The olive tree (Olea europaea
L.) is affected by several diseases including anthracnose, a major concern in most olive-producing countries, which is able to destroy the entire production [1
]. Anthracnose is caused by diverse species of fungi belonging to genus Colletotrichum
]. Some Colletotrichum
species, previously classified as Colletotrichum acutatum
and then included within the C. acutatum
complex (Colletotrichum nymphaeae, Colletotrichum fiorinae, Colletotrichum godetiae, C. acutatum, Colletotrichum rhombiforme
, and Colletotrichum simmondsii
), highly prevail in areas where the disease occurs epidemically [3
]. In Portugal, the species C. nymphaeae, C. acutatum
, and C. godetiae
together reach levels of over 95% [6
The disease typically affects fruits near maturation and, consequently, the quality of the fruits and oil obtained (high acidity, off-flavor, reddish color, and a considerable reduction of polyphenols, α-tocopherol, and β-sitosterol) [7
]. Under moist conditions, infected fruits develop dark, necrotic, circular, sunken lesions with an abundant production of orange-colored masses of spores on the surface, leading to premature fruit drop, as well as fruit rot; in dry weather, mummification occurs, frequently leading to total yield losses [2
]. The pathogen can also be present on flowers, leaves, shoots, and branches and may cause blossom blight, chlorosis, and necrosis of the leaves in the early spring and severe defoliation and wilting in the late spring and early summer, as well as dieback of the branches, with the latter being associated with toxins produced by the pathogen [1
In addition to environmental conditions (e.g., humidity, rain, and temperature), the virulence of the pathogen, the maturity and integrity of the fruits, and the olive cultivar have also been associated with the disease incidence [3
]. In Portugal, the main olive oil cultivar, Galega vulgar, is known to be very susceptible to anthracnose, Cobrançosa is moderately tolerant, and Azeiteira is considered to be resistant [10
Conidia from Colletotrichum
spp. germinate from acervuli on tree mummified fruits, leaves, and twigs and are dispersed through rain during the fall when the fruits begin to ripen, becoming the primary inoculum of the disease [1
]. The pathogen sporulates on the surface of rotten fruits, and the spores give rise to secondary infection cycles. Moral et al. [14
] showed that in the spring, leaves, shoots, flowers, and young fruits become infected, but the infection remains latent and may be an important source of inoculum for autumn epidemics [12
]. The complete disease cycle of olive anthracnose is still not fully understood [9
]. It is not known if the pathogen can travel from latent infected branches or other infected organs to other organs, such as flowers and fruits, which were not directly infected. If that happens, once a plant becomes infected, the plant may act as a reservoir and will not be dependent on a new infection to initiate the disease. If so, fungicide treatments before flowering would protect flowers from early infections but would not be efficient for already existing latent infections. At present, the control of the disease is based on the application of copper products or penetrating products, such as trifloxystrobin, during autumn, and no systemic products are used. In this context, the study of internal infections and their possible circulation on vascular organs would be extremely useful for the improvement of management choices, namely the timing and the use of systemic fungicides.
Building on the latter knowledge, the aim of the present study was to understand aspects of Colletotrichum colonization and the primary infection of olive anthracnose. For that, the presence of Colletotrichum spp. was evaluated in the interior of different organs from three major Portuguese olive cultivars with different degrees of susceptibility to olive anthracnose, grown in different sites. The following hypotheses were tested: (i) there would be significant differences in the presence of Colletotrichum spp. in different plant organs; (ii) there would be significant differences in the presence of Colletotrichum spp. in different cultivars; and (iii) there would be significant differences in the presence of Colletotrichum spp. in different sites.
2. Materials and Methods
2.1. Study Area and Sample Collection
The sampling was carried out during 2016 in three important olive oil-producing sites within the Alentejo region (southern Portugal), all influenced by the Mediterranean climate: In Vidigueira (38°10’01.17” N, 7°44’16.75” W), the altitude is 156 m above sea level, the mean temperature is 15.0 °C, the annual rainfall is approximately 600 mm, and the soils are of granite origin. In Monforte (39°4’3.99” N, 7°28’13” W), the altitude is 376 m above sea level, the mean temperature is 16.5 °C, the annual rainfall is approximately 660 mm, and the soils are mostly of schist and calcareous origin. In Elvas (38°54’31.34” N, 7°8’43.52” W), the altitude is 220 m above sea level, the mean temperature is 16.3 °C, the annual rainfall is approximately 598 mm, and the soils are mostly of schist and calcareous origin. All the olive trees sampled were of medium size (with ages ranging from 10 to 30 years) and were planted at a spacing of 7 × 5 m. The sampled olive groves occupied an area of 320,000 m2 in Monforte, 150,000 m2 in Vidigueira, and 30,000 m2 in Elvas, and all were produced under an intensive regime. All the experimental olive groves included programmed applications of fungicide and insecticide products, such as copper hydroxide, trifloxystrobin, deltamethrin, and dimethoate.
