Diversity of Botryosphaeriaceae species associated with canker and dieback of avocado ( Persea americana ) in Italy

Summary. Increased branch canker and dieback were observed in commercial avo-cado ( Persea americana ) orchards in Sicily, Italy. Surveys were conducted in 2021 and 2022 on 11 orchards to investigate etiology of the disease. Seventy-five plants from four orchards, showing branch canker and dieback, were sampled. Isolations from woody diseased tissues revealed the presence of fungi ( Botryosphaeriaceae ). Identification of the isolates was achieved by morphological and multi-loci phylogenetic analyses (Maximum Parsimony and Maximum Likelihood) of the ITS, tef1-α, and tub2 loci. Botryosphaeria dothidea, Lasiodiplodia citricola , Macrophomina phaseolina, Neofusicoccum cryptoaustrale , and Neofusicoccum luteum were identified. Representative isolates collected from the orchards, characterized based on the tub2 locus and identified as N. parvum, were excluded from this study, since this species has already been reported in our territory . Pathogenicity tests were conducted on potted, asymptomatic, 2-year-old avocado trees using mycelial plugs. These tests showed that all the Botryosphaeriaceae species characterized in this study were pathogenic to avocado. This is the first report of L. citricola , M. phaseolina and N. cryptoaustrale causing can-ker and dieback on avocado trees, and is the first record of these fungi causing branch disease on avocado in Italy.


INTRODUCTION
Avocado (Persea americana L.) is a tree native to Mexico and has spread to many tropical and subtropical regions (Bost et al., 2013).Consumption of avocado fruit and new plantings of avocados has considerably increased (Bost et al., 2013).The greatest production is in Mexico, followed by Colombia and the Dominican Republic (FAOSTAT, 2022).In Europe, Spain was the first country to develop commercial production of avocados (Pérez-Jiménez, 2008).In Italy, avocado production is spread in the Southern regions, mainly in Sicily, where the cultivated area has increased in the last 10 years (Migliore et al., 2017).In Sicily, avocado provides good agricultural diversification as an alternative crop to citrus (Guarnaccia et al., 2016).
Several diseases can affect avocados, and several fungi taxa have been associated with different symptoms.Traditionally, root diseases have been considered the most important limiting factors for avocado production.Among these, those caused by Phytophthora cinnamomi and Rosellinia necatrix are considered the most important and widespread diseases of avocado, leading to serious losses, especially in the Mediterranean regions where avocado production is well established (Zentmyer, 1980;López-Herrera and Melero-Vara, 1992;Fiorenza et al., 2021).In recent years, species of Nectriaceae have also been shown to be important, especially in Australia where different taxa have been associated with crown root rot disease (Parkinson et al., 2017).In Italy, recent studies have shown the presence of Nectriaceae spp.causing a complex of root symptoms (Vitale et al., 2012;Aiello et al., 2020b).
In recent decades, increased research has been carried out on canker diseases of fruit and nut crops (Moral et al., 2019;Guarnaccia et al., 2022a).These diseases have been re-discovered as important and limiting for perennial crops, especially because they cause polyetic epidemics, a complex of pathogen taxa are involved, and most of the causal agents are polyphagous and live as latent pathogens.Among the taxa associated and responsible for shoot, branch and trunk cankers and dieback, Botryosphaeriaceae is a widely investigated group of fungi (Batista et al., 2021).Botryosphaeriaceae includes fungi that can be pathogens, saprobes and endophytes (Slippers and Wingfield, 2007;Phillips et al., 2013), and can be severe threats to fruit, nut, ornamental and forest trees (Slippers and Wingfield, 2007;Moral et al., 2019).DNA-based tools, especially multi-locus phylogeny, have shown that many genera and species within the Botryosphaeriales have been described, synonymized, and reaccommodated (Zhang et al., 2021).
In Italy, the first investigations of avocado branch and trunk canker were reported in 2016, showing the presence of Botryosphaeriaceae (N.parvum), Diaporthaceae (D. foeniculacea and D. sterilis) and Glomerellaceae (Colletotrichum gloeosporioides and C. fructicola) (Guarnaccia et al., 2016).Studies on avocado canker diseases in Italy have continued, and in 2018, a new species Neocosmospora persea was described, causing branch and trunk canker, which was also later reported in Greece (Guarnaccia et al., 2018(Guarnaccia et al., , 2022b)).More recently, the new species Neopestalotiopsis siciliana and Ne.rosae were reported as causing stem lesions and dieback on avocado (Fiorenza et al., 2021).
An increased incidence of shoot and branch canker has been observed in Sicilian avocado orchards since 2016.The present study has investigated the diversity of Botryosphaeriaceae associated with symptomatic trees.The aims of the study were: (i) to characterize the Botryosphaeriaceae recovered from symptomatic avocado samples, and (ii) to test their pathogenicity to this host.

