Sample collection
During the growing season of 2021, visibly infected leaves and stems with distinctive symptoms of late blight were picked up from potato plants. The blighted samples were collected from the areas of the Seed Potato Production Center (SPPC) in Ibb Governorate in Yemen (Fig. 1). Our experimental research on plants; including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation. Leaves and stems with a single, fresh, nicely sporulating lesion were selected from four different plants/field (two lesions from each plant). The collected fresh leaves and stems were transferred from the open field to the laboratory under cold conditions − 4°C [9] in transparent polyethylene bags. A total of 54 leaves and 17 stems, which were collected from 10 fields, were used. A list of the 71 isolates used in this study, including isolate name, location of collection, host, and sampling year, is presented in Table 1.
Isolation and purification of P. infestans
This trial was conducted at the laboratory of microbiology, College of Science, University of Sana'a, Yemen. Isolation of P. infestans was conducted using the tuber-slice method as described by Tumwine et al. [10]. The collected leaf samples were carefully washed under tap water in order to remove the dust, insect eggs, and soil remains, and then dried on Whatman filter paper at room temperature. Moreover, healthy potato tubers were sterilized by rinsing in 1% sodium hypochlorite (NaClO) for 2 min and were transferred to Whatman filter paper at room temperature for drying. Subsequently, blighted potato leaves were cut into small pieces and were placed into healthy, sterilized potato tubers in a Petri dish, which were incubated at 18°C in darkness for seven days for sporulation enhancement. A thick white growth of sporangiophore with plenty of zoosporangia covered the tuber slice surface within 5–6 days.
The white mycelium of P. infestans was picked up using a glass needle, and it was transferred to V-8 agar medium (100 ml of V8 juice, 0.05 g ß-sitosterol, 1g CaCO3, and 15 g agar; the final volume was adjusted to 1 L with distilled water and autoclaved at 15 psi for 20 minutes) amended with 100 mg/L ampicillin, 20 mg/L rifampicin, and 50 mg/L nystatin [11]. The purified cultures of P. infestans were kept at − 4°C [12].
Morphological Characterization
To identify P. infestans isolates, the Laboratory Protocols of Phytophthora species from the American Phytopathological Society were utilized, according to Ivors [13]. This approach was utilized to develop important morphological features, including colony morphology, sporangium and oospore development. Moreover, scanning electron microscopy was used in accordance with Bozzola [14]. This method allows for the study of microbes on the substrate's surface, hyphae morphology, and spore production.
Samples prepared by tacking a disc with approximately 0.5 cm of a solid medium containing mycelia of P. infestans were mounted on copper stubs (approximately 2.5 x 2.5 cm) in a double carbon tape pasted on an aluminum foil film. In a sputter, the specimen was coated with gold for 5 min, then, all morphological characters were recorded. P. infestans micrography was taken under scanning electron microscopy (SEM QUANTA-FEG 250 with field emission gun, FEI Company Netherlands) at the National Research Center, Egypt.
Extraction of DNA, PCR Amplifcation and Sequencing
P. infestans isolate was cultured on V8 medium and incubated at 18°C for two weeks [15]. At Assiut University's Molecular Biology Research Unit, fungal DNA was extracted using a Patho-gene-spin DNA/RNA extraction kit (Intron Biotechnology Company, Korea). Sequencing and polymerase chain reaction (PCR) were carried out at the SolGent Company in Daejeon, South Korea. In order to amplify the ITS region of the rRNA gene, the universal primers ITS1 (forward) and ITS4 (reverse) were used. Primers have the following composition: ITS1 (5' - TCCGTAGGTGAA CCTGCGG − 3'), and ITS4 (5'- TCCTCCGCTTATTGATATGC − 3'). With the addition of ddNTPs to the reaction mixture, the purified PCR product was sequenced using the same primers [16].
Pathogenicity test:
Koch's postulates were applied to confirm that the isolated microorganism was the actual cause of late blight by using a modified detached-leaf assay as described by Karki [17]. Three plugs of P. infestans were cut and re-inoculated in a new (V-8) media plate in the dark at 18°C. The plates were kept facing downward to avoid any moisture development on the plugs. The sporangia from a 10–14 day old were taken off the plate by flooding the plate with 5 ml ice cold sterilized water (to expedite the release of zoospores) and mixing properly with a spreader. Then, the plate was kept at 4°C for 2 to 4 h to release zoospores. Zoospores were collected by filtering through two layers of sterile cheesecloth and diluted in 20 ml of ice-cold, sterilized water. The motile zoospores were counted by using a hemocytometer under a microscope [17].
Healthy, full-grown compound leaves from 6–10-week-old plants were collected. The abaxial side of each leaflet was inoculated with 4–6 droplets per leaflet and placed in a Petri dish filled with sterilized water agar media. Thereafter, the Petri dishes were incubated in a room with natural light at a temperature of 21°C and monitored daily. After 5 days, the leaves were assessed visually for the appearance of symptoms (Fig. 2). Samples from the incubated pathogen were isolated for morphological characterization under scanning electron microscope.