The sampled olive trees belonged to three different cultivars (Galega vulgar, Cobrançosa, and Azeiteira) and did not present any visible anthracnose symptoms. At each site, the area of olive trees from each cultivar was divided into several plots, and three experimental plots with ten olive trees each (totaling 30 olive trees per cultivar) were randomly selected by a uniform probability function. A total of 270 trees was sampled (3 sites × 3 cultivars × 30 trees per cultivar). The sampling was repeated at 3 different periods, for each type of plant organ; 2-year stems (early spring), flower buds (late spring), and immature fruits (early fall), totaling 810 samples (270 trees × 3 periods). For each tree and for each plant organ, 50 samples were collected around the whole tree at 1.5 m above the ground. The sampling was always made before the applications of the chemical products. The samples were transported to the laboratory in a refrigerated basket, stored at 4 °C, and processed within the next 48 h.
2.2. Isolation of Colletotrichum spp.
To suppress the epiphytic micro-organisms on the field-collected samples, 2-year stems, flower buds, and immature fruits were surface disinfected. The disinfection involved a sequence of 3-min immersions in 96% ethanol, 3% sodium hypochlorite solution, 70% ethanol, and 3 times in ultra-pure water, respectively, and then, the samples were dried in sterile Whatman paper. The 2-year stems were cut into 0.5 cm2 sections; the flower buds and immature fruits were cut lengthwise and placed (6 pieces per plate) on 9-cm diameter Petri dishes containing potato dextrose agar medium (PDA) (Merck, Darmstadt, Germany). The entire procedure was performed inside a sterile laminar airflow chamber. The plates were subsequently incubated in darkness at 23–25 °C for 4 days.
Colletotrichum spp. were selected by morphological characteristics, such as the rate of growth, mycelium color, texture, nature of the growing margin, and color of the reverse side. The shape of the conidia was observed under an Olympus BX-50 compound microscope (1000× magnification). The fungus was then isolated by transferring a colony to a new (PDA) plate for growing. Mycelia from isolated Colletotrichum spp. were ground in liquid nitrogen and used in DNA extraction for further identification of the species.
2.3. Fungal DNA Extraction and Identification
The DNA extraction was performed using the Cetyltrimethylammonium ammonium bromide (CTAB) method [16
], with some modifications as described previously [17
]. The DNA concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA).
The fungal isolates were identified by PCR amplification of the internal transcribed spacer (ITS
) region (ITS1, 5.8S rRNA, ITS2), part of the β-tubulin 2
) gene, and the intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH
) by using primers ITS1 and ITS4 [18
], T1 and T22 [19
], and GDFfwd and GDFrev [20
], respectively. The PCR reactions were performed in a total volume of 50 μL, containing 30–80 ng of genomic DNA, 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs (Fermentas, Thermo Scientific, Waltham, MA, USA), 0.2 μM of each primer, and 2.5 U of DreamTaq DNA polymerase (Fermentas). The amplification reactions were carried out in a Thermal Cycler (BioRad, Hercules, CA, USA) with an initial temperature of 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s; 55 °C for 45 s (for ITS
), 58 °C for 55 s (for tub2
), and 56 °C for 55 s (for GAPDH
); and 72 °C for 60 s, as well as a final extension at 72 °C for 10 min.
The amplified products were analyzed by agarose gel electrophoresis. The PCR products were purified using DNA Clean & Concentrator (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions and sequenced in both directions by Macrogen (Madrid, Spain). The search for homologous sequences was done using basic local alignment search tools (BLAST) at the National Center for Biotechnology Information (NCBI). The analysis of the ITS
, and GAPDH
sequences was carried out using CLUSTAL W in MEGA software version 7.0 [22
]. The phylogenetic relationships were inferred using neighbor joining (NJ), and the trees were produced using the minimum evolution, maximum parsimony, and maximum likelihood methods in the MEGA 7 software. The bootstrap analyses with 1000 replicates were performed to evaluate the significance of the interior branches.