Field surveys and fungal isolation
Surveys were conducted in Sicily (Italy) during 2020 and 2021 in the main avocado production areas (Catania, Messina, and Siracusa provinces).Eleven orchards were investigated and selected for sampling.Samples (three to ten plants from each site) of symptomatic branches, trunks and shoots were collected, and brought to the Plant Pathology laboratory, Dipartimento di Agricoltura, Alimentazione e Ambiente, Sezione di Patologia Vegetale, University of Catania.Small sections (0.5 cm 2 ) of symptomatic tissues were surface disinfected for 1 min in 1.5% sodium hypochlorite solution (NaOCl), rinsed in sterile distilled water, dried on sterile absorbent paper and placed on potato dextrose agar (PDA; Lickson) amended with 100 mg L -1 of streptomycin sulphate (Sigma-Aldrich) to prevent bacterial growth, and incubated at 25±1°C for 7 d.Isolation frequency of Botryosphaeriaceae was calculated using the formula: F = (N Bot /N Tot ) × 100, where F is the frequency of Botryosphaeriaceae; N Bot is the number of woody fragments from which Botryosphaeriaceae were isolated; and N Tot is the total number of woody fragments from which fungi were isolated.Single hyphal tip cultures on PDA were obtained.Thes isolates are maintained in the Plant Pathology collection of the University of Catania.

Morphological and culture characters of isolates
Representative isolates of each morphologically different group of isolates were transferred onto Technical Agar (AT, 1.2% Agar Technical, Biolife) supplemented with autoclaved pine needles (Smith et al., 1996), and were incubated at room temperature under UV light.The size, colour, and shape of conidia produced by the isolates were examined.After 14 d, pycnidia were observed with a stereoscope, and were mounted in 100% lactic acid.Fifty conidia from each representative isolate were measured (length and width), using an Olympus-BX61 fluorescence microscope coupled to an Olympus DP70 digital camera.Measurements were captured using the software analySIS 3.2 (Soft Imaging System GmbH).Dimensions are reported here as averages.

DNA extraction and PCR analyses
The representative isolates were cultivated on PDA for 7 d, and genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation) following the manufacture's protocol.The quality of the DNA was determined using a Nanodrop Lite Spectrophotometer (Thermo Fisher Scientific), and was diluted to 5 ng μL -1 with nuclease-free water.The internal transcriber spacer region (ITS) of the nuclear ribosomal RNA operon was amplified with primers ITS5 and ITS4 (White et al, 1990), and the primers EF1-728F and EF1-986R (Carbone and Kohn, 1999) were used to amplify part of the translation elongation factor 1 alpha locus (tef1-α), and primer sets Bt2a and Bt2b (Glass and Donaldson, 1995) were used for the partial beta tubulin locus (tub2).The amplifications were each carried out in a final volume of 25 μL using One Taq® 2× Master Mix with Standard Buffer (BioLabs), according to the manufacturer's instructions, on an Eppendorf Mastercycler (AG 22331).The PCR consisted of initial 30 s at 94°C, followed by 35 cycles at 94°C for 30 s, 50-52°C (ITS), 57-59°C (tef1-α), or 52°C (tub2) for 1 min, followed by 68°C for 1 min, and 5 min at 68°C.All PCR products were visualized on 1% agarose gels (90 V for 40 min), stained with GelRed®, purified, and sequenced by Macrogen Inc. Forward and reverse DNA sequences were assembled and edited using AliView software (Larsson, 2014), and were submitted to GenBank.Sixty-two isolates were sequenced (amplifying the tub2 locus only), and based on these preliminary results only 23 representative isolates were considered for further locus sequencing and phylogenetic analyses.

