Citrus production in Botswana is a small but rapidly growing industry, mainly consisting of small-scale farmers, producing grapefruit, Eureka lemons, mandarins, and Valencia oranges for the local market. In 2022 Botswana joined the Citrus Growers’ Association of Southern Africa, in part to improve access to international export markets. Accurate information about the status of pests associated with a commodity in an exporting country is needed to gain international market access. Phyllosticta citricarpa, the cause of citrus black spot (CBS), is present in most citrus-producing countries including South Africa (Coetzee et al., 2022), but is a regulated pest for some trading partners. A survey was therefore conducted in a manner that is relevant to the biology of the pathogen and in accordance with relevant International Standards forPhytosanitary Measures (ISPMs) in the nine districts of Botswana in 2022. Valencia oranges with CBS-like symptoms were detected only in the Tuli Block. The Tuli Block is part of western Botswana located between Zimbabwe and South Africa and is known for citrus farming. Fruits with symptoms, sunken, brown necrotic lesions, with a reddish-brown border (Figure 1a), were surface sterilised by immersion in 1.25% sodium hypochlorite for five minutes, followed by rinsing twice in sterile water before air drying. Small fragments of several lesions per fruit were placed onto potato dextrose agar (PDA) and incubated for 10 to 14 days at 25°C. On PDA, colonies with a dark centre and submerged mycelial growth with irregular margins forming a plectenchymatous crust, were regarded as putative P. citricarpa isolates (Figure 1b). Isolates were transferred onto oatmeal agar plates and incubated at 25°C to differentiate P. citricarpa from other Phyllosticta species. The plates were examined for the presence of a yellow halo around colonies after seven days (Figure 1c) (Baayen et al., 2002; Food and Agriculture Organization, 2014). Details are in the caption following the image FIGURE 1 Open in figure viewer PowerPoint Citrus black spot on Valencia oranges from Tuli block in Botswana: a) Symptoms on fruit; b) Phyllosticta citricarpa growing on potato dextrose agar; c) Phyllosticta citricarpa growing on oatmeal agar, forming a characteristic yellow halo Molecular identification was conducted on twelve putative P. citricarpa isolates. DNA was isolated from mycelia of two-week-old colonies grown on PDA plates. Mycelia were scraped from the plates and lyophilised before extraction, using the Promega Wizard？ Genomic DNA Purification Kit (Madison, USA). Real-time PCR to detect P. citricarpa was done using probes described by Hu et al. (2014). Results obtained from the morphological and molecular analyses confirmed the presence of P. citricarpa on Valencia oranges in the Tuli Block. To fulfil Koch's postulates, detached, surface sterilised oranges were wounded and inoculated with P. citricarpa agar plugs from two-week-old cultures of four isolates (four fruits per isolate). Fruits inoculated with clean agar plugs were used as negative controls. Fruits were incubated in a moisture chamber for three weeks at 25°C. CBS symptoms were observed only on inoculated fruits and CBS could be reisolated from the lesions. The twelve isolates were subjected to high-throughput sequencing (HTS, NCBI Bioproject number: PRJNA991338). Their sequences were deposited in Genbank (Accession Nos. SAMN36292249-SAMN36292260) and analysed together with data from 41 previously sequenced isolates from Southern Africa (Bioproject number: PRJNA825332; Coetzee et al., 2022). The phylogenetic tree placed the Botswana isolates closest to those from the Eastern Cape province in South Africa, indicating a possible dissemination pathway between these two countries (Figure 2). Mating types were determined using the primer sets, MAT1-1-1 and MAT1-2-1 (Wang et al., 2016), and in silico PCR based on the HTS. Both mating types, in c. 1:1 ratio, are present in Botswana, implying that both sexual and asexual reproduction may play a role in the epidemiology of the disease in Botswana. Details are in the caption following the image FIGURE 2 Open in figure viewer PowerPoint UPGMA phylogenetic tree (1000 bootstrap replications, bootstraps < 70 are not shown) based on Nei distances between isolates as calculated by read mapping and variant calling from the high-throughput sequence data from 12 Botswana Phyllosticta citricarpa isolates and 41 isolates from Southern Africa (HTS data available under NCBI Bioproject numbers PRJNA991338 and PRJNA825332). Isolates from Botswana are shown in bold. ES: Eswatini; EC: Eastern Cape; KZN: KwaZulu-Natal; LIM: Limpopo; MP: Mpumalanga; NW: North West, SA: South Africa This is the first report of CBS, caused by P. citricarpa, in Botswana. Knowledge of the geographic distribution of the disease in Botswana will help curb the spread of the disease and allow the country to establish and officially maintain CBS-pest free areas in accordance with ISPM 4 - Requirements for the establishment of pest free areas (Food and Agriculture Organization, 2017) to enhance trade.