Colletotrichum infections during flower development and fruit ripening in four olive cultivars

Summary. Olive anthracnose, caused by Colletotrichum , is an important disease in olive-growing regions, with the most destructive symptoms being fruit rot and blossom blight. Susceptibility of fruit to Colletotrichum increases with maturity, but differences between cultivars and Colletotrichum species have been reported, still information on flower susceptibility during development is scarce. The susceptibility of the olive cultivars Arbequina, Coratina, Frantoio and Picual was evaluated during flower development and fruit maturity to Colletotrichum acutatum s.s., C. nymphaeae , C. fioriniae , C. theobromicola and C. alienum . Susceptibility to anthracnose begins in early stages during flower development and increases during blossoming. Flowers of Arbequina, Coratina and Picual were susceptible, whereas those of Frantoio were moderately susceptible. Green fruit developed less anthracnose than mature fruit. At the green fruit stage, Arbequina and Frantoio were the most susceptible, Coratina was intermediate, and Picual was moderately susceptible, while no differences were found among the cultivars at mature fruit stages. No mayor differences were found among the Colletotrichum species with exception of C. theobromicola , which caused greatest severity at the green fruit stage. Future research should focus on developing anthracnose management strategies to minimize the disease progress from early stages of flower development and fruit ripening, especially in the most susceptible olive cultivars.

The most common host symptom caused by anthracnose is fruit rot at the ripening stage.Fruit rot causes yield losses and deterioration in oil quality (Moral et al., 2014;Leoni et al., 2018).Infections can also occur from flowering to fruit ripening.During bloom, species of Colletotrichum can infect host plant calices, petals, stamens, and pistils causing flower collapse known as blossom blight (Moral et al., 2009;Talhinhas et al., 2018;Filoda et al., 2021;Moreira et al., 2021).Each infected flower is usually covered with an orange gelatinous mass of Colletotrichum conidia (Sergeeva et al., 2008;Moreira et al., 2021).If the flower is not destroyed, Colletotrichum infects the fruit set, remaining as latent until fruits ripen, when the typical soapy fruit is expressed (Moral et al., 2009;Talhinhas et al., 2018), although the importance of these infections for yield losses is unknown.
Infected fruits show depressed brown lesions that are rapidly covered with abundant orange gelatinous masses of Colletotrichum conidia.Fruits can be infected from green stages, and these infections remain latent infections until fruit maturation.The infected fruit can fall or remain mummified on trees, serving as a primary inoculum sources for subsequent infections in the next year (Moral et al., 2008;2014;Mosca et al., 2014;Talhinhas et al., 2018).
Studies have shown that susceptibility of olive cultivars to anthracnose can be variable (Moral and Trapero, 2009;Cacciola et al., 2012;Moral et al., 2014;2017), and depends on the Colletotrichum species (Talhinhas et al., 2015).Fruit susceptibility varies according to developmental stage (Moral et al., 2008;Moral and Trapero, 2009;Talhinhas et al., 2015;Moral et al., 2017).Nevertheless, information is limited on the susceptibility of different fruit maturity stages and different cultivars to Colletotrichum.Although flowers can be infected by Colletotrichum (Moral et al., 2014;Kolainis et al., 2020;Moreira et al., 2021), there have been no studies that indicate when in flower differentiation the flowers become susceptible.Knowing when the first infections occur at flowering and their potential incidence is important so anthracnose management strategies can be developed, including those based on fungicides.
The present study focused on evaluating the susceptibility to five Colletotrichum species of the Arbequina, Coratina, Picual and Frantoio olive cultivars, the major cultivars produced in Uruguay.The study concentrated on flower development and fruit ripening host stages.

Plant and fungal material
Apparently healthy olive flower panicles and fruit were collected from commercial orchards of the cultivars Arbequina, Coratina, Picual and Frantoio with scarce anthracnose.The panicles were collected at three different flowering stages, 1-swollen bud (BBCH51), 2final differentiation (BBCH55), or 3-beginning of flowering (BBCH61), and fruits were collected at two physiological maturity stages, 1-green fruit (BBCH80) or 2ripe fruit (BBCH89) (Sanz-Cortes et al., 2002).The collected samples were stored in nylon bags in coolers until processing in the laboratory.
For inoculum preparation, each isolate was grown on Potato Dextrose Agar (Oxoid Ltd) at 24°C under near UV-light with a 12-h photoperiod.After 7 d incubation, colony surfaces in culture plates were each flooded with 10 mL of sterile distilled water (SDW), and the scraped with a sterile spatel.The resulting conidium suspensions were filtered through layers of cheesecloth, and conidium concentration was adjusted to 1 × 10 6 conidia mL -1 with a hemacytometer.