Phylogeny
Sequences were read, assembled, and edited using MEGAX: Molecular Evolutionary Genetics Analysis across computing platforms (Kumar et al., 2018).The ITS, tub2 and tef1-α DNA sequence datasets were aligned using MEGAX.For comparison, 57 additional sequences were selected according to the most recent taxonomic classification of Botryosphaeriaceae genera and species involved in this study (Table 1).Two analyses were performed.Maximum parsimony analysis (MP) was performed in PAUP* (Phylogenetic Analysis Using Parsimony) version 4.0a (Swofford, 2002).The analysis of the combined dataset (ITS + tub2 + tef1-α) was obtained with the heuristic search function and tree bisection and reconstruction (TBR) as branch swapping algorithms with the branch swapping option set on 'best trees' only.Gaps were treated as 'missing', the characters were unordered and of equal weight, and Maxtrees were limited to 100.Tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency index (RC) were calculated.A total of 1000 bootstrap replicates were performed to test the robustness of the tree topologies.The best-fit model of nucleotide evolution for each locus, according to the Akaike information criterion (AIC), was evaluated using MrModeltest v. 2.4 (Nylander, 2004).The Maximum Likelihood analysis (ML) of the combined loci was performed in GARLI v.0.951 (Zwickl, 2006), and clade support was assessed by 1000 bootstrap replicates.Phyllosticta ampelicida (CBS 111645) and Phyllosticta citricarpa (CBS 102374) served as the outgroup taxa in both analyses.

Pathogenicity test
A pathogenicity test was conducted in a greenhouse, February to April 2022.Five 2-year-old asymptomatic avocado plants 'Hass' grafted on 'Zutano' rootstocks Table 1.Information of fungal isolates used in the phylogenetic analysis and their corresponding GenBank accession numbers.Isolates in bold font are from this study.The "T" superfix identifies type material.Species  were selected for each tested fungal species.Inoculations were each carried out using a mycelium plug (0.5 cm 2 ) from a 10-d-old culture of each of Botryosphaeria dothidea (AC7), Lasiodiplodia citricola (AC20), Macrophomina phaseolina (AC29), Neofusicoccum cryptoaustrale (AVORAM4), and Neofusicoccum lutem (AVF5).
Each inoculation site was first surface disinfected with a 70% ethanol solution.Two points of inoculation for each plant were made on the stem after removing a piece of bark with a sterile scalpel blade, placing the isolate mycelium plug onto the wound and covering it with Parafilm® (American National Can) to prevent desiccation.Three 2-year-old asymptomatic avocado plants were inoculated with sterile PDA plugs to serve as inoculation controls.The plants were moved to a greenhouse and regularly watered.Temperature in the greenhouse ranged from 18 to 27°C and humidity from 70 to 80%.The inoculated plants were monitored weekly for symptom development, and a final assessment was conducted 63 d after the inoculations.Lesion length measurements were recorded, and were statistically analyzed in Statistix 10 (Analytical Software, 2013) using analysis of variance (ANOVA).Mean differences were compared with the Fisher's protected least significant difference (LSD) test at α = 0.05.To fulfill Koch's postulates, re-isolations were carried out following the procedure described above, and each re-isolated fungus was identified through observation of morphological characteristics.

