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Article

Diversity and Pathogenicity of Botryosphaeriaceae and Phytophthora Species Associated with Emerging Olive Diseases in Italy

by
Benedetto T. Linaldeddu
1,*,
Giovanni Rossetto
1,
Lucia Maddau
2,
Thomas Vatrano
3 and
Carlo Bregant
1
1
Dipartimento Territorio e Sistemi Agro-Forestali, Università degli Studi di Padova, Viale dell’Università 16, 35020 Legnaro, Italy
2
Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy
3
Independent Researcher, Via Einaudi 9, 88021 Borgia, Italy
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(8), 1575; https://doi.org/10.3390/agriculture13081575
Submission received: 20 July 2023 / Revised: 31 July 2023 / Accepted: 3 August 2023 / Published: 7 August 2023
(This article belongs to the Special Issue Diseases and Disorders of Perennial and Ornamental Plants)
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Abstract

:
Extensive collar rot, sunken and bleeding cankers, shoot blight, and fruit rot symptoms on olive trees have recently been observed in several orchards in Italy. Since there is little information about the etiology of these diseases and given the high economic relevance of this iconic crop, a study was conducted from autumn 2017 to summer 2022, in four Italian regions, to define the occurrence, distribution and impact of the main pathogens involved. A total of 1064 symptomatic olive samples were collected and processed. Based on colony appearance, micromorphological analysis and DNA sequence data, thirty-eight species, including eighteen Botryosphaeriaceae species belonging to five genera and fifteen Phytophthora species, were isolated and identified, thirteen of which, Diplodia africana, D. fraxini, D. subglobosa, Dothiorella omnivora, Do. sarmentorum, Do. sempervirentis, Sardiniella urbana (Botryosphaeriaceae), Phytophthora cactorum, P. cinnamomi, P. citricola, P. crassamura, P. niederhauserii and P. pseudocryptogea, are reported here for the first time in olive trees. Pathogenicity tests performed on unripe drupes and on potted olive seedlings completed Koch postulates and highlighted that several species of Botryosphaeriaceae and Phytophthora represent a growing threat to olive trees.

1. Introduction

The olive tree (Olea europaea L. subsp. europaea var. europaea) is one of the first domesticated fruit crops and one of the most widespread species in Mediterranean agroecosystems [1]. Its domestication dates back to more than 5000 years ago [2], and it is supposed that current cultivars have originated from the wild Mediterranean olive (oleasters) [3,4]. Over time, human activities have largely contributed to its dissemination from Mediterranean countries to new cultivation areas such as Australia, California, Chile, China, South Africa and New Zealand [5,6,7,8,9]. In Europe, olive production reaches more than 14 million tons, and Italy is the second producer country after Spain, with 2.4 million tons in 2022 [10,11].
In Italy, olive orchards are present in all regions with an overall growing area of 1,156,334 ha, of which 29% is located in Apulia, followed by Calabria (16%), Sicily (14%), Tuscany (7%), Lazio (7%), Campania (6%), Abruzzo and Sardinia (4%) and Umbria (2%) and the rest is in the other regions [11].
Pests and diseases remain the major limiting factor in olive production in both traditional and emerging producing countries [12,13,14]. Among fungal diseases, the olive anthracnose caused by Colletotrichum spp., olive leaf spot caused by the ascomycetous fungus Venturia oleaginea, and verticillium wilt caused by the soil-borne fungus Verticillium dahliae are considered the most important olive diseases and the major limiting factor for olive oil production worldwide [15,16,17,18]. In addition to these well-known diseases, for which management strategies are available, since 2014, a complex wilting syndrome has been recorded in different olive orchards, initially in Veneto and Lombardy and subsequently in other Italian regions, including Sardinia and Calabria. Preliminary field surveys have highlighted a complex symptomatology characterized by the simultaneous presence of leaf chlorosis, shoot blight, progressive branch dieback, drop of unripe drupes, and root and collar rot. These diseases have a great impact on reducing plant productivity and vitality [19]. The symptoms observed on olive trees in northern Italy resemble those reported by Sánchez-Hernanádez et al. [20] in Spain, Úrbez-Torres et al. [14] in California and Carlucci et al. [21] in southern Italy, in which several species of Phytophthora and Botryosphaeriaceae were associated with declining olive trees.
Species in the family Botryosphaeriaceae are emerging as important olive pathogens causing leaf spot, fruit rot and branch dieback in different countries and continents [14,22]. In addition to Botryosphaeria dothidea, the causal agent of Dalmatian disease [23], Neofusicoccum mediterraneum, N. luteum, and N. parvum have recently been reported in Australia, California, Croatia, Italy, Spain, and Uruguay as emerging olive pathogens [21,24,25,26,27,28]. At the same time, over the last decade, reports of root and collar rot and sudden-death symptoms in young and mature olive trees caused by Phytophthora spp. have increased worldwide [20,29,30,31].
Therefore, given the growing expansion of the new wilting syndrome in various Italian olive orchards and the still limited information available about its etiology, a study was conducted in four Italian regions to isolate and identify the main species involved, as well as to evaluate their pathogenicity.

