was used (Saitou and Nei,
251
1987). The NJ tree and the statistical confidence of a particular group of sequences in
252
the tree, evaluated by bootstrap test (2,000 pseudoreplicates), were performed using the
253
computer program MEGA 6 (Tamura et al. 2013). Sequences derived in this study were
254
lodged at GenBank. GenBank accession numbers of the strains collected during this
255
study are listed in Table 1.
256
Pathogenicity tests
257
One representative isolate of each identified species was inoculated towards to
258
determine their pathogenicity capacity. In addition, one C. godetiae isolate (CH-21),
259
which belongs to the C. acutatum species complex and is the dominant species in the
260
Andalusian population of the pathogen (Moral et al., 2014), was selected to determine
261
pathogenicity on olive detached branches or potted plants (Table 1).
262
Page 11 of 46
12
Pathogenicity on detached branches. In March 2010, branch segments 14 to 18 cm long
263
and 1.0 to 1.5 cm in diameter were collected from ‘Gordal Sevillana’ trees from “The
264
Olive Cultivars Garden” orchard that belongs to the Andalusian Institute for Research
265
and Formation in Agriculture and Fishery (IFAPA in Spanish) and is located in
266
Mengíbar (Jaén province, Andalusia region). The branch segments were sealed at both
267
ends with Parafilm (Parafilm, Menasha, WI) to reduce desiccation, and bark surfaces
268
were sterilized with 70% ethanol at the point of inoculation, situated in the middle of
269
each branch. In order to remove the outer bark, yet leave the inner bark intact, 10-mm
270
diameter holes were made in the bark using a cork borer. In the cavities, a 10-mm
271
diameter mycelium plug of each isolate was inserted such that the inoculum was placed
272
in direct contact with vascular tissues as described by Moral et al. (2010), and then the
273
inoculated area was wrapped with Parafilm. A total of 11 representative isolates
274
belonging to six species were individually tested using five replicate branches per
275
isolate. The experiment was repeated three times and five branches were treated with
276
acidified PDA as control. Inoculated and control branches were then incubated in humid
277
chambers (plastic containers, 30 by 23 by 6 cm, with 100% RH obtained by 300 ml
278
water added) at room temperature (26 to 33°C) under continuous cool fluorescent lights
279
(350 µmol m
-2
s
-1
) for 30 days. Humid chambers were placed inside at room
280
temperature in a completely randomized design. For each branch, evaluation of the
281
lesion length and the length of the colonized bark surface bearing pycnidia, and
282
calculation of the relative (%) affected length of each branch were obtained according to
283
Moral et al. (2010). Small wood fragments that form the margin of the affected area of
284
each inoculated branch were plated on acidified PDA to isolate the pathogens.
285
Pathogenicity on potted plants. Five-year-old potted plants cv. Gordal Sevillana were
286
obtained from a commercial nursery. Each plant was inoculated with mycelium plugs as
287
Page 12 of 46
13
described above for detached branches. The same 11 isolates were inoculated using
288
three replicate plants per isolate and four branches per plant. After inoculation, plants
289
were placed in a greenhouse at 25 to 30°C in a completely randomized design and were
290
irrigated as needed. The area around the experiment was abundantly irrigated to
291
increase the RH. Lesion length and percentage (%) of dead branches were assessed 90
292
days after inoculation. The pathogens were isolated from the olive branches as
293
described above. The experiment was repeated three times and five branches from the
294
same plant were treated with acidified PDA as control in each replicate of the
295
experiment.
296
Evaluation of cultivar resistance
297
To evaluate the resistance of different table cultivars to N. mediterraneum, which was
298
the most virulent species of the previous experiments, branches and five-year-old plants
299
of 10 table cultivars were inoculated using the reference isolate N. mediterraneum
300
BOO071. In the same way, the resistance of cultivars to B. dothidea was evaluated
301
using the reference isolate B. dothidea BOO046 (Moral et al., 2010).
302
Branch Inoculation with N. mediterraneum. In March 2010, branch segments 14 to
303
18cm long and 1.0 to 1.5 cm in diameter were cut from 10 table olive cultivars growing
304
at “The Cultivar Garden” orchard (Table 4). Sampling and inoculation were conducted
305
as described above. Ten branches per cultivar were inoculated and the experiment was
306
conducted three times. Likewise, other 10 branches per replicate were treated with
307
acidified PDA as control. A total of 270 branches were inoculated with N.
308
mediterraneum and 90 branches were used as control. All the branches were incubated
309
in humid chambers as described above. Evaluation of lesion length and the length of the
310
colonized bark surface bearing pycnidia, and calculation of the relative (%) affected
311
Page 13 of 46
14
length of each branch were obtained according to Moral et al. (2010). The pathogen was
312
re-isolated from the branches as described above.
313
Plant Inoculation with N. mediterraneum. Five-year-old potted plants of seven table
314
cultivars were obtained from a commercial nursery in Cordoba province from Andalusia
315
(Table 4). Each plant was inoculated with N. mediterraneum using mycelial plugs as
316
described above. Three replicate plants and four branches of each one per cultivar were
317
inoculated. Five branches from the same plant were treated with acidified PDA as
318
control in each replicate (plant) of the experiment. Inoculated plants were then
319
incubated in a greenhouse from 25 to 30°C in a completely randomized design, and
320
irrigated abundantly to increase the RH. The percent (%) of dead branches and the
321
lesion length of inoculated branches were assessed 90 days after inoculation. The
322
experiment was repeated four times. The pathogen was isolated from the branches as
323
described above.
324
Fruit Inoculation with B. dothidea. In September 2010, immature fruit of 10 table
325
cultivars were collected from “The Cultivar Garden” orchard (Table 4). Fruits were
326
washed, disinfested, and inoculated according to Moral et al. (2010). Inoculated and
327
control fruit (PDA plugs without mycelia) were incubated in humid chambers as
328
described above. Disease severity was assessed every two weeks for two months using
329
the 0 to 5 rating scale previously described by Moral et al. (2008a). Disease severity
330
index (DSI) was calculated according to the formula: DSI = (Σni ×i)/ N, where i
331
represents severity (0 to 5), ni is the number of fruit with the severity of i, and N is the
332
total number of fruit (Moral et al., 2008a). The Area Under the Disease Progress Curve
333
(AUDPC) was calculated by trapezoidal integration of DI values over time. There were
334
Page 14 of 46
15
three replicates (humid chambers) per treatment and 30 fruit per replicate. Treatments
335
were arranged in a completely randomized design.
336
Statistical analysis
337
Analysis of variance (ANOVA) and means comparison tests were conducted to
338
determine the differences in virulence among isolates and in resistance among cultivars.