Field surveys and fungal isolations
Disease was observed on 2 to 10-year-old avocado plants 'Hass', grafted on different rootstock cultivars ('Zutano', 'Duke 7', and 'Dusa') in Sicily (Italy).All the sampled plants showed symptoms of shoot and branch canker, and dieback emerging within the green canopies (Figure 1 A to D). Occasionally, a white powder was present on the surfaces of the lesions (Figure 1 E).It was also possible to observe the infections starting from pruning wounds (Figure 1 F and G).The bark of cankered shoots was cracked, darkly discoloured, and/or slightly sunken (Figure 1 H).Cankers were reddish-brown under the bark, and variable in shape.Necrotic lesions and internal discolouration were observed at the grafting points of young plants (Figure 1 I).Isolations frequently (41%) yielded Botryosphaeriaceae-like fungi, and Botryosphaeriaceae were detected in all the samples analyzed.
A total of 106 Botryosphaeriaceae isolates were collected and stored.Of these, 62 isolates (59%) were processed for DNA extraction, PCR, and sequencing.A  preliminary screening based on the tub2 locus was conducted on all 62 isolates, and this showed that representative isolates from seven orchards (orchard numbers 5 to 11, Table 2) were N. parvum.Since this fungal species was already characterized and reported in a preliminary study (Guarnaccia et al., 2016), these isolates were excluded from further locus sequencing and phylogenetic analyses, but the N. parvum isolates were collected and stored, since the present investigation showed that this fungus predominated in Sicilian avocado orchards.A total of 23 isolates derived from four orchards (orchards numbers 1 to 4) were fully characterized, as these isolates were previously unreported in Italy.These 23 isolates were from 75 young (2 to 4-year-old) plants showing typical symptoms of canker and dieback.More details of the collected and characterized isolates are summarized in Table 2.
According to these results, five species isolated from avocado in this study were identified, including: B. dothidea, L. citricola, M. phaseolina, N. cryptoaustrale, and N. luteum (Figure 2).The ITS, tub2, and tef1-α sequences generated in this study were deposited in GenBank (Table 1).

Morphological and cultural characteristic of the isolates
Observing pure cultures on PDA, a total of six groups of Botryosphaeriaceae-like fungi were observed: Isolate AC5 (B.dothidea) had olivaceous colonies that became grey with black reverse sides.Conidia were hyaline, fusiform and measured 23.2 × 5.6 µm.
Lasiodiplodia citricola AC20 had colonies with abundant aerial mycelium that became smoke grey to olivaceous-grey or iron-grey on the surfaces and greenish grey to dark slate blue on the reverse sides.Conidia were initially hyaline, aseptate, ellipsoid to ovoid and becoming pigmented, verrucose and ovoid, and measured 21.3 × 13.1 µm.
Macrophomina phaseolina isolate AC29 had grayish fluffy aerial mycelium on the colony surfaces, which were purplish grey on the reverse sides.Abundant microsclerotia were produced on pine needles in AT medium.Conidia were 25.0 × 10.5 µm.
The colonies of isolate AVORAM4 (N.cryptoaustrale) were initially white with fluffy aerial mycelium, changing to straw-yellow after 3 d incubation and then to pale olivaceous-grey.Conidia were hyaline, smooth with granular contents, aseptate, fusiform, and measured 20.0 × 6.0 µm.
Isolate AVF5 (N.luteum) was initially white with fluffy aerial mycelium and changed to yellow after 3-4 d incubation, after which the colour changed to pale olivaceous-grey from the middle of the colonies to the irregular margins.Conidia were hyaline, thin walled, aseptate, smooth, ellipsoidal, and measured 19.5 × 5.5 µm.
Isolates of N. parvum had white fluffy aerial mycelium that became grey and then black with the age.Conidia were hyaline, non-septate, and subglobose, with obtuse apices, and measured 18.2 × 6.1 µm.
Table 2. Information on fungal isolates collected and processed in this study from 11 avocado orchards.* identifies the representative isolates preliminarily identified based on the tub2 locus.¥ identifies the isolates fully characterized (ITS + tub2+ tef1-α) and included in the phylogenetic analyses.× indicates that the representative isolates were excluded from the phylogenetic analyses, because they were identified as Neofusicoccum parvum in the preliminary tub2 locus characterization.Orchard