2. Materials and Methods

2.1. Field Surveys and Sampling

From summer 2017 to autumn 2022, the health status of olive trees belonging to 18 cultivars was monitored in thirty-eight orchards located in Veneto and Lombardy (northern Italy), Sardinia (center) and Calabria (south) (Table 1). The olive trees were checked for the presence of symptoms on fruits (necrotic depressed spot and rot), stem and branches (dieback, exudates and cankers) and at the collar and root system (exudates, necrosis and loss of fine roots). A total of 1064 symptomatic samples, including necrotic fruits (299 unripe and 266 ripe drupes), cankered branches (366) and necrotic roots, including the rhizosphere (133), were randomly chosen for laboratory diagnostic analysis (Table 1).
In order to estimate and measure the amount of plant disease symptoms [32], premature fruit drop was estimated in 2020 in an organic farm in Veneto, site 17. In particular, a field survey was conducted from May to September on eight mature olive trees chosen at random. Under the canopy of the selected trees, a square-shaped plot (1 × 1 m) was established. Every two weeks in the period from June to September, the drupes fallen into the plots were collected and counted, and the plot surface was cleaned. The occurrence and incidence of wilted branches with sunken cankers were monitored in the period from 2017 to 2021 on 160 olive trees in site 27 (Sardinia). The disease incidence was evaluated as the number of symptomatic trees among the total number of olive trees monitored [32]. The symptomatic branches were cut every year in February immediately after the disease-incidence survey.
In site 10 (Veneto), all plants were visually checked for the occurrence of typical Phytophthora symptoms such as poor growth, leaf chlorosis, shoot blight, bleeding cankers, and collar and root rot. The disease incidence was estimated as the number of symptomatic trees out of the total number of trees. In addition, the same plants were monitored for the presence of branch canker and dieback, as well as symptoms on fruits.

2.2. Isolation of Fruit Rot Agents

In the laboratory, olive fruits were surface-disinfected with 70% ethanol for 30 s, rinsed in sterile distilled water, and then air-dried in an aseptic condition. Ten fragments (4 × 4 mm) of symptomatic tissues (epicarp and mesocarp) for fruits were cut aseptically, placed onto potato dextrose agar (PDA, Oxoid Ltd., Basingstoke, UK) in Petri dishes, and incubated at 25 °C in the dark for 7 days. In order to obtain pure cultures, hyphal tips from emerging colonies were sub-cultured onto half-strength PDA dishes, labelled and incubated at 25 °C in the dark.

2.3. Isolation of Branch Canker Agents

Branch samples were initially disinfected with ethanol (70%) for 30 s, and then the outer bark was removed with a sterile scalpel. Longitudinal and cross sections were made to observe internal disease symptoms. Isolations were performed from ten small fragments (4 × 4 mm) of the inner bark and xylem cut aseptically from the margin of the necrotic lesions. All fragments were placed onto 90 mm Petri dishes containing PDA and incubated at 25 °C for 7 days in the dark. Pure cultures were obtained and stored as reported previously.

2.4. Isolation of Collar and Root Rot Agents

Phytophthora isolations were performed using the method described by Linaldeddu et al. [33]. In particular, collar, roots and rhizosphere samples were flooded separately in 1 L of distilled water in plastic boxes. Young cork oak and elder leaves were placed as baits on the clean water surface after 24 h. Boxes were maintained at 18 °C under natural daylight for 5 days. Leaves showing dark spots or Phytophthora sporangia were cut in small fragments (4 × 4 mm), air dried and then placed in Petri dishes containing PDA supplemented with 100 mL/L of fresh carrot juice, 0.013 g/L of pimaricin and 0.05 g/L of hymexazol (PDA+) [34].
Isolations were also performed from collar and root tissue samples taken from the margin of inner bark necrotic lesions. The small fragments obtained with a sterile scalpel were aseptically placed onto 90 mm Petri dishes containing PDA+. The dishes were incubated in the dark at 18 °C and examined every 12 h. Pure cultures obtained from hyphal tips were stored on PDA and carrot agar (CA) at 20 °C in the dark.

2.5. Identification of the Isolates

Fungal and Phytophthora isolates were initially grouped in morphotypes on the basis of colony growth characteristics, including surface and reverse colony appearance observed after 7 days of incubation on PDA and CA at 20 °C and 25 °C in the dark. In addition, morphometric data of conidia and sporangia recorded using the software Motic Images Plus 3.0 paired with a Moticam 10+ camera connected to a Motic BA410E microscope (Motic, Wetzlar, Germania) were used for the identification of the isolates. To enhance sporangia production, the CA plugs of each isolate were placed in Petri dishes containing unsterile pond water and fine root samples of oaks, whereas the production of conidia was enhanced through the exposure of the fungal colonies to natural sunlight on the laboratory bench for one month.
Furthermore, DNA sequence data analysis was used to confirm the identification of all isolates at species level. Genomic DNA from the mycelium of pure culture was extracted, as reported by Linaldeddu et al. [33]. For all isolates, the complete internal transcribed spacer (ITS) region of the rDNA was amplified and sequenced using the primers ITS1 and ITS4 [35]. Polymerase chain reactions (PCRs) were performed in 50 µL reaction mixtures using the GoTaq® Hot Start Green Master Mix (Promega, Milan, Italy) and a SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc, Waltham, MA, USA). Amplification conditions were as follows: an initial denaturation step at 94 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 25 s, annealing at 54 °C for 35 s and extension at 72 °C for 45 s, and a final elongation step of 8 min at 72 °C. PCR products were purified using the MonarchTM PCR & DNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Both strands were sequenced using their BMR Genomics DNA sequencing service (www.bmr-genomics.it, accessed on 31 July 2023). Sequences were edited with FinchTV v1.4.0 (Geospiza, Inc., http://www.geospiza.com/finchtv, accessed on 31 July 2023) and compared with sequences of ex-type culture deposited in GenBank (http://blast.ncbi.nlm.nih.gov, accessed on 31 July 2023). New sequences were deposited and are available in GenBank.