339
For the different analysis conducted, dependent variables were lesion length to evaluate
340
differences in virulence among isolates; lesion length and pycnidia colonization or dead
341
branches (%) to evaluate differences in resistance among cultivars by using detached
342
branches or potted plants, respectively; and AUDPC to evaluate differences in
343
resistance among cultivars by using detached fruits. When ANOVA showed significant
344
differences, the treatment means were compared according to Tukey’s HSD test at P =
345
0.05. The non-pathogenic isolates were excluded from the statistical analysis. The data
346
were logarithmically transformed to meet the homogeneity of the variances or
347
normality. Because the data of cultivar resistance to N. mediterraneum did not show
348
homogeneity of variances, even with the logarithmic transformation, they were analyzed
349
using the Kruskal-Wallis nonparametric test and the cultivars were compared according
350
to Dunn´s test at P = 0.05. Linear regression was used to study the relationship between
351
dependent variables of this study. In addition, we studied the relation between fruit
352
resistance to B. dothidea and the susceptibility to fruit rot caused by Colletotrichum
353
species (Moral et al., 2014). All the data were analyzed by using Statistix 10 (Analytical
354
software, 2013).
355
356
357
358
Page 15 of 46
16
RESULTS
359
Collection of fungal isolates
360
In total, 120 fungal isolates from affected olive trees showing twig-branch dieback were
361
recovered in Spanish olive orchards. Overall, olive twig death was usually associated
362
with cankered stems. Amongst the collected isolates, five fungal groups were clearly
363
differentiated, the most frequent being Botryosphaeriaceae species (mainly N.
364
mediterraneum), followed by Cytospora, a Phoma-like species (two sub-groups), and
365
Diaporthe species. Representative isolates from each of the five fungal groups were
366
selected for further studies. In addition, a fungal group of isolates that were frequently
367
isolated from affected orchards in Tunisia was included.
368
Morphological characterization
369
All studied isolates showed mycelial growth and sporulation within 14 days of
370
incubation on olive leaves except one Phoma-like isolate, CH-12, which showed very
371
poor mycelial growth, but developed some pycnidia around the original mycelium plug.
372
Based on their appearance in culture, the isolates were again assigned to five main
373
fungal groups (Table 2). The first group comprised a Botryosphaeriaceae species with a
374
Neofusicoccum anamorph (isolates BOO071 and CH-06), this species was characterized
375
by floccose to felted aerial mycelium showing colors that varied from light to olive
376
green on acidified PDA. Conidia were hyaline, aseptate, and fusiform. Pycnidia were
377
dark, ostiolate, globose, and erumpent. Two groups (Group 2 and 3) were clearly
378
differentiated, belonging to Phoma-like isolates: Group 2 (isolates CH-04 and CH-16)
379
was characterized by brown to yellowish mycelium with a regular margin. Conidia were
380
dark, ellipsoid or ovoid, slightly flattened, and thin-walled. Pycnidia were irregular,
381
branched, wooded, velvety, and sprawled; Group 3 (isolates CH-14 and CH-15),
382
Page 16 of 46
17
showed flat, fine, medium density to very dense mycelium, the color varied from white
383
to pink, or green to yellow, with a regular colony margin. Conidia were hyaline,
384
aseptate, and oblong. Pycnidia were dark, ostiolate, and globose. A fourth group
385
included a Diaporthe species that was characterized by having white, cottony, slow-
386
growing, raised mycelium with a regular colony margin. In this fourth group, two types
387
of hyaline conidia were observed: oblong, slightly fusiform, and ligulated conidia (α);
388
and filiform and curved conidia (β). Pycnidia were dark, ostiolate, globose, erumpent,
389
and velvety. Finally, Cytospora isolates formed a fifth group characterized by white,
390
flat, felty, texture uniform mycelium with an irregular colony margin. Conidia were
391
hyaline, aseptate, and with an alantoide form. Pycnidia were dark, erumpent, ostiolate,
392
tuberculate, globous, and velvet. (Tables 2 and 3; Fig. 1).
393
Molecular characterization
394
To confirm the identification based on morphology, BT, EF, ITS, and LSU
395
regions were obtained for all isolates studied. The combined alignment of BT and ITS
396
included 18 taxa (including the outgroup G. resinaceum) (Fig. 2). In this phylogenetic
397
tree, combined sequences of the genera Colletotrichum (Isolate CH-21), Cytospora
398
(Isolate CH-13), Diaporthe (Isolates CH-01 and CH-03), Neofusicoccum (Isolates CH-
399
06 and BOO071) and Nothophoma (Isolates CH-4, CH-14, and CH-15) formed five
400
well-supported clades, which Bootstrap Support ranged from 94 to 100%. Each clade
401
incorporated one representative isolate of C. godetiae (CBS 127561), Cy. pruinosa (Fr.)
402
Sacc. (CBS 118555), Diaporthe sp. (UCR1395), N. mediterraneum (UCD720SJ) and
403
No. quercina (Syd.) Q. Chen & L. Cai (CBS 633.92) for Colletotrichum, Cytospora,
404
Diaporthe, Neofusicoccum and Nothophoma clades, respectively. However, the isolates
405
CH-12 and CH-16 clustered in a sub-clade close to Leptosphaeria biglobosa Shoemaker
406
Page 17 of 46
18
& H. Brun (CBS 532.66) with a low BS value (BS = 74%) and were hypothesized as a
407
Phoma-like species. Subsequently, phylogenetic analysis on the LSU single-locus
408
alignment was analyzed for the elucidation of the organismal phylogeny of the doubtful
409
isolates (Fig. 3). This alignment was successful in identifying all of the isolates. The
410
isolates CH-01 and CH-03 are grouped together in a clade (BS = 99%) with a
411
representative isolate of Diaporthe sp. (PHAg); the isolate CH-13 clustered (BS =
412
100%) with a representative isolate of Cy. pruinosa (CBS119207); and the isolates CH-
413
12 and CH-16 clustered (BS = 93% and BS=100%, respectively) with a representative
414
isolate of Co. incompta (CBS 467.76). The rest of the isolates formed the same clades
415
as those observed in the combined alignment of BT and ITS. Finally, sequences of EF
416
and its single-locus alignment were not helpful in identifying our Phoma-like isolates.
417
Pathogenicity tests
418
Pathogenicity on detached branches. Necrotic lesions on ‘Gordal Sevillana’ branches
419
were observed two weeks after inoculation. In general, vascular tissues showed sunken
420
and sectorial necrotic lesions in the affected wood. Eventually, pathogens belonging to
421
the Cytospora, Phoma-like, or Diaporthe genus produced cankers on the bark of the
422
inoculated branches. Only the two N. mediterraneum isolates (BOO071 and CH-06), a
423
Cytospora sp. isolate (CH-13), and a Co. incompta isolate (CH-16) caused visible
424
lesions on detached branches. Both N. mediterraneum isolates were the most virulent (P
425
< 0.05), causing an average length of necrosis of 15.93 ± 3.04 cm and 13.51 ± 5.96 cm,
426
respectively (Fig. 4). The remaining fungal species (C. godetiae, Phoma-like sp., and
427
Diaporthe sp.) did not cause lesions on inoculated branches. Further, no lesions were
428
observed in control branches. The species N. mediterraneum, Cy. pruinosa, and Co.
429
incompta were isolated from affected tissues, and C. godetiae, Diaporthe sp., and No.
