Occurrence of Leaf Spot Caused by Diaporthe phaseolorum on Bletilla striata in China.

Abstract

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant widely distributed in eastern and southern Asia. In April of 2020, a leaf spot disease on B. striata was observed in plant nurseries in Guilin, Guangxi Province, China. Disease incidence was estimated at approximately 20% (n = 150 plants) across the survey area (~ 0.3 ha). The initial symptoms were small, reddish to brown spots, circular or irregular in shape. Subsequently, they developed into large dark brown, irregular lesions. As the lesions coalesced, leaves withered and defoliated. To isolate the causal agent, eighteen small pieces (~ 5 mm2) were collected from the margin of the necrotic lesions on Chinese ground orchid, surface disinfected (2 min in 1% NaOCl, and rinsed three times in sterile water), and placed on potato dextrose agar (PDA) at 26°C for 3 days. Hyphal tips were transferred to PDA to obtain pure cultures. Twelve isolates were obtained, of which eight isolates had similar morphological characteristics. After 7 days growth on PDA, colonies were grayish-white, fluffy, with white aerial mycelium. After 3 weeks, colonies formed white aerial mycelial mats, and pycnidia developed. The α-conidia were abundant, hyaline, aseptate, ellipsoidal to fusiform, measuring 4.6 to 6.7 μm × 2.1 to 3.0 μm (n = 55), whereas the β-conidia were hyaline, long, slender, straight or curved, measuring 10.3 to 17.2 μm × 0.9 to 1.8 μm (n = 59). Morphological features were similar to Diaporthe sp. (Santos et al. 2011, Udayanga et al. 2015). For further molecular identification, DNA was extracted from the mycelia of the representative isolate BJ26.3 following the CTAB (cetyltrimethylammonium bromide) method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial translational elongation factor subunit 1-α (EF-1α), calmodulin (CAL) , histone H3 (HIS3), β-tubulin (TUB) genes were amplified and sequenced using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R, CAL-228F/CAL-737R, CYLH3F/H3-1b, and Bt2a/Bt2b, respectively (White et al. 1990, Guarnaccia et al. 2018). The obtained sequences were deposited in NCBI GenBank under the following accession numbers: OK560457, OK539595, OK539592, OK506726, OK539598. BLAST analysis of the deposited sequences showed 99 to 100% identity with accession numbers KC343177 (563/566 bp), KC343903 (521/523 bp), KC343419 (423/427 bp), KC343661 (340/340 bp), KC344145 (658/662 bp) of D. phaseolorum CBS 127465 (Guarnaccia et al. 2018). In addition, a phylogenetic analysis using concatenated sequences confirmed BJ26.3 as D. phaseolorum. Furthermore, pathogenicity tests were carried out on 1.5-year-old B. striata plants. Healthy leaves on three plants (1 leaf per plant) were inoculated with 5 × 5 mm mycelial discs of strain BJ-26.3 from 3-day-old PDA cultures. Another three plants treated with sterile water served as the control. All plants were covered with transparent plastic bags and maintained in a greenhouse at 26°C with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained healthy. The experiments repeated three times showed similar results. Finally, D. phaseolorum was consistently re-isolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. To our knowledge, this is the first report of D. phaseolorum causing leaf spot of B. striata worldwide. This study might provide important information for growers to manage this disease.