Crop rotation affects soil properties and soil microbial diversity and structure. Currently, it is not well understood how soil microbial diversity changes following different crop rotation systems of industrial hemp, an ancient and economically important crop. Therefore, these changes were analyzed in this study. Our results showed that different rotation systems significantly affected the wilt disease incidence, plant height, yield, soil physicochemical properties and soil microbial communities in the greenhouse. The rotation systems used in this study significantly reduced the plant mortality and increased the yield compared with a monoculture system. The levels of alkaline hydrolysis and available phosphorus in the soil decreased significantly compared with a monoculture cropping system. Using MiSeq high-throughput sequencing, we showed that the soil diversity and number of bacteria and fungi were significantly higher for rotation systems and controls compared to the monoculture system. The relative abundance of pathogens increased with a monoculture system. Redundancy analysis suggests that soil properties may also affect the soil microbial composition. Taken together, different rotation systems used in this study significantly decreased the disease incidence, increased plant yields and increased soil microbial diversity compared with monoculture for industrial hemp. We believe that applying these rotation systems is an efficient and eco-friendly approach to control soil borne pathogenic diseases and increase floral yields.
Industrial hemp (Cannabis sativa L.) is an ancient and economically important crop used in food, medicine, textile, and paper industries (Chandra et al. 2017). In July 2021, an estimated 30% of the industrial hemp plants showed wilted leaves and root rot in the greenhouse at the Modern Agriculture Demonstration Area Management Center, Harbin City, Heilongjiang Province, China. Initially, the diseased plants exhibited green and reversible wilting of lower canopy leaves. Upon progression the plants showed irreversible wilting. The epidermal tissue of root and rhizome showed slight cracks and the vascular bundle exhibited light brown discoloration, and then died. Six randomly selected disease plants were collected. Small fragments (5 mm) were cut from the infected roots, surface-sterilized with 70% ethanol for 30s and 1% sodium hypochlorite for 5 min and rinsed three times in sterile H2O. Then the small pieces were embedded on potato dextrose agar at 25℃ for 4 days and sub-cultured by hyphal tipping to isolate the fungus. A single-spore culture was obtained by monosporic isolation. The colonies were characterized by an abundant white cottony mycelium, which became gray or purple with age. The macroconidia were transparent, short to medium in length, straight to slightly curved, septate 0 to 4, 16.8 to 26.6 µm long × 3.5 to 4.1 µm wide. The apical cells were long and tapering to a point and the basal cells were notched. Microconidia were elliptic or kidney-shaped, and septate 0 to 4. The conidia were 4.2 to 11.3 µm long × 3.5 to 5.5 µm wide (n = 50). The morphological characteristics were very similar to those of Fusarium oxysporum (Leslie and Summerell 2006). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1) and RNA polymerase II beta subunit (RPB2) genes were amplified and sequenced with the primers ITS1/ITS4, EF-1/EF-2 (Uwaremwe et al. 2020), and 5f2/7c (O'Donnell et al. 2010). The 520 bp (ITS), 948 bp (TEF1), and 861 bp (RPB2) sequences were deposited in GenBank with acce. nos. MZ722998, OK180473 and OK180474, respectively. NCBI BLAST analysis showed 98 to 100% similarity with the sequences of F. oxysporum. Moreover, the sequences alignment similarity for the six isolates were 100%. Based on the morphological and molecular characteristics, the isolates were identified as F. oxysporum. For the pathogenicity test, 20 seedlings were inoculated 30 ml of a conidial suspension (106 conidia/ml) using the root dip method. Another set of 20 seedlings were inoculated with the same quantity of sterile distilled water as the controls. After inoculation, all seedlings were maintained in a greenhouse at 25°C ± 2, with a relative humidity of 60 to 70% and a 16 h light/8 h dark cycle. This test was repeated twice. The leaves of the inoculated seedlings gradually became yellow and exhibited wilting within 15 to 20 days, the epidermal tissue of root showed light brown discoloration. Eventually the plants were dead within 40 to 50 days after inoculation. The control seedlings did not show any wilt symptoms. F. oxysporum was re-isolated from the infected root tissues to fulfill the Koch's postulates. In addition to F. oxysporum, F. brachygibbosum, Pythium aphanidermatum, F. solani, and F. equiseti have also been reported to cause wilt symptoms of industrial hemp (Zamir et al. 2018). To our knowledge, this is the first report of Fusarium wilt on C. sativa caused by F. oxysporum in the Northeast China.
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