2.6. DNA Extraction
DNA was extracted using a modified CTAB-protocol [20
]. Stem internodes, hypocotyls, roots, axillary buds, apices, whole seedlings or seeds were placed in aluminum foil and frozen in liquid nitrogen. Special precautions (changing gloves and disinfesting cutting tools) were taken to prevent cross-contamination of samples. The samples were groundto powder with a mortar and pestle and collected into 1.5 mL Eppendorf tubes. CTAB extraction buffer (600 µL, 100 mM Tris pH 8, 1.4 M sodium chloride, 20 mM EDTA, 2% CTAB, 0.5% mercaptoethanol) was added, and the tubes were vortexed and incubated at 65 °C for 60 min. Chloroform-isoamyl alcohol (400 µL, 24: 1) was added, the tubes were vortexed for 1 min and centrifuged at 11,200 rpm for 15 min. The supernatant was transferred into a new 1.5 mL Eppendorf tube, 500 µL of chloroform-isoamyl alcohol (24: 1) and 400 µL of CTAB extraction buffer were added, the tubes were vortexed for 1 min and centrifuged at 11,200 rpm for 15 min for the second time. The supernatant was transferred into a new 1.5 mL Eppendorf tube, 500 µL of isopropanol was added, the tubes were vortexed and placed in −20 °C overnight. The tubes were centrifuged at 11,200 rpm for 15 min, and the DNA pellet was rinsed with 500 µL ice-cold 70% ethanol for 5 min in room temperature. The tubes were centrifuged at 11,200 rpm for 15 min, and the DNA pellet was dissolved in 50-µL HPLC water, incubated at 65 °C for 5 min and placed in −20 °C. DNA was similarly extracted from sporangia of P. belbahrii.
Detection of the pathogen was done with the following specific primers of the genomic ribosomal DNA (ITS1) of P. belbahrii
This pair produces a single DNA band of approximately 134 base pairs in size. The total volume of the reaction mixture was 15 µL and contained 7.5 µL Hy-Taq Ready Mix (2×), 0.3 µl of each Bas primer, 5.9 µL of nuclease-free water, and 1 µL of DNA sample (80–100ng/µL) or HPLC water for negative controls. PCR amplification was performed in the MultiGeneTM OptiMax Thermal Cycler (Labnet International Inc., Edison, NJ, USA) with the following cycle parameters: Initial denaturation at 95 °C for 5 min, 30 cycles of denaturation at 95 °C foe 30 sec, annealing at 63 °C for 30 sec, extension at 72 °C for 30 sec and termination at 72 °C for 5 min. The PCR products were separated by 1% agarose gel electrophoresis in TBE buffer (40 mM of Tris-borat, pH 8.0, 1 mM of EDTA) at 130 V for 40 min and stained with ethidium bromide. The Gene Direx (Taoyuan, Taiwan) 100 bp DNA ladder was run on the same gel. The gel was placed under UV light in order to identify the DNA bands.
2.9. Seed Infection and Transmission
Multiple experiments were conducted to study seed infection and disease transmission via basil seeds. Three cultivars of basil were studied during 2012–2017: ‘Peri’ (Genovese cutting type, Volcani Center for Agricultural Research, Israel), ‘Sweet Basil‘ (Genovese type, Genesis Ltd., Ashalim, Israel), and ’Genoveser’ (broad Italian leaf type, Ball Straathof, South Africa).
Two seed samples (Peri) were obtained in summer 2012 from Hishtil Nurseries Ltd. (Petach Tikva, Israel). The first sample was harvested from healthy basil plants and the other from BDM-infected basil plants. Seed samples were assayed by PCR (3 × 50 seeds per sample) for the presence of P. belbahrii and other seeds (~1000 seeds per sample) were grown in pasteurized soil in growth chambers to the 4-leaf stage, and examined for symptoms of BDM and for the presence of P. belbahrii by PCR.
In Mid-March of 2016 and 2017, about five hundred 12-leaf basil plants (‘Sweet Basil’) were planted in a 6 × 50 m net houses (covered with a 50 mesh white plastic net) located at BIU farm on campus. The plants were spray inoculated with spore suspension of isolate Knafo 3 of P. belbahrii
in early April in both years, after they had reached the 18–20 leaf stage. Symptoms including sporulation were first observed at 7 and 9 days post inoculation in 2016 and 2017, respectively. Temperature was favorable for disease development [21
] in both years. The disease reached the top of the plants in mid-June at about flowering time.
To examine seed infection and BDM transmission, seeds (including the petals and calyx) were harvested in July of both years. In 2016, about 5000 seeds were collected from about 100 infected plants. The floral parts were removed and two subsamples of 150 seeds were taken for DNA extraction. The other seeds (about 4700) were sown in pasteurized soil in pots, 10 seeds per pot. About 1000 commercial seeds (Sweet Basil and Peri) were similarly sown as controls. Plants were grown in a growth chamber at 22 °C (60%–70% RH, 14 h light a day, 60 µmole.m−2 s−1). When they reached the 4-leaf stage, plants were examined for symptoms of BDM and then assayed by PCR, 15 samples of 10 plants per sample from each seed batch, for the presence of pathogen in their tissues.
In 2017, about 13,700 seeds were harvested from about 200 infected plants. They were divided into two batches: seeds of one batch (~9400 seeds) were separated from their floral parts (petals and calyx) and sown in pasteurized potting mixture without disinfection, 10 seeds per pot. Seeds of the other batch (~4300) were similarly sown, but with their petal and calyx (carrying sporangia) attached. About 1000 commercial seeds (Sweet Basil and Peri) were similarly sown as controls. Plants were grown as above and when reached the 4–6 leaf stage they were examined for symptoms and sporulation of BDM and then taken for PCR assays, 5–22 samples of 10 plants per sample from each seed batch.
In 2018, seeds were collected from the following five basil cultivars growing at Ashalim in southern Israel: Emily 42, Emily 47, Emily 201, Edwina 5 (broaden Italian leaf), and an experimental line 1241 (Genovese type). Plants were all heavily infected with an isolate called “M” of P. belbahrii at time of harvest. Unlike isolate Knafo 3, isolate M was pathogenic to also line 1241, which carries the Pb1 gene for resistance.
To detect P. belbahrii in contaminated seeds (seeds collected from infected plants), two samples of 150 from each cultivar in each year were taken for DNA extraction. Seeds of one sample were disinfected to remove sporangia attached to their surface (one min soaking in 3% hypochlorite followed by washing with sterile water) before extraction. Disinfection did not affect seed germination. Seeds of the other sample were extracted with no previous treatment.