2.6. Pathogenicity Assays

Pathogenicity trials were performed according to Koch’s rules on asymptomatic unripe fruits (cv. Rasara) and on 3 year-old-seedlings (cv. Leccino) using pure cultures of one representative isolate of the main species obtained. Three different assays were performed.
In the first assay, artificial inoculations of asymptomatic unripe olive fruits (cv. Rasara) collected at the end of July from an organic orchard located in Veneto (Arquà Petrarca) were performed on 12 cm Petri dishes under controlled conditions. Forty fruits per fungal isolate were initially surface disinfected with 90% ethanol and then wounded with a sterile needle (depth of 1 mm) and inoculated with a 3-mm-diameter mycelium PDA plug taken from an active 5-day-old colony. Forty fruits wounded in the same way were inoculated with a sterile PDA plug and used as controls. Inoculated and control fruits were incubated in a climatic chamber at 25 °C in the dark with 100% relative humidity for 7 days. Four fungal species were used in this assay. At the end of the experimental period, the occurrence of a necrotic lesion was assessed, and the size of the area was measured using Assess 2.0 [36].
The second assay was conducted on 3 year-old-seedlings (cv. Leccino) using pure cultures of ten fungal species. Five branches per fungus were used in the artificial inoculations. The outer bark surface of each branch was initially disinfected with 90% ethanol and then inoculated with an agar-mycelium plug (3 × 3 mm) in the center of a wound, of the same size, made with a sterile scalpel. The inoculation site was protected with a moist cotton ball and wrapped with aluminum foil. Five branches, inoculated with a sterile PDA plug, were used as controls. The assay was conducted in the period from April to July 2019 in a cold greenhouse with the temperature ranging from 6.1 °C to 37.6 °C. At the end of the experimental period (122 days), the length of necrotic lesions caused by each fungal species was measured and recorded after the removal of the outer bark.
Finally, in the third assay, five olive seedlings of the same cultivar and age were inoculated with thirteen Phytophthora species. The assay was performed at the collar by placing a mycelial agar plug of 5 mm diameter on a wound of the same size made with a sterile cork borer. The inoculation point was protected as previously reported, and the seedlings were kept in the same experimental conditions. Seedlings were regularly watered, and after one hundred and twenty-two days, the presence, nature and size of the external and internal symptoms were detected on all inoculated seedlings.
For each bioassay, re-isolation of the fungal and Phytophthora species was performed from twenty small fragments of inner bark or epicarp tissues cut around the margin of the necrotic lesions and placed onto PDA (fungi) or PDA+ (Phytophthora). Growing colonies were sub-cultured onto PDA, incubated in the dark at 20 °C for 7 days, and identified on the basis of colony appearance and DNA sequence data.

2.7. Data Analysis

The results of the pathogenicity test were checked for normality and then subjected to analysis of variance (ANOVA). Significant differences among mean values were determined using Fisher’s least significant differences multiple-range test (p = 0.05) after one-way ANOVA using XLSTAT 2008 software (Addinsoft, Paris, France).

3. Results

3.1. Field Surveys

In all orchards, the widespread presence of severe diseases with a strong impact on productivity and vitality of olive trees was observed. In addition to the sporadic presence of some common olive tree diseases, such as peacock’s eye disease and Cercospora leaf spot, several plants, regardless of age or variety, showed a complex symptomatology on the drupes, branches and root system (Figure 1, Figure 2 and Figure 3).
An anomalous unripe fruit drop was found in June, July and early August. The symptomatic drupes were characterized by the initial presence of small sunken necrosis, which progressively expanded up to the stalk, causing an intense carpoptosis (Figure 1a–g). Disease incidence was estimated at up to 100% in several sites, mainly in 2017 and 2019; in the monitored plot (site 17), the number of fallen drupes per square meter varied from 115 ± 28 (19 June 2020) to 214 ± 107 (31 July 2020), with total loss of production in August. Symptoms on ripe fruits varied depending on the fungal species involved but can basically be separated into two main groups: light brown soft rot and reddish-brown rot (Figure 1h,i). In the latter case, the infected fruits wrinkled, mummified and remained attached to the tree. On the epicarp of the mummified fruits, the occurrence of black pycnidia of B. dothidea, which represents an inoculum source for the next spring infection, was constantly observed (Figure 1i).
Furthermore, a wilting syndrome was observed in all of the monitored sites (Figure 2). Seasonal dynamics of wilting symptoms on twigs and branches include several stages: initially, olive trees show irregularly scattered light-red necrotic areas of the canopy as the wilted leaves rapidly turn to a rusty color; subsequently the necrotic areas expand and merge, affecting large portions of the canopy. In autumn, the leaves fall, leaving the branches bare (Figure 2a,b). On symptomatic branches, extensive sunken cankers characterized by inner bark and internal wood necrosis were often observed. In the cross section, sunken cankers showed the typical V-shaped necrotic sector caused by Botryosphaeriaceae (Figure 2k). In some cases, cankers on the stem and at the collar showed red-brown exudations. At site 27, the incidence of olive trees with new branch dieback symptoms was found to vary over the five years of investigation from a minimum of 11% in 2018 to a maximum of the 27% in 2020.
Finally, in the monitored orchards, several plants showed leaf chlorosis, poor growth and often sudden-death symptoms. The latter symptom was particularly frequent in early autumn. Plants with sudden-death symptoms were easily recognizable as the dark yellow leaves remained attached to the branches even for an entire growing season. The plants characterized by sudden death showed large, necrotic, inner-bark lesions at the collar and main roots, as well as loss of fine roots (Figure 3). Disease incidence was very variable among sites. In site 10, 20% of olive trees showed these typical Phytophthora disease symptoms.

