First Report of Root Rot of Aloe barbadensis Caused by Pythium spinosum in China

Aloe barbadensis is a perennial evergreen succulent herb with many uses including medical care, cosmetology, and health remedies. It is considered a promising new economic plant resource. In June 2014, 1-year-old aloe plants that were cultivated directly into a field sandy loam soil in a greenhouse in Weihai City (Shandong Province) contained up to 5% stunted plants with symptoms of water-soaked lesions on roots and stem bases. The plants were irrigated using furrow irrigation. The stunted plants first appeared in low-lying areas that were prone to water accumulation. Fragments of 1 mm in size were excised from water-soaked roots and stem bases of diseased plants, dipped in a 0.2% calcium hypochlorite solution for 10 min, placed on V8 medium (200 ml of V8 [Campbell’s Soup], 15 g of agar, 0.2 g of CaCO₃, and 1 liter of distilled water), and incubated in the dark at 28°C for 5 days. A Pythium sp. was consistently isolated and produced hyphal swellings, oogonia, antheridia, and oospores. Most of oospores were globose, smooth, and plerotic, with some being aplerotic. The dimensions of hyphal swellings, oogonia, and oospores, respectively, ranged from 9.0 to 22.0 (average 14.5) µm, from 13.3 to 22.3 (average 18.4) µm, and from 12.8 to 19.5 (average 16.5) µm. Finger-like projections were uniformly distributed on the walls of the oogonia, and the antheridia were shaped like sticks or curved rods. Isolates of the Pythium sp. were tentatively identified as P. spinosum using morphological characteristics (van der Plaats-Niterink 1981). Five of the isolates were molecularly identified further. Genomic DNA was extracted from the isolates of the Pythium sp. using a DNA extraction kit (OMEGA, U.S.A.). The cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region rDNA were amplified and sequenced using primers FM55/FM52R (Long et al. 2012) and ITS1 /ITS4, respectively (White et al. 1990). The five COI sequences were aligned and were identical for all five isolates, as well as the five ITS sequences. BLASTn analysis of the 558-bp COI sequence (accession no. MG931976) resulted in a 100% identity with that of the P. spinosum strain PPR18604 (accession no. GU071817) from grapevine in South Africa, and the 931-bp ITS sequence (accession no. MG214650) showed 100% identity to GenBank accession number AY598701, which belongs to P. spinosum. Koch’s postulates were conducted by first producing inoculum for one of the isolates (SDLH-1). Inoculum was produced by first soaking corn kernels for 24 h in water, followed by autoclaving for 2 h at 121°C. Isolate SDLH-1 was grown on corn meal agar medium for 10 days, after which agar plugs were transferred to the sterilized corn kernels, which were incubated at 28°C for approximately 15 days, until the corn kernels were covered in white hypha. Ten healthy 1-year-old aloe plants were transplanted into a sterilized sandy loam potting soil that was artificially infested with the corn inoculum (2 g of inoculum per 100 g of sandy loam mixture). Inoculated and noninoculated control plants were maintained in a greenhouse at 28°C and 80% relative humidity with a 12-h photoperiod, and the experiment was repeated once. All 20 inoculated plants exhibited symptoms that were similar to those observed in the greenhouses within 4 weeks. P. spinosum was recovered only from the water-soaked roots and stem bases of inoculated plants, and the reisolated cultures were identified based on morphological characteristics and sequencing of the COI gene. No symptoms were observed on the control plants. To our knowledge, this is the first report of root rot on A. barbadensis caused by P. spinosum in China and worldwide. Identification of the pathogen will assist in devising strategies to protect this important plant from the pathogen and to prevent yield losses.