Pathogenicity test
The pathogenicity showed that all the Botryosphaeriaceae species in this study were pathogenic to avocado plants, and produced similar symptoms to those observed in the field.All the inoculated species produced external and internal discolouration lesions.The inoculation controls did not show any symptoms (Figure 3).After 15 d, all the inoculated trees showed dark discolouration of the outer layers of bark.In detail, N. luteum isolate AVF5 produced the longest lesions (mean = 55.0 mm), followed by N. cryptoaustrale isolate AVO-RAM4 (50.9 mm), M. phaseolina isolate AC29 (43.6 mm), B. dothidea isolate AC7 (37.8 mm) and L. citricola isolate AC20 (31.4 mm).All the inoculated fungi produced lesion lengths that were statistically different from the controls (P < 0.05), and only lesions from Neofusicoccum sp. were significantly different compared to those from L. citricola (Figure 4).Re-isolations showed gave colonies with the morphological characteristics the same as the inoculated species, fulfilling Koch's postulates.

DISCUSSION
This study has elucidated the diversity of Botryosphaeriaceae species causing avocado canker and dieback in commercial orchards in Italy.The species characterized were B. dothidea, L. citricola, M. phaseolina, N. cryptoaustrale, and N. luteum.Neofusicoccum parvum was also constantly encountered during field surveys.This species had been characterized in a previous study (Guarnaccia et al. 2016), and was here characterized only on the basis of tub2 locus, and excluded from the phylogenetic analysis.This research confirms that N. parvum was the predominant Botryosphaeriaceous species associated with canker and dieback symptoms of avocado in Sicilian orchards.
Macrophomina phaseolina is widely distributed and is a serious threat to different crops (Baird et al., 2003;Sarr et al., 2014).This pathogen causes charcoal rot of soybean (Sarr et al., 2014), chickpea (Dell'Olmo et al., 2022), sunflower (Bokor, 2007), sorghum (Sharma et al., 2014), and strawberry (Koike, 2008).It has also been reported to cause diseases on woody hosts, such as grapevine (González and Tello, 2011;Nouri et al., 2018), olive (Sergeeva et al., 2005), pistachio (Nouri et al., 2020), and almond (Inderbitzin et al., 2010).Macrophomina phaseolina was thought to be one of the pathogens causing avocado root rot in Australia (Poudel et al., 2021), but it has not been recorded as causing canker on this host.Based on previous studies in Italy, on fruit and ornamental hosts showing typical symptoms of Botryosphaeriaceae, including canker and dieback of woody tissues, M. phaseolina has not been previously isolated.This is the first report of M. phaseolina on avocado.Further investigations are required need to clarify the geographic extent this species in Italy, and its association with different host plants.
Neofusicoccum cryptoaustrale was detected in only one of the sampled avocado orchards.This fungus was first described Eucalyptus trees in South Africa (Crous et al., 2013;Pavlic-Zupanc et al., 2017), and was reported on ornamental and fruit crops, including Pistacia lentiscus (Linaldeddu et al., 2016), Olea europea (van Dyk et al., 2021;Hernández-Rodríguez et al., 2022), and mangrove species (Osorio et al., 2017).This fungus formed a cryptic sister species with N. australe (Crous et al., 2013).Results of the present study showed that the isolates from avocado clustered with the type isolate of N. cryptoaustrale (CBS 122813), close to the well supported clade of N. australe.We do not exclude that the present study isolates identified as N. cryptoaustrale could be re-accommodated following progress with multi-locus phylogeny.Neofusicoccum luteum is well known as a canker pathogen of avocado, and has been reported to cause branch canker and stem-end rot on avocado in California (McDonald et al., 2009;2011;Twizeyimana et al., 2013;Avenot et al., 2022), Australia (Tan et al., 2019), New Zealand (Hartill, 1991;Hartill and Everett 2002), and Chile (Tapia et al., 2020).This fungus was also identified in California as the main cause of stem-end rot in harvested avocado fruit (Twizeyimana et al., 2013).
Despite of the diversity of Botryosphaeriaceae identified in the present study, N. parvum was the most prevalent species associated with canker and dieback of avocado, since it was detected from seven sampled locations with a high isolation frequency, as was previously reported in Italy by Guarnaccia et al. (2016) and in Spain by Arjona-Girona et al. (2019).Neopestalotiopsis also came from symptomatic tissues showing cankers and discolouration, but was not included in this study since it was already reported and described by Fiorenza et al. (2022b).Pathogenicity tests showed that representative isolates caused lesions on healthy plants.These data demonstrated that all the inoculated fungi were pathogenic to avocado, and that the isolates characterized as N. cryptoaustrale and N. luteum were the most virulent compared those of B. dothidea, M. phaseolina, and L. citricola.
Botryosphaeriaceae species have been reported as pathogens of the ornamentals to the agricultural crops in Italy, especially in Sicily (Ismail et al., 2013;Guarnaccia et al., 2016;Aiello et al., 2020aAiello et al., , 2022;;Gusella et al., 2020Gusella et al., , 2021Gusella et al., , 2022;;Bezerra et al., 2021;Fiorenza et al., 2022a;Costanzo et al., 2022).Of the studies in Italy, Botryosphaeriaceae have been commonly encountered in different hosts and environments.Presence of contiguous susceptible hosts and the polyphagous behaviour of this pathogen family can guarantee inoculum survival in nurseries, open fields, and urban areas.The fungi characterized in the present study have also been described on other hosts.Botryosphaeriaceae (including those detected in this study) are endophytes, able to induce latent infections (Slippers and Wingfield, 2007).It is possible to detect the levels of latent infections using qPCR (Luo et al., 2017;2019;2020;2021).
The orchards investigated in the present study contained mainly young avocado trees (2 to 4-year-old).Presence of Botryosphaeriaceae spp.within the tissues in young trees indicates that most of the infections may originate nurseries, and then spread once the trees are transplanted in open fields.In Sicily, avocado trees are imported from other Mediterranean countries, because there are no nurseries specialized in avocado propagation.For these reasons, monitoring of latent infections, and attention during nursery propagation, are needed to avoid or limit Botryosphaeriaceae infections and new sources of inoculum.
This study presents updated results on the association of Botryosphaeriaceae species causing canker and dieback on avocado in Italy.The surveys and analyses have elucidated the diversity of this group of fungi involved in avocado canker diseases.Further studies are required to elucidate the epidemiology, control, and latent pathogenic status of Botryosphaeriaceae on avocado.This study is also the first to report L. citricola, M. phaseolina and N. cryptoaustrale causing canker and dieback on avocado trees, and is the first report of the recorded fungi causing branch disease on avocado in Italy.
Botryosphaeriaceae species associated with canker and dieback of avocado (Persea americana)

