A method for directional evolution breeding of salt-tolerant varieties of morellomycetes polysporus isolated strains

Abstract

The application discloses a salt-tolerant variety directional evolution breeding method of morey morel isolated strains, and relates to the technical field of morel breeding, which takes the morey morel isolated strains as the starting material, and directional breeding is carried out through multiple cycle iterations, each cycle including five directional evolution steps and one refreshing step. In the directional evolution step, hypha blocks are picked from the edges of the hypha in twelve directions, and are inoculated on PDA plates with the NaCl concentration increasing by 0.1% in mass percentage as a step to culture and screen; in the refreshing step, the salt-tolerant culture obtained through screening is cultured into the original strain and the cultivated strain, and then is used for field mushrooming on the saline-alkali soil with the corresponding salt concentration, and the ascospores in the high-yield area are collected to form the starting strain for the next cycle. The application utilizes the high spontaneous mutation rate and the multinucleate characteristics of morey morel, and under the condition of not using mutagen, the existing commercial high-yield strain is directionally transformed into a strain tolerating 2.0% NaCl in mass percentage and maintaining high-yield performance.

Description

Technical Field

[0001] This invention belongs to the field of morel breeding technology, and particularly relates to a method for the directional evolution and selection of salt-tolerant varieties of multispore isolated morel strains. Background Technology

[0002] Morel mushrooms are edible fungi belonging to the genus *Morchella* in the family Moridiaceae of the phylum Ascomycota. They differ fundamentally from most edible fungi belonging to the phylum Basidiomycota, such as shiitake and oyster mushrooms, in their phylum Basidiomycota. Currently, the morel strains used on a large scale in production mainly come from two sources: one is cultures isolated from tissue blocks at the base of the morel stipe, whose genetic composition is identical to the parents, essentially representing asexual reproduction; these are called tissue-isolated strains. The other is cultures obtained by collecting large ascospores ejected from mature morel fruiting bodies; after germination, these cultures are mixtures of numerous ascospore offspring, called multispore strains. Multispore strains are essentially mixtures of numerous ascospores produced by a single morel fruiting body, meaning they consist of numerous offspring produced sexually by a pair of parent strains. These offspring are related as siblings, collectively forming a genetically diverse population, rather than a genetically homogeneous individual. While these offspring share most of their genetic material, differences still exist, resulting in rich genetic diversity within the population. Furthermore, morel mycelial cells are multinucleated, with each cell typically containing 20-40 nuclei. The DNA in these nuclei can mutate and evolve independently, further enhancing the genetic heterogeneity within the mycelium. These characteristics provide a unique biological basis and feasibility for treating multispore-isolated morel strains as population-based manipulation objects and continuously improving strain performance using the principles of directed evolution.

[0003] Meanwhile, morel mushrooms are highly prone to mutation during subculture, partly due to the high rate of spontaneous mutations and chromosomal rearrangements in their genome. Approximately five consecutive asexual subcultures can lead to the accumulation of unfavorable mutations, resulting in decreased strain viability. However, it is precisely this high genetic mutation rate that allows multisporous morel species to spontaneously generate a vast number of diverse mutants within the population without any mutagenic treatment. This provides an exceptionally rich pool of candidates for directed evolution under specific selection pressures. Morel mushrooms thrive in neutral to alkaline soil environments, tolerating alkaline pH up to 9.0. The organic nutrients required for fruiting are primarily provided by exogenous nutrient bags, resulting in low requirements for soil organic matter content. Based on these characteristics, saline-alkali land, unsuitable for most crops, is considered an excellent soil for morel cultivation due to its alkaline pH and low organic matter content, which morel mushrooms prefer. However, while saline-alkali soils possess suitable pH levels and low organic matter content, they also exhibit excessively high salinity, with sodium chloride content typically exceeding 2% (w / w). Current commercial morel varieties are completely unable to grow and produce fruiting under these salinity levels. Therefore, there is an urgent need in production to select morel strains capable of tolerating sodium chloride concentrations above 2% (w / w).

[0004] However, existing methods for breeding salt-tolerant morel strains mainly rely on collection and domestication from wild resources. Finding naturally salt-tolerant strains from wild populations is like searching for a needle in a haystack, a highly luck-dependent approach. This method not only heavily relies on the collection of large amounts of wild germplasm resources and extensive field trials, which is time-consuming and labor-intensive, but even if materials with satisfactory salt tolerance are occasionally found, their yield and commercial traits are often unsatisfactory and cannot be directly applied to commercial production. Currently, systematic techniques for the targeted breeding of salt-tolerant morel strains are lacking, especially efficient breeding methods that directly modify currently market-recognized high-yielding, multi-spore commercial strains to retain their original high-yielding properties while adding strong salt tolerance (tolerance to 2% w / w or higher NaCl). Summary of the Invention

[0005] The technical problem this invention aims to solve is to address the shortcomings of existing salt-tolerant morel strains, which mainly rely on wild resource collection and domestication, resulting in low screening efficiency and difficulty in directly transforming existing high-yielding commercial strains into salt-tolerant high-yielding strains. This invention provides a method for the directed evolution and breeding of salt-tolerant morel multispore isolates. This method can, without the use of mutagens, directionally transform existing high-yielding multispore strains into strains that can tolerate NaCl concentrations of 2.0% or higher by mass while maintaining high yield performance.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A method for directional evolutionary breeding of salt-tolerant varieties of morel multispore isolates includes the following steps:

[0008] The starting material was a multispore isolate of morel mushrooms, which was derived from a mixture of ascospores ejected from the fruiting bodies of morel mushrooms.

[0009] Targeted breeding is carried out through a cyclical iterative process, with each cycle including several directional evolutionary steps and a refresh step.

[0010] The directed evolution step is as follows: On the culture plate of the multispore isolated strain, mycelial agar blocks are picked from multiple directions from the edge of the mycelium and inoculated into the center of multiple PDA plates containing a preset NaCl concentration. The mycelium is cultured until it spreads from the center to two-thirds of the radius of the culture plate. The plate with the best growth is selected for the next step. The NaCl concentration of each directed evolution step in the same cycle is increased step by step with a fixed step size.

