Identification of Fusarium oxysporum Causing Leaf Blight on Dendrobium chrysotoxum in Yunnan Province, China

Abstract

Leaf-blight disease caused by the Fusarium oxysporum is an emerging problem in Dendrobium chrysotoxum production in China. Symptoms of leaf blight were observed on seedlings of D. chrysotoxum cultivated in a nursery in Ruili City, Yunnan Province, China. In this study, we isolated the Fusarium sp. associated with leaf-blight disease of D. chrysotoxum from the diseased seedlings. A pathogenicity test was performed to fulfill Koch's postulates to confirm the pathogenicity of isolated strains and identified using morphological and molecular techniques. The results revealed that all four isolated Fusarium sp. isolates (DHRL-01~04) produced typical blight symptoms followed by marginal necrosis of leaves on the D. chrysotoxum plants. On the PDA medium, the fungal colony appeared as a white to purplish color with cottony mycelium growth. Microconidia are oval-shaped, whereas macroconidia are sickle-shaped, tapering at both ends with 2-4 septations. The phylogenetic trees were construed based on internal transcribed spacer (ITS), translation elongation factor (EF-1α), and RNA polymerase subunit genes RPB1 and RPB2 genes, respectively, and blasted against the NCBI database for species confirmation. Based on the NCBI database's blast results, the isolates showed that more than 99% identify with Fusarium oxysporum. To our knowledge, this is the first comprehensive report on the identification of Fusarium oxysporum as the causal agent of Dendrobium chrysotoxum leaf blight in Yunnan Province, China, based on morphological and molecular characteristics.

Keywords: Koch’s postulates; internal transcribed spacer (ITS); morphological characteristics; pathogenicity; phylogenetic analysis.

Figure 1

Symptoms of leaf blight on D. chrysotoxum seedlings in the nursery beds. (A) Early water-soaked corrugated lesions on the stem and graft interface; (B) yellowing of leaves and marginal necrosis; (C) plant leaves fall off. Arrows indicated leaves fall off the stem; Circles indicated lesions in (A,C).

Figure 2

Morphological characteristics of Fusarium oxysporum isolated from diseased Dendrobium chrysotoxum plants. The colony growth on PDA medium (A, B), microconidia (C), macroconidia (D), and conidia chain bar = 10 μm and macroconidia (E–G).

Figure 3

One percent agarose gel image of PCR-amplified products of ITS, EF-1α, RPB1, and RPB2 gene of four isolates from Dendrobium chrysotoxum. Marker—2000 bp DNA marker (D2000, Tiangen, Beijing, China), DHRL-01~DHRL-04 were the gene PCR production of 4 isolates, and N-C—negative control.

Figure 4

Maximum-likelihood phylogenetic tree generated from ITS (A), EF-1α (B), RPB1 (C), and RPB2 (D) sequences analysis of isolated strains of DHRL-01~04, respectively. Colletotrichum fructicola and Plectosphaerella cucumerina were used as the outgroup of ITS. Colletotrichum fructicola and Aspergilus nidulans were used as the outgroup of EF-1α, Pseudogymnoascus destructans, and Aspergilus tanneri were used as the outgroup of RPB1, Aspergilus tanneri and Aspergilus tanneri were used as the outgroup of RPB2. Scale bars indicate the number of substitutions per site. Bootstrap values are expressed as percentages based on 1000 replicates. Different background colors represent different clusters in the phylogenetic tree.

Figure 5

Symptoms produced on Dendrobium chrysotoxum plants inoculated with Fusarium oxysporum. (a,b) yellowing, marginal necrosis, and, later, expansion to the bottom leaves after 15 days post-inoculation with Fusarium oxysporum DHRL-01. (c) Whole leaves became blighted and dropped after 30 days post-inoculation with Fusarium oxysporum DHRL-01, and (d) healthy plants as control (inoculated with sterilized ddH₂O) was only physical damage in leaves.