Apr 19, 2023
First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona.
Accepted in “Plant Disease” April 2023
Neeraja Singh†, Jeff Klingenberg, and Bindu Poudel-Ward
Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with the foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in the areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface-disinfested with 1 % sodium hypochlorite (NaOCl) for 2 min, rinsed three times with sterile water and were plated onto potato dextrose agar (PDA). Fungal isolate (Yuma) displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod regime. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width x 114 µm length (n=30). For molecular identification, genomic DNA was isolated from sclerotia using the DNeasy Plant kit (Qiagen). The internal transcribed spacer (ITS), translation elongation factor- 1α (TEF- 1α), calmodulin (CAL), and β-tubulin (β-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O’Donnell and Cigelink, 1997), respectively. A BLAST search of sequences revealed 98.7 to 100-% identity to Macrophomina phaseolina sequences (MK757624.1, KT261797.1, MK447823.1, MK447918.1). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina(Holliday and Punithaligam, 1970). Sequences were submitted in the GenBank under accession numbers OP599770.1 (ITS), OP690156.1 (TEF-1 α), OP612814.1 (CAL), and OP690157 (β-TUB). Pathogenicity assay was performed on 9-week-old stevia plants, var. SW2267, grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth (pH 5.6) at 28°C with 12 h of daylight. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty-healthy-plants were inoculated by soil drenching 50 ml of sterile water containing 105 microsclerotia/ml per pot. Five non-inoculated plants were kept as control. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on inoculated plants, whereas the control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequencing analysis. Although M. phaseolina has been reported earlier on stevia in North Carolina (Koehler and Shew, 2018), this is a first report from Arizona. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in Arizona in coming years.
Supplementary Figure. Signs of naturally occurring charcoal rot infection on Stevia
rebaudiana caused by Macrophomina phaseolina (A, B); root necrosis (C); discoloration
of root tissue in the cross-section (D); microsclerotia underneath the bark tissue (E);
microsclerotia from a pure culture (F).
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