Sample collection and isolation
Ten daylily leaves with anthracnose disease symptoms were collected from the Shanghai Institute of Technology, China. According to the method used by Zhou et al, (2015), the characteristics of daylily leaves suspected to be infected with anthracnose were observed and recorded. The samples were stored in sterile polythene bags and brought to the laboratory for stereomicroscope (Koppace, 3.5X-180X, China) examination. The isolation and purification of fungal isolates were performed based on the tissue separation method combined with the single spore separation method. The lesion margin pieces were cut into square pieces with sides of 2–3 mm and sterilized with 75% ethanol (CH3CH2OH) for 30 s, and rinsed three times with distilled sterile water in an ultra-clean workbench. Excess moisture was blotted from diseased patches with filter papers, and they were placed on the PDA medium (potato dextrose agar medium: potato 200g, glucose 10g, agar powder 10g, and added distilled water constant volume to 1L) plate, which was then placed in an incubator at 25 ± 1°C in the dark for seven days. Spores were removed from the lesion margin pieces using sterilized dissect needle, transferred to clear WA medium (water agar medium: agar powder 10g, and added distilled water constant volume to 1L), and then scattered single spores were transferred to another WA medium slant and purified until single spores were obtained. The pure cultures were obtained and stored on PDA slants at 4℃.
After culturing on PDA at 25 ± 1 ℃ in the dark for 7 d, the macroscopic colony characters and microscopic characteristics of two fungal isolates, including the colony, mycelial appressoria, conidia, and conidial appressoria, were initially observed. The conidia were placed on the glass coverslip of a microscope slide. Mycelial appressoria were observed using the slide culture technique. The morphological characteristics of conidia were observed and a morphological map was constructed under the light microscope (Leica, DM4 B, Germany). The colony diameter of isolates from 1 d to 7 d after inoculation was assessed using the cross measurement method (Sun et al., 2020b). Fifty conidia were randomly selected to measure their size at 60-fold magnification.
DNA extraction, PCR amplification, and sequencing
The genomic DNA of two fungal isolates was extracted from fresh mycelia grown on PDA for 7 d using the UNIQ-10 Column Fungal Genomic DNA Isolation Kit, following the manufacturer’s instructions (Sangon, Shanghai, China). The quality of DNA was analyzed by 1.5% (w/v) agarose gel electrophoresis and Nanodrop One, and stored at −20℃ for PCR analysis.
ITS, ACT, GAPDH, and TUB2 genes of the isolated DNA were amplified and sequenced using their primer pairs: ITS1/ITS4 (ITS, Gardes and Bruns, 1993), GDF1/GDR1 (GAPDH, Guerber et al., 2003), ACT-512F/ACT-783R (ACT, Carbone and Kohn 1999), and T1/T2b (TUB2, Glass and Donaldson, 1995), respectively (Table S1). PCR amplification was carried out using Taq DNA polymerase in a PCR Thermal Cycler (Bio-Rad, S1000, America) in a total volume of 30 μL. The reaction conditions were pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at a temperature depending on the primers for 30 s, extension at 72 ℃ for 2 min, cycling for 35 times; and extension at 72 ℃ for another 5 min. All PCR products were purified with the anPrep Column PCR Product Purification Kit, according to the manufacturer’s instructions. All the above kits and primers were provided by Sangon, Shanghai, China. The purified PCR products were sequenced (Sangon, Shanghai, China). The consensus sequences were deposited in GenBank (Table 1).
Sequence analysis and phylogenetic tree construction
A multigene phylogenetic tree was constructed using the method described by Liu et al. (2020). The homology of sequences from the isolates was compared with the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by blastn and further analyzed using Clustal (https://www.ebi.ac.uk/Tools/msa/clustalo/). The maximum likelihood method was used to construct the multigene phylogenetic tree based on ITS, GAPDH, ACT, and TUB2 genes by MEGA7.0 with 1000 bootstrap replicates. Alternaria alternata (strain: AA2-8) was used as the outgroup. GenBank accession numbers of all the isolates used in the phylogenetic analyses are listed in Table 1.