430
Page 18 of 46
19
quercina were isolated from asymptomatic branches. A significant and positive linear
431
correlation (P < 0.001; R
2
= 0.724) was observed between the length of necrosis and
432
pycnidia production, the bark surface inoculated with N. mediterraneum isolate
433
BOO071 being that producing the highest number of pycnidia of the pathogen (data not
434
shown).
435
Pathogenicity on potted plants. The first symptoms were observed 30 days after
436
inoculation with mycelium plugs on the xylem tissues of the cv. Gordal Sevillana.
437
Symptoms—mainly vascular necrotic lesions—were similar to those observed in
438
detached branches. Only the N. mediterraneum isolates, BOO071 and CH-06, and the
439
Co. incompta isolate, CH-06, were able to induce the typical dieback symptoms and
440
cankers that affected the development of the plants, where the BOO071 isolate was the
441
most virulent (Fig. 4). The remaining fungal species (C. godetiae, Cy. pruinosa,
442
Diaporthe sp., and No. quercina) did not cause any lesions on inoculated plants as with
443
the control branches. The species N. mediterraneum and Co. incompta were consistently
444
isolated from affected tissues while C. godetiae, Cy. pruinosa, Diaporthe sp. and No.
445
quercina were isolated distantly from points away from the inoculation point on
446
asymptomatic branches.
447
Evaluation of cultivar resistance
448
Branch inoculation with N. mediterraneum. Olive branches of the nine inoculated
449
cultivars showed vascular necrotic tissues after two weeks of inoculation. Symptoms
450
observed were the same as those described above. According to the lesion on detached
451
and inoculated branches, the cv. Gordal Sevillana was significantly (P < 0.05) the most
452
susceptible to the pathogen (necrosis length 15.72 ±4.62 cm), followed by ‘Santa
453
Caterina’ y ‘San Agostino’, which did not show significant differences (P > 0.05)
454
Page 19 of 46
20
among them. Conversely, the cv. Manzanilla Cacereña was the least susceptible
455
(necrosis length 6.19 ± 2.11 cm) followed by ‘Verdial de Huévar’ and ‘Morona’, which
456
did not show significant (P > 0.05) differences among them (Table 4). For the whole of
457
cultivars, a weak but significant negative correlation (R
2
= 0.190; P = 0.0001) between
458
branch diameter and length of necrosis was observed, showing that branch susceptibility
459
increases when the diameter of branches decreases. Finally, a significant positive linear
460
correlation (R
2
= 0.1359; P < 0.001) was observed between the length of necrosis and
461
pycnidia production (data not shown), the bark surface of cv. San Agostino being the
462
most occupied by pycnidia.
463
Plant inoculation with N. mediterraneum. Olive plants showed the first symptoms at 14
464
days after inoculation, while the first dead branches were observed at eight weeks. In
465
the potted plant trials, the cvs. Manzanilla Cacereña and Gordal Sevillana showed a
466
high percentage of dead branches, 83.33 and 62.50 %, respectively, while the ‘Aloreña
467
de Atarfe’, ‘Hojiblanca’ and ‘Verdial de Huévar’ did not produce any dead branches
468
(Fig. 5; Table 4). According to necrosis length, the cvs. Manzanilla Cacereña and
469
Gordal Sevillana were also the most susceptible to the pathogen (necrosis lengths 10.6 ±
470
3.27 cm and 10.38 ± 2.52 cm, respectively) without significant differences between
471
them. The remaining of cultivars, ‘Aloreña de Atarfe’, ‘Hojiblanca’, ´Manzanilla de
472
Sevilla’, ‘Morona’, and ‘Verdial de Huévar’, were significantly less susceptible
473
(necrotic lesion ranged 3.06-5.25 cm) than the previous two cultivars and formed a
474
homogeneous group (P > 0.05). When we studied the relationship between the
475
percentage of dead branches of the different cultivars and their canker lengths (from
476
detached branches and branches in vivo), the percentage of dead branches was observed
477
to be significantly (R
2
= 0.965; P < 0.001) related to the canker lengths of inoculated
478
Page 20 of 46
21
potted plants, whereas the relationship was not significant (P = 0.8740) when detached
479
branches were used.
480
Fruit Inoculation with B. dothidea. When immature olive fruits were inoculated with B.
481
dothidea, the first symptoms were observed at 21 days after inoculation. These were
482
small depressed-necrotic lesions surrounding the inoculation point that advanced until
483
they covered the entire surface of the fruit. Olive ‘Hojiblanca’, ‘Manzanilla de Sevilla’,
484
and ‘Morona’ fruit were the most resistant to B. dothidea, although, there was an
485
extensive overlap for the AUDPCs showed by the fruit of the different cultivars (Table
486
4). The resistance to B. dothidea was not correlated (P = 0.6968) with the resistance to
487
fruit rot caused by C. godetiae.
488
DISCUSSION
489
Six fungal species belonging to six different genera were isolated from olive showing
490
branch dieback symptoms in Spain and Tunisia. Affected trees showed defoliation
491
and/or leaf wilting in young twigs, and darker sunken areas along affected branches,
492
which revealed perennial cankers under the bark. Cankered zones presented a well-
493
defined dark line of demarcation between infected and healthy tissues. Dieback diseases
494
characterized by perennial cankers affecting branches and trunks have been studied in
495
many different perennial hosts worldwide, including olives (Moral et al., 2010; Úrbez-
496
Torres et al., 2013). The current work elucidates the etiology of olive branch dieback in
497
Spain, with a special emphasis on the pathogenicity of associated fungi. Moreover, the
498
resistance of the main table cultivars against the main causal agents of the disease has
499
also been evaluated.
500
Morphological characters (conidial and mycelial) were useful to separate the isolates in
501
four different groups, Botryosphaeriaceae, Cytospora, Phoma-like and Phomopsis, in
502
Page 21 of 46
22
agreement with those described for each genus (Chen et al., 2015; Liu et al., 2015;
503
Niekerk et al. 2004; Phillips, 2013). Nevertheless, taxa of Phoma-like species are
504
morphologically difficult to distinguish. This is also true for Ascochyta species. Both of
505
these genera have in the past been linked to Didymella sexual morphs. Recently, Chen
506
et al. (2015) clarified the generic delimitation in Didymellaceae by combining multi-
507
locus phylogenetic analyses using ITS, LSU, RNA polymerase II second largest subunit
508
(rpb2) gene, partial gene regions of β-tubulin (tub2), and morphological observations.
509
LSU, in particular, has been described as one of the most helpful regions to identify
510
species belonging to the Didymellaceae family. Here, the BT, ITS, and LSU regions
511
were needed to identify the species of each genus, LSU being especially helpful to
512
identify Phoma-like species. Conversely, sequences of EF were unhelpful for
513
identification. The ITS and EF combined phylogenetic analysis was useful in describing
514
the diversity of species that affect olive trees in Spain and Tunisia, confirming the five
515
described groups based on morphology.
516
Concerning the Botryosphaeriaceae group, only one species, N. mediterraneum, was
517
recovered, the prevalent pathogen in this work. The current study corroborates the
518
presence of this species in lignified tissues of olive trees in Spain, suggested as the main
519
causal agent of the disease (Moral et al., 2010; Romero et al., 2005). In addition, N.
