Asian ginseng (Panax ginseng) is an economically important perennial herb, mainly cultivated in Jilin Province, China. In September 2013, Asian ginseng plants in Jilin showed rusty root symptoms. Typical symptoms included rusty superficial lesions of irregular shapes and margins. Ten symptomatic roots were collected from each of five fields for investigation. To isolate the pathogen, root epidermal tissues with typical lesions were excised, surface-sterilized, and placed on potato dextrose agar (PDA) amended with 50 μg/ml tetracycline. After incubation at 20 ± 1°C in the dark for a week, 18 single-spore isolates out of 50 samples were obtained and identified as Ilyonectria robusta (A.A. Hildebr.) A. Cabral & Crous based on morphological characters and DNA sequence analysis (1). After incubating 7 days on PDA in the dark at 20°C, colonies were cottony to felty in texture and orange white to brownish grey in color with average diameters of 60 ± 3 mm. Isolates were cultured on synthetic nutrient-poor agar for conidial measurements. Macroconidia formed on simple conidiophores predominately, with mostly one and occasionally up to three septa, and were cylindrical with both ends broadly rounded. Macroconidia varied in size depending on the number of cells as follows: one-septate, 7.0 ± 0.6 × 27.7 ± 2.7 μm; two-septate, 7.3 ± 0.7 × 33.3 ± 2.1 μm; three-septate, 7.4 ± 0.6 × 33.4 ± 2.2 μm. Microconidia that formed on complex conidiophores were ellipsoid to ovoid and ranged in size from aseptate 3.7 ± 0.5 × 8.7 ± 1.1 μm to one-septate 5.0 ± 0.6 × 13.1 ± 1.6 μm. Brown chlamydospores were abundantly produced on PDA, globose to subglobose in shape, and in size of 10.9 ± 1.3 × 11.8 ± 1.5 μm (n ≥ 30 observations per structure for each measurement). The isolates were further classified by amplifying and sequencing the ITS1-5.8S rRNA-ITS2 region and histone H3 gene with primer pairs ITS5 and ITS4 (4), and H3-1a and H3-1b (3), respectively. Sequences of the two loci (GenBank Accession Nos. KM015300 and KM015299) showed 100% identity among the three examined isolates and the published I. robusta isolates (JF735268 and JF735517). To confirm the pathogenicity, bare roots of 3-year-old Asian ginseng were inoculated with mycelial plugs of three isolates of I. robusta selected randomly. Four roots were inoculated as replicates for each isolate with pathogen-free agar plugs as a control. One week post-inoculation in the dark at 20 ± 1°C, all the inoculated ginseng roots showed light-brown to dark-brown lesions. I. robusta was recovered from symptomatic roots and confirmed by analyzing the DNA sequence of the histone H3 gene. The inoculation experiment was repeated, and both trials showed the same results. The ginseng tissue under the control agar plugs remained symptomless, and no fungi were isolated. To our knowledge, this is the first report of I. robusta causing rusty root of P. ginseng in China (1,2,5). References: (1) A. Cabral et al. Mycol. Prog. 11:655, 2012. (2) I. Erper et al. Eur. J. Plant Pathol. 136:291, 2013. (3) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (5) X. Lu et al. Plant Dis. 98:1580, 2014.
In northeastern China, Asian ginseng (Panax ginseng) roots exhibited reddish brown lesions of various sizes, irregular shapes, and diffuse margins, typical of rusty root disease. The lesions remain superficial, smooth, and limited to the epidermal and peridermal tissues. In September 2013, 10 symptomatic roots were collected from each of three fields in Jilin and Heilongjiang provinces. One piece of symptomatic skin tissue from each root was excised, surface-disinfested in 1% NaClO for 3 min, rinsed three times with sterile water, and then placed on tetracycline-amended (50 μg/ml) potato dextrose agar. After incubation at 22 ± 1°C in the dark for a week, small olivaceous black colonies developed from the symptomatic tissue from five of the 30 samples. No spores were observed. A single hyphal tip of each colony was transferred to a fresh V8 agar plate to purify the culture. Two-week-old colonies on V8 agar were olivaceous gray, and 42 to 46 mm in diameter with an outer white margin (3 to 5 mm wide). Conidia produced in V8 broth after 3 weeks with a 12-h photoperiod were straight and hyaline, cylindrical or subcylindrical with no or one septum. Mature conidia were 12.8 to 21.8 × 2.2 to 4.5 μm (mean 18.2 × 3.0 μm, n = 100 conidia for each of three isolates). Three isolates selected randomly were further identified by analyzing the partial sequences of the ITS region of rDNA with primers ITS4 and ITS5 (5), and partial sequences of β-tubulin with the primers tub2F and tub2R (1). Sequences of the three isolates (GenBank Accession Nos. KJ149287, KJ149288, and KJ149290 to 93) showed 99% to 100% homology with previously identified and deposited Rhexocercosporidium panacis isolates (DQ2499992 and DQ457119) for both loci (3). Therefore, the three isolates were identified as R. panacis and deposited in China General Microbiological Culture Collection Center (CGMCC3.17259 to 61). Pathogenicity of R. panacis in Asian ginseng was investigated using these three isolates as described previously with slight modifications (4). Bare roots of 3-year-old Asian ginseng were surface-disinfested as described above, and inoculated with mycelial plugs (4 mm diameter) cut from the margin of actively growing colonies of the isolates on V8 agar. Three mycelial plugs were placed on each root at 3-cm intervals and four roots (replicates) were inoculated for each isolate. Four additional roots were inoculated with non-colonized agar plugs as control. The treated roots were placed on moist filter paper in an enamel tray. The plates were sealed with plastic wrap to prevent desiccation and incubated in the dark at 18 ± 1°C. Four weeks post inoculation, all the inoculated ginseng roots showed red-brown lesions, which turned to dark red or black over time. R. panacis was recovered from symptomatic roots for all isolates and confirmed by ITS sequence analysis. The mock-inoculated control roots remained symptomless and no R. panacis was isolated. The inoculation experiment was repeated and showed the same results. R. panacis was reported in 2006 to infect roots of Panax quinquefolius (2,3,4). To our knowledge, this is the first report of R. panacis causing rusty root of P. ginseng. References: (1) P. R. Hirsch et al. Mycol. Res. 104:435, 2000. (2) Z. K. Punja et al. Can. J. Plant Pathol. 35:503, 2013. (3) R. D. Reeleder. Mycologia. 99:91, 2007. (4) R. D. Reeleder et al. Phytopathology 96:1243, 2006. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.
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