Astragalus membranaceus is a perennial plant in the family of Leguminosae. It is one of the most important traditional Chinese medicines. Root rot has become a serious constraint to commercial production of this medicinal plant in northeastern, northern and northwestern China. In June 2020, A. membranaceus plants in a field at the village of Sada, Minhe County, Haidong City, Qinghai Province (102°51′30.8″E, 36°19′8.58″N, H:2437m), showed symptoms in the form of a large number of brown longitudinal cracks or turtle cracks in the root, and weak growth potential of the aboveground part, with yellowing and falling off of the leaves in severe cases. Root rot seriously affects the quality and yield of A. membranaceus. The investigation was performed in the area of field was 3,996 m2, and the incidence of root rot reached 10%. Five A. membranaceus root rot samples were collected. To isolate the fungi, rotted root pieces (about 0.5 cm) were obtained with the sterile scissors from the junction of infected and healthy tissues. The symptomatic roots were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plate in incubator for 2 days at 25°C. After that the surface of root was moldy, abundant conidiophores and conidia formed. Ten isolates were obtained by single spore isolation technique. The mycelia were white or light yellow on PDA. Primary conidiophores were Verticillium-like with two to three levels. The stipe length measured 41.91 to 97.62 μm. Secondary conidiophores were penicillate with two or three whorls of phialides. The stipe length measured 36.67 to 65.98 μm. Conidiogenous cells are slender cylindrical. Conidia were colorless, round or oval, and aseptate, with an average length and width of 6.5 to 8.5×2.5 to 3.5 μm. Chlamydospores were round or oval, with an average length and width of 9.91 to 16.21×8.07 to 13.64 μm. The identity of ten isolates was confirmed by sequencing the internal transcribed spacer (ITS) locus using the primers ITS1F/ITS4 (sequence length was 526 bp), the translation elongation factor 1-alpha (EF-1α) gene using the primers EF-1H/EF-2T (sequence length was 567 bp) (O'Donnell et al. 1998), and the sequences included ITS1, 5.8S ribosomal RNA gene, ITS2, and large ribosomal RNA gene using the primers F1/F2 (sequence length was 1010 bp) (Cao et al, 2020). BLAST analysis of all the three sequences of isolates, HQ-1, HQ-1-1, HQ-2-1, HQ-3-1, HQ-4, HQ-6, HQ-6-1, HQ-7-1, HQ-9-1, and HQ-10-1, in the Genebank database resulted in matches of 99.2% or more for Clonostachys rosea (NCBI accession Nos. MZ433169-MZ433178 (ITS), MZ451390-MZ451399 (TEF1-α), and MZ433195-MZ433204 (ITS 1, 5.8 S rRNA, ITS 2, and LSU gene). Ten isolates were inoculated on their host for pathogenicity test. The isolates were grown on PDA six days for sporulation. The healthy semi-annual seeding stage root were inoculated by treating them with 10 mL of conidia suspension (1 × 105 conidial/mL). A group of 10 seedlings of the same age was treated with sterile water to serve as the control. Inoculated root were moved into artificial climatic box at 25℃, relative humidity was 70%, with 12 h light / 12 h dark condition. Typical symptoms first appeared as brown tiny spot about 3 days, extended and cracked for 5-7 days, and mycelia were observed on the surfaces of the lesions. Uninoculated plants remained healthy. Isolates were re-isolated from the symptomatic root, and the colonial morphology of the recovered isolates were the same with the original isolate. The assays were conducted three time (Fig S1). These results showed that these isolates maintained their pathogenicity to their own hosts. To our knowledge, this is the first report of C. rosea causing root rot of A. membranaceus in China. Clonostachys rosea has been previously reported to cause root rot of soybeans in the United States (Bienapfl et al, 2012). The disease must be considered in existing management practices, and our finding provided foundation for preventing or curing this disease.