The pathogenicity of the two fungal isolates was tested on one-year-old healthy daylily ‘Rosy Rhino’ × ‘Spacecoast Storm’ hybrids. Green, full-length leaves from living daylily plants were collected by cutting the leaves at the base of a division. The cut leaves were immediately rinsed under running water and air-dried in an upright position in a glass beaker with the cut ends immersed in distilled water. In an ultra-clean workbench, distilled water was used to wash the impurities on the surfaces of the leaves. The leaves was then sterilized with 70 % ethanol for 30 s to sterilize the surface, and rinsed three times with distilled sterile water to remove the residual alcohol on the leaf surfaces. To help delay leaves senescence, leaves were cut into 4-5 cm sections and placed in 9 cm borosilicate glass culture dishes covered with filter paper moistened with 0.1 µg/mL gibberellic acid solution (Buck, 2013). The daylily leaves were treated three ways: non-wound, stab wound, and excised wound treatments. In the non-wound treatment, no mechanical damage was done to the leaves. In the stab treatment, the leaves were stabbed using a self-made 5-needle plum-blossom needle to stab the central position of two side of the leaves veins. In the excised wound treatment, field pruning was applied by cutting a small opening about 2 cm long on one side of the leaves.
A mycelial plug with an area of about 0.25 cm2 was taken from the edge of the active growing colony of each isolate with a hole punch (Gao et al., 2020b), and the mycelial plug was inoculated on the leaf wounds with the fungal mycelia facing the leaf. A moist filter paper larger than the mycelial plug was placed on the surface of the it to keep the moisture. For the controls, only the PDA medium and wet filter paper were placed on the leaves without mycelial plug. The experimental design consisted of six replicate dishes (each with a single leaf segment) for each treament and each isolate of daylily. Borosilicate glass culture dishes were placed in a light incubator and moisturized at 25 ℃ with 12 h photoperiod. All experiments were repeated twice. The onset time and pathogenicity of leaves were observed and recorded every day. If the typical symptoms produced by artificial inoculation were the same as the original symptoms under natural conditions, and the control leaves did not develop the disease, then the infected plants were inoculated for the re-separation of pathogenic fungi, and Koch’s postulates were verified (Liu et al., 2020).
Fungicide susceptibility analysis
Fungicide susceptibility analysis of the two fungal isolates from daylily was carried out (Arikan, 2007). The sensitivity of the isolates to four fungicides was determined by the mycelial growth rate method (Liang et al., 2015). All fungicides were provided by Chinese companies, and the detailed information is shown in Table 2. According to the concentrations of fungicides used in production, the experimental concentrations of the fungicides were designed by the fractional dose method. The fungicides were prepared into a solution and added to the PDA medium. The solution was spread to the whole medium by gently shaking the dishes.
A mycelial plug (0.25 cm2) was obtained by a hole punch, and it was inoculated on the middle of a borosilicate glass culture dish with a diameter of 9 cm that was filled with PDA medium with fungicides, with the fungal mycelia facing down the agar. PDA plates with the same amount of sterile water were used as controls, and each fungicide and concentration had three independent replicates. PDA plates were incubated in a constant temperature incubator at 25 ± 1 ℃ for 7 d in the dark. For each plate, the colony diameter from 1 d to 7 d was assessed by using the cross measurement method. The percentage of growth inhibition in response to each fungicide at varying concentrations was calculated with the control plate as a reference (Liang et al., 2015). The calculation was as follows:
inhibitory effect (%) = [(Fungal colony diameter of the control − fungal colony diameter of the treatment) / (Fungal colony diameter of the control − 0.5)] ×100%
Through the linear regression relationship between the mass concentration pair value (X) and the probability value of inhibition rate (Y), the toxicity regression equation was Y= a X + b. The mass concentration EC50 (effective inhibiting concentration) values in the effective inhibition were obtained (Ma et al., 2009), then the sensitivity of each fungicide to the pathogen was compared. This experiment was conducted twice.