Crossbreeding is the most commonly used method in breeding of Lentinula edodes, however low fruiting rate of the hybrids has always caused troubles and barriers for breeders. An early screening method of the fruiting ability could make the breeding work more efficient. In this paper, a rapid and high-throughput laccase activity detection method based on agar diffusion principle was developed. In this way, we investigated the constitutive and inducible extracellular laccase activity of 36 strains in a breeding population of L. edodes on different media and performed a correlation analysis with fruiting ability of these strains. The results showed the laccase activity of mycelium cultured in non-induced medium for 8 d could be used as an early screening index to judge whether it had fruiting ability at the later stage. Early rapid and simple screening method for hybrid populations was established based on laccase activity characteristics of mycelia. 127 strains from another 5 different hybrid populations were used to verify the early screening method. From the validation results, the early screening method was effective, but the appropriate screening threshold was needed to select according to the cross population, which would greatly to improve the breeding efficiency of L. edodes.

Lentinula edodes (Berk.) Pegler was popular in Southeast Asia not only because of its delicious taste, but also the characteristics of high-protein and low-fat content (Li, et al., 2018). According to the statistics of Chinese Edible Fungi Association (CEFA), the total production in China reached 10.43 million tons in 2018, which was an increase of 82.2% in the last decade. Excellent cultivars are the basis and guarantee for obtaining high-yield and high-quality fruit bodies in cultivation. Breeding is an effective way to obtain excellent varieties. Conventional breeding methods in mushrooms include systematic selection, hybridization, mutation, and protoplast fusion (Ikegaya, 1997). Among them, hybridization through mating with different monokaryons is still the most variety-producing method in breeding of L. edodes (Ha, Kim, & Ro, 2015).

As a typical two-factor, tetrapolar basidiomycete, the basidiospores of L. edodes have four different mating types (AxBx, AyBy, AxBy, and AyBx) with two separately mating locus A and B. Before the crossing, single spore isolations of two dikaryotic parental strains were collected and monokaryotic mycelium were germinated by single spore isolations. In breeding, two compatible monokaryons were inoculated closely then the mycelia fused to form the dikaryotic hyphae. Only monokaryons with different locus of mating factor A and B could form the fertile progenies (Brown & Casselton, 2001). If the monokaryons come from only one parent, it is selfing. After the crossing, usually clamp-connection could be easily observed in L. edodes to distinguish the dikaryons with monokaryons (Xiong et al., 2014). The dikaryon strains were screened for excellent hybrid progenies through multiple turns of cultivations. However, the hybrid dikaryon progeny populations were usually large (300-500). And depending on different parents, the breeding progenies no matter selfing or crossing, which mating by compatible single spore isolations, formed normal fruit bodies usually account for 30-70% (Turner, Wright, Ward, Osborne, & Self, 1975). Cultivation and screening are time-consuming and labor-consuming. At present, the whole cultivation cycle of L. edodes was divided into four stages, including vegetative mycelial growth and colonization of the growth substrate, light-induced brown film formation, primordium initiation, and fruiting body development, took at least 4-6 mo (Tang et al., 2013). Formation of normal fruiting bodies was affected by poor growth at any stage. No efficient selection system has been established. If an early screening method can be developed based on the physical or chemical properties of mycelia, the breeding process of L. edodes would be remarkably accelerated.

Laccase (Lcc; EC 1.10.3.2), which is a kind of lignocellulose-degrading enzyme, were divided into constitutive laccases and inducible laccases according to their synthesis stages and environmental impacts. Constitutive laccases were synthesized at all stages of mycelium growth, while inducible laccases synthesis generally occurred to secondary metabolism stage which was easily affected by the external environment such as inducers (Soden & Dobson, 2001). Laccase had been reported to be associated with the rapid growth of cell and the formation of primordia and the fruit body development in many basidiomycetes (Das, Sengupta, & Mukherjee, 1997; Ohga, Cho, Thurston, & Wood, 2000; Zhao & Kwan, 1999). It was also reported to participate in a series of biological processes, such as mycelia growth (Turner et al., 1975), brown film formation and fruiting body development in L. edodes (Leatham, 1985; Tang et al., 2013). At present, there are many methods to determine fungal laccase activity, but the original method is not suitable for the detection of large quantities of strains.

In this study, a rapid and high-throughput laccase activity detection method based on agar diffusion principle was developed. In this way, we investigated the constitutive and inducible extracellular laccase activity of 36 strains in a breeding population of L. edodes on different media and performed a correlation analysis with fruiting ability of these strains. Early rapid and simple screening method for hybrid progenies was established based on laccase activity characteristics of mycelia. The method was validated by 127 strains from another 5 hybrid populations, which would greatly to improve the breeding efficiency of L. edodes.