[0011] The refresh step is as follows: Select the plate with the best growth from the plates obtained in the last directed evolution step of this cycle, pick out mycelial agar blocks from multiple directions from the edge of the mycelium on the plate, and inoculate them onto multiple original culture media. After the mycelium has fully grown and formed sclerotia, transfer them to the culture medium for further culture to obtain multiple culture media. Conduct field fruiting experiments on saline-alkali soil with NaCl content corresponding to the target salt tolerance level of this cycle, set up multiple replicate plots, count the fruiting yield of each culture, select the plot corresponding to the culture with the highest average yield, collect the ascospores ejected from the fruiting bodies, prepare a spore suspension, and inoculate it onto PDA plates containing the corresponding NaCl concentration to form the multispore isolated strain for the start of the next cycle.

[0012] Multiple cycles were repeated until a multi-spore strain of morel mushrooms that could tolerate the target NaCl concentration was obtained.

[0013] Preferably, each cycle contains 5 directed evolution steps, and the NaCl concentration in each directed evolution step increases progressively in a fixed step size of 0.1% by mass, with the cumulative increase in NaCl concentration in each cycle being 0.5% by mass.

[0014] Preferably, in the directed evolution step, when picking mycelial agar blocks from the edge of the mycelium on the multispore isolated strain culture plate, a 1 cm × 1 cm agar block covered with mycelium is picked from each of the 12 directions from 1 o'clock to 12 o'clock, with the center of the plate as the center, and inoculated into the center of 12 PDA plates containing the same NaCl concentration.

[0015] Preferably, in the refresh step, when picking agar blocks of mycelium from the edge of the plate, a 1 cm × 1 cm agar block covered with mycelium is picked from each of the 12 directions from 1 o'clock to 12 o'clock, with the center of the plate as the center, and inoculated onto 12 portions of original culture medium.

[0016] Preferably, the original culture medium is formulated as follows by weight percentage: 35% rice husk, 20% humus, 40% water, 2% dipotassium hydrogen phosphate, 2% calcium carbonate, and 1% calcium sulfate; the cultivated culture medium is formulated as follows by weight percentage: 35% wheat, 15% rice husk, 5% humus, 40% water, 2% dipotassium hydrogen phosphate, 2% calcium carbonate, and 1% calcium sulfate.

[0017] Preferably, in the refresh step, the culture conditions after inoculation of the original culture medium are 15℃ for 20 days; the inoculation ratio of the original culture medium to the culture medium for the transfer of the original culture medium is 1:100 by weight, and the culture is carried out at 15℃ for 20 days after inoculation.

[0018] Preferably, in the refresh step, the field mushroom production experiment is set up with 12 cultivars, each cultivar is assigned 5 replicate plots, each plot is 40 square meters in area, and each plot is arranged in a randomized block designation; one-way ANOVA and Duncan's multiple comparison method are used to statistically analyze the differences in average yield among the cultivars, and the cultivar with the highest average yield and marked as 'a' by Duncan's method or with no significant difference from 'a' is selected for ascospore collection.

[0019] Preferably, in the refresh step, the specific operation of collecting ascospores is as follows: randomly select robust morel fruiting bodies from each plot corresponding to the cultivation species with the highest average yield, collect the ejected ascospores to make a mixed suspension, concentrate it, and drop it onto the center of a PDA plate containing the corresponding concentration of NaCl, so that it can germinate and form a multispore isolated strain; wherein, the NaCl concentration contained in the plate in the refresh step is the same as the NaCl concentration in the last directed evolution step of the current cycle.

[0020] Preferably, the target NaCl concentration is 2.0% by mass or higher; the cycle is terminated when all plates fail to grow normally in the directed evolution step of a certain cycle.

[0021] Preferably, the morel mushroom variety is either *Morchella esculenta* or *Morchella esculenta*.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] First, this invention directly uses existing commercially available high-yielding morel strains as starting materials and transforms them into salt-tolerant strains through directed evolution. This avoids the risky operation of blindly selecting from wild resources and can directly endow high-yielding strains with salt tolerance characteristics, significantly shortening the breeding cycle.

[0024] Secondly, this invention fully utilizes the high spontaneous mutation rate of the morel genome and the biological characteristics of the mycelial cells being multinucleated (containing 20-40 nuclei in a single cell). No chemical mutagen or physical mutagenesis method is used in the entire breeding process, making the operation simple and safe, and avoiding the tedious steps of repeatedly optimizing the mutagenesis conditions.

[0025] Third, this invention manages and screens the entire multispore isolate of morel as a genetically diverse population. When picking mycelium from 12 directions at the edge of the colony, mycelial fragments carrying different nuclear genotype combinations can be captured from different spatial locations of the same colony, effectively expanding the sources of genetic variation available for screening and increasing the probability of obtaining superior salt-tolerant individuals.

[0026] Fourth, this invention sets a refresh step after the five directed evolution steps in each cycle, which forces the strains that have undergone continuous asexual passage under high salinity pressure to complete a sexual reproduction process. Through sexual reproduction, the unfavorable mutations accumulated by continuous asexual passage are eliminated, the degenerative trend of the strains is reversed, the population vitality is restored, and healthy starting materials are provided for the next round of pressure screening under higher salinity, thus ensuring the sustainability of multiple rounds of iterative cycles.

