Preparation and pathogenicity evaluation of mononuclear hyphae of Athelia bombacina

Abstract: 【Objective】This study aimed to establish a method for the preparation of mononucleated mycelium of Athelia bombacina and evaluate its pathogenicity, so as to lay a foundation for the follow- ing-up study on molecular biology【. Methods】The mycelia were inoculated on PDB medium and cul- tured in shaking table at 25°Cand 120 r·min-1 for 1, 2, 3 and 4 d. Fresh mycelia with a mass of 0.5 g were collected and washed twice with sterile water and twice with 0.6 mol·L-1 mannitol osmotic stabi- lizer. 5 mL of 1.5% snailase, 1.5% driselase, 1.5% cellulase and 1.5% lyase were added respectively. The mycelia were hydrolyzed at 30 °C and 120 r · min-1, for 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 h, the num- ber of protoplasts was observed and calculated with the blood cell counting plate, and the type of cell wall lyase with high protoplast yield and the enzymolysis time were determined. The effects of age, ly- ase and enzymolysis time on the protoplast preparation of fruit rot fungus HG-AB20170418 were studied, and the differences of morphology, growth rate and pathogenicity between the monokaryon hyphae and the original hyphae were compared and analyzed. Finally, the heterozygosity of hyphae before and  after the treatment was evaluated.【Results】The highest yield of protoplasts was obtained with lyso- zyme as lysozyme, and the enzymolysis effect of cellulase was very weak. The enzymolysis time of the lyase and the snail enzyme was shorter, and the optimal enzymolysis time was 2.0 h, while the enzymol- ysis time of the driselase and the cellulase was longer. After 3 days of culture, a large number of proto- plasts could be observed under the microscope, and all the hyphae were lysed in a short time. After 4 days of culture, a large number of hyphae could still be seen in 2.5 hours under the action of the optimal lyase. The original strain grew radially on the PDA, but the mycelial growth was spiral. On the 3rd and 5th day of culture, the colony diameter of the original strain was 37.54 mm and 65.00 mm, respectively. The growth rate was significantly lower than that of the original strain. There was no significant difference in the growth rate among different strains. There were no lock-like joint structure under biological microscope after mononuclear treatment, however, a large number of lock-like joint structures were ob- served under the microscope of the original strain. After inoculation, of the strains with mononuclear treatment , all the fruit of Huangguan pear produced disease spots on the third day, but the pathogenici- ty of different strains was significantly different. On the 5th and 10th day after inoculation, the diameter of infected spots with original strain was 4.39 mm and 6.34 mm respectively. The diameter of lesion of mononuclear strain ABD-3 was similar to that of the original strain. There were two main peaks before the treatment, and the heterozygous rate was 1.96%. The complex genome with high heterozygosity was not conducive to the later sequencing, but only one main peak appeared after the treatment, and the heterozygous rate was about 0.00%, it was a simple genome.【Conclusion】The optimal conditions for the preparation of mononucleated hyphae were that the hyphae of A. bombacina were cultured in PDB for 3 days, a large number of protoplasts could be obtained from A. bombacina mycelia after 1.5% lyso- zyme and 1.5% snail enzyme lysis for 1.5 h. The growth rate of the regenerated mycelia was significant- ly lower than that of the original strain, but the mononucleared mycelia with the same pathogenicity as the original strain could be obtained. The genomes of the monocytic hyphae obtained from the above protoplasts were simple. The establishment of this method laid a foundation for the subsequent molecular biology research of A. bombacina.