The dikaryotic parental strains used in this study were 7 commercial cultivars preserved in Edible Fungi Institute of Shanghai academy of agricultural science (IEF, SAAS). Cultivars ‘Shenxiang215’, ‘HuxiangF2’, ‘Qihe7’, ‘L808’, ‘Zunhua168’, ‘L60’ and ‘L238’ were cultivated in China for years. Single spore isolations of these cultivars were collected, and the mating types of each cultivar’s single spore isolations were detected by cross mating method (Raper, Krongelb, & Baxter, 1958). The population named as the prefix of ‘SH’ was crossed by ‘Shenxiang215’ and ‘HuxiangF2’ and 36 hybrid progenies were obtained, which were used for the development of early screening methods.

In this study, we constructed other 5 hybrid populations by cultivars ‘Qihe7’ vs ‘L808’, ‘L808’ vs ‘L60’, ‘L60’ vs ‘L238’, ‘Shenxiang215’ vs ‘Qihe7’, and ‘Shenxiang215’ vs ‘Zunhua168’ respectively by crossing of their single spore isolations. 127 strains were screened from these populations for the verification of early screening methods for hybrid populations. The 5 hybrid populations were named as the prefix of ‘QL8’, ‘L86’, ‘L62’, ‘SQ’, and ‘SZ’ respectively.

The mycelia cultivation on both non-induced and induced culture media. Non-induced culture was performed on PDA (Potato Dextrose Agar, BD Difco, USA), and 20 mL of PDA medium was quantitatively added into plastic petri dishes. After inoculation, the culture was conducted in the dark at 23 °C, with five replicates of each strain. In the induced media vanillic acid which indicated laccase induction capability in many basidiomycetes (Piscitelli et al., 2011) was added to the PDA at a concentration of 0.2 mmol/L, and the cultivation method was as same as the non-induced PDA.

A big tube (φ30 × 300 mm) filled with sawdust-based substrate was used in this study. The ingredient of the sawdust-based substrate in dry weight was 79% sawdust, 20% bran and 1% gypsum. Then the substrate stirred with tap water to 55% moisture content, and autoclaved at 121 °C for 3 h. After cooled to 25 °C, a 20 mm diameter colony disk which grown on the PDA was punched and inoculated into the tube. The tubes were cultivated in an incubator at 23 °C in the dark, with five replicates per strain.

1 g of substrates were dug out from the tubes and mixed with 5 mL of double distilled water and shaken at 28 °C for 2 h by ultrasonic wave. The mixture was filtered by two layers of gauze, and the filtrate was centrifuged under 2430 g at 4 °C for 10 min. The supernatant was the crude enzyme solution.

In this study, a new method based on agar disk diffusion was established to achieve a rapid detection of laccase activity. Guaiacol, which we adopted as detection substrate of laccase, was dissolved in 1% (w/v) low melting agarose at final concentration of 2 mmol/L as the detection media. Media was added into 24 - well cell culture plate in a volume of 2 mL to prepare a detection plate. 5 mm diameter agar disk (taken according to the operation mode in Fig. 5) or 300 μL crude enzyme solution was added to the center of the cell of the detection plate. Agar diffusion and enzyme reaction were performed in the incubator at 28 °C for a certain time. After incubation, the agar disks were removed with an inoculation needle, and was determined spectrophotometrically at 465 nm in a 16 − points (4 × 4) matrix mode (Tecan M200pro, Männedorf, SUI). Three agar disks repeat for each stain. A control well without guaiacol was set for each strain. The Supplementary Fig. S1 showed the plates before and after detection. One-unit enzyme active in this study was defined as 0.001 OD value increases per hour of 1 mg mycelia. Laccase activity (U/mg) = ΔOD⁄(0.001 × t×m) or (ΔOD × 5000)⁄(0.001 × t × 300 × 1000). ΔOD was the OD value of agar disk on the detection media minus on the blank control. t was incubation time. m was the mycelia weight on the agar disk.

All strains were cultivated in polypropylene bags filled with solid medium consisting of (%): 79 sawdust, 20 wheat bran and 1 plaster in Shanghai Guosen Biotechnology Co., Ltd. (Shanghai, China). After inoculation with fungal mycelium, the bags were kept in the dark at 22-24 °C and 65-70% relative humidity for 30 d, and then kept in the light at 22-24 °C and 75-85% relative humidity for 60 d before transfer to a ventilated field at 16 °C and 90% relative humidity for 9 d. Each strain was inoculated with 9 bags and randomly placed. The fruiting ability were observed. The primordia can be formed in the later cultivation process, indicating that it has fruiting ability, otherwise it does not have fruiting ability.

