Characterization and pathogenicity of Pleurostoma richardsiae causing decline of mango trees in Southern Italy

Summary. Mango trees ( Mangifera indic a) showing symptoms of twig and branch die-back, internal wood necroses, and decline, were surveyed in an orchard in Palermo province (Eastern Sicily, Italy). A Pleurostoma -like fungus was consistently isolated from symptomatic wood tissues. Based on morphology and phylogenetic analysis of ITS and tub2 sequences, the fungus was identified as Pleurostoma richardsiae . A pathogenicity test was conducted by inoculating stems of 2-year-old mango seedlings with mycelium plugs and conidium suspensions of a representative isolate. Two months after inoculation, necrotic lesions were observed around the inoculation points, and P. richardsiae was reisolated from the necrotic tissues. This is the first report of P. richardsiae causing dieback and decline of mango trees.

Mango (Mangifera indica L.; Anacardiaceae) is a fruit tree crop that is native to India and Southeast Asia (Mukherjee, 1953).Mango is widely cultivated especially in tropical and subtropical regions.In recent years, its cultivation has increased in the Mediterranean basin, due to the popularity of the fruit among European consumers and good productivity, and adaptability of the species to different environments.In Italy, the cultivation of subtropical crops is mainly concentrated in the coastal regions of Sicily (Palermo, Messina, and Catania provinces), where climate and soil conditions are suitable (Lauricella et al., 2017).The growing interest in the mango fruit is due to their high content of bioactive compounds beneficial for human nutrition and health, which makes it attractive for direct consumption and useful for food and pharmaceutical industries (Maharaj et al., 2022;García-Mahecha et al., 2023).
In Italy, few pre-and post-harvest fungal diseases of mango have been reported.The most production-limiting diseases include canker and shoot blight caused by Botryosphaeriaceae spp.(Aiello et al., 2022), and fruit decay and stem-end rot caused by Colletotrichum spp.(Ismail et al., 2015).Stem-end rot of fruit caused by Neofusicoccum spp.has also been occasion-ally observed (Ismail et al., 2013a).Leaf spots caused by Pestalotiopsis uvicola and P. clavispora (Ismail et al., 2013b), and wilt caused by Verticillium dahliae (Ahmed et al., 2014), have also been reported.
Since 2021, dieback of twigs and branches was observed on 6-year-old mango trees ('Glenn' grafted on 'Gomera 3' rootstocks) during surveys of mango orchards in Bagheria (Palermo province, Italy).Affected trees had declining vegetation vigour during the year, followed by rapid dieback of twigs and branches, often leading to tree death (Figure 1).During a survey in May 2023, examination of cross-sections of branches and twigs of declining and low vigour trees revealed irregular, brown to black wood necroses, co-occurring in some cases with black spots and reddish-brown to black streaking of the inner wood tissue (Figure 2).Further observations detected canopy thinning and dry leaves hanging from the twigs.The sudden decline observed after years of slow and stunted growth made disease incidence variable, with few plants showing dieback at each season, and disease incidence based on these symptoms was approx.10%.
Twigs and branches showing internal necroses were randomly collected from five plants, and were kept in plastic bags and taken to a laboratory for pathogen isolation and further analyses.Small wood fragments (3 × 3 × 3 mm, n = 160) from the margins of necrotic or apparently healthy tissues were surface sterilized in a 1.2% sodium hypochlorite solution for 60 s, and then rinsed once in sterile distilled water for 60s.The fragments were air dried in a laminar-flow cabinet on sterile paper, placed on potato dextrose agar (PDA, Lickson) amended with lactic acid (APDA; containing 1 mL L -1 of 98% [vol/ vol] lactic acid), and were then incubated at approx.25°C under natural light for 10 d.One type of fungal colony consistently grew from the symptomatic mango tissues, with isolation frequency of 60 to 72%.From twelve representative Pleurostoma-like colonies, single-conidium isolates were obtained, and were stored in the collection of the Dipartimento di Agricoltura, Alimentazione e Ambiente, section Patologia Vegetale, University of Catania.
Two representative isolates (CP23, CP28) were grown on acidified Malt Extract Agar (AMEA) at 25 ± 1°C for 21 d in the dark, to study colony morphology (texture, density, obverse and reverse colour, and margin), according to Vijaykrishna et al. (2004).Mycelium samples were mounted on microscope slides in lactic acid.Lengths and widths of conidia (n = 30) were measured at 100× magnification, using a Zeiss Axiolab 5 microscope and Zeiss Axiocam 208 color, using the software Zen Core (v.35.96.03000), and average dimensions and length/width ratios of the dimension means were calculated.
All collected fungal isolates were grown on PDA for 14 d, and mycelium was then removed with a sterile scalpel.Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation).The obtained DNA was stored at 4°C for further analyses.Two gene regions were amplified and sequenced.The internal transcriber spacer region (ITS) of the nuclear ribosomal RNA operon was amplified with the primers ITS5 and ITS4 (White et al., 1990), and the primers Bt2a and Bt2b were used for the partial beta tubulin gene (tub2) (Glass and Donaldson, 1995).PCRs were carried out in a total volume of 25 µL, using One Taq ® 2X Master Mix with Standard Buffer (BioLabs), according to the manufacturer's instructions.PCR conditions were set as follows: 30 s at 94°C; 35 cycles, each of 30 s at 94°C, 1 min at 52°C, and 1 min at 68°C; and 5 min at 68°C.PCR products were visualized on 1% agarose gels (90 V for 40 min) stained with GelRed® Nucleic Acid GelStain (Biotium), were purified, and then sequenced in both directions by Macrogen Inc. (Seoul, South Korea).The obtained forward and reverse DNA sequences were assembled, edited and aligned using MEGA X (Kumar et al., 2018).All sequences of the ITS and tub2 gene regions obtained were deposited in the National Centre of Biotechnology Information (NCBI) GenBank database (Table 1).