2.4. Multivariate Data Analysis
Multivariate analyses were performed to detect significant differences in the total number of olive trees showing the presence of Colletotrichum
spp. in three different plant organs “2-year stems, flower buds, and immature fruits”; in three cultivars, “Galega vulgar, Cobrançosa, and Azeiteira”, and in three sites “Vidigueira, Monforte, and Elvas”. The statistical analyses of the data were performed using the PRIMER v6 software package [23
] with the PERMANOVA add-on package [24
]. The total number of olive trees with the presence of Colletotrichum
spp. was calculated using the dataset from different plant organs, in each cultivar, and at each site. A three-way permutational analysis of variance (PERMANOVA) was applied to test the hypothesis that significant differences existed in the total number of trees with Colletotrichum
spp. among “2-year stems, flower buds, and immature fruits”, among “Galega vulgar, Cobrançosa, and Azeiteira”, and among “Vidigueira, Monforte, and Elvas”. The PERMANOVA analysis was carried out following the two-factor design: organs: “2-year stems, flower buds, and immature fruits” (3 levels, fixed); cultivars: “Galega vulgar, Cobrançosa, and Azeiteira” (3 levels, random); and sites: “Vidigueira, Monforte, and Elvas” (3 levels, random, nested in cultivars). The total data were square root transformed in order to scale down the importance of highly abundant replicates and increase the importance of the less abundant ones in the analysis of similarity. The PERMANOVA analysis was conducted on a Bray–Curtis similarity matrix [25
]. The null hypothesis was rejected at a significance level <0.05 (if the number of permutations was lower than 150, the Monte Carlo permutation p was used).
A principal component analysis (PCA) of the presence of Colletotrichum spp., was performed to explore patterns in the multidimensional data by reducing the number of dimensions with minimal loss of information. The PCA ordination was based on each of the three sites “Vidigueira”, “Monforte”, and “Elvas” and on each of the three cultivars “Galega vulgar”, “Cobrançosa”, and “Azeiteira”. Prior to the calculation of the PCA, the ordination data were checked for normal distribution and, if necessary, were log (X + 1) transformed prior to analysis, and then, the data were normalized by subtracting the mean and dividing by the standard deviation for each variable.
The relative contribution of the presence of Colletotrichum spp. in 2-year stems, flower buds, and immature fruits to the average of similarity and dissimilarity between the a priori defined groups; sites (Monforte, Vidigueira, and Elvas); and cultivars (Galega, Cobrançosa, and Azeiteira) was calculated using the two-way crossed similarity percentage analysis (SIMPER, cut-off percentage: 100%).
Although the disease cycle of anthracnose has been studied for several years [2
], some aspects are still not yet fully understood. It is known that the anthracnose pathogen infects several parts of the olive tree: buds, flowers, sepals, pedicels, peduncles, leaves, petioles, leaf scars, shoots, twigs, receptacles, and fruits [9
]. However, there is lack of information about the presence of the pathogen in the interior of the plant organs, as well as the impact it may have on the initiation and development of the disease. In addition, there is not a clear relation between the Colletotrichum
species identified (within the C. acutatum
complex) and the olive cultivars with different degrees of susceptibility.
In this study, the presence of Colletotrichum
spp. was determined in the interior of 2-year stems, flower buds, and immature fruits of anthracnose asymptomatic olive trees from three different cultivars with different susceptibilities to the pathogen, grown in three different sites in Alentejo, the largest olive producing region in Portugal. Initially, 68 Colletotrichum
spp. isolates were selected for their morphological and cultural characteristics: the color of the colonies, which varied from pink to grey and orange and the shape of the conidia, which presented a fusiform shape [27
]. The ITS
regions from all 68 Colletotrichum
isolates were first used for PCR and sequencing due to their easy amplification when compared to alternative genes; however, the analysis of ITS
-rDNA resulted in sequences highly similar to five different Colletotrichum
species, and the analysis was not able to discriminate among them; thus, β-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH
) genes were amplified and sequenced, because both genes have previously shown good results in Colletotrichum
species identification [6
]. The results based on the single alignment and phylogenetic analysis of both the β-tubulin
sequences, allowed for the categorization of all the isolates into two species (C. nymphaeae
and C. godetiae
) within the C. acutatum
complex. The prevalence of the species belonging to the C. acutatum
complex over the Colletotrichum gloesporioides
complex, as observed here, has been associated with areas where anthracnose is endemic and more aggressive [4
]. This was also shown in other studies performed in Spain, Italy, Tunisia, and in the Alentejo region (Portugal) where an incidence of 100% C. acutatum
over C. gloesporioides
was observed [2
], associated with losses of over 90% [2
To the best of our knowledge, this was the first time that the C. godetiae
species was detected in Alentejo. A previous study revealed that all the isolates from the Alentejo region, were identified as C. nymphaeae
]. Despite the low incidence of C. godetiae
in Alentejo, as well as in Ribatejo (<6%) and Beira Baixa (<3%), this species showed high incidences in other Portuguese regions, showing predominance over other Colletotrichum
species in the Trás-os-Montes region [6
]. In other countries, such as Italy, Montenegro, and Greece, C. godetiae
is the most frequent Colletotrichum
species, leading some authors to suggest that this species is the most frequent olive anthracnose pathogen in the central Mediterranean [2