[0027] Fifth, the final strain obtained by the method of this invention not only significantly improved its salt tolerance from less than 0.5% NaCl to 2.0% NaCl, but also showed no significant difference in yield on normal, salt-free soil compared to the original strain, and its yield on 2.0% saline-alkali soil was comparable to that of the original strain on salt-free soil, thus achieving the synergistic preservation of salt tolerance and high yield. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0030] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0031] This invention provides a method for the directed evolution and selection of salt-tolerant varieties of Morus alba multispore isolates. The core concept of this method lies in fully utilizing the characteristic that Morus alba multispore isolates are essentially genetically diverse populations composed of numerous homologous ascospore progeny, and the unique biological attributes of Morus alba mycelial cells being multinucleated, with each cell containing 20-40 nuclei, and the DNA of different nuclei capable of independent mutation and evolution. The entire multispore strain is considered as a unit for directed evolution. Simultaneously, taking advantage of the high spontaneous mutation rate of the Morus alba genome, individuals with enhanced salt tolerance are continuously enriched and screened from the population by applying progressively increasing salinity selection pressure, without using any chemical mutagens or physical mutagenesis methods. The entire breeding process consists of multiple cycles, each cycle including 5 directed evolution steps and 1 refresh step. These cycles can continue until the target salt tolerance concentration is reached or the strain can no longer tolerate higher salt concentrations. The five directed evolution steps are responsible for asexual selection and salt stress adaptation of the mycelium under gradually increasing NaCl concentrations. The refresh step forces the strain to complete a full sexual reproduction process (from vegetative growth to ascospore production), using sexual reproduction to eliminate unfavorable mutations accumulated from continuous asexual passages, reversing the degenerative trend of the culture, restoring population vitality, and providing healthy starting material for the next round of stress selection. The salt concentration of the refresh step is consistent with that of the fifth directed evolution step in the same cycle, which is equivalent to giving the strain a rest period under high salt pressure, allowing it to complete sexual reproduction and degenerative repair while maintaining its current salt tolerance level.

[0032] Under the above-described overall inventive concept, the key operational elements involved in this method will be described in detail below.

[0033] The starting material for this invention is a multispore isolate of *Morchella esculenta*, derived from large quantum cysts ejected from the mature fruiting bodies of a target commercially high-yielding *Morchella esculenta* variety. The collected ascospores are prepared into a spore suspension, inoculated onto PDA plates for germination and culture, yielding a mixed mycelial culture composed of numerous spore progeny. Before the first cycle, the multispore isolate is activated on PDA plates containing 0.1% NaCl (mass percentage) to adapt to initial selection pressure, resulting in a vigorous mycelial culture for subsequent selection in the directed evolution step.

[0034] The standard procedure for each directed evolution step is as follows: From the PDA plate containing the culture of the current multispore isolated strain, taking a 1cm × 1cm agar block covered with mycelium at each of the 12 directions corresponding to the 1 o'clock to 12 o'clock positions, and inoculating the 12 agar blocks at the leading edge of the mycelial spread, these 12 agar blocks are placed in the center of 12 fresh PDA plates containing the specified NaCl concentration. The plates are then incubated at 15°C until the mycelium spreads outwards to 2 / 3 of the plate's radius (i.e., covering 2 / 3 of the radius from the center). Subsequently, the density, growth rate, and colony uniformity of the mycelial growth are comprehensively evaluated from these 12 plates, and the plate with the best growth is selected for the next step. Within each cycle, the NaCl concentration for the five directed evolution steps increases progressively in increments of 0.1% (w / w), cumulatively increasing the salt tolerance concentration by 0.5% (w / w) throughout the entire cycle.

[0035] The standard procedure for the refresh step is as follows: Select the best-growing plate from the 12 plates obtained in step 5 of this cycle's directed evolution. On this plate, pick a 1cm x 1cm agar block covered with mycelium from each of the 12 directions along the edge of the circular mycelium growing on its surface. Inoculate these 12 agar blocks onto 12 primary culture media. Each primary culture medium contains 1000g of the following formula: 35% (w / w) rice husks, 20% (w / w) humus, 40% (w / w) water, 2% (w / w) dipotassium hydrogen phosphate, 2% (w / w) calcium carbonate, and 1% (w / w) calcium sulfate. Incubate at 15℃ for 20 days after inoculation to allow the morel mycelium to fully grow and form sclerotia. After 20 days, transfer the primary culture medium covered with morel mycelium and sclerotia to the cultivation medium at a 1:100 (w / w) inoculation ratio. The culture medium for the spawn was formulated as follows: 35% (w / w) wheat, 15% (w / w) rice husks, 5% (w / w) humus, 40% (w / w) water, 2% (w / w) dipotassium hydrogen phosphate, 2% (w / w) calcium carbonate, and 1% (w / w) calcium sulfate. The culture was continued at 15℃ for 20 days, resulting in 12 spawn samples, numbered 1 to 12, corresponding to mycelial blocks picked from 12 different directions on the aforementioned plate.

[0036] The above 12 cultivars were used for field fruiting trials. A saline-alkali soil plot with a NaCl content corresponding to the target salt concentration of this cycle's refresh step was selected and randomly divided into 12 × 5 = 60 experimental plots, each with an area of ​​40 square meters. Each cultivar was assigned 5 plots as replicates, and cultivation was carried out according to a randomized block design. Field management was conducted using conventional morel cultivation methods, including furrowing, sowing, covering with soil, and placing exogenous nutrient bags. After fruiting, the fresh mushroom yield of each plot was calculated in grams per square meter (g / m²). 2The data were recorded in units of 12 cultivars, each with five plots. One-way ANOVA was used to statistically analyze the yield data, and Duncan's multiple comparison method was used to label the significance of differences between group means (significance labels a, b, c, d, etc., where label a indicates the highest mean significantly better than other groups, or groups with no significant difference from the highest mean share the label a). Five plots corresponding to the cultivar with the highest average yield were selected. From each plot, 20 healthy, normally developed morel fruiting bodies were randomly selected, totaling 100 fruiting bodies. Ascospores ejected from these 100 fruiting bodies were collected, and a multispore suspension was prepared. After concentration, 500 μL of this suspension was added to the center of a fresh PDA plate containing the corresponding NaCl concentration for the current cycle. Germination and culture resulted in the formation of a next-generation morel multispore isolate tolerant to this salt concentration, which served as the starting strain for the next cycle.

[0037] The above operation is repeated, with the salt tolerance concentration increased by 0.5% (w / w) in each cycle, until the target salt tolerance concentration (e.g., 2.0% w / w NaCl) is reached or the strain fails to grow normally on all 12 plates in the directed evolution step. The method of the present invention is further illustrated below through specific embodiments.

