First report of Lasiodiplodia theobromae causing dieback in custard apple (Annona squamosa) tree in China.

Abstract

Custard apple (Annona squamosa Linn) is popular for its sweet taste and rich aroma. Hainan province is the major production area of custard apple in China. In September 2020, wilting of leaves and branches, discoloration of the vascular system and dieback of trees were observed in plantings in Lingao County of Hainnan Province, China (Fig. 1a-c). The incidence of dieback in three orchards was at least 19%, and affected samples were brought to the laboratory. Fragments of approximately 5 mm in length were obtained from five diseased branches, which were collected from different plants and orchards. Fragment surface were sterilized in 75% ethanol for 1 min, and 1% mercury chloride for 1 min, then rinsed three times with sterile distilled water. These tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated at 28°C for 3 days. Fungal colonies were transferred to fresh PDA plates, and single-spore cultures were obtained. We isolated twenty-six fungal strains, of which twenty-three isolates were morphologically identified as Lasiodiplodia species (Phillips et al. 2013). The colony morphology was initially round and white, then turned to grey or black after 5-7 days at 28°C, and formed pycnidia for 20 days (Fig. 1d). The immature conidia were ellipsoid, colorless, hyaline and unicellular, becoming brown, bicellular with longitudinal striations at maturity (Fig. 1e). Mature conidial size was: 26.61±1.57×14.87±1.14 µm (n=60). For molecular identification, genomic DNA of three isolates (HSYF01, HSYF02 and HSYF03) was extracted using E.Z.N.A.® HP Plant DNA kit (Omega Bio-Tek). The internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin (TUB) regions were amplified using the primers ITS1/4, EF1-728F/986R and Bt2a/Bt2b, respectively (Carbone and Kohn 1999; Glass and Donaldson 1995; White et al. 1990). A BLAST search of ITS, EF1-α and TUB gene sequences (Accession nos. MW625913-MW625918, MW876481-MW876483) had in 99.8% (493bp out of 494 bp), 99.31% (286bp out of 288 bp) and 100% (372bp out of 372 bp) identity to CBS164.96 of the L. theobromae (Accession nos. AY640255, AY640258 and KU887532). The identity of the putative pathogen isolates was further confirmed by phylogenetic analysis (Fig. 2). Ten healthy 1-2-year-old custard apple trees were used to confirm pathogenicity. Custard apple plants were wounded approximately 15-20 cm below the tips with a sterilized scalpel. Each cut was inoculated with a 5 mm agar plugs with mycelium from the PDA cultures. The wound site was moisturized with wet cotton wool and wrapped with laboratory film (Silveira et al. 2018). Two seedlings treated with sterile agar plugs served as a controls. The pathogenicity test was repeated twice. After 14 days of incubation in a glasshouse, all inoculated seedlings had characteristic discoloration and necrotic lesions starting from the apical branches, (Figs. 1f and 1g). The stems exhibited browning and vascular streaking of the wood from the inoculation point (Figs. 1h), while the control seedlings remained symptomless. Typical colonies of L. theobromae were isolated and identified from all inoculated seedings, fulfilling Koch´s postulates. Although, postharvest fruit stem-end rot on custard apple caused by L. theobromae was previously described (Hu et al. 2003; Meng et al. 2017), this is the first report of L. theobromae causing dieback in mature custard apple trees in China.