Flower inoculation
Flowers in either the Stage 1-swollen bud (BBCH51) or Stage 2-final differentiation (BBCH55) were each surface-disinfected by dipping for 1 min in 1.0% NaClO solution, and then rinsed three times with SDW.For the Stage 3-beginning of flowering (BBCH61) flowers, surface disinfection was not possible due the sensitivity of flower petals to NaClO.After air-drying, the flowers were dipped in respective isolate conidium suspensions for 30 s, placed in transparent plastic trays containing moistened filter paper, and then incubated at 24°C with a 12 h photoperiod.Control treatments inoculated with SDW were included for each cultivar and flower phenological stage.Three repetitions for each cultivar, phenological stage and Colletotrichum isolate were used.Each repetition consisted of at least eight swollen buds for Stage 1, two panicles each with at least 15 undeveloped flower buds for Stage 2, and two panicles with at least ten open or semi-open flowers for Stage 3.Each experiment was performed using a completely randomized design with factorial arrangement.
Anthracnose incidence was assessed periodically until the plant material was destroyed or until 100% incidence was achieved, after 12 d for swollen buds, 6 d for final differentiation, or days for beginning of flowering.Incidence was calculated as the percentage of affected buds or flowers in the total number of buds or flowers evaluated.In each evaluation, the initial symptoms and their evolution was recorded as the number of necrotic buds or flowers, and the presence of gelatinous mass of Colletotrichum conidia.

Fruit inoculation
Fruits at the Stage 1-green fruit (BBCH80) or Stage 2-ripe fruit (BBCH89) were surface-disinfected by dip-ping for 3 min in 1% NaClO solution, and then rinsed three times with SDW.After air-drying, the fruits were dipped in respective isolate conidium suspensions for 30 s, and were then placed into plug seedling trays, one fruit in each hole.The plug seedling trays were enclosed in moistened transparent nylon bags, and were then incubated at 24°C with a 12 h photoperiod.Control treatments inoculated with SDW were included for each cultivars and fruit phenological stages.Four repetitions of fruits per cultivar, phenological stages and Colletotrichum isolates were used.Each repetition consisted of five fruits, and each experiment was carried out using a completely randomized design with factorial arrangement.

Statistical analyses
Flower anthracnose incidence was plotted for the three flower phenological stages of the four olive cultivars.A regression curve was fitted considering the significance of the regression and the coefficient of determination (R 2 ), based on average incidence of the 15 Colletotrichum isolates and evaluation time.Anthrac- nose incidence values were also used to calculate the area under the disease progress curve (AUDPC) for each Colletotrichum species, using the following formula: where I = incidence (%) at ith observation, ti = time (d) at the ith observation, and n = the total of number of observations.The AUDPC data were analyzed for normality with the Shapiro-Wilk test, and for homogeneity with the Levene test.These data were subjected to ANOVA with a factorial arrangement, with olive cultivar and Colletotrichum species as factors.the treatments means were compared using Tukey's test (P ≤ 0.05).
Fruit anthracnose severity values were used to calculate the McKinney's Index (Moral et al., 2017), using the following formula: where i = the severity of symptoms (0 to 5), n i = the number of fruits with the severity of i, and N = the total number of evaluated fruits.
The McKinney's Index values at the two maturity stages and for the four olive cultivars were plotted.A regression model was adjusted for each Colletotrichum species considering the statistical significance of the regression and the coefficient of determination (R 2 ).Anthracnose severity values were used to calculate the AUDPC for each Colletotrichum species using the formula outlined above.The AUDPC data were analyzed for normality with the Shapiro-Wilk test, and for homogeneity with the Levene test.The AUDPC was transformed to when necessary, to comply with normality assumptions.Then, the AUDPC data were subjected to ANOVA, with a factorial arrangement, with olive cultivar and Colletotrichum species as factors.The treatments means were compared using by Tukey's test (P ≤ 0.05).Statistical analyses were carried out using the programs InfoStat version 2016 (http://www.infostat.com.ar) and SigmaPlot version 12.0 (http://www.sigmaplot.co.uk).

