| Morchella is a world-famous fungus for both food and medicine.In recent years,the cultivation industry of Morchella has developed rapidly,but it is still unable to achieve sustainable and stable yield,mainly because the mechanism of mushroom emergence has not been thoroughly studied.Conidia may play an important role in the life history of Morchella,which is one of the main directions of basic research on Morchella recently.However,due to the difficulty of obtaining and germinating conidia in indoor culture,the research on conidia of Morchella is scarce at present.In this study,the single factor test was used to explore the conditions for the production of large numbers of conidia in laboratory.After obtaining conidia successfully,fluorescence staining was performed to observe and count the nuclear quantity distribution of Morchella conidia.Then,a large number of single conidia were isolated and cultured by microspore single-spore selection method,and several Morchella monospore strains were obtained and preserved.At the same time,the theoretical germination rate of Morchella conidia was calculated.Finally,the mating type genes of Morchella conidia strains were identified.The main results are as follows:(1)On the basis of the outdoor cultivation pattern,several groups of single factor experiments were conducted to explore the best culture container,medium components,mulch type,mulch thickness and mulch time(that is,bacterial age)to produce Morchella conidia indoors.Finally,a feasible culture system for obtaining a large number of Morchella conidia was established.The specific contents of the system are as follows.A hexagonal glass jar with bottom diameter of 7.6 cm,top diameter of 6.6 cm,height of 9.8 cm and volume of 280 m L is used as the culture container.Soil and PDA medium are used as the culture substrate.After inoculation with Morchella ascospore monospora strain A67 at 20℃ for 20 days,the surface of the colony is covered with 1 cm of sterile moist river sand.Then incubated in a temperature regulating incubator and water is supplemented by spraying at the right time.Temperature regulation simulates the diurnal variation trend of air temperature in outdoor winter,which is controlled from 8: 00 to 10:00 to 10℃.Then it turns to 12℃ from 10:00 to 12:00,16 ℃ from 12:00 to 14:00,12℃from 14:00 to 16:00,10℃ from 16:00 to 18:00,6℃ from 18:00 to 8:00(the next day),and so on.The conidia of Morchella could be produced after 12-16 days of temperature adjustment culture,and a large number of Morchella conidia could be obtained after 5-7 days later.(2)Observed under visible light,it was found that the conidiospore stalks of Morchella were finer than vegetative mycelia,most of which were spindle-shaped,with the top bending outwards about 15°-30°.Several small peduncles were born at the end of secondary hyphae,generally with 4-6rounds,which produced colorless,globular Morchella conidia at the top of the peduncle.It was observed after DAPI fluorescence staining that the diameter of Morchella mycelium was about 7 μm-13 μm,and there were pore membranes between the cells.Each cell contained multiple mature nuclei with a diameter of 1 μm-2 μm.The diameter of conidia generally ranged from 3.4μm to 5.3 μm,and the average diameter of uninucleate conidia was 4.2 μm,which was slightly smaller than that of binuclear spores(average diameter 4.8μm)and trinuclear spores(average diameter 5.1 μm).Among the 70403 conidia of Morchella obserived,there were 6277 dinucleated conidia,accounting for 8.92%.Only 21 trinuclear conidia were observed,accounting for 0.03%.Only one tetrakaryotic conidia was found and the morphology was not clear.It was also found that with the extension of collection time,the proportion of binuclear conidia gradually increased,and the proportion of binucleate conidia in a single quadant increased from 7.71% to 11.41%.While the number of polykaryotic conidia decreased gradually with the extension of sampling time,and the proportion of polykaryotic conidia in the sample decreased from 0.05% to 0.01%.(3)Among the 5234 selected conidia of Morchella,89 germinated strains were recorded as C1-C89 according to the order of germination time.The colony morphology of 51 strains cultured on PDA medium were obviously different from that of Morchella,so they were removed.The remaining 38 strains were extracted from DNA and identified by ITS,among which only 17 strains were identified as Morchella monoconidia by Blast comparison on NCBI.They were C2,C3,C9,C11,C12,C13,C14,C15,C20,C25,C48,C90,C91,C92,C93,C94 and C96,and the germination rate of Morchella conidia on PDA medium was only 0.32%.(4)MAT1-1-1 and MAT1-2-1 mating genes of monospora proascospora strains A67 and 17 strains which were identified as the monospora strains of Morchella(C2,C3,C9,C11,C12,C13,C14,C15,C20,C25,C48,C90,C91,C92,C93,C94 and C96)were amplified and separated by 1% agarose gel electrophoresis.The results showed that there was only one mating type gene in all of A67 and 17 monoconidia strains,and which was MAT1-1-1 only.The purpose of this study is to solve the problem that it is not easy to obtain materials in the research process of Morchella conidia,to explore the possibility of Morchella conidia germination,to explore the relationship between conidia mating type and female mycelium mating type.Then explore the possible important role of conidia in Morchella mushroom formation.This study provides a theoretical basis for the practical significance of producing a large number of conidia in artificial cultivation of Morchella.At the same time,it also lays a foundation for further revealing the mechanism of mushroom emergence mechanism of Morchella,and increases the possibility of successful cultivation of Morchella indoors. |
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