First report of seedling stem blight of mango caused by Neofusicoccum parvum in Italy

Seedling blight of mango (Mangifera indica L.) was observed in a commercial nursery located in Messina province (eastern Sicily, Italy) during winter of 2021. More than 30% of 3,000 seedlings, about three to six months old, of mango cv. Gomera 3 showed symptoms of basal stem blight. The symptoms started from seed, led to the decline and subsequent death of the plants. Necrotic lesions appeared at crown level two months after sowing. The stem tissues of ten symptomatic plants were cut, surface sterilized, dipped in 1.5% sodium hypochlorite for 1 min and transferred onto potato dextrose agar medium (PDA) and incubated at 25°C for four days. Approximately 60% of stem tissues developed very similar fungal colonies, resembling to Botryosphaeriaceae. A total of four representative isolates were collected through single hyphal-tip and stored at 4 °C. The internal transcriber spacer region (ITS) was amplified with primers, ITS5/ITS4 (White et al., 1990), and EF1-728F and EF1-986R (Carbone and Kohn, 1999) were used to amplify part of the translation elongation factor 1alpha gene (tef1-α), and primers Bt2a/Bt2b (Glass and Donaldson, 1995) were used for the partial β-tubulin (tub2). The obtained sequences were deposited in GenBank with accession numbers: ON911292-95 for the ITS, ON933621-24 for tef1-α and ON933625-28 for tub2.To compare the results, 50 additional sequences were selected and inserted in the alignment according to the recent literature on the Botryosphaeriaceae (Bezerra et al., 2021; Zhang et al., 2021). Maximum parsimony analysis (MP) of concatenated dataset (ITS + tef1-α + tub2) was performed in PAUP v.4.0a. Clade support was assessed by 1,000 bootstrap replicates and Botryosphaeria dothidea was used as an outgroup. Our isolates clustered within the group of Neofusicoccum parvum (71% bootstrap value) (ex-type CMW9081). Based on these results, and morphological data (50 conidia length × width average: 18.1 × 6.6, respectively) our isolates (named MC) were identified and confirmed as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Pathogenicity tests were also conducted on 18 mango cv. Gomera 3 seedlings. The crown roots of each seedling were mechanically wounded and a mycelial plug of the isolate MC14 was placed onto them and covered with soil. Controls (three seedlings) were inoculated with sterile PDA only. Seedlings were maintained in a growth chamber with a 12 hrs photoperiod at 25°C ± 1°C and watered regularly. After five days, stem lesions appeared externally (1.6 cm) and one month after the inoculation, all the inoculated seedlings died. However, controls did not show any obvious symptoms. Re-isolations were conducted as described above and fulfilled Koch's postulates confirming pathogenicity. Among the diseases affecting mango plants, Botryosphaeriaceae represent a serious threat in Sicily as reported by Aiello et al., 2022. The endophytic behaviour of Botryosphaeriaceae is well known, making them latent pathogens (Slippers and Wingfield, 2007). In Italy, N. parvum was detected in mango orchards since 2013 (Ismail et al. 2013), but symptoms of seedlings stem blight have never been reported in the nursery. In Sicily, an increase of Botryosphaeriaceae infection has been observed recently, especially in nurseries, where N. parvum has been identified as a most destructive pathogen (Aiello et al., 2020; Gusella et al., 2021). To our knowledge, this is the first report worldwide of N. parvum causing mango seedling blight. The high incidence of infected seedlings detected in this study highlights the potential risk during propagation in the nursery, representing a significant source of inoculum for the field.