First Report of Bismarck Palm Anthracnose Caused by Colletotrichum siamense in China

: Bismarck palm ( Bismarckia nobilis Hildebr. & H. Wendl.) is one of the most sought after palms. A severe disease of Bismarck palm was discovered in Wenchang, Hainan Province, China, in July 2010. The main symptoms were russet to gray-white lesions at the tip of leaflets


Introduction
Bismarckia palm (Bismarckia nobilis Hildebr. & H. Wendl.) is a popular ornamental plant, which is usually used for landscapes, green belts and home gardens [1]. B. nobilis is one of the most sought after palms grown in the Coconut Grant View Garden (CGVG), which is located in Wenchang, Hainan Province, China. There are more than 100 species of palm that are grown as both palmae germplasms and ornamental plants in the CGVG. In July 2010, a severe leaf disease emerged on B. nobilis in the CGVG). The aim of this study was to determine the etiological agent of the disease of Bismarck palm.

Fungal Isolation and Morphological Characterization
Diseased (mature and typically symptomatic) Bismarck palm leaves were collected from the CGVG. Pieces (3-5 × 3-5 mm) of leaflet tissue from the margins of lesions were immersed in 70% ethanol and rinsed twice with sterile distilled water. They were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark.
A BLAST search of the nucleotide database of GenBank indicated that the related amplicons of the ACT, CHS1, GAPDH, HIS, ITS and TUB2 genes of the representative isolate BWZ2. Molecular phylogenetic trees were constructed based on the close-neighbor-interchange algorithm (Maximum Parsimony) by MEGA version 7 [7] using six combined loci. Colletotrichum novae-zelandiae was used as an outgroup.

Pathogenicity Testing
To fulfill Koch's postulates, three isolated leaflets (about 10 cm in length) cut from 3-year-old Bismarck palms were wounded with a sterilized scalpel, and 6 × 6 mm mycelial plugs cut from the edge of colonies and grown on PDA at 25°C for 7 d were attached to the wound. Control leaflets were inoculated with plain PDA plugs. All the leaflets were then incubated in petri dishes (Ф = 15 cm) with a relative humidity of approximately 90% at 25°C. The pathogenicity was tested twice.

Symptoms, Morphological and Cultural Characterization
In July of 2010, this severe leaf disease was observed with an incidence of 100% (4 out of 4 plants were infected; 19.5489ºN, 110.7909ºE) and 89.2% (37 of 41 plants were infected; 19.5490ºN, 110.7904ºE) on B. nobilis in the CGVG ( Figure 1A). The initial visible symptoms were russet to gray-white lesions at the tip of leaflets ( Figure 1B), and the irregular-shaped lesions surrounded by reddish to brown halos then gradually extended to base of the compound leaf ( Figure  1C and D). At the advanced stage, entire infected leaves dried up ( Figure 1B).
A fungus was consistently obtained. It produced grayish white, cottony aerial mycelia ( Figure 1E), with peach to orange conidial masses ( Figure 1F) at the inoculum point on the PDA plates. The colonies were initially white and later turned pale brownish to pinkish, while the bottom of plates was pale yellowish to pinkish. The conidia were hyaline, one-celled, and cylindrical with obtuse to slightly rounded ends that were aseptate and ranged from 12.5 to 17.5 × 5.0 to 7.5 µm (n =100) ( Figure 1G). The hyphae grew at a temperature of 10-35°C, and the optical temperature was 25-30°C. The morphological characters were consistent with those of Colletotrichum siamense ([3, 8-10].

Pathogenicity Test
After 7 d, typical gray-white lesions surrounded by brown halos were observed on all the inoculated leaflets but not on the control leaflets ( Figure 1F). The fungus was reisolated from the diseased leaflets and was morphologically identical with strain BWZ2. No visible symptoms were observed in the control leaflets.

Discussion and Conclusion
Bismarckia palm adapts well to the changing environments [1]. So far no major disease has been reported on it. The causal agent was identified as C. siamense Prihastuti, L. Cai & K. D.
Hyde based on morphological [2,[8][9][10] and molecular results. In the genus Colletotrichum, C. siamense is the most widely distributed species, being observed on nearly all continents with up to 228 records in references. In addition, C. siamense presents the most diverse host range, being found in at least 103 wild and cultivated hosts [11].
Colletotrichum siamense is the primary type of anthracnose disease in tropical fruits [4,9]), and it has been cited as a pathogen of areca palm (Areca catechu L.) [12] (Cao et al, 2020), Cornus hongkongensis [13] and Pharbitis purpurea [14] in China. However, it has not been reported on Bismarck palm anywhere in the world [15]. To the best of our knowledge, this is the first report of Bismarck palm anthracnose caused by C. siamense in China.