Flower infections
Typical anthracnose symptoms and signs developed in flowers at the three phenological stages in the four cultivars inoculated with the Colletotrichum species.No symptoms were observed in control treatments (Figure 1, a to c).Initial symptoms consisted of brownish colouration of the swollen buds or the flower buds at final differentiation, and necrotic lesions on flower petals (Figure 1, a.2 to c.2).The affected organs then quickly blighted, and were covered with orange-salmon coloured gelatinous masses containing abundant Colletotrichum conidia (Figure 1, a.3 to c.3). Accelerated detachment of swollen buds, flower buds and open flowers was observed in comparison with the control treatments, where the buds or flowers remained attached for longer periods.
Almost all graphs of blossom blight incidence fitted exponential curves (Figure 2 and Supplementary Table 1).For the AUDPC variables, statistically significant interactions were found between olive cultivars and Colletotrichum species inoculated in the three flowering phenological stages (Table 2).The lowest anthracnose incidence was observed at the swollen bud stage, and no major differences were recorded among the four olive cultivars.In this stage, the first symptoms appeared approx.4 to 7 d after inoculation, whereas the greatest anthracnose incidence was recorded at 12 d, and ranged between 20 and 40% (Figure 2).
At final differentiation stage, the first symptoms were observed at 3 d after inoculation, with the least average incidence (2.0%) in the Frantoio cultivar and the greatest (17%) in the Arbequina cultivar.Incidence then progressed rapidly, and 6 d after inoculation reached 59 for Frantoio and 100% for Arbequina.At Stage 3beginning flowering greatest anthracnose incidence was recorded.Two d after inoculation, incidence ranged from 8% for Frantoio to 50% for Picual, and 2 d later between 45% for Frantoio and 91% for Arbequina (Figure 2, Table 3).
Except for C. alienum inoculated onto cv.Arbequina at bud swollen stage, all five Colletotrichum species infected flowers at all three phenological stages.Nevertheless, in some specific species-cultivar-phenological stage combinations, anthracnose symptoms were not visible until the second evaluation, for example, the culivars Coratina and Picual inoculated with all Colletotrichum species at the swollen bud stage (Figure 2).