3.2. Fungal and Fungal-Like Species Associated with Symptomatic Samples

A total of 886 fungal and fungal-like isolates were obtained from the 1064 samples processed; in particular, Botryosphaeriaceae and Peronosporaceae (oomycetes) were the most abundant families, with 752 and 121 isolates, respectively. All fungal and Phytophthora isolates showed mycelial growth and conidia or sporangia production within 30 days. Overall, twenty-three fungi and fifteen Phytophthora species were identified based on morphological features and DNA sequence data. The ITS sequence of a representative isolate of each species was deposited in GenBank (Table 2). The complex symptomatology detected in the monitored orchards is due to the attack, often simultaneous, of various pathogens. In particular, the severe drop of unripe drupes is caused mainly by B. dothidea. The early attack of B. dothidea causes necrotic sunken lesions that progressively expand up to the stalk, determining the premature fall of drupes. In addition to B. dothidea, in the rot symptoms detected in autumn, thirteen other Botryosphaeriaceae species are involved, albeit with less frequency (Table 2). It is interesting to note that the assemblage of Botryosphaeriaceae species associated with symptomatic drupes changes during the growing season, with several species occurring on mature drupes, including the invasive species N. australe and N. parvum (October–November).
From the 366 branch samples with sunken canker and dieback symptoms, eighteen species of Botryosphaeriaceae (Botryosphaeria dothidea, Diplodia africana, D. fraxini, D. mutila, D. olivarum, D. sapinea, D. seriata, D. subglobosa, Dothiorella iberica, Do. omnivora, Do. sarmentorum, Do. sempervirentis, Neofusicoccum australe, N. cryptoaustrale, N. luteum, N. mediterraneum, N. parvum and Sardiniella urbana), two Diatrypaceae (Eutypa lata and Eutypa leptoplaca), two Togniniaceae (Phaeoacremonium iranianum and Phaeoacremonium scolyti) and one species of Apiosporaceae (Apiospora arundinis) were isolated. Neofusicoccum parvum was the dominant species associated with canker and dieback symptoms in Lombardy, Veneto and Sardinian olive orchards (Table 2). However, the etiology of woody samples was extremely complex, since from the symptomatic branch tissues, several other species were constantly isolated, such as B. dothidea, Diplodia fraxini, D. olivarum, D. seriata, D. subglobosa and Dothiorella sarmentorum (Table 2).
Isolation performed from 133 symptomatic collar and root samples yielded a total of 121 colonies belonging to fifteen Phytophthora species (Table 2). The most frequently isolated species were Phytophthora plurivora and P. palmivora, followed by P. acerina and P. pini (Table 2). Phytophthora palmivora was isolated in 7 out of 27 monitored orchards, including Veneto, Lombardy, Sardinia and Calabria, whereas the other dominant species were isolated in one or two regions. In particular, P. plurivora and P. acerina were widespread in Veneto and Lombardy, P. plurivora and P. heterospora in Sardinia and P. crassamura, P. oleae and P. palmivora in Calabria (Figure 4).

3.3. Pathogenicity

In the first assay, all four Botryosphaeriaceae species tested were shown to be pathogenic on unripe olive fruits (cv Rasara) under laboratory conditions. A dark-brown, necrotic lesion surrounding the inoculation point was observed 48 h after inoculation, while 7 days later, the necrotic area in some cases extended over the entire surface. Control drupes remained asymptomatic. Neofusicoccum parvum produced the most extensive lesions, followed by D. olivarum, B. dothidea and D. mutila (Table 3).
In the pathogenicity assay on branches, all inoculated species, except Apiospora arundinis, proved to be pathogenic on olive seedlings, though with significant differences in aggressiveness (Table 4). The most aggressive species was N. parvum; at 122 days after inoculation, the isolate N2 caused significantly longer lesions that were externally visible as sunken cankers similar to those observed in the field (Table 4). In addition, even the cankers caused by D. fraxini, D. olivarum and D. subglobosa were characterized by sunken bark lesions, a symptom congruent with field observations.
In the inoculation at the collar, the inner bark necrotic lesions caused by P. pini, P. plurivora and P. acerina were consistently larger compared with those of other species and controls, confirming the aggressiveness of these species on olive trees (Table 5). Also, P. cactorum and P. palmivora caused extensive lesions, whereas P. inundata caused only a small brown discoloration, which did not differ statistically from the control.
On all control seedlings, a very small brown discoloration was visible in correspondence to the inoculation point, but the wound had begun to heal.
All fungal and Phytophthora species were successfully re-isolated from symptomatic tissue, thus fulfilling Koch’s postulates.

