Stem-end rot (SER) caused by Lasiodiplodia theobromae is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC50 values ranged from 0.0006 to 16.4131 µg/ml. A total of 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruits. The mycelial growth, conidia germination and ability to grow at temperatures ranging from 12–35℃ of resistant isolates decreased, suggesting fitness penalties . The experiment showed that after treatment with 10 µg/ml prochloraz, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damages of membranes in the sensitive isolates were more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette subfamily ABCG gene were cloned, but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than those in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

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.