Identification and comparison of biological characteristics and pathogenicity of different mating types of V. dahliae isolated from potato and sunflower

Sample collection

A total of 374 samples (exhibiting diseased basal stems) were collected from fields located in the Inner Mongolia Autonomous Region, Hebei Province, Shanxi Province, Shaanxi Province, Jilin Province, Liaoning Province and Heilongjiang Province (Supplemental Table 1). Among them, 167 samples were collected from potato and 207 from sunflower. The samples were stored in a 4 °C refrigerator before isolation. Apart from collecting diseased plants for pathogen isolation, some commercial sunflower seeds and potato tubers were also purchased from the market for V. dahliae isolation. All plant samples and seeds were collected in compliance with the regulations of the Chinese Academy of Sciences and all state laws regarding biological sample collection within the borders of China.

Disease samples collected from the various regions were done under the supervision and permission of the various extension officers of the Agriculture Academy of Sciences of Inner Mongolia Autonomous Region, Hebei Province, Shanxi Province, Shaanxi Province, Jilin Province, Liaoning Province and Heilongjiang Province and the individual farmers cultivating the land respectively for research purposes only.

Culture media

Water agar medium (WA) used for the isolation of pathogenic fungi and single spore purification constituted 15 g of agar in 1000 mL of distilled water. For the cultivation of V. dahliae isolates, potato dextrose agar medium (PDA), which constituted 200 g potato, 20 g glucose, 15 g agar, and 1000 mL distilled water, was used, as well as complete medium (CM), which constituted 6 g yeast extract, 6 g caseinacid hydrolyzed, and 10 g sucrose in 1000 mL distilled water. These media were all freshly prepared and contained 100 mg/mL kanamycin (AMRESCO, Cat. No: K0408) to restrict the growth of contaminants.

Isolation and purification of pathogens

The vascular tissues of the basal stem were cut vertically into 3–5 mm slices, dipped in 75% ethanol for 3–5 s, dipped in 0.1% NaClO for 30 s and rinsed twice with sterile double-distilled water. The slices were dried with sterilized filter paper in a lamina flow hood and then placed on water agar (WA) medium. Three days later, the edge of the growing colony surrounding the tissue slices was cut out and transferred onto a new PDA plate for culturing. The isolates cultured on PDA plates were washed with sterile water after the colony had grown to 2/3 of the petri dish. The conidiospore suspension was prepared with sterilized distilled water and adjusted to a concentration of 1.0 × 10⁶ conidia/mL using a hemocytometer. A 100 µL aliquot of the conidiospore suspension was drawn on freshly prepared WA medium, spread evenly and cultured at 25 °C for 2 days. The monospore colonies were cut and transferred onto PDA medium to obtain a pure culture.

The protocol above was also used for isolating pathogens from the sunflower seeds and tubers with a little modification. The seed hulls of sunflower seed samples were taken off, the radicles were destroyed (to prevent germination) then seeds were surface sterilized in 75% ethanol for 30 s then 0.1% NaClO for 30 s and rinsed twice with sterile double-distilled water for three minutes. The surface sterilized seeds were placed on sterilized filter paper and allowed to air dry before placing them on freshly prepared PDA media.

The potato tubers were cut across transversely into thin sheets of 2 mm, surface sterilized in 75% ethanol for 30 s then 0.1% NaClO for 1 min and rinsed twice with sterile double-distilled water for three minutes. The surfaced sterilized potato tuber samples were then placed on sterilized filter paper and allowed to air dry before they were cultured on freshly prepared PDA media.

DNA isolation and PCR amplification

DNA extraction was carried out using the CTAB protocol as described by Doyle²⁵. Isolates were cultured on PDA medium at 25 °C for 2 weeks and then scratched off the mycelium carefully from the medium into 2.0 mL Eppendorf tubes. The mycelium samples were placed in liquid nitrogen in preparation for tissue lysis using TissueLyser LT (QIAGEN Hilden, Germany). The powdered mycelium was mixed with extraction buffer (100 mM Tris–HCl pH 8.0; 20 mM EDTA-Na 2; 1.4 M NaCl; 2% cetyltrimethyl ammonium bromide and incubated at 65 °C for 30 min. Phenol–chloroform-isoamyl alcohol (25:24:1) was added and centrifuged. Isopropanol was used for DNA precipitation.

DNA was used as a template for PCR amplification with specific primers synthesized by Beijing Housheng Botai Technology Co., Ltd. and listed in Table 2. The 25 µl PCR system contained 1 µl of each primer (10 µM), 0.5 µl Taq DNA polymerase (Tiangen, Beijing, China), 2 µl of dNTPs (2.5 mM), 2.5 µL of 10X PCR buffer, 18 µl of distilled water and 2.0 µl of DNA template. PCR was performed in a Gene Pro Thermal Cycler (BIOER) with the following procedure for all primer pairs: 94 °C for 5 min, followed by 35 cycles of 94 °C for 40 s, 56 °C for 40 s, 72 °C for 40 s, and 72 °C for 10 min for extension.

The amplicons were separated on a 1.0% agarose gel stained with GelView (BioTeke, Beijing, China) and then observed and photographed under UV light. Amplicons were sent to Beijing Housheng Botai company for sequencing. Sequencing results were subjected to BLAST on the NCBI website and compared with the available data in GenBank to confirm the species of the isolates.

Mating-type and physiological race identification

The purified isolates were cultured on PDA medium for 5–7 days at 25 °C. Their genomic DNA was extracted and amplified with specific primers (listed in Table 1) that could identify different mating types and physiological races of V. dahliae, which were isolated from both sunflower and potato.

Morphological comparison of two mating-type strains of V. dahliae

Four isolates from the different mating types, MAT1-1 (P48 and S11) and MAT1-2 (P50 and S12), of both potato (P48 and P50) and sunflower (S11 and S12) were randomly selected for macro- and micromorphological comparison. The selected isolates were cultured on freshly prepared PDA medium for two weeks. The cultured plates were washed with sterilized water to prepare conidiospore suspensions (1 × 10⁶ conidia/mL), and then 2 μL was pipetted onto the center of the PDA culture medium for smearing. The plates were kept in an incubator for 7 days at a temperature of 25 °C. The colony morphology was observed physically 7 days post inoculation (dpi). A conidiospore suspension was made to observe the conidia and hyphal structures using an optical microscope.

Pathogenicity comparison among different isolates

Seedlings of both potato and sunflower were grown under greenhouse conditions and used to ascertain the pathogenicity of isolates of both MAT1-1 and MAT1-2 of V. dahliae recovered from both hosts. The selected isolates were cultured in CM media to produce the conidiospore suspension. We prepared 20 plants for each isolate for inoculation, and each plant was inoculated with 200 mL of conidiospore suspension (1 × 10⁶ conidia/mL) using the root dipping method²⁶ (Alkher et al. 2009). 20 plants were inoculated with water as control. The entire experimental setup was repeated three times, and symptoms were recorded with the criteria listed in Table 2 after 21 dpi.

The disease index was calculated according to the formula below²⁷ (Xiao et al. 1998):