[0038] It should be noted that the preparation of PDA plate culture medium, aseptic operation techniques, and routine cultivation management measures for morel mushrooms used in the following examples are all common techniques and methods in the field, and will not be described step by step. The NaCl concentration percentages mentioned in the examples are all mass percentages (w / w), i.e., the number of grams of NaCl contained in 100g of culture medium or soil. Mycelial growth rate was determined using the cross-sectional method to measure colony diameter. Each treatment had three replicate plates, and the results are expressed as mean ± standard error (Mean ± SE).

[0039] Example 1: Salt-tolerant directional selection of morel strain ZSTR801

[0040] The starting strain in this embodiment was *Morchella sextelata* cultivar ZSTR801, isolated by Sichuan Yibin Zhengde Biotechnology Co., Ltd. in 2016, and preserved aseptically in water-sealed test tubes at 4°C. Before starting targeted breeding, the mycelial growth rate of the starting strain on PDA plates with different NaCl concentrations was first measured (measured 4 days after inoculation, the same below), and the results are shown in Table 1.

[0041] Table 1. Mycelial growth rate of the starting strain of *Morchella esculenta* ZSTR801 under different NaCl concentrations.

[0042] 0.0 7.2±0.8 0.1 7.0±0.6 0.2 6.6±0.7 0.3 5.1±0.4 0.4 2.3±0.2 0.5 Stop growing

[0043] As shown in Table 1, the initial strain of *Morchella esculenta* ZSTR801 grew vigorously in the absence of NaCl. As the NaCl concentration increased, the mycelial growth rate gradually decreased, and growth completely stopped at a concentration of 0.5%, indicating that its initial salt tolerance limit was lower than 0.5% NaCl.

[0044] First cycle: The multispore isolate of the ZSTR801 starting strain was activated and cultured on PDA plates containing 0.1% NaCl. In directed evolution step 1, agar blocks were picked from the edge of the mycelium of the activated plate in 12 directions and inoculated into the center of 12 PDA plates containing 0.1% NaCl, and cultured at 15°C until the mycelium spread to 2 / 3 of the radius. The plate with the best growth was selected, and mycelial blocks were picked from the edge of the plate in 12 directions and entered into directed evolution step 2, inoculated into PDA plates containing 0.2% NaCl. This process was repeated, with directed evolution step 3 inoculated into plates containing 0.3% NaCl, directed evolution step 4 inoculated into plates containing 0.4% NaCl, and directed evolution step 5 inoculated into plates containing 0.5% NaCl.

[0045] After the directed evolution step 5 was completed, the best-growing plate was selected from 12 plates containing 0.5% NaCl, and 12 cultivars were prepared according to the aforementioned refresh procedure. A saline-alkali soil with a NaCl content of 0.5% (w / w) was selected and divided into 60 plots for field fruiting experiments. The yield statistics of the 12 cultivars are shown in Table 2.

[0046] Table 2. Yield and variance analysis of 12 cultivation samples of *Morchella esculenta* ZSTR801 in the first cycle (unit: g fresh mushrooms per square meter)

[0047] 1# 574 585 535 593 512 559.8 34.8 bcd 2# 576 442 596 515 579 541.6 63.6 bcd 3# 490 573 469 457 566 511.0 54.7 cd 4# 531 537 612 608 652 588.0 52.3 bc 5# 681 655 635 690 711 674.4 29.8 a 6# 587 538 623 686 566 600.0 57.2 ab 7# 561 624 542 581 494 560.4 48.0 bcd 8# 652 501 648 529 512 568.4 75.2 bcd 9# 559 559 671 507 556 570.4 60.4 bcd 10# 580 545 641 679 538 596.6 61.5 b 11# 581 689 657 568 532 605.4 65.3 ab 12# 515 505 412 548 536 503.2 53.7 d

[0048] As shown in Table 2, cultivar #5 had the highest average yield, reaching 674.4 g / m³. 2 Furthermore, after being labeled as 'a' by Duncan's multiple comparisons, it showed no significant difference from 6# and 11#, but was significantly higher than the other cultivated species. Therefore, five plots corresponding to cultivated species 5# were selected, and 20 robust fruiting bodies were randomly selected from each plot, collecting ascospores from a total of 100 fruiting bodies. The ascospore suspension was prepared, concentrated to 500 μL, and dropped onto the center of a PDA plate containing 0.5% NaCl. Germination resulted in the formation of a 0.5% NaCl-tolerant morel strain ZSTR801 multispore isolate, which entered the second cycle.

[0049] Second cycle: Using the 0.5% NaCl-tolerant multispore isolates obtained in the first cycle's refresh step as starting material, directed evolution steps 1-5 were carried out in the same manner, with NaCl concentrations of 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%, respectively. The refresh steps were conducted in a field fruiting experiment on saline-alkali soil with a NaCl content of 1.0% (w / w). The yield statistics of the 12 cultivars are shown in Table 3.

[0050] Table 3. Yield and variance analysis of 12 cultivation samples of *Morchella esculenta* ZSTR801 in the second cycle (unit: g fresh mushrooms per square meter)

[0051] 1# 478 399 232 406 439 390.8 94.1 bc 2# 376 306 434 488 284 377.6 85.5 bc 3# 329 502 457 453 418 431.8 64.8 b 4# 305 408 412 410 370 381.0 45.9 bc 5# 383 432 307 484 492 419.6 76.7 b 6# 402 314 375 333 329 350.6 36.6 bc 7# 420 293 373 341 237 332.8 70.8 bc 8# 347 526 281 487 379 404.0 101.0 bc 9# 368 373 254 221 241 291.4 73.2 c 10# 636 574 639 722 628 639.8 53.0 a 11# 316 453 323 423 349 372.8 61.7 bc 12# 533 303 334 266 328 352.8 104.2 bc

[0052] As shown in Table 3, the average yield of cultivar 10# was the highest, at 639.8 g / m³. 2 Duncan's multiple comparison marker was 'a', significantly higher than other cultivars. Ascospores were collected from five plots corresponding to cultivar 10#, and a spore suspension was prepared and dropped onto the center of a PDA plate containing 1.0% NaCl to form a multi-spore isolate resistant to 1.0% NaCl, which then entered the third cycle.