520
mediterraneum was the most virulent species from this study; although, differences in
521
virulence between tested isolates were observed. These differences were only observed
522
when pathogenicity was evaluated on potted plants, but they were not observed on
523
detached branches. This result suggests that there is a possibility of finding genetic
524
diversity within N. mediterraneum populations with geographic and host preferences. In
525
fact, the teleomorph of N. mediterraneum was reported for the first time in the world in
526
the Andalusian region (Moral et al., 2015), which invites us to further research its
527
Page 22 of 46
23
genetic diversity. In addition, studies conducted in Spain by Romero (2012), in which
528
olive plants were inoculated with B. dothidea, D. corticola, Dot. iberica and N.
529
mediterraneum showed that only N. mediterraneum was able to induce cankers on
530
plants with a high level of virulence. In other olive-growing areas, a broad range of
531
species belonging to the Botryosphaeriaceae (D. mutila, D. seriata, Dot. iberica, L.
532
theobromae, N. luteum, and N. parvum) has been associated with olives showing branch
533
dieback (Sergeeva et al., 2009; Taylor, 2001; Úrbez-Torres et al., 2013).
534
Among all of the fungal taxa isolated from the symptomatic wood of olives in this
535
study, Phoma-like species were the most diverse. All of the Tunisian isolates in this
536
study were identified as Phoma-like species. In concordance with this result, today only
537
Phoma sp. and P. fungicola have been reported as causing branch dieback of olive trees
538
in this country (Rhouma et al., 2010; Taieb et al., 2014). Our Tunisian isolates were
539
identified as No. quercina and Co. incompta, but the latter was pathogenic to both
540
detached branches and potted plants. In agreement with this result, Co. incompta has
541
also been described as causing branch dieback on olives in Croatia (Ivic et al., 2010),
542
Greece (Malathrakis, 1979), and Italy (Tosi and Natalini, 2009; Tosi and Zazzerini,
543
1994). Concerning negative pathogenicity results obtained for the No. quercina isolates
544
(both Spanish and Tunisian), previous surveys on olive orchards in California revealed
545
that the occurrence of Phoma species in olives is very low (Úrbez-Torres et al., 2013),
546
leading us to hypothesize that it has limited pathogenicity in olives. It is well known
547
that the Phoma genus contains plant pathogenic species as well as numerous saprobic
548
and endophytic species associated with a wide range of hosts (Chen et al., 2015).
549
Species in Diaporthe were the third most prevalent fungi isolated in our study with two
550
isolates identified as Diaporthe sp. To date, species of this genus have only been
551
Page 23 of 46
24
associated with olive branch dieback in California (Úrbez-Torres et al., 2013). In our
552
study, pathogenicity tests revealed that all isolates of Diaporthe sp. were not pathogenic
553
on olive branches. However, these results contrast with those obtained by Úrbez-Torres
554
et al. (2013) who demonstrated the pathogenicity of some Diaporthe isolates on olive
555
branches. Because Diaporthe is known to be cosmopolitan and found primarily as
556
endophytes, parasites, and saprotrophs in a wide range of hosts (Udayanga et al., 2011),
557
this difference in pathogenicity in olives between both countries could be due to
558
variability in isolate virulence. Many other possible factors such as the age of the host,
559
and/or differences in cultivar susceptibility could also be explanatory. Thus, a broader
560
pathogenicity study using a higher number of isolates of this genus and different plant
561
cultivars will be required to further clarify the pathogenic role of Diaporthe sp. as a
562
causal agent of branch dieback on olive.
563
Cytospora pruinosa was isolated with the least prevalence. In addition, this was the
564
least virulent species to detached branches among all the pathogenic fungi tested in this
565
study and found to not be pathogenic to the potted plants. To our knowledge, this is the
566
first report of Cy. pruinosa causing olive branch dieback in Spain. To date, only the
567
species Cy. oleina has been associated with olive branch dieback in Greece (Rumbos,
568
1988). Our results suggest that although many fungi such as Diaporthe are pathogenic
569
to a wide range of crops, they would only be able to induce cankers on hosts such as
570
weakened or stressed olive trees. Notably, no relationship was found between the dead
571
branches (%) of inoculated plants and canker length on detached branches. This
572
reinforces the idea that inoculation in vivo is essential to the characterization of fungal
573
pathogens or, even to identify differences in virulence among isolates belonging to the
574
same species. In addition, the non-pathogenic isolates were recovered from
575
asymptomatic tissues which indicates that they are common endophytic fungi in olive.
576
Page 24 of 46
25
Pathogenicity of C. godetiae, the main causal agent of olive anthracnose in Andalusia
577
(Moral et al., 2014), was also evaluated on lignified tissues. This isolate was included to
578
test the hypothesis that the syndrome of dead branches is caused by phytotoxins
579
produced by this pathogen often present in rotten fruits (Moral et al., 2009). The
580
pathogenicity test showed that C. godetiae was not pathogenic to olive branches. The
581
results support that the isolates of the complex species C. acutatum do not cause the
582
typical dieback symptoms through direct infection on lignified tissues, since the toxin
583
produced by the pathogen is the main cause in inducing twig-branch dieback (Moral et
584
al., 2009). However, a wide collection of Colletotrichum spp. should be tested for
585
pathogenicity to elucidate the role of their toxins in branch dieback of olive.
586
Disease resistance of olive cultivars offer an economically sound alternative to chemical
587
control with minimal environmental impact, which can be integrated in pest
588
management strategies. Here, we conducted the first comparison of cultivar resistance
589
to N. mediterraneum causing cankers on olive worldwide, and to B. dothidea causing
590
fruit rot of olives in Spain. Trials conducted on detached branches showed that ‘Gordal
591
Sevillana’ was the most susceptible cultivar to N. mediterraneum followed by ‘Santa
592
Caterina’ and ‘San Agostino’; whereas ‘Manzanilla Cacereña’ was the most resistant,
593
followed by ‘Verdial de Huévar’ and ‘Morona’. Detached branches could be under huge
594
stress and does not behave physiologically in the same way as a branch attached to a
595
tree. Thus, results obtained from detached branches could not be absolutely
596
representative of cultivar resistance. In this way, cultivar resistance was also evaluated
597
by using potted plants. In this case, inverted results than those obtained by using
598
detached branches were observed on potted plants where ‘Manzanilla Cacereña’ was the
599
most susceptible cultivar to N. mediterraneum, followed by ‘Gordal Sevillana.’ ‘Verdial
600
de Huévar’ was the most resistant cultivar. The results last are in concordance with
601
Page 25 of 46
26
those obtained by Romero (2012) who indicated that ‘Gordal Sevillana’ was more
602
susceptible than ‘Picual’ when live plants were inoculated with N. mediterraneum.
603
Concerning B. dothidea, results revealed that ‘Aloreña de Atarfe’ was the most
604
susceptible cultivar; whereas ‘Hojiblanca’ and ‘San Agostino’ were the most resistant.