Fruit infections
Characteristic anthracnose symptoms and signs developed in fruits at the two phenological stages (green and ripe) of the four inoculated cultivars, and also in some fruit not inoculated with Colletotrichum species (Figure 1, d and e).Symptoms in uninoculated fruits could have been from natural latent Colletotrichum infections.At the green fruit stage, initial symptoms consisted of small, depressed, 1-2 mm necrotic lesions scattered  throughout each fruit (Figure 1, d.2).Symptoms then progressed into depressed brown lesions, which were quickly covered with orange gelatinous masses of Colletotrichum conidia, known as "soapy fruit" (Figure 1, d.3).On ripe fruit, symptoms developed more quickly and consisted of typical soapy fruit (Figure 1, e.2 and e.3).
Fruit anthracnose severity at both green and ripe fruit phenological stages, fitted sigmoidal curves (Figure 3 and Supplementary Table 1).For AUDPC variables, significant interactions were found between olive cultivars and inoculated Colletotrichum species inoculated, at both green and ripe fruit phenological stages (Table 2).On green fruit, the first symptoms were observed between 5 and 14 d after inoculation.In this stage, Arbequina and Frantoio reached severity values close to 50% at 30 d after inoculation, and Coratina 35 d after inoculation, whereas Picual reached 50% severity at 50 d after inoculation (Figure 3).At the mature fruit stage, the first symptoms were observed 4 d after inoculation.Anthracnose evolution occurred more quickly compared to that at the green fruit stage.For all cultivars and Colletotrichum species, severity was an average of 77% at 12 d after inoculation, and 92% at 18 d after inoculation (Figure 3).
The five Colletotrichum species were able to infect fruits at two phenological stages in the four olive cultivars.Colletotrichum theobromicola caused the greatest AUDPC values at the green fruit stage (Table 3).Green fruit inoculated with this species developed symptoms earlier ( 5  ment, susceptibility increased, and symptoms and signs progressed rapidly.Symptoms included brown colouration of the flower organs that progressed to collapse of inflorescences, known as blossom blight.Affected organs were rapidly covered by typical orange-salmon coloured gelatinous masses, corresponding to Colletotrichum conidia (Sergeeva et al., 2008;Iliadi et al., 2018;Moreira et al., 2021).
Results of the present study are in similar to those of Kolainis et al., (2020), who observed that in the olive cultivars Koroneiki and Kalamon, the first anthracnose symptoms appeared 2 d after inoculation of detached flowers at beginning of flowering stage.In contrast, Moral et al., (2009) inoculated flowers of Arbequina, Hojiblanca and Picual cultivars at the same phenological stage, but the first symptoms were visible 5 d after inoculation.These differences could be because Moral et al., (2009) utilized attached flowers, whereas in the present study and that of Kolainis et al. (2020), detached flowers were used.
For the four olive cultivars evaluated, some differences were observed in anthracnose susceptibility at different flowering stages.No major variations were observed at the swollen bud stage, but at final differentiation and beginning of flowering, Frantoio was the least susceptible cultivar to Colletotrichum, Arbequina was the most susceptible, and Coratina and Picual were of intermediate susceptibility.Similar results were obtained by Moral et al., (2009), who found that Arbequina was more susceptible than Hojiblanca and Picual when inoculated at the beginning of flowering.
In previous research, we demonstrated that species C. acutatum s.s., C. nymphaeae, C. fioriniae, C. theobromicola and C. alienum caused typical blossom blight at beginning of flowering (Moreira et al., 2021).In the present study, although the incubation period could be variable, isolates of these five Colletotrichum species infected olive from the early stages of flower development.
Results recorded here for behaviour of inoculated green and ripe olive fruit were generally in agreement  with previous research.Olive fruits can be infected by Colletotrichum at different stages during fruit ripening, but the susceptibility increases with ripening (Moral et al., 2008;2009;Sergeeva, 2014;Chattaoui et al., 2016;Moral et al., 2017).We also observed differences in symptom development.While on ripe fruit the typical soapy fruit symptoms were observed from the beginning, on green fruit initial symptoms consisted of small and depressed necrotic lesions that later progressed into the typical soapy fruit.In addition, at the green stage, the first symptoms were visible between 5 and 7 d after inoculation, whereas on ripe fruit symptoms were seen earlier, between 1 and 5 d post inoculation.Similar results were obtained by Moral et al., (2008), who observed first symptoms at 7 d on detached leaves, and at 4 d on ripe fruit, after Colletotrichum inoculation.
For susceptibility of the different olive cultivars, at the green fruit stage Picual was the least susceptible and Arbequina and Frantoio were the most susceptible cultivars to Colletotrichum spp., while Coratina developed disease with intermediate susceptibility.These results are similar to other studies, where Arbequina was susceptible to moderately susceptible, Coratina was moderately susceptible (Andres 1991; Moral and Trapero, 2009;Bartolini and Cerreti, 2013), and Picual was moderately resistant or resistant to anthracnose (Moral and Trapero, 2009;Talhinhas et al., 2015;Moral et al., 2017).
On mature fruit, the four olive cultivars were all highly susceptible to anthracnose.These results showed some discrepancies with other studies, where Frantoio was found to be highly resistant to anthracnose in Spain (Moral et al., 2008;Moral and Trapero, 2009;Moral et al., 2017).Nevertheless, in accordance with the present results, Frantoio showed high susceptibility to anthracnose in Argentina (Andres, 1991) and Italy (Loprieno and Tenerini, 1960).
Although the differences in anthracnose susceptibility found among cultivars at the green fruit stage were not recorded for ripe fruit, this was not surprising.Moral and Trapero (2009) reported that green fruits were more resistant to Colletotrichum than ripe fruit, probably because of greater concentrations of phenolic compounds in green than ripe fruits.However, when maturity is reached, all olive cultivars can become diseased, and develop complete rot regardless of their early fruit susceptibilities (Moral et al., 2008).
Colletotrichum theobromicola differed substantially from the other Colletotrichum species inoculated in this study, being the most aggressive pathogen at the green fruit stage.This was surprising since this species, together with C. alienum, was isolated from a low proportion of olives with typical anthracnose symptoms in Uruguay (Moreira et al., 2021).In Australia, inoculated detached green stage fruit was more affected by C. theobromicola and C. gloeosporioides s.s than C. aenigma, C. cigarro, C. queenslandicum, C. siamense and C. karstii (Schena et al., 2014).Regarding the international occurrence of C. theobromicola, This pathogen has been reported affecting olive in Argentina, Australia, and Uruguay (Schena et al., 2014;Lima et al., 2020;Moreira et al., 2021), but not in countries of the Mediterranean basin, where most olives are cultivated.Schena et al., (2014) mentioned that new diseases are expected to emerge as consequence of climate change, and suggested that C. theobromicola could play an important role in olive anthracnose disease with these changes.
For ripe fruit, behaviour of the five Colletotrichum species was similar, except for C. alienum which was less aggressive when it was inoculated on the cultivars Arbequina and Frantoio.In Portugal, Talhinhas et al., (2015)  The present study is the first in which anthracnose susceptibility has been evaluated at different host flower development stages.Based on these results, the risk of anthracnose occurrence starts at early stages of f lower development.This would allow development of improved disease management decisions.For example, the most opportune time to initiate preventive fungicide applications should be at early stages of flower development, to minimize yield losses caused by olive anthracnose.For olive cultivars, Frantoio was moderately susceptible to anthracnose, whereas Picual, Coratina and Arbequina were susceptible cultivars during flowering.However, the present study results indicate that at the green fruit stage, Frantoio and Arbequina were the most susceptible cultivars, Coratina had an intermediate susceptibility, and Picual was least susceptible.In mature fruit, no differences were found among the assessed cultivars.In this study we confirmed that Arbequina, the main olive cultivar produced in Uruguay, was one of the most susceptible to Colletotrichum during flowering and fruit ripening.Future research should focus on improving anthracnose management strategies to minimize the impacts of this disease during flower development and fruit maturity, especially in those olive cultivars that are most susceptible to this disease.

