First report of wilt and root rot on bell pepper (Capsicum annuum) caused by Thielaviopsis ethacetica

In March 2022, sudden wilting, black root rot and death were observed on bell pepper (Capsicum annuum) plants grown in a glasshouse at Tan Ha commune, Lam Ha District, Lam Dong, Vietnam (11°44′46.5″N 108°11′45.8″E), with a disease incidence of about 10% (Figure 1). A stem-streaming test was performed but no bacterial streaming was recorded, thus indicating Ralstonia solanacearum, the bacterium causing pepper bacterial wilt, was not responsible. Diseased roots were surface sterilised (Chu et al., 2022). Small sections of symptomatic roots were placed on potato dextrose agar (PDA) amended with chloramphenicol (50 mg/l). Fungal colonies that developed from diseased tissue were transferred to PDA plates. Five similar pure cultures with grey-black mycelia were obtained from hyphal tips (Figure 2). They produced cylindrical primary conidia (7–11 × 3–6 µm, n = 20), oblong secondary conidia (8–16 × 5–9 µm, n = 20), and aleurioconidia (14–20 × 7–11 µm, n = 20) (Figure 3). These morphological characteristics are consistent with the Thielaviopsis paradoxa (syn. Ceratocystis paradoxa) complex (de Beer et al., 2014; Mbenoun et al., 2014).

FIGURE 1. Natural disease symptoms in bell pepper in the glasshouse: (a) wilting and (b-c) black root rot

FIGURE 2. Colony morphology of Thielaviopsis ethacetica isolate TROC-2 on potato dextrose agar medium after five days at 25°C. (a) front; (b) reverse

FIGURE 3. Conidia of Thielaviopsis ethacetica isolate TROC-2: (a) cylindrical primary conidia, (b) obovoid-subglobose thick-walled aleurioconidia in chains, and oblong secondary conidia in (c) isolation and (d) chains. Scale bars = 10 µm

The ITS region from three representative isolates, TROC-1, TROC-2, and TROC-3, was amplified using primers ITS1 and ITS4 (White et al., 1990). BLASTn analysis of the ITS sequences of three isolates, TROC-1, TROC-2, and TROC-3 (Genbank Accession Nos. OP068111, OP068112, and OP068113, respectively), showed 99.8% identity to Thielaviopsis ethacetica (formerly Ceratocystis ethacetica; de Beer et al., 2014) type strain ICMP 13062 (KX954597). The phylogenetic tree grouped TROC-1, TROC-2, and TROC-3 with the epitype strain and other strains of T. ethacetica (Figure 4), thus confirming the identity of the three isolates as T. ethacetica.

FIGURE 4. Neighbour-joining phylogenetic tree constructed from the ITS reference sequences of 17 strains representing species of the Thielaviopsis paradoxa complex (Mbenoun et al., 2014). The bootstrap consensus tree is inferred from 1,000 replicates. The bootstrap support values above 50% are given at the nodes. The scale bar represents nucleotide substitutions per site. The tree is rooted by Ceratocystis virescens (KC305133)

Ten sixty-day-old bell pepper plants grown in a greenhouse in 10-cm-diameter pots, containing a 4:1 w/w mixture of field soil and sand, were used to confirm the pathogenicity of isolate TROC-2. TROC-2 was cultured in a mixture of rice hulls and millet (Chu et al., 2022). A mixture containing TROC-2 mycelia or the pathogen-free mixture was mixed with the soil in the pots. After seven days, all the plants in inoculated pots showed wilting, death, and black root rot (Figure 5), identical to those observed in the field, while plants in control pots remained symptomless. A fungus with the same morphology as TROC-2 was recovered from infected roots, thus fulfilling Koch's postulates.

FIGURE 5. Symptoms in bell pepper grown in the greenhouse seven days after artificial inoculation (control: healthy plants; TROC-2: plants inoculated with Thielaviopsis ethacetica isolate TROC-2 showing wilting, death, and root rot)

Thielaviopsis paradoxa and T. ethacetica have been reported to cause rot in pineapple and cacao, trunk and bud rot in oil palm, and sett rot in sugarcane (de Beer et al., 2014; Borges et al., 2019). However, to our knowledge, this is the first report of T. ethacetica as the causative agent of wilt and root rot of bell pepper. Further studies are needed on the origin of T. ethacetica and its distribution in bell pepper and other Capsicum species, thereby informing quarantine decisions to prevent the entry and spread of this pathogen.

ACKNOWLEDGEMENTS

V.-C. Han and H.H. Vo contributed equally to this work.