First Report of Leaf Black Spot Caused by Alternaria alternata on Rosemary (Rosmarinus officinalis L.) in China
Siliang Huang1, Siqing Liu1, Di Yang2, Chanjuan Du2, Xueling Zheng1, Yuying Li1, Aili Tao1, Yunfeng Ye3*, Gang Fu2*
1 Nanyang Normal University, Nanyang 473061, China
2 Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences / Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China Ministry of Agriculture and Rural Affairs / Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Nanning 530007, China
3 Horticultural Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
*Co-corresponding author: fug110@gxaas.net (Gang Fu), yeyunfeng111@126.com (Yunfeng Ye)
Rosemary (Rosmarinus officinalis L.) is an aromatic, evergreen, medicinally important shrub and widely used for cooking, tea, cosmetics as well as medicinal materials. It is grown in many countries including China that had more than 9300 hm2 of commercial cultivation area in 2021. In March 2020, a leaf spot disease sporadic occurred in field rosemarry plants in Nanyang City (32º51 ́ N, 111º36 ́ E), Henan Province, China. The disease outbreaked in September with a disease incidence of 57-83%. Symptoms initially appeared as small brown leaf spots that gradually expanded into dark blackbrown irregular lesions. Most of the spots started from the leaf tip or leaf margin, and gradually spread to the leaf base, resulting in heavy defoliation especially on rainy days. Diseased leaf segments (1×3 mm) were surface-sterilized by dipping in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar, then incubated at 28°C in the dark for 5 days. Twelve fungal isolates with the same morphological characteristics were obtained from nine affected leaves. The fungal colonies were initially white and turned gray brown withflocculent aerial mycelia and a whorled back. Conidia were frequently born in a long chain, with a short beak, brown or light-brown, 13.2 to 48. 7 (average 26.1) × 4.0 to 13.1 (average 8.0) μm in size (n=148) with 0 to 8 transverse and 0 to 3 longitudinal/oblique septa. Phenotypic features of the isolates agreed with those of Alternariaalternata (Simmons et al. 2007). Two isolates Aa1 and Aa2 were randomly selected for molecular and pathogenicity tests. DNA was extracted from mycelia. Partial sequences of internal Page 1 of 5transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) were amplified using the primer pairs ITS1/ITS4 and EFI-728F/EFI-986R (Wei et al. 2022), respectively. The GenBank accession nos. were OK036714 and OK036715 for ITS, and ON951980 and ON951981 for TEF1-α of Aa1 and Aa2, respectively, with a maximal identity of greater than 99% to multiple A. alternata strains. In the neighbour joining phylogenetic tree of the amplified ITS and TEF1-αsequences both Aa1 and Aa2 clustered with A. alternata strains, clearly separating them from other Alternaria spp. For pathogenicity test, conidial suspensions (1×106 spores /mL) of Aa1 and Aa2 were separately sprayed on healthy one-year-old rosemary plants (n=3) with their leaves slightly wounded with a sterilized needle. Control plants (n=3) were sprayed with sterile water. Both inoculated and control plants were incubated at 90% RH, 28 °C. After 14 days, all the inoculated leaves showed black brown lesions similar to those on naturally affected field plants, whereas controls remained symptomless. Fungal cultures with the same phenotypic features as the inocula were constantly re-isolated from the infected leaves. A. alternata was reported as pathogen causing foliar necrosis on rosemary in Italy (Perello et al.1995) and leaf spot (or leaf blight) onmultiple plant species such as Actaea dahurica (Hai et al. 2022), and Ligustrum japonicum (Wei et al. 2022) in China. This is the first report of A. alternata causing leaf black spot on rosemary in China.
References:
Hai F. S., et al. 2022. Plant Dis. https://doi.org/10.1094/PDIS-02-22-0396-PDN
Perello A., et al. 1995. Investig. Agraria. Prod. Prot. Veg.
Simmons E. G., et al. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Center, Utrecht, The Netherlands.
Wei F., et al. 2022. Plant Dis.https://doi.org/10.1094/PDIS-01-22-0068-PDN
e-Xtra items：
Fig.1 Leaf black spot symptoms on Rosmarinus officinalis and the morphology of A. alternata. (a) natural symptoms; (b) black spot symptoms observed on the 14th day after being inoculated with A. alternata; (c) negative control; (d) colony; (e)conidia; (f) conidia in chains
Fig.2 Phylogenetic trees inferred from maximum likelihood analysis based on the sequences of ITS and TEF1-α. The values (from 1,000 replicates) are indicated at the branch nodes as the percentages supported by bootstrap. The scale bar represents a genetic distance of 0.05 substitutions per nucleotide position. (a) phylogenetic tree based on ITS sequence; (b) Page 2 of 5phylogenetic tree based on TEF1-α sequence
Funding:This research was financially supported by the National Natural Science Foundation of China (NSFC31960520); the Special Project of Basic Scientific Research of Guangxi Academy of Agricultural Sciences (2021YT069,31960520).
Fig.1 Leaf black spot symptoms on Rosmarinus officinalis and the morphology of A. alternata. (a) natural symptoms; (b) black spot symptoms observed on the 14th day after being inoculated with A. alternata; (c) negative control; (d) colony; (e)conidia; (f) conidia in chains 1058x698mm (72 x 72 DPI) Page 4 of 5
Fig.2 Phylogenetic trees inferred from maximum likelihood analysis based on the sequences of ITS and TEF1-α. The values (from 1,000 replicates) are indicated at the branch nodes as the percentages supported by bootstrap. The scale bar represents a genetic distance of 0.05 substitutions per nucleotide position. (a) phylogenetic tree based on ITS sequence; (b) phylogenetic tree based on TEF1-α sequence 803x322mm (72 x 72 DPI) Page 5 of 5
链接：https://apsjournals.apsnet.org/doi/epdf/10.1094/PDIS-09-22-2165-PDN