The obtained sequences were first compared with those available in GenBank.BLASTn searches of ITS and tub2 sequences showed 99% similarity with the sequences of Pleurostoma richardsiae (Nannf.)Réblová & Jaklitsch (Pleurostomataceae, Calosphaeriales) isolate CBS 270.33 (GenBank accession No. MT153151.1),and 100% similarity with P. richardsiae isolate KARE1881 (GenBank accession No. MT735027.1).Phylogenetic analysis based on Maximum Parsimony (MP) was conducted on a concatenated dataset of ITS and tub2, including a total of 29 taxa, based on Lawrence et al. (2021).Multiple alignment was conducted in MEGA X. Maximum Parsimony analysis was carried out using Phylogenetic Analysis Using Parsimony (PAUP*) version 4.0a (Swofford, 2002), and Phaeoacremonium minimum CBS 246.91 and Phaeoacremonium novae-zelandiae CBS 110156 were used as the outgroups.The MP parameters were set as follows: 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 unordered and of equal weight, and Maxtrees were limited to 100.MP scores including tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency index (RC) were calculated.A total of 1,000 bootstrap replicates were performed to test the robustness of the tree topology.The MP analysis of the combined dataset showed that of 1574 total characters, 512 were parsimony informative, 268 were parsimonyuninformative, and 794 were constant.In total, 100 trees were retained.Tree scores were: TL = 1874, CI = 0.688, RI = 0.649, and RC = 0.446.The MP analysis showed that the isolates from Bagheria clustered within the group of P. richardsiae, with strong support (bootstrap support 100) (Figure 3).
To assess pathogenicity of P. richardsiae, isolate CP28 was inoculated onto 2-year-old potted healthy plants of mango 'Gomera 3', which were maintained in a growth chamber at 25 ± 1°C with a 12 h photoperiod.The stem of each plant was surfaced disinfected with 70% ethanol and wounded with a sterilised 5 mm diam.cork borer.Agar plugs (5 mm diam.)taken from 30-d-old fungal cultures growing on AMEA at 25 ± 1°C, or 20 µL of conidium suspension (1 × 10 5 conidia mL -1 ), were placed into each stem wound.A total of six plants were inoculated, with three plants per inoculation method and two inoculation sites per plant along the stem.Control plants were inoculated with AMEA plugs and sterile distilled water.After inoculation, the wounds were sealed with Parafilm ® , and the plants were maintained at 25 ± 1°C in a growth chamber.After 2 months, bark was removed with a sterile blade, and lengths of lesions (upward and downward from inoculation points) were measured, and means were calculated.Re-isolations were carried out (as described above) to determine fulfilment of Koch's postulates, and proportions (%) of Pleurostoma-like colonies were determined.
Pathogenicity tests confirmed that P. richardsiae was pathogenic to mango trees, causing reddish-brown to black necrotic lesions in the wood of all the inoculated plants, and these lesions were visible 2 months after inoculation (Figure 4, b and c).Control plants did not show any symptoms, except those due to wound oxidation (Figure 4, a and d).Mean lesion length from P. richardsiae isolate CP28 was 2.25 ± 1.29 from conidium suspension inoculations, and 2.85 ± 0.47 cm from mycelium plug inoculations.Re-isolation frequency was 80%, and these fungal colonies matched the originally inoculated P. richardsiae isolate as indicated by colony and conidium morphology.
Pleurostoma richardsiae has been reported from many countries as the cause of diseases in important crops.The fungus is a severe vascular pathogen of olive in Brazil, California, Croatia, Greece, Italy, Spain and South Africa (Carlucci et al., 2013;Nigro et al., 2013;Markakis et al., 2017;Ivic et al., 2018;Canale et al., 2019;Spies et al., 2020;Agustí-Brisach et al., 2021;Lawrance et al., 2021;van Dyk et al., 2021).This fungus was also reported as a pathogen of grapevine in Brazil, California, Italy, Spain, and South Africa (Halleen et al., 2007;Rolshausen et al., 2010;Carlucci et al., 2015;Pintos Varela et al., 2016;Canale et al., 2019).It was sometimes isolated from esca-affected vines, although its role in the disease has not yet been confirmed (White et al., 2011).Avocado and nut crops have also been reported to be hosts of this pathogen (Olmo et al., 2015;Markakis et al., 2017;Sohrabi et al., 2020).Several species of Botryosphaeriaceae, several Phaeoacremonium spp.and Fomitiporia mediterranea, have been isolated with P. richardsiae from woody tissues of grapevine (Pintos Varela et al., 2016;Raimondo et al., 2019), almond (Sohrabi et al., 2020), andolive (Spies et al., 2020;van Dyk et al., 2021), showing the same symptoms.However, only P. richardsiae was isolated from symptomatic twig and branch tissues of mango trees in the present study.This is the first report of P. richardsiae causing disease on mango trees.Future epidemiological studies are needed to assess the presence of P. richardsiae in mango orchards in other areas of Italy, and to evaluate possible effects of climate change on the emergence of new pathogen-host interactions.It is known that climate change may alter plant physiology and increase susceptibility to disease, and may also widen plant pathogen host ranges (Guarnaccia et al., 2023;Joachin et al., 2023).To date, Botryosphaeriaceae that cause canker and shoot blight of plants are considered the most damaging trunk pathogens for mangoes in Italy (Aiello et al., 2022).The present record of severe damage of mango caused by P. richardsiae deserves attention for the risks it could pose to this important fruit tree crop.