[0053] Third cycle: Using the 1.0% NaCl-tolerant multispore isolate obtained in the second cycle as the starting material, the NaCl concentrations for directed evolution steps 1-5 were 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%, respectively. The refresh step involved field fruiting trials on saline-alkali soil with a NaCl content of 1.5% (w / w). The yield statistics for the 12 cultivars are shown in Table 4.

[0054] Table 4. Yield and variance analysis of 12 cultivation samples of *Morchella esculenta* ZSTR801 in the third cycle (unit: g fresh mushrooms per square meter)

[0055] 1# 546 373 557 479 601 511.2 88.8 b 2# 542 300 370 308 372 378.4 97.4 cd 3# 488 443 501 351 510 458.6 65.4 bcd 4# 326 355 459 316 517 394.6 88.9 bcd 5# 511 578 392 411 438 466.0 77.2 bcd 6# 559 504 338 429 365 439.0 92.7 bcd 7# 626 709 594 576 638 628.6 51.3 a 8# 585 366 471 473 572 493.4 89.0 bc 9# 355 446 517 511 488 463.4 66.7 bcd 10# 445 376 374 246 357 359.6 71.9 d 11# 539 329 627 432 443 474.0 113.3 bcd 12# 357 493 516 514 645 505.0 102.3 bc

[0056] As shown in Table 4, cultivar #7 had the highest average yield, at 628.6 g / m³. 2 Duncan's multiple comparison marker was 'a', significantly higher than other cultivars. Ascospores were collected from five plots corresponding to cultivar 7#, and spore suspensions were prepared and dropped onto the center of a PDA plate containing 1.5% NaCl to form a multispore isolate resistant to 1.5% NaCl, which then entered the fourth cycle.

[0057] Fourth cycle: Using the 1.5% NaCl-tolerant multispore isolate obtained in the third cycle as the starting material, the NaCl concentrations for directed evolution steps 1-5 were 1.6%, 1.7%, 1.8%, 1.9%, and 2.0%, respectively. The refresh step involved field fruiting trials on saline-alkali soil with a NaCl content of 2.0% (w / w). The yield statistics for the 12 cultivars are shown in Table 5.

[0058] Table 5. Yield and variance analysis of 12 cultivation samples of *Morchella esculenta* ZSTR801 in the fourth cycle (unit: g fresh mushrooms per square meter)

[0059] 1# 521 496 659 460 598 546.8 80.6 bc 2# 649 694 704 682 802 706.2 57.4 a 3# 448 392 515 502 432 457.8 50.8 c 4# 648 493 630 545 552 573.6 64.2 b 5# 673 518 506 688 540 585.0 88.2 b 6# 500 732 568 463 600 572.6 104.2 b 7# 619 484 564 746 648 612.2 97.5 ab 8# 614 497 607 616 393 545.4 98.8 bc 9# 454 637 606 650 615 592.4 79.3 b 10# 513 573 563 496 559 540.8 34.1 bc 11# 562 679 617 520 542 584.0 64.1 b 12# 597 616 559 548 541 572.2 32.7 b

[0060] As shown in Table 5, cultivar #2 had the highest average yield, reaching 706.2 g / m³. 2 The Duncan multiple comparison marker was 'a', showing no significant difference from '7#', but significantly higher than the other cultivated species. Ascospores were collected from five plots corresponding to cultivated species '2#', and a spore suspension was prepared and dropped onto the center of a PDA plate containing 2.0% NaCl, forming a 2.0% NaCl-tolerant multispore isolate of *Morchella esculenta* ZSTR801. Thus, the final selected strain of *Morchella esculenta* ZSTR801, tolerant to 2.0% NaCl, was obtained.

[0061] Example 2: Salt-tolerant selective breeding of Morel strain ZSTR701

[0062] The starting strain in this embodiment was Morchella eximia cultivar ZSTR701, isolated by Sichuan Yibin Zhengde Biotechnology Co., Ltd. in 2015, and preserved aseptically in water-sealed test tubes at 4°C. The baseline data of mycelial growth rate of the starting strain on PDA plates with different NaCl concentrations are shown in Table 6.

[0063] Table 6. Mycelial growth rate of the *Morchella esculenta* ZSTR701 starting strain at different NaCl concentrations.

[0064] 0.0 6.9±0.7 0.1 6.7±0.7 0.2 5.4±0.6 0.3 2.0±0.2 0.4 Stop growing

[0065] As shown in Table 6, the initial salt tolerance of the *Morchella esculenta* strain ZSTR701 was slightly lower than that of ZSTR801, and it stopped growing at a concentration of 0.4% NaCl.

[0066] Using the same directed evolution cycle operation method and refresh step operation procedure as in Example 1, four cycles of salt tolerance directed selection were carried out on Morel ZSTR701. The results of field fruiting trials for each cycle refresh step are as follows.

[0067] Table 7 shows the yield and variance analysis of the 12 cultivars in the first cycle. Cultivar #11 had the highest average yield, at 637.4 g / m³. 2 The genus *A* was identified as *a* by Duncan multiple comparisons, significantly higher than other cultivated species. Ascospores were collected from five plots corresponding to cultivated species 11# to form a multispore isolate resistant to 0.5% NaCl, which was then introduced into the second cycle.