4. Discussion

This study represents the most comprehensive investigation to date on Botryosphaeriaceae and Phytophthora-related diseases on olive trees in Italy. The results obtained have allowed us to clarify the complex etiology of the emerging diseases affecting olive trees in four Italian regions achieving new insights into the symptomatology caused by Botryosphaeriaceae and Phytophthora attacks on this host. Specifically, the symptoms caused by the main isolated species often overlapped at site level and in the same tree, making a clear separation often difficult. The often-synergic attack of Botryosphaeriaceae and Phytophthora species in some cases causes serious economic losses due to tree decline, mortality and fruit yield reduction.
Based on ITS sequence data, twenty-three fungal species belonging to eight genera, including Apiospora, Botryosphaeria, Diplodia, Dothiorella, Eutypa, Neofusicoccum, Phaeoacremonium and Sardiniella, were identified.
Amon the fungal species, Botryosphaeriaceae were the main species isolated from olive trees with branch cankers and fruit rots; this is in accordance with other recent studies in which members of this family were found to be the most aggressive species isolated from olive trees in California, Croatia, Italy, Spain, South Africa, Tunisia and New Zealand [14,21,25,28,37,38]. Field surveys and pathogenicity tests have shown that N. parvum and D. olivarum are the main pathogens involved in olive cankers and dieback in the monitored orchards, although several other species, and in particular, D. fraxini, D. olivarum, D. seriata and D. subglobosa, can play an important role in the etiology of the symptoms observed at a local scale. Neofusicoccum parvum and Diplodia olivarum have recently been detected as the key agents of a new wilting syndrome on oleaster trees in Italy [39]. Species within the genus Neofusicoccum and Diplodia have been reported as pathogens causing cankers, dieback and mortality on several woody hosts worldwide [40]. The diversity and pathogenicity of Botryosphaeriaceae species associated with branch dieback of olive trees detected in this study suggest that no one single agent is involved in the etiology but that it is the result of multiple infections of different Botryosphaeriaceae species, which are variable in assemblages among regions and sites. The co-occurrence of different Botryosphaeriaceae associated with cankers with a V-shaped necrotic sector and dieback symptoms is a recurring aspect in the pathosystems in which these pathogens are involved in both forestry and agriculture [41,42,43,44,45]. The complex etiology detected in this study is in accordance with the results obtained by Urbez-Torres et al. [14] in California. Among the species isolated from cankered branches, the presence of D. fraxini and D. subglobosa, two aggressive pathogens involved in the ash dieback etiology, emerges [46,47]. This is the first report of these two Diplodia species as olive pathogens.
Further investigations are needed to establish the pathogenicity of S. urbana, Do. omnivora, Do. sarmentorum, Do. sempervirentis and D. africana on olive trees. Sardiniella urbana is a rather rare pathogenic species known only on hackberry [48]. This represents the first report of S. urbana on olive. Given that the olive orchard sampled in Veneto was surrounded by hackberry, it is likely that high inoculum pressure resulted in a few infections of the olive trees by this pathogen. The genus Dothiorella was established by Saccardo [49] and currently encompasses several endophytic, saprobic and plant pathogenic species associated with a wide range of woody hosts worldwide, including olive [50]. This is the first report of D. africana, Do. omnivora, Do. sarmentorum and Do. sempervirentis on olives.
Until 2005, B. dothidea was the only Botryosphaeriaceae species known as the causal agent of olive drupe rot, “Dalmation disease” [23]. Lazzizera et al. [22], during an extensive survey of drupe rot in southern Italy, found that in addition to B. dothidea, other species of Botryosphaeriaceae, namely D. olivarum, N. australe, N. vitifusiforme, N. parvum and N. mediterraneum, cause the same symptoms on olive drupes. The most common species was B. dothidea, which was isolated from 34% of the rotted drupes. This finding agrees with the results obtained in this study. Field surveys conducted in this study showed that B. dothidea invades the drupes in the early stage of fruit setting, and new infection can occur during all olive fruit growth and developmental stages. On unripe fruits, B. dothidea was the dominant species, while in the last stages of drupe development, several Botryosphaeriaceae species can cause fruit rot. However, a clear difference in symptomatology was noted between attacks on drupes of B. dothidea and N. parvum (Figure 1h,i). These differences were also confirmed in pathogenicity assays. Unlike the results of Lazzizera et al. [22], on ripe drupes, we found almost exclusively pycnidia of B. dothidea and rarely those of N. australe. All Botryosphaeriaceae species isolated from drupes are known to produce bioactive secondary metabolites [51,52,53,54]. The identification of the compounds, including pigments that could cause serious risks for human health and significant loss in the commercial value of oil, have several practical implications for health care and the improvement in olive oil quality in Mediterranean countries.
Diatrypaceae and Togniniaceae are often reported as weak or opportunistic olive pathogens in different countries [55,56]. In the pathogenicity assay, Eutypa lata, E. leptoplaca and Phaeoacremonium iranianum have been confirmed to be pathogenic in olives, although their role in these emerging diseases appears marginal. Eutypa leptoplaca is reported here for the first time as an olive pathogen. This species has been reported as a grapevine pathogen in California [57].