605
These results complement those obtained by Latinovic et al. (2013) who described
606
highly resistant native cvs. Crnjaka and Gloginja. It is interesting to note that the
607
resistance of green olive fruit to B. dothidea is not correlated with the resistance of ripe
608
fruit to C. godetiae, showing that different resistance mechanisms are at work in both
609
types of fruits (Moral et al., 2014).
610
Cultivar resistance to Botryosphaeriaceae species affecting lignified tissues of perennial
611
crops has also been studied in mango (Mangifera indica L) and pistachio (Pistachio
612
vera L.) (Partiff et al., 2003). According to our results, the Spanish cultivars
613
‘Hojiblanca’, ‘Manzanilla Cacereña’, and ‘Verdial de Huévar’ showed a good level of
614
resistance to branch dieback, making it a potential alternative tool against both branch
615
dieback types.
616
This study has resulted in significant information regarding the etiology of branch
617
dieback of olive as well as cultivar resistance to N. mediterraneum and B. dothidea. The
618
variation found among fungal species recovered from affected olive trees with cankers
619
and branch dieback symptoms should be taken into account to develop optimum control
620
strategies. Therefore, further research is needed to elucidate the role of many fungal
621
species associated with branch dieback of olives, as well as the environmental and
622
cultural practices that influence the disease.
623
624
625
Page 26 of 46
27
ACKNOWLEDGMENTS
626
This research was funded by the Spanish Ministry of Education and Science (project
627
AGL2004-7495) and by the Andalusian Regional Government (Project P08-AGR-
628
03635). Both projects were co-financed by the European Union FEDER Funds. J.M.
629
holds a Marie Skłodowska Curie fellowship launched by the European Union’s H2020
630
(Contract number 658579). C.A.B. is the holder of a ‘Juan de la Cierva-Formación’
631
fellowship from MINECO. The authors thank J. A. Layosa and F. Luque for her skillful
632
technical assistance. They also thank C. Cunningham, K. Tomari, and T. J. Michailides
633
for critical review of the manuscript.
634
LITERATURE CITED
635
Barranco, D., Fernández-Escobar, R., and Rallo, L., eds. 2008. El cultivo de olivo. Junta
636
de Andalucía & Mundi-Prensa, Madrid.
637
Barranco, D., and Rallo, L. 2000. Olive cultivars in Spain. HortTechnology 10:107-110.
638
Barnett, H. L., and Hunter, B. B. 1998. Illustrated genera of imperfect fungi. 4th edition.
639
APS Press. St. Paul, MN.
640
Carbone, I., Anderson, J. B., and Kohn, L. M. 1999. A method for designing primer sets
641
for the speciation studies in filamentous ascomycetes. Mycologia 91:553-556.
642
Carlucci, A., Lops, F., Cibelli, F., and Raimondo, M. L. 2015. Phaeoacremonium
643
species associated with olive wilt and decline in southern Italy. Eur. J. of Plant.
644
Pathol. 141:717–729.
645
Carlucci, A., Raimondo, M. L., Cibelli, F., Phillips, J. L., and Lops, F. 2013.
646
Pleurostomophora richardsiae, Neofusicoccum parvum and Phaeoacremonium
647
Page 27 of 46
28
aleophilum associated with a decline of olives in southern Italy. Phytopathol.
648
Mediterr. 52:517-527.
649
Chen, Q., Jiang, J. R., Zhang, G. Z., Cai1, L., and Crous, P. W. 2015. Resolving the
650
Phoma enigma. Stud. Mycol. 82:137-217.
651
652
Crous, P. W., Wingfield,
M. J., Guarro,
J., Hernández-Restrepo, M., Sutton, D. A.,
653
Acharya, K., Barber,
P. A., Boekhout,
T., Dimitrov,
R. A., Dueñas, M.,
Dutta, A.
654
K.,
Gené, J.,
Gouliamova,
D. E., Groenewald,
M., Lombard,
L., Morozova, O. V.,
655
Sarkar,
J., Smith, M. Th., Stchigel,
A. M., Wiederhold,
N. P., Alexandrova,
656
Antelmi, V. I., J. Armengol,
J., Barnes,
I., Cano-Lira,
J. F., Castañeda Ruiz,
R. F.,
657
Contu,
M., Courtecuisse,
Pr. R., da Silveira, A. L., Decock,
C. A., de Goes,
A.,
658
Edathodu,
J., Ercole,
E., Firmino,
A. C., Fourie,
A., Fournier,
J., Furtado,
E. L.,
659
Geering,
A. D. W., Gershenzon,
J., Giraldo, A., Gramaje,
D., Hammerbacher,
A.,
660
He,
X. L., Haryadi, D., Khemmuk,
W., Kovalenko, A. E., Krawczynski, R.,
661
Laich, F., Lechat, C., Lopes, U. P., Madrid, H., Malysheva, E. F., Marín-Felix,
662
Y., Martín, M. P., Mostert, L., Nigro, F., Pereira, O. L., Picillo, B., Pinho, D. B.,
663
Popov, E. S., Rodas Peláez, C. A., Rooney-Latham, S., Sandoval-Denis, M.,
664
Shivas, R. G., Silva, V., Stoilova-Disheva, M. M., Telleria, M. T., Ullah, C.,
665
Unsicker, S. B.,
van der Merwe, N. A., Vizzini, A., Wagner, H. G., Wong, P. T.
666
W., Wood, A. R., and Groenewald, J. Z. 2015. Fungal Planet description sheets:
667
320–370. Persoonia 34:167-266.
668
Dhingra, O. D., and Sinclair, J. B. 1995. Basic plant pathology methods. 2nd edn. CRC
669
Press, Boca Raton, FL, USA.
670
Page 28 of 46
29
Eldesouki, I. E. 2013. Interacciones de Batrocera oleae Gmel. (Mosca del olivo) con
671
Botryosphaeria dothidea Moug. (Escudete de la aceituna) y de Phloeotribus
672
scarabaeoides Bern. (Barrenillo del olivo) con Verticillium dahliae Kleb.
673
causante de la Verticilosis del olivo. PhD Thesis, University of Córdoba,
674
Córdoba, Spain.
675
Felsenstein, J., 1985. Confidence limits on phylogenies: An approach using the
676
bootstrap. Evolution 39:783-791.
677
Glass, N., and Donaldson, G. C. 1995. Development of primer sets designed for use
678
with the PCR to amplify conserved genes from filamentous Ascomycetes. Appl.
679
Environ. Microbiol. 61:1323-1330.
680
Iannotta, N., Noce, M. E., Ripa, V., Scalercio, S., and Vizzarri, V. 2007. Assessment of
681
susceptibility of olive cultivars to the Bactrocera oleae (Gmelin, 1790) and
682
Camarosporium dalmaticum (Thüm) Zachos & Tzav.-Klon. attacks in Calabria
683
(Southern Italy). J. Environ. Sci. Health 42:789-793.
684
Ivic, D., Ivanovic, A., Milicevic, T., and Cvjetkovic, B. 2010. Shoot necrosis of olive
685
caused by Phoma incompta, a new disease of olive in Croatia. Phytopathol.