Angelica sinensis is a perennial plant in the Apiaceae and a source of one of the most important traditional medicines in China. This medicinal herb has been mainly cultivated in Gansu, Sichuan, Yunnan, Shanxi, Hubei, and Qinghai Provinces. In June 2020, angelica plants in a field in the village of Sada, Minhe County, Haidong city, in Qinghai Province (102°83′00.00″E, 36°31′93.00″N, elevation:2437 m), displayed symptoms of yellowing, stunting, root rot, and wilting(Fig. S1). Severe brown discoloration of vascular tissue in the stems of infected plants was observed. Root rot seriously affected the quality and yield of A. sinensis. The incidence of root rot in the fields reached 15% in 2020 and 18% in 2021. Fungi were isolated from diseased roots using a single spore method. The identity of isolates was confirmed by sequencing the nuclear ribosomal internal transcribed spacer (ITS) using the primers ITS1/ITS4 (White et al, 1990) and the translation elongation factor 1-alpha (TEF1-α) gene using the primers EF-1/EF-2 (O'Donnell et al. 1998). BLAST analysis of the ITS and TEF1-α sequences of isolates DG-1, DG-1-1, DG-1-2, DG-2, DG-2-1, DG-5, and DG-5-1 in the NCBI database showed 99% to 100 % nucleotide identity to those of Clonostachys rosea (NCBI accession Nos. MZ424803-MZ424809 (ITS), and MZ451383-MZ451389 (TEF1-α)). BLAST analysis of the ITS and TEF1-α sequences of isolates, DG-3, DG-3-1, and DG-4 in the NCBI database produced matches of 99.1% or better to Fusarium acuminatum (NCBI accession Nos. MZ424810-MZ424812 (ITS), and MZ441148-MZ441150 (TEF1-α)). Conidia of C. rosea were hyaline, round or oval, aseptate, with an average length and width of 6.5-8.5×2.5-3.5 μm. Chlamydospores were round or oval, with an average length and width of 9.9-16.2×8.1-13.6 μm. Conidiophores of C. rosea were hyaline, septate, Verticillium-like, conidiogenous cells were slender and cylindrical. Macroconidia of F. acuminatum were hyaline, falciform, 0-5 septate, with an average length and width of 22.5-39.5 × 2.5-4.5 μm. Microconidia were rare. Chlamydospores were oval, with an average length and width of 9.5-14.3×8.1-11.8 μm. Conidiophores of F. acuminatum were hyaline, septate, branched (Fig. S2). A pathogenicity test was performed by inoculating a conidial suspension of 1×105 per ml in 0.025% Tween 20 onto roots of A. sinensis. Inoculated roots were incubated in a humid and dark chamber at 26°C for 24 h, and then with 12 h light/12 h dark for six days. The pathogenicity assay was repeated three times for each isolate. Root rot was observed seven days after inoculation and mycelia were observed on the surface of the lesions (Fig. S1). Two isolates were recovered from the lesions and named DG-2-D and DG-3-1-D. The colonial morphology of these two isolates and the original isolates DG-2 and DG-3-1 was identical after six days of inoculation on PDA (potato dextrose agar) (Fig. S2). The two isolates recovered were identified as C. rosea and F. acuminatum, respectively, by amplifying and sequencing a portion of the TEF1-α gene. To our knowledge, this is the first report of C. rosea causing root rot of A. sinensis in China. Fusarium acuminatum was reported to cause root rot of A. sinensis in Gansu province, China in 2021 (Niu et al. 2021). Clonostachys rosea has been previously reported to cause root rot of soybean in the United States (Bienapfl et al. 2012). The disease must be considered in existing management practices, and our findings provide a foundation for management this disease.
Maize (Zea mays L.) is one of the most important crops in the world, it can be divided into forage, grain, and industrial maize according to its use (Barrière, 2017). Maize for silage is harvested, including all green plants in the suitable period, chopped, and processed; then, it is suitable for silage fermentation to feed cattle, sheep, and other herbivorous livestock (Kolver et al., 2001;Qi et al., 2021).Generally, maize for silage has high levels of metabolizable energy, protein, and starch, with lower levels of fibre, low pH and
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