Fifteen Phytophthora species belonging to six out the twelve major Phytophthora clades were identified. The genus Phytophthora over the last three decades has expanded rapidly to reach the current 220 species [58], of which 15 have been isolated in this study. The presence of the fifteen species in the Italian regions was discontinuous, suggesting that different Phytophthora species can cause the same symptoms on olive trees. In particular, in north Italy, three species within clade 2, P. acerina, P. plurivora and P. pini, were widespread. This confirms the results of a preliminary study conducted in Veneto (north Italy) [30]. Phytophthora plurivora, like some other species in clade 2, is a polyphagous pathogen whose host range comprises plants of different families, including Betulaceae, Fagaceaea and Oleaceae in forest ecosystems, horticulture, and less frequently in agriculture [30,59,60,61,62].
Phytophthora acerina was originally described in 2014 on Acer pseudoplatanus in Lombardy (north Italy), and a few years later, in 2018, causing sudden death to olive trees in Veneto [30] and Alnus glutinosa in Sardinia in 2020 [63]. The high virulence displayed by P. acerina on inoculated olive seedlings, as well as on Alnus spp., proves it to be an extremely aggressive pathogen. The host and geographic range are still unknown; recently, it has been isolated in Slovenia along the Italian border (Bregant, Linaldeddu and Ogris unpublished data) and in the Marche region (central Italy) (Bregant, Linaldeddu and Murolo unpublished data).
Phytophthora pini was originally described as a new species in 1925 but without a Latin description (Leonian, 1925). In 2011, Hong et al. [64] examined ex-authentic cultures of P. pini, proving that they were identical to P. citricola subgroup I. Therefore, Phytophthora pini has been resurrected to distinct species status and formally redescribed with a Latin description. Many aspects related to the ecology of this pathogen are unknown, and to properly evaluate its potential impact on olive and oil production in the Mediterranean region, further studies are necessary.
In addition to P. plurivora, in Sardinia, other two species P. heterospora and P. crassamura were frequently isolated. Phytophthora heterospora is a sister species of P. palmivora, formally described on olive trees and other hosts in 2021 [31]. In the compared pathogenicity assay, P. heterospora proved to be moderately aggressive on olives, although less so than P. palmivora. Phytophthora heterospora is common on nursery plants [65] but has recently also been isolated from declining oleaster and narrow-leaved mock-privet (Phillyrea angustifolia, family Oleaceae) in different natural ecosystems in Sardinia (Linaldeddu, unpublished data). Phytophthora crassamura was formally described as a distinct species in 2015 from different host species of the Mediterranean maquis in the archipelago of La Maddalena [66], but it is actually widespread. Recently, it has been reported in several restoration sites in California [67] and globe artichoke plants in the main growing area in Sardinia [68]. Almost all findings of P. crassamura came from geographic areas characterized by a Mediterranean climate. Phytophthora crassamura was the dominant species in the Calabrian olive groves. In this region, two other species, P. oleae and P. bilorbang, were constantly isolated from symptomatic trees.
Phytophthora oleae was originally reported, causing fruit rot in olive orchards in Italy and root rot in wild-olive trees in Spain [69,70]. In this study, P. oleae emerged as one of the most common pathogens involved in root rot symptoms. Its ability to infect roots, stem and also fruits highlights the risk posed by this species on olive trees.
Phytophthora bilorbang was formally described in Australia in 2012 [71], but it was previously informally designated as Phytophthora taxon oaksoil [72] and is known to occur in France [73] and Oregon [74]. In the present study, P. bilorbang was isolated in both Veneto and Calabria olive orchards. This is consistent with a previous record [75], in which this pathogen was reported on olive trees (cv. Nera di Gonnos) with leaf chlorosis, severe defoliation, and root rot symptoms in an experimental orchard in Calabria. Phytophthora bilorbang, like several other members of clade 6, has a prevalently aquatic lifestyle and is frequently recovered from streams and riparian ecosystems as an opportunistic pathogen [71]. Given its ecology and moderate pathogenicity, the innovative olive growing systems (intensive and super-intensive) could favor the attacks of this opportunistic species.
The other Phytophthora species were recovered less frequently. Among these, Phytophthora cactorum, P. cinnamomi, P. citricola and P. niederhauserii were detected for the first time on declining olive trees worldwide. The discovery of P. niederhauserii in Sardinian olive orchards is of particular concern due to its high virulence [76] and the recent discovery from the same oleaster trees showing the new wilting syndrome caused by N. parvum ([39]; Linaldeddu unpublished).
The current trend of discovering an increasing number of pathogenic Phytophthora species in agricultural crops [77] emphasizes how important efforts need to be made to develop appropriate management strategies to limit the impact on the quality and quantity of agricultural productions. Our finding has contributed to expanding knowledge on the biodiversity of Phytophthora species associated with olive trees in Italy, which currently include sixteen species.
Among the species evaluated in the pathogenicity tests, Apiospora arundinis (syn. Arthrinium arundinis) and P. inundata were the only species that did not cause any disease symptoms on inoculated seedlings, and both were re-isolated with a low frequency. Apiospora arundinis has been detected from branch cankers together with N. parvum, which in the pathogenicity test reproduced the symptoms observed in nature, suggesting an endophytic behavior of A. arundinis on olive trees. Phytophthora inundata is reported as an opportunistic pathogen of woody hosts in riparian ecosystems that is able to cause sporadic disease outbreaks on susceptible hosts after soil flooding or waterlogging [78].