[0068] Table 7. Yield and variance analysis of 12 cultivation samples of Qimei Morel ZSTR701 in the first cycle (unit: g fresh mushrooms per square meter)

[0069] 1# 597 580 462 426 418 496.6 85.7 b 2# 531 480 404 565 409 477.8 71.8 bc 3# 441 610 383 365 425 444.8 97.3 bc 4# 352 436 513 515 425 448.2 68.2 bc 5# 433 364 376 427 470 414.0 43.6 bc 6# 360 459 383 586 456 448.8 88.3 bc 7# 699 557 425 479 351 502.2 133.3 b 8# 472 425 456 577 464 478.8 57.7 bc 9# 608 426 393 619 527 514.6 103.0 b 10# 421 235 324 468 380 365.6 90.2 c 11# 573 690 698 580 646 637.4 59.1 a 12# 438 511 595 436 610 518.0 83.0 b

[0070] The second cycle involved a field trial of the refresh step on saline-alkali soil with 1.0% NaCl. The yields and variance analysis of the 12 cultivars are shown in Table 8. Cultivar #3 had the highest average yield, at 656.8 g / m³. 2 Duncan's multiple comparison marker was 'a', showing no significant difference from '4#', but significantly higher than the other cultivated species. Ascospores were collected from the five plots corresponding to cultivated species '3#' to form a multispore isolate resistant to 1.0% NaCl, which entered the third cycle.

[0071] Table 8. Yield and variance analysis of 12 cultivation samples of Qimei Morel ZSTR701 in the second cycle (unit: g fresh mushrooms per square meter)

[0072] 1# 461 411 483 583 561 499.8 71.3 bcd 2# 466 588 408 404 595 492.2 93.9 bcd 3# 755 568 677 648 636 656.8 67.9 a 4# 456 688 617 552 494 561.4 93.4 ab 5# 493 615 439 542 554 528.6 66.3 bc 6# 386 515 486 423 443 450.6 51.0 bcd 7# 458 360 391 492 335 407.2 66.1 d 8# 488 567 595 613 445 541.6 72.1 b 9# 587 459 498 612 498 530.8 65.3 bc 10# 357 555 446 398 376 426.4 79.2 cd 11# 441 650 412 529 477 501.8 93.6 bcd 12# 468 665 499 478 631 548.2 92.6 b

[0073] The third cycle involved a field trial of the refresh step on saline-alkali soil with 1.5% NaCl. The yields and variance analysis of the 12 cultivars are shown in Table 9. Cultivar #5 had the highest average yield, at 653.0 g / m³. 2 Duncan's multiple comparison marker was 'a', showing no significant difference from '11#', but significantly higher than the other cultivated species. Ascospores were collected from the five plots corresponding to cultivated species '5#' to form a multispore isolate resistant to 1.5% NaCl, which entered the fourth cycle.

[0074] Table 9. Yield and variance analysis of 12 cultivation samples of Qimei Morel ZSTR701 in the third cycle (unit: g fresh mushrooms per square meter)

[0075] 1# 412 392 586 492 384 453.2 85.7 cd 2# 457 467 466 497 505 478.4 21.2 cd 3# 379 639 468 487 456 485.8 95.0 cd 4# 352 549 422 429 469 444.2 72.2 cd 5# 547 670 762 641 645 653.0 76.8 a 6# 443 590 502 515 426 495.2 65.1 cd 7# 400 389 354 471 312 385.2 59.0 d 8# 395 360 566 467 450 447.6 78.8 cd 9# 544 454 356 350 478 436.4 83.0 cd 10# 534 449 480 588 573 524.8 59.5 bc 11# 482 650 584 698 645 611.8 83.1 ab 12# 608 490 459 392 390 467.8 89.5 cd

[0076] The fourth cycle involved a field trial of the refresh step on saline-alkali soil with 2.0% NaCl. The yields and variance analysis of the 12 cultivars are shown in Table 10. Cultivar #7 had the highest average yield, at 635.6 g / m³. 2Duncan's multiple comparison marker was 'a', significantly higher than other cultivated species. Ascospores were collected from five plots corresponding to cultivated species #7, and spore suspensions were prepared and dropped onto the center of a PDA plate containing 2.0% NaCl to form the final selected strain of *Morchella esculenta* ZSTR701, which is resistant to 2.0% NaCl.

[0077] Table 10. Yield and variance analysis of 12 cultivation samples of Qimei Morel ZSTR701 in the fourth cycle (unit: g fresh mushrooms per square meter)

[0078] 1# 369 428 527 478 410 442.4 61.4 b 2# 519 379 618 500 511 505.4 85.0 b 3# 358 377 169 382 309 319.0 88.7 c 4# 596 455 476 476 372 475.0 80.1 b 5# 619 500 560 425 484 517.6 74.3 b 6# 362 523 395 501 451 446.4 68.3 b 7# 682 509 684 658 645 635.6 72.7 a 8# 574 400 579 466 402 484.2 88.4 b 9# 461 452 576 375 430 458.8 73.6 b 10# 395 583 445 458 439 464.0 70.6 b 11# 449 523 478 527 376 470.6 62.0 b 12# 620 534 566 392 537 529.8 84.4 b

[0079] Experimental Example 1: Verification of the salt tolerance of the final selected strain

[0080] To verify the salt tolerance of the final strains obtained after four cycles of directional selection, the initial and final selected strains of *Morchella esculenta* ZSTR801 and *Morchella esculenta* ZSTR701 were inoculated onto PDA plates containing 0.0%, 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% NaCl, respectively. After incubation at 15°C for 4 days, mycelial growth rate and colony diameter were measured. The results are shown in Tables 11 and 12.

[0081] Table 11. Mycelial growth rates of the initial and final strains of *Morchella esculenta* ZSTR801 at different NaCl concentrations.

[0082] 0.0 7.2±0.8 7.1±0.6 0.5 Stop growing 6.4±0.6 1.0 Stop growing 7.4±0.7 1.5 Stop growing 7.1±0.6 2.0 Stop growing 6.9±0.5 2.5 Stop growing Stop growing

[0083] Table 12. Mycelial growth rates of the initial and final strains of *Morchella esculenta* ZSTR701 at different NaCl concentrations.