Figure 1 .
Figure 1.Decline of mango trees 'Glenn' grafted on 'Gomera 3' , caused by Pleurostoma richardsiae in an orchard located in Bagheria (Palermo, Italy).(a) Dieback of twigs and branches showing necroses of inner wood tissues.(b) Severe dieback of a mango tree.

Figure 2 .
Figure 2. Details of symptoms on a mango tree 'Glenn' .(a to d) Cross-sections of branches and twigs of declining trees with irregular, brown to black wood necroses.(e) Cross-section of a twig showing reddish-brown to black streaking in the inner wood tissue.(f) Longitudinal section of branch showing internal wood necrosis.

Figure 3 .
Figure 3.A parsimonious tree generated from maximum parsimony analysis of the two-gene (ITS + tub2) combined dataset.Numbers beside the clades represent parsimony bootstrap values from 1,000 replicates.Isolates in bold font were obtained in the present study.The bar indicates the number of nucleotide changes.

Figure 4 .
Figure 4. Pathogenicity test on mango seedlings 'Gomera 3' .(a) Agar plug inoculation control.(b) Stem inoculated with Pleurostoma richardsiae isolate CP28, showing a brown to black lesion.(c) An internal lesion produced by P. richardsiae isolate CP28 around the inoculation point.(d) Internal tissue of a control stem with no symptoms around the agar inoculation point.(e) Colony of P. richardsiae isolate CP22 grown on acidified malt extract agar for 30 d at 25°C.

Table 1 .
Fungal isolates from mango used in phylogenetic analysis.Isolates in bold font were obtained in the present study.
a Isolates linked to type specimens.b ITS = internal transcribed spacer; tub2 = beta-tubulin.