5. Conclusions

In conclusion, in this study, the high diversity of Botryosphaeriaceae and Phytophthora species has been associated with olive tree diseases in Italy. The complexity of the etiology detected in the monitored orchards, together with the results from the pathogenicity assays, highlight that several Botryosphaeriaceae and Phytophthora species could represent a new emerging threat to the olive and oil industry in Italy. The overlap of symptoms caused by different pathogens suggests that only through a holistic diagnostic approach is it possible to identify the species involved.
Olive groves are long-term investments that require adaptive management strategies. The findings obtained in this study emphasize that new management strategies need to be developed to protect this iconic fruit crop. Currently, there are a few effective chemical products to prevent or cure the attacks of Botryosphaeriaceae and Phytophthora on olive trees.

Author Contributions

Conceptualization, B.T.L.; methodology, B.T.L. and C.B.; investigation, B.T.L., C.B., G.R., L.M. and T.V.; data curation, B.T.L., L.M. and C.B.; writing—original draft preparation, B.T.L.; writing—review and editing, B.T.L., C.B., G.R., L.M. and T.V.; funding acquisition, B.T.L. and L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Regione Veneto and Servizio Fitosanitario Regionale del Veneto, by the Land Environment Resources and Health (L.E.R.H.) doctoral course (University of Padova), and by Fondo di Ateneo 2020, an internal funding from the University of Sassari.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank Chiara Padovan, Roberto Callegaro (Frantoio Evo del Borgo), Enzo Gambin (AIPO), Bruno Bernardi, and Sergio Carraro (Regione Veneto) for assistance during field surveys and sampling.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Overview of the symptoms observed on unripe and ripe drupes: necrotic lesions caused by Botryospharia dothidea on unripe fruits (a–c), necrosis that progressively expands up to the stalk (red arrow) (d–f), extensive fruit drop observed in July (g), drupe with the typical light brown soft rot caused by Neofusicoccum parvum (h), and reddish-brown mummified drupes with black pycnidia of B. dothidea (i). From the left to right colony, morphology of B. dothidea, Diplodia mutila, Diplodia olivarum, Diplodia seriata and N. parvum after 7 days growth at 25 °C on PDA in the dark.
Figure 1. Overview of the symptoms observed on unripe and ripe drupes: necrotic lesions caused by Botryospharia dothidea on unripe fruits (a–c), necrosis that progressively expands up to the stalk (red arrow) (d–f), extensive fruit drop observed in July (g), drupe with the typical light brown soft rot caused by Neofusicoccum parvum (h), and reddish-brown mummified drupes with black pycnidia of B. dothidea (i). From the left to right colony, morphology of B. dothidea, Diplodia mutila, Diplodia olivarum, Diplodia seriata and N. parvum after 7 days growth at 25 °C on PDA in the dark.
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Figure 2. Overview of symptoms observed on the olive tree canopy: extensive dieback with phylloptosis (a,b), shoot blight (c), twig dieback (d,e), sunken cankers on twigs and branches (f–j), and V-shaped necrotic sector in cross section (k). From top to bottom, colony morphology of Botryosphaeria dothidea, Diplodia fraxini, Diplodia mutila, Diplodia olivarum, Diplodia seriata, Diplodia subglobosa, Dothiorella sarmentorum and Neofusicoccum parvum after 7 days growth at 25 °C on PDA in the dark.
Figure 2. Overview of symptoms observed on the olive tree canopy: extensive dieback with phylloptosis (a,b), shoot blight (c), twig dieback (d,e), sunken cankers on twigs and branches (f–j), and V-shaped necrotic sector in cross section (k). From top to bottom, colony morphology of Botryosphaeria dothidea, Diplodia fraxini, Diplodia mutila, Diplodia olivarum, Diplodia seriata, Diplodia subglobosa, Dothiorella sarmentorum and Neofusicoccum parvum after 7 days growth at 25 °C on PDA in the dark.
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Figure 3. Overview of Phytophthora-related symptoms observed on olive trees: chlorosis and increased transparency of the canopy (a,b), trees with sudden death syndrome (c–e), root rot and loss of fine roots on 6-year-old plants (f), and stem canker and inner bark necrotic lesion at the collar (g–j). From left to right, the colony morphology of Phytophthora acerina, P. bilorbang, P. crassamura, P. oleae, P. palmivora, P. pini and P. plurivora after 7 days growth at 20 °C on CA in the dark.
Figure 3. Overview of Phytophthora-related symptoms observed on olive trees: chlorosis and increased transparency of the canopy (a,b), trees with sudden death syndrome (c–e), root rot and loss of fine roots on 6-year-old plants (f), and stem canker and inner bark necrotic lesion at the collar (g–j). From left to right, the colony morphology of Phytophthora acerina, P. bilorbang, P. crassamura, P. oleae, P. palmivora, P. pini and P. plurivora after 7 days growth at 20 °C on CA in the dark.
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Figure 4. Isolation frequency and distribution of the 12 most common Phytophthora species isolated in this study.
Figure 4. Isolation frequency and distribution of the 12 most common Phytophthora species isolated in this study.
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Table 1. Details of the monitored orchards and number of symptomatic samples collected per site.
Table 1. Details of the monitored orchards and number of symptomatic samples collected per site.
Italian
Region		SitesCultivarType of Samples
Unripe DrupesRipe DrupesStem/BranchesCollar/Roots
Veneto1Casaliva6-1012
2Grignano--153
3Grignano3-15-
4Grignano2-13-
5Grignano--19-
6Grignano--51-
7Grignano--42
8Grignano-4132
9Leccino, frantoio, Trepp, favarol--21-
10Leccino, grignano, trepp16351720
11Grignano--142
12Grignano--8-
13Grignano12-75
14Grignano--6-
15Arbequina, arbosana10-316
16Rasara, leccino, matosso, itrana-1572
