First report of Rhizopus stolonifer causing Rhizopus bunch rot on grapes in Pakistan

During June 2016, a postharvest survey of table grapes was carried out in the main fruit markets of Attock (33°46'07.9"N 72°21'43.0"E) and Jehlum (32°56'22.3"N 73°43'31.4"E) districts of Punjab Province. At the time of sampling, two cultivars (King’s Ruby and Perlette) were sampled at five different locations of both districts. Disease incidence % of bunches averaged 3 to 4 %. Some infected fruit appeared water-soaked, light brown and covered by fluffy mycelium consisting of erect sporangiophores with black sporangia on the top (Figure 1). Symptomatic tissue pieces were surface-sterilized with 0.1% NaOCl for 30 sec, rinsed three times with sterile distilled water, dried on filter paper for 45 sec and incubated on potato dextrose agar (PDA) at 25°C. After 1 day, mycelium on PDA was transferred to a fresh PDA plate and incubated at 25°C with a 12-h photoperiod. Within 3 days, white to yellow colonies with black aerial sporangia were formed (Figure 2). A total of 64 isolates were examined morphologically. Sporangiophores were erect, light brown and 623 to 3800 µm long. One to three rhizoids were observed opposite to each sporangiophore. Sporangia were black, globose to sub-globose, 91 to 124 μm in diameter. Columellae were conical to cylindrical and 86 to 187 μm long × 72 to 205 μm wide. Sporangiospores were hyaline to light dark grey, globose, ellipsoidal in shape and 6.8 to 12.4 × 3.6 to 12.5 μm (Figure 3). These features were identical to the description of Rhizopus stolonifer (Ehrenb.) Vuill (Liou et al., 2007). For molecular identification, the ITS1-5.8S-ITS2 region of two representative isolates (Rizo 05 and Rizo 07) was amplified with primers ITS1/ITS4 (White et al., 1990). Sequence comparison of two isolates Rizo05 and Rizo 07 (Accession no. MH348205  and MH356272) revealed 100% identity with previously reported isolates of Rhizopus stolonifer (Accession no. MG865992, KU729185, HM051076, and MF374842). To complete Koch’s postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of Rizo 05 and Rizo 07 were pipetted onto three non-wounded and four wounded asymptomatic grape berries (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries to serve as a negative control. Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice (Ghuffar et al., 2018). Black to light brown, fluffy mycelium with the original symptoms was observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control (Figure 4). The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Previously, Rhizopus stolonifer has been reported as a pathogen on grapes in Chile (Latorre et al., 2002). To our knowledge, this is the first report of Rhizopus stolonifer causing bunch rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against Rhizopus rot of grapes in Pakistan.