Isolation, identification, and pathogenicity
The pathogen was isolated from the stump of a cut tree with symptoms described previously. Portions of infected tissue (about 0.5 cm in diameter) were placed on the surface of petri plates with potato dextrose agar (PDA; KASVI) and incubated at 26°C under white fluorescent light with a 12 h photoperiod for 48 h. Subsequently, the hyphal tips were isolated from emerging colonies and transferred to a new plate with PDA under the conditions previously described.
For morphological identification, slides were prepared from fungal structures from a seven-day old colony using cotton blue and clear lactophenol. The observations and measurements were made with a light microscope (Leica DM1000) coupled to a camera (Leica DFC295) and the Leica Application Suite Core software (LAS V3.7). The intervals were generated from 30 measurements.
Molecular identification was performed from mycelium grown on PDA plates for nine days at 25°C. Genomic DNA was extracted using a DNA isolation kit (Norgen, Ontario, Canada) according to manufacturer’s instructions. Five regions were targeted for PCR amplification and sequencing, including actin (ACT), calmodulin (CAL), histone (HIS3), translation elongation factor 1-α (TEF1), and 5.8S nrDNA of the internal transcribed spacer region (ITS). Amplifications were performed with an automated thermal cycler (MJ Research PTC–200) using a PCR master mix (Thermo Fisher Scientific, MA, USA). The reaction was carried out at final volume of 25 µL, composed of 12.5 µL of PCR master mix, 1 µL (10 pmol) of each primer pair, 1 µL of DNA (30 ng/µL), and 9.5 µL of sterile distilled water. Primers and PCR programs used for the amplification are described in Victoria Arellano et al., (2021). Amplicons were purified with a PCR cleaning product (Mebep Bioscience, Shenzhen, China) and the PCR fragments were sequenced in both directions using the BigDye Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems, USA) following the manufacturer’s protocol. The products were analyzed on an ABI Prism 3730 XL DNA sequencer (Applied Biosystems). Contigs were generated by comparing (forward and reverse sequences) and manually editing in MEGA (Molecular Evolutionary Genetics Analysis, version 11) when necessary (Tamura et al., 2021). Consensus sequences were compared in megablast (NCBI), to determine the percentage of similarity of the pathogen for each analyzed locus (Altschul et al., 1990). The sequences derived in this study were deposited in GenBank.
The pathogenicity test followed Koch’s postulates. Four sets of three healthy one year old Butia odorata plants were inoculated by needle injection. Three of these sets were inoculated using three different concentrations of spores (106, 107, and 108 conidia mL− 1), and the remaining set was inoculated with sterile distilled water. All the concentrations were applied in the same volume (2 mL) in the bulb of the plant. Plants were transferred to a greenhouse maintained at 24 to 28°C, and 85 ± 5% relative humidity, and checked for symptoms every 8 h. Fifteen days after inoculation, fungal reisolation was performed from infected necrotic tissue. The original fungi isolated were compared morphologically with those previously inoculated.