17Rasara5050757
18Frantoio, leccino-542
19Frantoio, leccino, Grignano-862
20Frantoio, leccino-105-
21Leccino, grignano, Frantoio-332
22Frantoio, leccino, pendolino-105-
23Leccino, frantoio, Grignano-1073
24Frantoio, leccino-552
Lombardy25Frantoio-653
26Frantoio-532
Sardinia27Frantoio1001001010
28Bosana--10-
29Semidana--57
30Pizz’e carroga100---
Calabria31Carolea---5
32Biancolilla---5
33Nocellara del Belice---5
34Frantoio---3
35Carolea---3
36Carolea---3
37Carolea---3
38Carolea---2
Table 2. Accession numbers deposited in GenBank and number of isolates of each species obtained from unripe drupes (UD), ripe drupes (RD), stem and branches (SB) and roots (R).
Table 2. Accession numbers deposited in GenBank and number of isolates of each species obtained from unripe drupes (UD), ripe drupes (RD), stem and branches (SB) and roots (R).
Fungal SpeciesAccession
Numbers		Type of SamplesSites
UDRDSBR
Apiospora arundinisOR2848452-2-1, 9
Botryosphaeria dothideaOR2848462087316-6, 10, 15–23, 25–27, 30
Diplodia africanaOR284847-1--27
Diplodia fraxiniOR2848482-15-10, 17
Diplodia mutilaOR284849323812-6, 9, 10, 12, 16–21, 27
Diplodia olivarumOR28485092415-1, 6, 7, 9, 11, 12, 21, 24, 27, 30
Diplodia sapineaOR284851-1--27
Diplodia seriataOR28485231847-1–4, 6–8, 10, 11,13, 15–17, 27, 30
Diplodia subglobosaOR284853-414-1–3, 9, 15, 17, 20
Dothiorella ibericaOR2848541-2-10, 16
Dothiorella omnivoraOR284855--1-16
Dothiorella sarmentorumOR284856-311-1–6, 17
Dothiorella sempervirentisOR284857--1-6
Eutypa lataOR284858--3-9, 16
Eutypa leptoplacaOR284859--2-1, 9
Neofusicoccum australeOR284860-291-27
Neofusicoccum cryptoaustraleOR284861-114-27, 29
Neofusicoccum luteumOR28486225--27
Neofusicoccum mediterraneumOR284863357-27, 28
Neofusicoccum parvumOR28486471890-1, 2, 4, 5, 6, 8–10, 15–28
Phaeoacremonium iranianumOR284865--2-14
Phaeoacremonium scolytiOR284866--2-9, 14
Sardiniella urbanaOR284867-1--17
Phytophthora acerinaOR284868---148, 10, 24, 27
Phytophthora bilorbangOR284869---610, 15, 38
Phytophthora cactorumOR284870---411, 17
Phytophthora cinnamomiOR284871---219
Phytophthora citricolaOR284872---131
Phytophthora crassamuraOR284873---1029, 31–33, 36
Phytophthora heterosporaOR284874---527, 29, 35
Phytophthora inundataOR284875---429, 31, 32
Phytophthora nicotianaeOR284876---215
Phytophthora niederhauseriiOR284877---410, 27
Phytophthora oleaeOR284878---631, 32, 34
Phytophthora palmivoraOR284879---157, 25–27, 33, 34, 37
Phytophthora piniOR284880---1211, 15, 18
Phytophthora plurivoraOR284881---311, 6, 8, 12, 27
Phytophthora pseudocryptogeaOR284882---531, 33, 36, 37, 38
Table 3. Mean lesion size (cm2) ± standard deviation caused by the four Botryosphaeriaceae species on unripe drupes and percentage of positive re-isolations.
Table 3. Mean lesion size (cm2) ± standard deviation caused by the four Botryosphaeriaceae species on unripe drupes and percentage of positive re-isolations.
Fungal SpeciesDrupe Lesion Size 1Positive Re-Isolation (%)
Botryosphaeria dothidea2.4 ± 0.8 b100
Diplodia mutila2.3 ± 0.9 b100
Diplodia olivarum2.7 ± 2.1 b100
Neofusicoccum parvum6.3 ± 2.6 a100
Control0.0-
1 Values with the same letter do not differ significantly at p = 0.05, according to LSD multiple range test.
Table 4. Mean lesion length (mm) ± standard deviation caused by the ten fungal species on olive branches and the percentage of positive re-isolations.
Table 4. Mean lesion length (mm) ± standard deviation caused by the ten fungal species on olive branches and the percentage of positive re-isolations.
Fungal SpeciesLength Necrotic
Lesion 1		Wilting SymptomsPositive
Re-Isolation (%)
Apiospora arundinis1.2 ± 0.4 efno20
Botryosphaeria dothidea6.1 ± 1.2 deno100
Diplodia fraxini33.5 ± 6.5 byes100
Diplodia olivarum14.6 ± 2.6 cno100
Diplodia seriata6.2 ± 1.3 dno100
Diplodia subglobosa13.6 ± 2.6 cno100
Eutypa lata6.1 ± 1.2 deno80
Eutypa leptoplaca5.8 ± 1.2 deno80
Neofusicoccum parvum58.2 ± 10.1 ayes100
Phaeoacremonium iranianum5.2 ± 1.2 deno60
Control0.2 ± 0.4 fno-
1 Values with the same letter do not differ significantly at p = 0.05, according to LSD multiple range test.
Table 5. Mean lesion length (mm) ± standard deviation caused by the thirteen Phytophthora species on olive seedlings and percentage of positive re-isolations.
Table 5. Mean lesion length (mm) ± standard deviation caused by the thirteen Phytophthora species on olive seedlings and percentage of positive re-isolations.
Phytophthora SpeciesLength Necrotic Lesion 1Positive Re-Isolation (%)
Phytophthora acerina27.7 ± 4.9 a100
Phytophthora bilorbang13.8 ± 5.9 c100
Phytophthora cactorum20.1 ± 5.8 b100
Phytophthora cinnamomi12.6 ± 2.1 c100
Phytophthora citricola7.3 ± 3.8 d100
Phytophthora crassamura4.8 ± 2.3 de100
Phytophthora heterospora11.3 ± 4.2 c100
Phytophthora inundata2.4 ± 0.5 e80
Phytophthora oleae7.2 ± 2.5 d100
Phytophthora palmivora19.1 ± 4.1 b100
Phytophthora pini29.5 ± 5.4 a100
Phytophthora plurivora28.7 ± 3.5 a100
Phytophthora pseudocryptogea13.1 ± 3.8 c100
Control1.6 ± 1.2 e-
1 Values with the same letter do not differ significantly at p = 0.05, according to LSD multiple range test.
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Linaldeddu, B.T.; Rossetto, G.; Maddau, L.; Vatrano, T.; Bregant, C. Diversity and Pathogenicity of Botryosphaeriaceae and Phytophthora Species Associated with Emerging Olive Diseases in Italy. Agriculture 2023, 13, 1575. https://doi.org/10.3390/agriculture13081575

AMA Style

Linaldeddu BT, Rossetto G, Maddau L, Vatrano T, Bregant C. Diversity and Pathogenicity of Botryosphaeriaceae and Phytophthora Species Associated with Emerging Olive Diseases in Italy. Agriculture. 2023; 13(8):1575. https://doi.org/10.3390/agriculture13081575

Chicago/Turabian Style

Linaldeddu, Benedetto T., Giovanni Rossetto, Lucia Maddau, Thomas Vatrano, and Carlo Bregant. 2023. "Diversity and Pathogenicity of Botryosphaeriaceae and Phytophthora Species Associated with Emerging Olive Diseases in Italy" Agriculture 13, no. 8: 1575. https://doi.org/10.3390/agriculture13081575

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