First Report of Cladosporium tenuissimum Associated with Leaf Spot of Alfafa (Medicago sativa L.) in China

Alfalfa (Medicago sativa L.) is a leading forage crop that is known as the “king of forage.” It has been widely cultivated all over the world for its high nutritional quality and high biomass production (Liu et al. 2018). Since 2015, an unknown leaf spot symptom with approximately 18% disease incidence has been observed on M. sativa at a pratacultural science experimental field of Southwest University, 105.58°E, 29.40°N, Chongqing, China. Infected leaves gradually exhibited white to gray irregular necrotic lesions. In severe cases, some leaf spots coalesced, covering more than 50% of the leaf area, eventually causing the leaves to fall off. The leaves with the spot symptoms were collected in field, surface sterilized with 70% ethanol for 5 s followed by 0.1% HgCl₂ treatment for 4 min, and rinsed in sterile water three times. Thereafter, leaf samples (2 × 2 mm) from margins of individual lesions were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. The diameter of the fungal colonies was 4.2 cm (3.4 to 5.3 cm) (n = 6). From a top-down view, colonies on PDA usually exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Colony color ranged from olive-brown to dark green; margins varied from gray-olivaceous to white without prominent exudates, and sporulation was profuse. From a bottom-up view, the colony color appeared almost dark. Hyphae were branched, 2 to 5 μm wide, pale olivaceous-brown, often irregular in outline, sometimes constricted at the septa. Conidiophores were subcylindrical-filiform, unbranched, 320 μm (46 to 510 μm) × 3.4 μm (2.5 to 4.1 μm) (n = 30), tips often unilaterally swollen. Ramoconidia were zero to one septate, smooth walled, 17.5 μm (9.3 to 24.6 μm) × 3.8 μm (2.8 to 5.0 μm) (n = 30). Conidia were single celled, smooth, olive-brown, and elliptical to limonifor, 4.5 μm (2.8 to 6.1 μm) × 3.0 μm (2.2 to 3.7 μm) (n = 100). Based on morphological and growth characteristics, the features of associated fungal pathogens were consistent with those of Cladosporium tenuissimum (Bensch et al. 2010, 2012). To further confirm its identity, three different genomic DNA regions—the internal transcribed spacer (ITS) regions, partial translation elongation factor-1 alpha (EF), and actin (ACT)—were amplified and sequenced. Fungal genomic DNA was extracted, and the ITS, EF, and ACT were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R (Bensch et al. 2012; Jo et al. 2018). The ITS sequence showed 99.6% identity with that of C. tenuissimum (MH383051.1). DNA sequences from EF and ACT were 100% identical to those of C. tenuissimum (MF473728.1 and LN834588.1). The representative sequences were deposited in GenBank (accession nos. MK311328, MK341044, and MK341045). Based on morphology and DNA sequence analysis, the associated fungus was demonstrated to be C. tenuissimum. Pathogenicity trials were conducted in a greenhouse at 25 ± 3°C under natural daylight conditions. Fungal isolates were grown on PDA for 7 days. Spores were harvested and resuspended in sterile water (2.0 × 10⁶ conidia/ml). Six pots of M. sativa (cv. Eureka) were inoculated by spraying on the leaves with conidial suspension. Another six control pots were sprayed with sterile water. All pots were covered with plastic bags containing wet cotton wool for 48 h. Seven days after inoculation, the inoculated plant leaves showed leaf spot symptoms that were similar to those previously observed in the field. From the leaf spots of inoculated plants, C. tenuissimum was successfully isolated again. Control plants did not exhibit any disease symptoms. To our best knowledge, this is the first report on the occurrence of C. tenuissimum causing leaf spot on M. sativa in China. So far, this pathogen has not been found to be able to cause plant death. However, it can damage the leaves of alfalfa and affect forage quality and yield. Understanding its etiology may help to control this plant fungal pathogen, thus reducing economic losses.