[0084] 0.0 6.9±0.7 6.9±0.5 0.5 Stop growing 7.0±0.6 1.0 Stop growing 7.0±0.5 1.5 Stop growing 6.4±0.7 2.0 Stop growing 6.8±0.6 2.5 Stop growing Stop growing

[0085] As shown in Tables 11 and 12, the initial strain of *Morchella esculenta* ZSTR801 ceased growth at a NaCl concentration of 0.5%, while the final selected strain grew normally within a NaCl concentration range of 0.5% to 2.0%, with a mycelial growth rate comparable to or even slightly higher than that of the initial strain under salt-free conditions, until growth ceased at a NaCl concentration of 2.5%. Similarly, the initial strain of *Morchella esculenta* ZSTR701 ceased growth at a NaCl concentration of 0.4%, and the final selected strain also grew vigorously within a NaCl range of 0.5% to 2.0%, ceasing growth at 2.5%.

[0086] The colony diameter data 4 days after inoculation (Tables 13 and 14) further visually demonstrate the differences before and after selection. The initial strains showed no visible growth on salt plates, and the colony diameter was the size of the inoculated agar block itself (approximately 7 mm); while the final selected strains achieved colony diameters of 25-30 mm at various salt concentrations after 4 days, indicating that they had fully adapted to the high-salt environment.

[0087] Table 13. Colony diameter of *Morchella esculenta* ZSTR801 starting and final strains 4 days after inoculation.

[0088] 0.0 28.8±3.2 28.4±2.4 0.5 Stop growing 25.6±2.4 1.0 Stop growing 29.6±2.8 1.5 Stop growing 28.4±2.4 2.0 Stop growing 27.6±2.0 2.5 Stop growing Stop growing

[0089] Table 14. Colony diameter of *Morchella esculenta* ZSTR701 starting and final strains 4 days after inoculation.

[0090] 0.0 27.6±2.8 27.6±2.0 0.5 Stop growing 28.0±2.4 1.0 Stop growing 28.0±2.0 1.5 Stop growing 25.6±2.8 2.0 Stop growing 27.2±2.4 2.5 Stop growing Stop growing

[0091] The above results indicate that, after four cycles of directional selection using the method of this invention, the salt tolerance of *Morchella esculenta* ZSTR801 and *Morchella esculenta* ZSTR701 was significantly increased from less than 0.5% NaCl to 2.0% NaCl. Furthermore, the mycelial growth activity of the selected strains within the salt tolerance range remained essentially consistent with that of the initial strains under salt-free conditions, and no negative effect of a significant decrease in growth rate due to increased salt tolerance was observed.

[0092] Experimental Example 2: Field Yield Comparison Before and After Breeding

[0093] To verify whether the final strain acquired salt tolerance while retaining the high-yielding performance of the original strain, a field comparison experiment was conducted on the initial strains of *Morchella esculenta* ZSTR801 and *Morchella esculenta* ZSTR701, along with the final selected strain, in normal soil with 0% NaCl and saline-alkali soil with 2% NaCl. Each treatment had five replicate plots, each 40 square meters, with consistent cultivation management practices. The yield statistics and t-test analysis are as follows.

[0094] Table 15 Comparison of the yield of *Morchella esculenta* ZSTR801 before and after breeding on soils with different salinities (yield unit: g / m³) 2 )

[0095] Starting strain - 0% saline soil 739 703 625 746 607 684.0 64.5 Starting strain - 2% saline soil 0 0 0 0 0 0.0 0.0 Final strain - 0% saline soil 772 606 668 725 701 694.4 62.3 Final strain - 2% saline soil 692 747 599 769 702 701.8 65.6

[0096] Independent samples t-tests were performed on the data in Table 15. Under 0% saline soil conditions, the average yield of the initial strain was 684.0 g / m³. 2 The final average yield of the strain was 694.4 g / m³. 2The difference between the two was not significant (P=0.802), indicating that the final strain after selection fully retained the high-yielding performance of the original strain in normal soil. Under 2% saline soil conditions, the initial strain failed to produce fruit at all, with a yield of zero; the final strain had an average yield of 701.8 g / m², which was comparable to the yield level of the initial strain in 0% saline soil, and the difference was extremely significant (P<0.001), proving that the selected strain has the ability to produce fruit normally and obtain high yields in 2% saline-alkali soil.

[0097] Table 16 Comparison of the yield of *Morchella esculenta* ZSTR701 before and after breeding on soils with different salinities (yield unit: g / m³) 2 )

[0098] Starting strain - 0% saline soil 671 691 550 575 634 624.2 60.6 Starting strain - 2% saline soil 0 0 0 0 0 0.0 0.0 Final strain - 0% saline soil 590 680 565 606 717 631.6 64.1 Final strain - 2% saline soil 618 552 676 727 621 638.8 66.0

[0099] The yield comparison results of *Morchella esculenta* ZSTR701 (Table 16) were highly consistent with those of *Morchella esculenta* ZSTR801. On 0% saline soil, the average yields of the starting and final strains were 624.2 g / m³. 2 and 631.6g / m 2 The differences were not significant (P=0.856). On 2% saline soil, the initial strain yielded zero, while the final strain achieved an average yield of 638.8 g / m³. 2 The differences were extremely significant (P<0.001). The above results fully demonstrate that the method of the present invention has successfully achieved the goal of directly transforming commercial high-yielding multispored strains into superior strains that are both tolerant to 2.0% NaCl and maintain their original high-yield levels. The breeding effect has been effectively verified in two different varieties.

[0100] The successful application of the method of this invention in morel breeding is closely related to the unique cellular biological characteristics of morels. Morel mycelial cells are multinucleated, with each cell containing 20-40 nuclei. The DNA of these nuclei can mutate and evolve independently. Therefore, even within the same cell of the same mycelial individual, genetics are not completely homogeneous, but rather constitute a microscopic "multinucleated population within a single cell." During the 12-directional edge picking operation, mycelial fragments picked from different directions of the colony may carry combinations of different nuclear genotypes, further enriching the sources of selectable genetic variation and providing an operational dimension for directed evolution that transcends ordinary mononuclear organisms. This concept of managing and screening the entire multisporous species and its multinucleated mycelium as a population is one of the important features that distinguishes this invention from conventional methods of directed evolution of microorganisms.