Figure 2 .
Figure 2. Mean incidences of olive blossom blight in flowers of four olive cultivars inoculated with five Colletotrichum species, at swollen buds (BBCH51/53), final differentiation (BBCH55), and beginning of flowering (BBCH61).Vertical bars indicate standard errors of means, each calculated from three replicates.Trend curves were graphed based on the average incidence at each evaluation time.The R 2 and estimations of the trend curves were obtained using SigmaPlot version 12.0 software.
d after inoculation) compared with the other Colletotrichum species (14 d after inoculation), and reached almost 100% severity about 15 d earlier than those inoculated with the other Colletotrichum spp.(Figure 3).In ripe fruit, the behaviour among the five Colletotrichum species was similar.Anthracnose indices close to 100% were reached at about 15 d after inoculation, except for the cultivars Arbequina and Frantoio inoculated with C. alienum, where severity indices were close to 50%.DISCUSSION This study assessed anthracnose susceptibility during flower development and fruit ripening in the four main olive cultivars grown in Uruguay, Arbequina, Frantoio, Coratina and Picual.That study used artificial inoculations with five Colletotrichum species, of detached olive panicles and fruits.The results indicated that the four olive cultivars assessed were are susceptible to Colletotrichum at all the evaluated phenological stages, although differences in phenological stages, olive cultivars, and Colletotrichum species were detected.The swollen bud stage was susceptible to Colletotrichum species.Although susceptibility of this phenological stage to anthracnose was low, we demonstrated that Colletotrichum can infect flowers from early stages during flower development.In later stages of flower develop-

Figure 3 .
Figure 3. Mean anthracnose severity indices in fruit of four olive cultivars inoculated with five Colletotrichum species at green (BBCH80) and ripe fruit (BBCH89) phenological stages.Severity values were used to calculate the McKinney indices.The incidence registered in the control treatments can be attributable to the latent infection of Colletotrichum spp.expression.Vertical bars correspond are standard errors of the means calculated based on four replicates.The R 2 and the estimations of trend curves were obtained using SigmaPlot version 12.0 software.

Table 1 .
Uruguayan Colletotrichum isolates used to evaluate the susceptibility of four olive cultivars at different phenological stages during flower development and fruit ripening.

Table 2 .
Analysis of variance of data of Areas Under Disease Progress Curves (AUDPCs) estimated based on anthracnose incidence and severity developed in olive flower and fruits, for four olive cultivars inoculated with five Colletotrichum species.The flowers were inoculated at three phenological stages and fruits at two stages.

Table 3 .
Mean Areas Under Disease Progress Curves (AUDPCs) estimated based on anthracnose incidence and severity developed in flower and fruits of four olive cultivars inoculated with five Colletotrichum species.The flowers were inoculated at three phenological stages and fruits at two stages.
found that C. acutatum s.s. and C. nymphaeae were more aggressive than C. gloeosporioides s.s. and C. rhombiforme on ripe fruit.In Italy Schena et al. (2017) observed that C. acutatum s.s. was more aggressive than C. godetiae on ripe fruit.In this work, C. acutatum s.s., C. nymphaeae and C. fioriniae were of similar aggressiveness.