First Report of Lasiodiplodia theobromae and Neofusicoccum parvum/N. ribis species Complex Causing Fruit Rot on Strawberry in the United States

Abstract

During the last three seasons, approximately 1% of strawberry (Fragaria × ananassa) with symptoms of fruit softening and discoloration were observed in three fields in Florida. Fruit eventually became black and mummified with pycnidia embedded in the tissue that produced hyaline, aseptate conidia. Eleven isolates recovered from infected tissue were purified by growing them on 2% water agar (WA) with sterilized pine needles (Pavlic et al. 2004) and transferring a single conidium to a new 2% WA plate, by spreading a conidial suspension over the medium and selecting a single germinating conidium 16 h later. DNA of the 11 isolates was extracted from 2-week-old colonies and PCR was performed following the conditions of White et al. (1990) and Carbone and Kohn (1999). Two sets of universal primers, ITS1F/ITS4 and EF1-728F/EF1-986R, were used to amplify and sequence the internal transcriber spacer (ITS) and partial translation elongation factor 1-alpha (EF1-α) regions, respectively. Sequences compared with the GenBank database using BLASTn showed 99 to 100% identity with ITS and EF1-α sequences within the Neofusicoccum parvum/N. ribis species complex or Lasiodiplodia theobromae: N. parvum (MH057203 and KM921768; n = 2), N. kwambonambiense (n = 3), N. ribis (n = 1), N. brasiliense (n = 2), and L. theobromae (MH049696 and KX580762; n = 3). All sequences were deposited in GenBank with following accession numbers: N. parvum/N. ribis species complex (MH809520 to MH809527 for ITS, and MH809508 to MH809515 for EF1-α), and L. theobromae (MH809517 to MH809519 for ITS, and MH809505 to MH809507 for EF1-α). Mycelia of both groups on V8 agar medium were initially white and aerial, but Lasiodiplodia mycelia became light gray after 3 to 5 days and Neofusicoccum mycelia turned dark gray to olivaceous. Conidia of the Neofusicoccum group were aseptate, hyaline, oblong, and 15.0 to 22.9 × 6.1 to 10.4 μm (n = 25). Lasiodiplodia conidia were similar but larger, 22.6 to 33.0 × 13.0 to 19.4 μm (young conidia) and when mature, became brown, one septate, with longitudinal striations, measuring 25.9 to 34.8 × 14.8 to 19.6 μm (n = 25). Similar morphology was previously reported (Alves et al. 2008; Pavlic et al. 2009). To confirm pathogenicity, four isolates (two from N. parvum/N. ribis species complex, and two L. theobromae) were selected and incubated for 2 weeks at 23°C under continuous light until formation of pycnidia. Twenty mature strawberry fruit of ‘Florida Radiance’ were inoculated in the field (five fruit per isolate), by wounding the fruit with a forceps and placing a single pycnidium inside the wound. Field conditions during the incubation period ranged from 16 to 29°C and 65 to 80% relative humidity. Disease incidence was assessed 6 to 10 days after inoculation, and the experiment was repeated once. After 10 days, fruit inoculated with the N. parvum/N. ribis were completely dark and covered with pycnidia, whereas fruit inoculated with L. theobromae had 80 to 100% of its surface covered with pycnidia. All 20 inoculated fruit showed the same symptoms as described above. Both fungi were reisolated from inoculated fruit, fulfilling Koch’s postulates. N. parvum and N. kwambonambiense have been previously reported to cause a postharvest disease of strawberry in Brazil (Lopes et al. 2014). To our knowledge, this is the first report of N. parvum/N. ribis species complex and L. theobromae causing fruit rot in strawberry fields in the U.S. This study sheds light on the possible broad distribution of these pathogens on different hosts as N. parvum was recently reported in the same area causing shoot blight and stem canker on pomegranate (KC and Vallad 2016).