686
Mediterr. 49:414–416.
687
Kimura, M. 1980. A simple method for estimating evolutionary rate of base
688
substitutions through comparative studies of nucleotide sequences. J. Mol. Evol.
689
16:111-120.
690
Kornerup, A., and Wanscher, J.H. 1963. Methuen Handbook of Colour. Methuen and
691
Co. Ltd., London.
692
Page 29 of 46
30
Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A.,
693
McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., López, R., Thompson, J.
694
D., Gibson, T. J., and Higgins, D. G. 2007. Clustal Wand Clustal X version 2.0.
695
Bioinformatics, 23:2947–2948.
696
Latinovic, J., Mazzaglia, A., Latinovic, N., Ivanovic, M., and Gleason, M. L. 2013.
697
Resistance of olive cultivars to Botryosphaeria dothidea, causal agent of olive
698
fruit rot in Montenegro. Crop Prot. 48:35-40.
699
Lazzizera, C., Frisullo, S., Alves, A., Lopes, J., and Phillips, A. J. L. 2008a. Phylogeny
700
and morphology of Diplodia species on olives in southern Italy and description
701
of Diplodia olivarum sp. nov. Fungal Divers. 31:63-71.
702
Lazzizera, C., Frisullo, S., Alves, A., and Phillips, A. J. L. 2008b. Morphology,
703
phylogeny and pathogenicity of Botryosphaeria and Neofusicoccum species
704
associated with drupe rot of olives in southern Italy. Plant Pathol. 57:948-956.
705
Liu J.K., Hyde K.D., Jones E. B. G., Ariyawansa H. A., Bhat D. J., Boonmee S.,
706
Maharachchikumbura S. S. N., Mckenzie E. H. C., Phookamsak R.,
707
Phukhamsakda C., Shenoy B. D., Abdel-Wahab M. A., Buyck B., Chen J.,
708
Chethana K. W. T., Singtripop C., Dai D. Q., Dai Y. C., Daranagama D. A.,
709
Dissanayake A. J., Doilom M., D'souza M. J., Fan X. L., Goonasekara I.D.,
710
Hirayama K., Hongsanan S., Jayasiri S. C., Jayawardena R. S., Karunarathna
711
S.C., Li W. J., Mapook A., Norphanphoun C., Pang K. L., Perera R.H., Peršoh
712
D., Pinruan U., Senanayake I.C., Somrithipol S., Suetrong S., Tanaka K.,
713
Thambugala K. M., Tian Q., Tibpromma S., Udayanga D., Wijayawardene N.N.,
714
Wanasinghe D. N., Wisitrassameewong K., Zeng X. Y., Abdel-Aziz F. A.,
715
Adamčík S., Bahkali A. H., Boonyuen N., Bulgakov T., Callac P., Chomnunti
716
Page 30 of 46
31
P., Greiner K., Hashimoto A., Hofstetter V., Kang J. C., Lewis D., Li X. H., Liu
717
X. Z., Liu Z. Y., Matsumura M., Mortimer P. E., Rambold G., Randrianjohany
718
E., Sato G., Sri-Indrasutdhi V., Tian C.M., Verbeken A., Von Brackel W., Wang
719
Y., Wen T. C., Xu J. C., Yan J. Y., Zhao R. L., and Camporesi E. 2015 —
720
Fungal diversity notes 1-110: taxonomic and phylogenetic contributions to
721
fungal species. Fungal Divers. 72:1-197
722
MAGRAMA, 2016. http://www.magrama.gob.es/es/agricultura/temas/producciones-
723
agricolas/aceite-oliva-y-aceituna-mesa/aceituna.aspx#para2
724
Malathrakis, N. E. 1979. Studies on a disease of olive due to fungus Phoma incompta
725
Sacc. & Mart. Ph.D. Thesis. University of Athens, Athens, Greece.
726
Moral, J., Bouhmidi, K., and Trapero, A. 2008a. Influence of fruit maturity, cultivar
727
susceptibility, and inoculation method on infection of olive fruit by
728
Colletotrichum acutatum. Plant Dis. 92: 1421-1426.
729
Moral, J., De Oliveira, R., and Trapero, A. 2009. Elucidation of the disease cycle of
730
olive anthracnose caused by Colletotrichum acutatum. Phytopathology 99:548-
731
556.
732
Moral, J., Eldesouki-Arafat, I., López-Escudero, F. J., Vargas-Osuna, E., Trapero, A.,
733
and Aldebis, H. K. 2016. Olive Escudete, caused by Botryosphaeria dothidea, as
734
Result of the Interaction fly-mosquito-fungus. Phytopathology 106 (S)
735
Moral, J., Luque, F., and Trapero, A. 2008b. First report of Diplodia seriata, the
736
anamorph of “Botryosphaeria” obtusa, causing fruit rot of olive in Spain. Plant Dis.
737
92:311.
738
Page 31 of 46
32
Moral, J., Muñoz-Díez, C., González, N., Trapero, A., and Michailides, T. J. 2010.
739
Characterization and pathogenicity of Botryosphaeriaceae species collected from
740
olive and other hosts in Spain and California. Phytopathology 100:1340-1351.
741
Moral, J., Pérez-Rodríguez, M., Michailides, T. J., and Trapero, A. 2015. First report of
742
the teleomorph of Neofusicoccum mediterraneum, a pathogen of olive.
743
Phytopathology 105 (S):97-98.
744
Moral, J., Xaviér, C., Roca, L. F., Romero, J., Moreda, W., and Trapero, A. 2014. La
745
Antracnosis del olivo y su efecto en la calidad del aceite. Grasas Aceites 65 (2)
746
doi: http://dx.doi.org/10.3989/gya.110913
747
Niekerk, J. M. van, Crous, P. W., Groenewald, J. Z., Fourie, P. H., and Halleen, F.
748
2004. DNA phylogeny, morphology and pathogenicity of Botryosphaeria
749
species on grapevines. Mycologia 96:781-798.
750
Parfitt, D.E., Arjmand, N., and Michailides, T. J. 2003. Resistance to Botryosphaeria
751
dothidea in pistachio. HortScience 38:529-531.
752
Phillips, A. J. L., Alves, A., Abdollahzadeh, J., Slippers, B., Wingfield, M. J.,
753
Groenewald, J. Z., and Crous, P. W. 2013. The Botryosphaeriaceae: genera and
754
species known from culture. Stud. Mycol. 76:51-167.
755
Rallo, L., Barranco, D., Caballero, J.M., Del Río, C., Martín, A., Tous, J., and Trujillo,
756
I., eds. 2005. Las variedades de olivo cultivadas en España. Consejería de
757
Agricultura y Pesca, Ministerio de Agricultura, Pesca y Alimentación and
758
Ediciones Mundi-Prensa, Madrid.
759
Page 32 of 46
33
Rehner, S. A., and Samuels, G. J. 1994. Taxonomy and phylogeny of Gliocladium
760
analysed from nuclear large subunit ribosomal DNA sequences. Mycol. Res. 98:
761
625-634.
