Quarantine identification of Albonectria rigidiuscula on the custard apple from Taiwan of China

Abstract:【Objective】Custard apple (Annona squamosa L.) is an edible tropical fruit, and is also called sugar apple or sweetsop. Custard apple is a very delicious fruit, valued for the flavor and texture of its pulp. Fruit also has high caloric value and sugar content. Owing to domestic market demand, Chi-na has expanded its imports. According to customs statistics, the domestic import of custard apple was135 000, 118 000, 80 000, 132 000 and 153 000 tons from 2015 to 2019. China imported 124 000 tons of custard apple in 2020, 95% of which was produced from Taiwan province. To import the custard ap-ple, it is required to apply from the plant quarantine authority for issuance of quarantine requirements and shall be in compliance with the quarantine requirements. Symptoms of the black irregular spots were observed in Fuzhou port. The experiment was undertaken to identify and characterize the patho-gen that caused black brown spot of custard apple.【Methods】Strain 2268-2-2-3 was isolated from cus-tard apple imported from Taiwan of China by using conventional PDA plate method. To identify strain 2268-2-2-3, the morphological characteristics, sequence alignment and its pathogenicity were analyzed.Fungal isolate was incubated on PDA for 14 days in the dark to observe the morphological characteris-tics, after which 40 mg fresh mycelia were collected and used for DNA extraction. The mycelia were ho-mogenized in the tube using the mixer mill MM 400 for 90 seconds at the frequency of 30 times per sec-ond. Genomic DNA extraction was conducted using Labserv Plant DNA Extraction kit with a KingFish-er mL machine, according to the instructions of the manufacturer. DNA concentrations were determined using a Nanodrop ND-1000 spectrometer. Then the DNA solutions were used as the templates for PCR.The internal transcribed spacer (ITS) of nuclear rDNA and translation elongation factor 1-alpha (EF1A) regions of the strain 2268-2-2-3 were amplified in PCR assays using universal primers ITS1/ITS4 and EF1-728F/EF1-986R. Nuclear ribosomal DNA (rDNA) region is relatively conserved within the species but variable among species, and thus is a useful region for distinguishing different species. Based on rD-NA sequences, some diagnosis protocols have been successfully developed for quarantine fungi. How-ever, in our experiment, we found it was difficult to identify the A. rigidiuscula by ITS sequence. Then, EF1A sequences belonging to the A. rigidiuscula and related species were further used to generate phy-logenetic trees with the neighbor joining (NJ) methods by using the MEGA 6.0. The phylograms were bootstrapped 1000 times to assess the degree of support for the pylogenetic branching indicated by the optimal trees. Trees were rooted with Fusarium circinatum (MW402083, MW402082) and Fusarium begoniae (MW401969, MW401968) as outgroup. To fulfill Koch’s postulates, strain 2268-2-2-3 was tested for pathogenicity to fruit of custard apple by artificial inoculation. The method included wound-ing of the fruits by sterile knife, inoculating into wounded fruits by placing agar of mycelium and ob-taining the sample from 10-day-old culture plates. The control was used by inoculating the wounded fruits with PDA agar. Inoculated fruits and controls were covered with sterile cotton wool and placed in the polyethylene bags. Each inoculated fruit was placed in a moist chamber and incubated at 25 ℃.Four fruits were used per treatment.【Results】The strain 2268-2-2-3 grew well on PDA medium. The mycelium was pink and could produce a large number of macroconidia and microconidia. Macroconid-ia were sickle-shaped or slightly curved columnar, with 5-9 septa, 52.98-76.33 μm long, and 5.38-8.56 μm wide. Microconidia were elliptic to cylindrical, with 0-1 septa, 4.95-10.38 μm long and 2.61-5.12 μm wide. There was no sexual stage during the cultural period. The ITS and EF1A sequences of strain 2268-2-2-3 was compared with those in GenBank database, showing that the sequence shared more than 98% homology with several sequences of A. rigidiuscula in GenBank, respectively. The phylogenetic tree based on EF1A sequences showed that the strain 2268-2-2-3 was gathered with other A. rigidiuscula iso-lates in a branch. The strain 2268-2-2-3 could infect sirikaya, forming dark brown irregular spots in the pathogenicity test.【Conclusion】A. rigidiuscula is an aggressive pathogen causing gall disease of cocoa and is listed as a quarantine fungus in China. However, identification and quarantine diagnosis of A. ri-gidiuscula based only on morphology is problematic due to its morphological plasticity and delayed ap-pearance of the fruiting bodies. Combined with the morphological characteristics, by using ITS and EF1A sequence analysis, the fungi were identified as A. rigidiuscula, which can cause dark brown irreg-ular spots on custard apple. To our knowledge, this is the first report of interception of A. rigidiuscula on custard apple in China, providing great values for quarantine practice in the ports.