First Report of Fruit Rot Caused by Botrytis eucalypti on Citrus sinensis in China

Lane Late navel is an emerging sweet orange (Citrus sinensis) variety in Zigui County, Hubei Province, China. The current extent of the production area is 5,700 ha. During the spring of 2017, over 2,000 ha were affected by a disease with symptoms similar to gray mold. Symptoms included light brown and slightly sunken lesions on the fruit and fruit drop, but spore masses were seldom found on the fruit surface. The disease incidence could reach 28.9% in waterlogged groves and 42.7% during postharvest transportation. The rind sections of symptomatic fruits collected from three different orchards in Zigui were surface sterilized with 2% NaClO for 1 min, rinsed in sterile distilled water three times, and then incubated on potato dextrose agar at 25°C in darkness for 3 to 5 days. Fungal colonies were initially white and then turned to gray. Isolates (YZU 171088, YZU 171089, and YZU 181069) selected from those three different orchards were transferred to water agar medium with sterilized C. sinensis fruit pieces and incubated at 22°C for 2 weeks to stimulate sporulation. Conidia were ellipsoid or ovoid, 9 to 14 × 6 to 9 μm. Conidiophores were straight, septate, 300 to 1,500 μm in length, and 8 to 13 μm in width. Sclerotia were not observed during cultivation. Five genes and primer pairs were used for molecular identification: G3PDH (glyceraldehyde-3-phosphate dehydrogenase, G3PDHfor/G3PDHrev), HSP60 (heat-shock protein 60, HSP60for/HSP60rev), RPB2 (DNA-dependent RNA polymerase subunit II, RPB2-5F/RPB2-7cR), and NEP1 and NEP2 (necrosis and ethylene-inducing proteins, NEP1for/NEP1revB and NEP2forD/NEP2revD) (Liu et al. 2016). The amplicons of those three isolates were sequenced. For each gene region, the amplicons among the three isolates were identical; thus, only the sequences of YZU 171088 were deposited in GenBank (accession nos. MH614610 to MH614614). The five sequences from each of the isolates showed 100% similarity with those of Botrytis eucalypti CERC 7170 (type strain) in a BLAST search except for one nucleotide in the HSP60 sequence. A phylogenetic tree was constructed based on the five genes using Mega 7.0 (1,000 bootstrap replicates, neighbor-joining method). The three isolates and B. eucalypti CERC 7170 located in one clade supported with 100% bootstrap values. The morphological and molecular evidence confirmed that the species was B. eucalypti, a pathogen that was first described from Eucalyptus in southern China (Liu et al. 2016). To conduct pathogenicity tests, 12 healthy C. sinensis fruits were surface sterilized with 2% NaClO for 2 min, rinsed with sterile distilled water three times, and then inoculated by injecting a conidial suspension (20 μl, 1.5 × 10⁵ conidia/ml) or inserting mycelial plugs (5-mm diameter) of the three isolates into artificial rind wounds (6 × 6 mm). Three mock inoculations with sterile distilled water and agar plugs were used as negative controls. All fruits were incubated in a moist chamber at 25°C. Light brown fruit rot symptoms and white mycelia were observed on both conidial suspension and mycelial plug inoculated fruits after 3 days, and lesions quickly expanded with dense and thick mycelia after 7 to 12 days on both the conidia- and plug-inoculated fruit, whereas the control fruit remained symptomless. The pathogenicity test was repeated twice. The same isolates were recovered from symptomatic fruit and identified by NEP1 sequence. To the best of our knowledge, this is the first report of B. eucalypti causing fruit rot on C. sinensis in the world. The widely distributed B. cinerea is known as the causal agent of citrus gray mold (Timmer et al. 2000). However, owing to the morphological and phylogenetic similarities of these two species, further studies on the differences of pathogenicity and disease management are required. Distribution frequency of B. eucalypti and other Botrytis spp. in orange groves and the existence of other possible coinfecting pathogens also need to be studied.