Figure 1 .
Figure 1.Symptoms of Botryosphaeriaceae on avocado trees observed in the field.A, Shoot dieback in the host canopy.B, Branch dieback.C and D, External canker (shoot canker).E, Canker with white powdery exudation.F and G, Infection originating from pruned wounds.H, bark cracking.I, infected grafting point.

Figure 2 .
Figure 2. One of 100 equally most parsimonious trees generated from maximum parsimony analysis of three-loci (ITS + tub2 + tef1-α) combined dataset of Botryosphaeriaceae species.Numbers before after slashes represent, respectively, parsimony and likelihood bootstrap values from 1,000 replicates.Phyllosticta ampelicida (CBS 111645) and Phyllosticta citricarpa (CBS 102374) were the outgroup taxa in both analyses.Isolates in bold font were generated in the present study.Bars indicate the numbers of nucleotide changes.

Figure 4 .
Figure 4. Mean lesion lengths (mm) resulting from the pathogenicity test of Botryosphaeria dothidea, Lasiodiplodia citricola, Macrophomina phaseolina, Neofusicoccum cryptoaustrale, and N. luteum on potted plants.Values are each for two inoculation points per plant for each fungal species.Control consisted of the same number of inoculation points.Vertical bars represent standard errors of the means.Bars accompanied with different letters indicate means that were significantly different (Fisher's protected LSD test; α = 0.05).

Table 1 .
(Continued).Isolates in bold font are from this study.The "T" superfix identifies type material.

Table 1 .
(Continued).Isolates in bold font are from this study.The "T" superfix identifies type material.