First report of Clavibacter nebraskensis, causing Goss's bacterial leaf blight on maize (Zea mays L.) in South Africa.

Abstract

Maize is a staple crop in South Africa and an important income source to both smallholder and commercial farmers (Gravelet-Blondin, 2015). Goss's bacterial leaf blight and wilt, caused by Clavibacter nebraskensis (Cn), is a significant maize disease in North America and a quarantine concern in unaffected regions, with seedborne transmission posing a risk of introduction (EPPO, 2024; Osdaghi et al. 2023). From February to April 2024, bacterial leaf blight symptoms, typical of Cn infection, were observed on maize (Zea mays L.) in the North-West, Mpumalanga and Gauteng provinces of South Africa. Lesions were tan, irregular, parallel to veins, with a shellac-like appearance and black water-soaked edges, showing characteristic "luminous freckles" when backlit. Symptomatic leaf samples were collected from 6 commercial maize fields. Eight samples from Carletonville and Potchefstroom (total of two fields) were evaluated at the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria, and another four samples from Delmas, Leslie, and Bapsfontein (total of four fields) were evaluated at Stellenbosch University's Plant Disease Clinic. DNA was extracted either directly from lesions or from cultures isolated from lesions. For direct DNA extraction, cetyltrimethylammonium bromide was used, followed by a Cn specific PCR with primer pair 1184F/R (McNally et al. 2016). Macerates from lesion edges were streaked out onto nutrient broth yeast (NBY) agar. DNA from a single culture, with yellow-orange mucoid colonies, was extracted with a Zymo Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA), and confirmed as Cn with previously mentioned PCR primers. Simultaneously, macerates from the lesion edges were streaked onto yeast dextrose chalk agar (YDC). Yellow-orange mucoid colonies developed after four day and were purified onto NBY and incubated at 25°C for 4 days. All isolates tested gram-positive, were coryneform, aerobic, and non-spore forming. Genomic DNA was extracted and the suspension amplified using the 27F/1492R primer pair (Lane, 1991), targeting the 16S rRNA gene. The product was sequenced and confirmed as Cn. Cultures are stored in the culture collections at Stellenbosch University Plant Pathology Department (STE-U) and at FABI (CMW and CMW-IA). At both facilities, cell suspensions at a final concentration of 107 cells/mL were used to inoculate the third leaf of V3 / V4 stage maize plants (P1513, Syngenta), by wounding the middle of the main leaf vein and applying a 25µL droplet. Typical Cn symptoms appeared 4 days post inoculation and Cn was reisolated from these lesions and confirmed with PCR to complete Koch's postulates. Four isolates were selected for high-throughput sequencing (NCBI Bioproject: PRJNA1184689). Assembled genomes (NCBI accession: CP173672-CP173675) were analysed on the Type Strain Genome Server (Meier-Kolthoff and Göker, 2019) and confirmed as Cn based on 16S rRNA and Genome Blast Distance Phylogeny. The genomes aligned to the Cn type strain NCPPB 2581 with 99.8% DNA-DNA hybridization on the GGDC 3.0 server (Meier-Kolthoff et al. 2022), exceeding the suggested 70% species threshold. Phylogenomic analysis based on Average Nucleotide Identity values also clustered these genomes with Cn isolates. This is the first report of this pathogen outside of North America.