]. These contrasting observations clearly suggest that environmental conditions could shape the population structure of olive anthracnose pathogens, and under unfavorable conditions to the disease, less virulent olive anthracnose pathogens, such as C. godetiae
, may emerge. The lower virulence of C. godetiae
when compared with C. nymphaeae
and the low disease severity observed in the Alentejo region during the sampling year (field observations) may have created the opportunity for C. godetiae
to appear. Nevertheless, the vast majority of the isolates were identified to be C. nymphaeae
(95.6%), corroborating that this species is the key pathogen in olive anthracnose in Portugal, as observed in other countries [32
All three cultivars showed the presence of Colletotrichum
spp.; Galega vulgar showed a significantly higher number of infected trees and higher percentages of infected organs, followed by Azeiteira and Cobrançosa, respectively. This result could be explained by the strong susceptibility of Galega vulgar to the disease [10
]. However, the relation between the presence of Colletotrichum
spp. and the development of anthracnose is not linear. In fact, a higher presence of the Colletotrichum acutatum
complex was found in Azeiteira when compared with Cobrançosa, despite Azeiteira being considered resistant to the disease and Cobrançosa moderately tolerant. This may mean that plants from the Azeiteira cultivar may present Colletotrichum
spp. without developing the disease. However, it cannot be ignored that information on the susceptibility of cultivars is sometimes discrepant due to factors such as the different ripeness times, as the susceptibility of fruit increases with ripeness, the misidentification of cultivars, the misidentification of the disease due to confusion with other pathogens that cause fruit rot, or the virulence of the pathogen populations [3
]. The low susceptibility to anthracnose of both of the early maturing cultivars Azeiteira and Cobrançosa is probably because the fruits are usually collected before the conditions are optimal for the development of the disease. Late maturing cultivars are more affected than early maturing ones [35
], which may also explain why Galega vulgar, a late maturing cultivar, is so susceptible to anthracnose. In addition, Galega vulgar, in contrast with Azeiteira and Cobrançosa, is very susceptible to the olive fly, which contributes to the existence of wounds that are, in turn, related to the increase of the rate of colonization of the fungus and the severity of the symptoms [1
]. The thinner epidermis cells of the Galega vulgar fruits, when compared with other less susceptible cultivars, also provide lower protection against pathogens [39
All the sites showed the presence of Colletotrichum spp. Elvas showed the highest number of infected trees, followed by Monforte and Vidigueira, respectively; however, these differences were not significant. Similarities in the environmental conditions and the management at the different sites may have contributed to these small differences.
All three of the tested plant organs presented Colletotrichum
spp. in their interior. The immature fruits showed a significantly higher presence of Colletotrichum
spp., followed by the flower buds and the 2-year stems, respectively. These results differ from the ones obtained by Moral et al. [9
], who showed that developing fruits were the plant organs that showed the lowest percentages of Colletotrichum
spp. (<1.5%) when compared with stems and flowers—very different from the 12.6% (34 trees with Colletotrichum
spp. in their fruits out of the 270 olive trees tested) obtained in this study. The sampling was performed following the dry spring of 2016, and the pathogen inoculum might have been even higher if the sampling was performed after a rainy season, as periodic rain events in the spring lead to high levels of the presence of the pathogen on vegetative organs [6
]. In addition, the severity of the disease in autumn is not correlated with the level of presence of Colletotrichum
spp. in vegetative organs but, instead, with the weather conditions in autumn.
Overall, our results suggest that the olive tree may serve as important source of inoculum. In addition, in our survey, no mummified fruits were observed, suggesting that they are not an essential source of inoculum, as already shown by other authors [6
]. The inoculum present inside the symptomless organs tested in this study may be responsible for the primary infection of Colletotrichum
spp. together with the latent infection of flowers and young fruits in the spring and summer, respectively, as suggested by Moral et al. [9
]. Trees become infected through the invasion of the mycelium from wounds or the peduncles and the petioles of the affected fruits and leaves, respectively [1
]; however, it had not been confirmed previously whether the fungus moves inside the plant, infecting other parts of the plant. It was interesting to verify that C. nymphaeae
2, characterized by a unique nucleotide mutation within the beta tubulin gene, was found in different organs of the same tree—in the 2-year stems and in recently formed vegetative organs, such as flower buds and immature fruits—which seem to suggest that the infection may be caused the same isolate, which has moved systemically inside of the plant. In addition, C. godetiae
was shown to be rare, but it was found in both the flower buds and fruits of the same tree.
Interestingly, Colletotrichum spp. was found simultaneously in different organs in seven trees, all of them belonging to cultivar Galega vulgar; this may help us to understand the high susceptibility of the cultivar, which may be associated with the greater ability of the fungus to move inside the plant.
All these observations, together with the fact that the highest percentages of the infected immature fruits were obtained in trees that also presented a high percentage of 2-year stem infections, seem to support the idea of the systemic movement of Colletotrichum spp. inside olive trees.