[0101] Furthermore, if a strain with a salt tolerance level exceeding 2.0% NaCl is required in actual breeding, additional cycles can be added according to the method described above. Specifically, after the fourth cycle, the NaCl concentrations for the five directed evolution steps in the fifth cycle are set to 2.1%, 2.2%, 2.3%, 2.4%, and 2.5% respectively, and the refresh step is carried out on saline-alkali soil with 2.5% NaCl; and so on, with each cycle cumulatively increasing the NaCl by 0.5% until all plates can no longer grow normally, indicating that the salt tolerance limit of the strain has been reached. The entire breeding process does not use any chemical mutagens or physical mutagenesis methods, is simple and safe to operate, and avoids cumbersome mutagenesis condition optimization steps and potential hazards to operators and the environment.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for directional evolutionary breeding of salt-tolerant varieties of Morchella multispore isolated strains, characterized in that, Includes the following steps: The starting material was a multispore isolate of morel mushrooms, which was derived from a mixture of ascospores ejected from the fruiting bodies of morel mushrooms. Targeted breeding is carried out through a cyclical iterative process, with each cycle including several directional evolutionary steps and a refresh step. The directed evolution step is as follows: On the culture plate of the multispore isolated strain, mycelial agar blocks are picked from multiple directions from the edge of the mycelium and inoculated into the center of multiple PDA plates containing a preset NaCl concentration. The mycelium is cultured until it spreads from the center to two-thirds of the radius of the culture plate. The plate with the best growth is selected for the next step. The NaCl concentration of each directed evolution step in the same cycle is increased step by step with a fixed step size. The refresh step is as follows: Select the plate with the best growth from the plates obtained in the last directed evolution step of this cycle, pick out mycelial agar blocks from multiple directions from the edge of the mycelium on the plate, and inoculate them onto multiple original culture media. After the mycelium has fully grown and formed sclerotia, transfer them to the culture medium for further culture to obtain multiple culture media. Conduct field fruiting experiments on saline-alkali soil with NaCl content corresponding to the target salt tolerance level of this cycle, set up multiple replicate plots, count the fruiting yield of each culture, select the plot corresponding to the culture with the highest average yield, collect the ascospores ejected from the fruiting bodies, prepare a spore suspension, and inoculate it onto PDA plates containing the corresponding NaCl concentration to form the multispore isolated strain for the start of the next cycle. Multiple cycles were repeated until a multi-spore strain of morel mushrooms that could tolerate the target NaCl concentration was obtained.

2. The method for directional evolution and breeding of salt-tolerant varieties of Morchella multispore isolated strains according to claim 1, characterized in that: Each cycle contains 5 directed evolution steps, with the NaCl concentration in each directed evolution step increasing in a fixed step size of 0.1% by mass. The cumulative increase in NaCl concentration in each cycle is 0.5% by mass.

3. The method for directional evolution and breeding of salt-tolerant varieties of morel multispore isolates according to claim 1, characterized in that: In the directed evolution step, when picking mycelial agar blocks from the edge of the mycelium on the multispore isolated strain culture plate, a 1 cm × 1 cm agar block covered with mycelium is picked from each of the 12 directions from 1 o'clock to 12 o'clock, with the center of the plate as the center. These blocks are then inoculated into the center of 12 PDA plates containing the same NaCl concentration.

4. The method for directional evolution and breeding of salt-tolerant varieties of morel multispore isolates according to claim 1, characterized in that: In the refresh step, when picking agar blocks of mycelium from the edge of the plate, take a 1 cm × 1 cm agar block covered with mycelium in each of the 12 directions from 1 o'clock to 12 o'clock, with the center of the plate as the center, and inoculate them onto 12 original culture media.

5. The method for directional evolution and breeding of salt-tolerant varieties of Morchella multispore isolates according to claim 1, characterized in that: The original culture medium is formulated as follows by weight percentage: rice husk 35%, humus 20%, water 40%, dipotassium hydrogen phosphate 2%, calcium carbonate 2%, and calcium sulfate 1%; the cultivated culture medium is formulated as follows by weight percentage: wheat 35%, rice husk 15%, humus 5%, water 40%, dipotassium hydrogen phosphate 2%, calcium carbonate 2%, and calcium sulfate 1%.

6. The method for directional evolution and breeding of salt-tolerant varieties of morel multispore isolates according to claim 1, characterized in that: In the refresh step, the original culture medium is cultured at 15℃ for 20 days after inoculation; the inoculation ratio of the original culture medium to the culture medium for the transfer of the original culture medium is 1:100 by weight, and the culture is carried out at 15℃ for 20 days after inoculation.

7. The method for directional evolution and breeding of salt-tolerant varieties of morel multispore isolates according to claim 1, characterized in that: In the refresh step, the field mushroom production experiment set up 12 cultivars, each cultivar was assigned 5 replicate plots, each plot area was 40 square meters, and each plot was arranged in a randomized block designation. One-way ANOVA and Duncan's multiple comparison method were used to statistically analyze the differences in average yield among the cultivars. The cultivar with the highest average yield and marked as 'a' by Duncan's method or with no significant difference from 'a' was selected for ascospore collection.

8. The method for directional evolution and breeding of salt-tolerant varieties of Morchella multispore isolated strains according to claim 1, characterized in that: In the refresh step, the specific operation of collecting ascospores is as follows: randomly select robust morel fruiting bodies from each plot corresponding to the cultivation species with the highest average yield, collect the ejected ascospores to make a mixed suspension, concentrate it, and drop it onto the center of a PDA plate containing the corresponding concentration of NaCl, so that it can germinate and form a multispore isolated strain; wherein, the NaCl concentration contained in the plate in the refresh step is the same as the NaCl concentration in the last directed evolution step of the current cycle.

9. The method for directional evolution and breeding of salt-tolerant varieties of morel multispore isolates according to claim 1, characterized in that: The target NaCl concentration is 2.0% or higher by mass; the cycle is terminated when all plates fail to grow normally in the directed evolution step of a certain cycle.

10. The method for directional evolution and breeding of salt-tolerant varieties of morel multispore isolates according to claim 1, characterized in that: The morel mushroom species are Morchella sextelata or Morchella eximia.