Screening And Rejuvenation Of Degraded Strains Of Auricularia Heimuer

Auricularia heimuer has a long history of cultivation,and it is the second largest edible fungus in China.After General Secretary Xi put forward the major instructions of " small fungus and large industry ",the black fungus industry ushered in greater policy support and social recognition,and will continue to play a greater role in rural revitalization.However,with the rapid development of Auricularia heimuer industry in China,there are also some industrial problems to be solved,among which the problem of strain degradation in production is prominent.Bacteria are important basic materials in the production of edible fungi,and play a "seed " role.The quality of strains plays a key role in the high-yield,high-quality and healthy development of the edible fungi industry.Therefore,it is very important to evaluate the quality of strains by systematic methods,screen the degraded strains efficiently and recover them effectively.In this study,18 strains of 6 Auricularia heimuer cultivars with different transfer times were systematically evaluated and screened from the biological level,protein level and DNA level by colony morphology observation,antagonistic reaction,esterase isozyme comparison and SRAP molecular marker analysis.The degraded strains were rejuvenated by tissue separation,mycelial tip separation and component-binding tip separation.The obtained rejuvenated strains were verified by mycelial growth rate determination and LBL decolorization method.The aim was to screen the degraded strains of Auricularia heimuer through comprehensive evaluation and explore the effective rejuvenation methods of the degraded strains.The main research results are as follows :1.Screening of degraded strains :Through colony morphology observation,it was found that there were differences in colony phenotype,hyphal germination days and hyphal growth rate among strains with different times of tube transfer.Among them,there were significant differences between A2,B2,C6 and D2 varieties,which showed that the hyphal density increased and the edge of the colony was not neat.It was difficult to grow full of PDA plates,and the color and whiteness of the colony decreased and there was pigment secretion.The mycelium germination days of some strains were slow to 4-5 days,and the mycelium growth rate was 0.57-1.70 mm / d except strain B2.Four degraded strains were screened from the colony phenotypic traits as A2 : 4 d,1.70 mm/ d.B2 : 2d,3.90 mm / d;C6 : 2d,1.45 mm / d;D2 : 5d,0.57 mm / d.Two antagonistic experiments were carried out among the strains of the same variety with different times of rotation.The antagonistic lines appeared among the strains of B,C and D varieties with different times of rotation,and there was no antagonistic line between the strains of A,E and F varieties with different times of rotation.According to the results of antagonistic experiments,three degraded strains were screened out as B2,C6 and D2,which had obvious antagonism with the other two strains with different times of rotation.Through the comparison of esterase isoenzyme bands,the differences of esterase isoenzyme bands among the strains of A,B,D and F four varieties with different times of tube transfer were found.Cluster analysis showed that the genetic similarity coefficient between the strains of A,B,D and F four varieties with different times of tube transfer was 0.24-0.88,and the genetic similarity coefficient of A2 and B2 was 0.56 and 0.24,respectively.It showed that the genetic distance was different from the other two strains of tube transfer.Eight pairs of primers with strong polymorphism were screened out from 153 pairs of SRAP universal primers for SRAP-PCR amplification.Cluster analysis showed that the genetic similarity coefficients of C,D and E strains with different tube transfer times were between0.79-0.93.When the genetic similarity coefficient of C6 was 0.79,C6 was clustered into two categories with the other two tube transfer times and formed a self-class.In summary,the differences of strains with different tube transfer times in the same variety at different levels showed different degrees of degradation.A total of six strains with different degrees of degradation were screened.Among them,D2 showed differences only at the biological level,indicating that the degree of degradation was relatively light.A2 and B2 changed at the protein level,and C6 changed at the DNA level,indicating that the degradation was serious.2.Rejuvenation of degraded strains :Based on the results of the earing test of six varieties in the early stage of the team,three varieties of A,B and F had fruiting body materials,and 15 tissue-isolated strains were obtained by three rejuvenation methods of tissue separation,tip separation and component combined with tip separation.Through the comparison of mycelial germination days and mycelial growth rates,three component rejuvenation strains A2 z,B2z and F6 z were screened.The tip separation was carried out for three consecutive times,and the growth rate was determined.It was found that the mycelial growth rate tended to be stable after two times.Finally,three tip and rejuvenation strains A2 t,B2t,and F6 t were obtained,and three components combined with tip separation and rejuvenation strains A2 k,B2k,and F6 k were obtained.Three strains of C6 t,D2t and E0 t were obtained by tip separation and rejuvenation of three varieties of seedless materials.The tip separation and rejuvenation method was optimized,and the tip separation of single mycelium was carried out by using a visual microscope.Amplification of 60-100 times can be successfully obtained to a single mycelium tip 1-2 mm part and the mycelium can survive,feasible.3.Verification of rejuvenation strains :The mycelial growth rates of the rejuvenation strains,the degenerated strains and the normal strains were determined.The results showed that the mycelial growth rates of the rejuvenation strains of A,B and F were higher than those of the degenerated strains.Among them,the differences between the rejuvenation strains A2 z,B2z and F6 z isolated from tissues and the degenerated strains were significant,and the F6 z was significantly higher than that of the normal strains,while the differences between the rejuvenation strains A2 z and B2 z and the normal strains were not significant.C,D,E three varieties of tip separation rejuvenation strains were significantly higher than degradation strains,C6 t and normal strains were not significantly different,D2 t and normal strains were significantly different,E0 t was significantly higher than normal strains.In conclusion,it is suggested that tissue separation method should be preferred in the case of fruiting body materials,and mycelium tip separation can be used to achieve the rejuvenation effect of mycelium growth when it is difficult to obtain fruiting body materials.The LBL decolorization reaction test was carried out on degraded strains,rejuvenation strains and normal strains.The color of the medium was observed,the pH value of the medium was measured,and the absorbance of the medium was measured to calculate the decolorization rate of the strain.The medium of the strain with strong mycelial activity was close to orange yellow and the pH value was small,while the medium was close to blue-green and the pH value was large.The decolorization rate was used to quantify the decolorization ability of the strain.The results showed that the rejuvenation effect of A,B and F was better through tissue separation,and the decolorization rate was A2 z : 89 %;B2z : 84 %;F6z : 78 %,significantly higher than degraded strains,and no significant difference with normal strains.C,D,E three varieties through the mycelium tip separation rejuvenation effect is better,decolorization rate C6 t : 51 %;D2t : 92 %;E0t : 87 %.The rejuvenation strains of D and E varieties were significantly higher than those of the degenerated strains and had no significant difference with the normal strains.It is concluded that it is feasible to isolate and rejuvenate mycelium tip in the case of difficult to obtain fruiting body materials,which is consistent with the verification results of mycelium growth rate.The LBL decolorization method not only evaluated the rejuvenation effect,but also verified the results of screening for degraded strains.In other words,as the mycelial growth activity decreased,the pH of LBL medium increased,the color deepened,and the decolorization rate of strains decreased.This method is simple and time-saving,which proves the feasibility of LBL decolorization method in the quality evaluation of Auricularia heimuer strains.