762
Rhouma, A., Triki, M. A., Krid, S., and Masallem, M. 2010. First report of a branch
763
dieback of olive trees in Tunisia caused by a Phoma sp. Plant Dis. 94:636.
764
Romero, M. A. 2012. Etiología, epidemiología y control del chancro de los Quercus
765
causado por Botryosphaeria spp. Ph.D. Thesis. University of Cordoba, Cordoba,
766
Spain.
767
Romero, M. A, Sánchez, M. E., and Trapero, A. 2005. First report of Botryosphaeria
768
ribis as a branch dieback pathogen of olive trees in Spain. Plant Dis. 89:208.
769
Rumbos, I. C. 1988. Cytospora oleina causing canker and dieback of olive in Greece.
770
Plant Pathol. 37:441-444.
771
Rumbos, I. C. 1993. Dieback symptoms on olive trees caused by the fungus Eutypa
772
lata. Bull. OEPP/EPPO Bull. 23:441-445.
773
Saitou, N., and Nei, M. 1987. The neighbor-joining method: A new method for
774
reconstructing phylogenetic trees. Molec. Biol. Evol. 4:406-425.
775
Sergeeva, V., Alves, A., and Phillips, A. J. L. 2009. Neofusicoccum luteum associated
776
with leaf necrosis and fruit rot of olives in New South Wales, Australia.
777
Phytopathol. Mediterr. 48:294-298.
778
Sutton, B. C. 1980. The Coelomycetes. Fungi imperfecti with picnidia acervuli and
779
stromata. Commonwealth Mycological Institute. Kew, UK.
780
Page 33 of 46
34
Taieb, S. K. H., Triki, M. A., Hammami, I., and Rhouma, A. 2014. First report of
781
dieback of olive trees caused by Phoma fungicola in Tunisia. J. Plant Pathol. 96:
782
117.
783
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 2013. MEGA6:
784
Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol.
785
30:2725-2729.
786
Taylor, R. K., Hale, C. N., and Hartill, W. F. T. 2001. A stem canker disease of olive
787
(Olea europaea) in New Zealand. New Zeal. J. Crop Hort. 29:219-228.
788
Tosi, L., and Natalini, G. 2009. First report of Eutypa lata causing dieback of olive trees
789
in Italy. Plant Pathol. 58:398.
790
Tosi, L., and Zazzerini, A. 1994. Phoma incompta a new olive parasite in Italy. Petria,
791
4:161-170.
792
Udayanga, D., Xingzhong, L., McKenzie, E. H. C., Chukeatirote, E., Bahkali, A. H. A.,
793
and Hyde, K. D. 2011. The genus Phomopsis: Biology, applications, species
794
concepts and names of common phytopathogens. Fungal Divers. 50:189-225.
795
Úrbez-Torres, J. R., Peduto, F., Vossen, P. M., Krueger, W. H., and Gubler, W. D.
796
2013. Olive Twig and Branch Dieback: Etiology, Incidence, and Distribution in
797
California. Plant Dis. 97:231-244.
798
Vilgalys, R., and Hester, M., 1990. Rapid genetic identification and mapping of
799
enzymatically amplified ribosomal DNA from several Cryptococcus species. J.
800
Bacteriol. 172:4238-4246.
801
White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct
802
sequencing of fungi ribosomal RNA genes for phylogenetics. Pages 315-322 in:
803
Page 34 of 46
35
PCR Protocols. A Guide to Methods and Applications. M. A. Innis, D. H.
804
Gelfand, J. J. Sninsky, and T. J. White, eds. Academic Press, San Diego, CA.
805
Zachos, D. G., and Tzavella-Klonari, K. 1983. Recherches sur les causes des infections
806
localisées ou géneéralisées des olives attaquées par le champignon
807
Camarosporium dalmatica. I. Influence de l´humidité, de la pression osmotique
808
et du pH des fruits. Ann. Inst. Phytopathol. Benaki 14:1-9.
809
Page 35 of 46
Table 1. Fungal isolates from olive trees located in Spain and Tunisia, and sequences from GenBank used in this study.
Species
Isolate
x
Host, cultivar
Symptoms
Collector
Origin
GenBank Accession no.
y
BT ITS LSU
Botryosphaeria
dothidea
BOO046 Olea europaea cv.
Santa Caterina
Dalmatian
disease
J. Moral Mengibar, Jaen,
Andalusia, Spain
GU292738 GU292626
n/a
Colletotrichum
godetiae
CH-21 Olea europaea cv.
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 &
M. Fischer
Finland n/a -n/a AF311003
Comoclathris
incompta
CH-12 Olea europaea cv.
Picual
Branch canker J. Moral Jaen, Jaen,
Andalusia, Spain
KU973707 KU973715
KU973728
CH-16 Olea europaea cv.
Meski
Branch canker A. Rhouma Tunisia KU973708 KU973716
KU973729
CBS 467.76 Olea europaea - M. M.
Averskamp
Greece n/a n/a GU238087
Cytospora pruinosa CH-13 Olea europaea cv.
Gordal Sevillana
Branch canker J. Moral Mengbar, Jaen,
Andalusia, Spain
KU973702 KU973711
KU973722
CBS 118555 Olea europaea cv.
africana
Branch canke Y. Li Wang South Africa KM034893 DQ243790
n/a
CBS 119207 - - J. Z.
Groenewald
- n/a n/a EU552121
Diaporthe sp. CH-01 Olea europaea cv.
Gordal Sevillana
Branch canker A. Trapero Sevilla, Sevilla,
Andalusia, Spain
KU973709 KU973717
KU973730
CH-03 Olea europaea cv.
Gordal Sevillana
Branch canker M. Pérez-
Rodríguez
Sevilla, Sevilla,
Andalusia, Spain
KU973710 KU973718
KU973731
Page 36 of 46
Species
Isolate
x
Host, cultivar
Symptoms
Collector
Origin
GenBank Accession no.
y
BT ITS LSU
UCR1395
Persea americana,
Hass
Fruit
M.
Twizeyimana
San Diego,
California, USA
JX898989
JX869042
n/a
PHAg
-
Branch canker
M. Pilloti
Rome, Italy
n/a
AY620999
AY621002
Ganoderma
resinaceum
GR145
-
-
C. L, Su
Shangai, China
DQ288101
KC311374
n/a
Leptosphaeria
biglobosa
CBS 532.66
Brassica
sp.
-
-
The Netherlands
KT389840
KT389541
KT389759
Neofusicoccum
mediterraneum
CH
-
06
Olea europaea
cv.
Gordal Sevillana
Branch canker
J. Moral
Sevilla, Sevilla,
Andalusia, Spain
KU973703
KU973712
KU973723
BOO071 Olea europaea cv.
Gordal Sevillana
Branch canker A. Trapero Arahal, Sevilla,
Andalusia, Spain
GU292757 GU292645
KU973724
UCD720SJ
Vitis vinifera
Branch canker
J. R. Úrbez
-
Torres
California, USA
GU799475
GU799452
n
/a
CBS 268.80 - - - - n/a n/a AY004336
Nothophoma
quercina
CH
-
04
Olea europaea
cv.
Gordal Sevillana
Branch canker
M. Pérez
-
Rodríguez
Sevilla, Sevilla,
Andalusia, Spain
KU973704
KU973713
KU973725
CH-14 Olea europaea cv.
Meski
Branch canker A. Rhouma Tunisia KU973705 KU973714
KU973726
CH
-
15
Olea europaea
cv.
Meski
Branch canker
A. Rhouma
Tunisia
KU973706
KU973720
KU973727
CBS 633.92 Microsphaera
alphitoides, Quercus sp.
- - Ukraine GU237609 GU237900
EU754127
x
CBS = Centraalbureau voor Schimmecultures, Utrech, The Netherlands; UCD = University California Davis-; UCR = University California Riverside
y
BT = β-tubuline, ITS = internal transcribed spacer, LSU = large subunit ribosomal RNA, and n/a = not available at the time of this publication. Sequences from Genbank used in the phylogenetic
analysis indicated in bold.
Page 37 of 46
Table 2. Morphological characters and mycelial growth of Colletotrichum, Cytospora, Diaporthe, Neofusicoccum and Phoma-like isolates
obtained from olive.
Species
Isolate
Mycelia
Obverse Reverse Growth
(mm/day)
y
Color Zonation Margin Color Zonation
Colletotrichum godetiae CH-21 Grey No Regular Dark grey 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 grey Yes 14.91
Neofusicoccum mediterraneum CH-06 Light green No Regular Grey 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
x
- - - - - -
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
x
Mycelia of this isolate was not optimum development for its characterization.
y
Single conidial cultures were grown on PDA for up to two weeks at 25 ± 2°C with a 12-h diurnal photoperiod of cool fluorescent light
(350 µmol m
–2
s
–1
).
Page 38 of 46
Table 3. Morphological characters of conidia and pycnidia of Colletotrichum, Cytospora, Diaporthe, Neofusicoccum and Phoma-like isolates obtained from olive.
Species
Isolate
Conidia Pycnidia
Length × Width (µm)
Length/Width
Morphology Color Morphology Color Diameter
(µm)
Colletotrichum
godetiae
CH
-
21
v
(13.05
-
) 14.35
(
-
15.65)
× (4.03-) 4.59 (-5.15)
w
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
Diaporhte
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
This isolate produces conidia in acervuli instead of in pycnidia
w
Mean and range values: Length × Width (µm). The extremes of the conidial measurements are shown inside parenthesis
x
Isolates with both conidia types, alpha and beta
y
Isolate with irregular pycnidia
z
Conidia were not observed for this isolate
Page 39 of 46
Tabla 4. Susceptibility of olive cultivar inoculated with Neofusicoccum mediterraneum on detached branches and potted plants and
Botryosphaeria dothidea on detached fruits.
Cultivar
Neofusicoccum mediterraneum Botryosphaeria dothidea
Detached branches Potted plants Fruits
Canker (cm)
Pycnidia
colonization (cm)
Canker (cm)
Dead
branches (%)
AUDPC
z
Aloreña de Atarfe
-
w
-
4.11 b
0.00
1.70 a
Ascolana Tenera
9.52 cd
x
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
y
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 Huévar 6.32 e 2.24 de 3.06 b 0.00 1.04 c
w
No symptoms were observed
x
Mean values with the same letter in a row are not significantly different according to Tukey’s honestly significant difference test
(P<0.05)
y
n/e: cultivars non evaluated in each experiment
z
AUDPC: Area under the disease progress curve
Page 40 of 46
A
AA
A
D
DD
D
G
GG
G
H
HH
H
I
II
I
J
JJ
J
B
BB
B
L
LL
L
K
KK
K
C
CC
C
E
EE
E
F
FF
F
Page 41 of 46
CH-03
UCR1395
CH-01
Diaporthe sp.
CH-13
CBS 118555
Cyt ospora pruinosa
CH-21
CBS 127561
Colletotrichum godetiae
CH-06
BOO071
UCD720SJ
Neofusicoccum mediterraneum
Leptosphaeria biglobosa
CBS 532.66 18
Phoma-like sp.
CH-12
Phoma-like sp.
CH-16
CH-04
CBS 633.92
CH-14
CH-15
Nothophoma quercina
Ganoderma resinaceum
GR145
100
44
100
100
99
97
75
98
75
97
74 99
96
95
0.1
Page 42 of 46
CH-04
CBS 633.92
CH-14
CH-15
Nothophoma quercina
CH-12
CH-16
CBS 467.76
Comoclathris incompta
Colletotrichum godetiae
CH-21
CH-13
CBS 119207
Cytospora pruinosa
PHAg
CH-01
CH-03
Diaporthe sp.
CBS 268.80
CH-06
BOO071
Neofusicoccum mediterraneum
Coltricia cinnamomea
Dai 2464
100
78
99
100
100
99
100
63
100
94
100
99
98
93
0.05
Page 43 of 46
Length
Length
Length
Length
of necrosis
of necrosis
of necrosis
of necrosis
(cm)
(cm)
(cm)
(cm)
a
aa
a
a
aa
a
a
aa
a
b
bb
b
b
bb
b
c
cc
c
b
bb
b
Potted plants
Potted plantsPotted plants
Potted plants
Detached branches
Detached branchesDetached branches
Detached branches
N. mediterraneum N. mediterraneum
Co
.
incompta
Co
.
incompta
C.
pruinosa
C.
pruinosa
Page 44 of 46
Figure 1. Two-weeks-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; B, C, Comoclathris incompta isolates CH-12 and CH-16, respectively;
D, Cytospora pruinosa isolate CH-13; E, F, Diaporthe sp. isolates CH-01 and CH-03,
respectively; G, Neofusicoccum mediterraneum isolate CH-06; H-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 cv. San
Agostino.
Figure 2. Phylogenetic analysis of taxa for the combined alignment of ITS and TUB
sequences. The evolutionary history was inferred using the Neighbor-Joining method
(Saitou and Nei, 1987). The optimal tree with the sum of branch length = 2.64571884 is
shown. The percentage of replicate trees in which the associated taxa clustered together
in the bootstrap test (2000 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 2-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 gamma distribution (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 dataset. Evolutionary analyses were
conducted in MEGA6 (Tamura et al., 2013).
Figure 3. Phylogenetic analysis of taxa for LSU 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 (2000
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 2-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 gamma
distribution (shape parameter = 1). The analysis involved 17 nucleotide sequences. All
Page 45 of 46
positions containing gaps and missing data were eliminated. There were a total of 782
positions in the final dataset. Evolutionary analyses were conducted in MEGA6
(Tamura et al., 2013).
Figure 4. Disease severity on detached branches and olive plants (cv. Gordal Sevillana)
two weeks and one month, respectively, after inoculation with Neofusicoccum
mediterraneum isolates BOO071 and CH-06, Cytospora sp. isolate CH-13 and
Comoclathris incompta isolate CH-16. For each isolate, mean values with the same
letter are not significantly different according to Tukey’s honestly significant difference
test (P < 0.05).
Page 46 of 46