Bivariate correlation analysis among laccase activity and fruiting ability was respectively performed by SPSS (IBM v23.0) software.

The 127 strains of five verification populations constructed in this study were incubated on non-induced PDA media at the 8th day to detect laccase activity. The laccase activity thresholds of strains which can used in early screening were certificated by these verification populations.

We established a method for detecting laccase activity based on agar disk diffusion in this study. This method was limited by the diffusion rate of the laccase from the agar disks to the detection media, and it is necessary to determine the appropriate diffusion and reaction time. The fruiting ability of 36 hybrid progenies of the test population ‘SH’ was shown in Table 1. 6 strains (2 strains with fruiting ability and 4 strains without fruiting ability) were selected for this experiment. The agar disks were punched out on the line at the 6th mycelia growth day on PDA, then the disks were placed in the 24 - well detection plate. After different diffusion and reaction hours, the disks were picked out and the absorbance value of each well was measured immediately. Laccase activity of 5 strains except strain SH327 increased with the time extension (Fig. 1). Absorbance value of SH096 and other 4 strains reached the upper limit of the microplate reader in 36 and 12-24 h respectively. SH327 was not detected any effective laccase activity during the entire measurement process, indicating that this strain may not secrete extracellular laccase on PDA medium. In the comparison of the different diffusion times of all strains, absorbance value of the strain with higher laccase activity reached the upper limit within 12 h, and the measurement diversity was smaller at 4-6 h, the best diffusion and reaction time was concluded as 6 to 12 h in this method. Taking into account that as the mycelia grow longer, the laccase activity would further increase, more absorbance values will exceed the upper limit of the machine, finally 8 h was used as the best diffusion and reaction time.

Fig. 1. The absorbance values of different diffusion and reaction time of the test strains.

By examining the characteristic curves of laccase activity changes of 36 hybrid progenies on non-induced PDA plates with increasing incubation days (Supplementary Fig. S2), we found that the curves could be divided into 3 different types of profiles (Fig. 2). The first type of profile (pp1) included 12 strains, represented by 3 strains in Fig. 2A, which was characterized by a rapid increase of laccase activity when were cultured 3-5 d. The laccase activity of all strains was more than 8 U/mg at the 7th day. And all absorbance values exceeded the upper limit at 8 d. The second type of profile (pp2) consisted of 16 strains (Fig. 2B showed 3 of them). Their characteristics were that the laccase activity started to increase when cultured were 6-7 d, and the enzyme activity was 1.17-8.13 U/mg by the 8th day. The third type (pp3) consisted of 8 strains (3 of shown in Fig. 2C). The laccase activity of these strains rose very slowly, or did not rise at all, and remained at a very low level (not more than 0.81 U/mg).

Fig. 2. The laccase activity profiles of partial test strains on PDA media. A, B and C represent three different types of characteristic curve pp1, pp2 and pp3 on PDA media.

After adding vanillic acid as laccase inducer to the PDA medium, the changes of their laccase activity with the cultivation days were continuously detected. Four different profiles of the laccase activities variation curves were appeared by all the test strains (Supplementary Fig. S3). Fig. 3 showed the profile curves represented by four typical strains. The first profile (vp1) represented by strain SH446 showed a consistent trend of laccase activity on the PDA medium with or without inducer. Vanillic acid induced laccase activity was slightly lower than the non-induced PDA medium, but there was no significant difference between them (Fig. 3A). The second profile (vp2) indicated that the presence of vanillic acid inhibited the production of extracellular laccase, and almost no laccase activity was detected on the induced PDA media (Fig. 3B). Compared with the second one, the third profile (vp3) showed that vanillic acid induced the extracellular laccase secretion, and laccase activity was significantly higher than that on non-induced PDA medium (Fig. 3C). The fourth profile (vp4) was represented by strain SH039, which showed that no extracellular laccase was produced in the presence or absence of inducer (Fig. 3D). It should be noted that, on the PDA medium supplemented with the inducers vanillic acid, the mycelial growth rate was inhibited in varying degrees compared with the non-induced PDA. After the addition of vanillic acid at 0.2 mmol/L, the inhibition degree ranged from 4.78% to 34.03%.

Fig. 3. Comparison of laccase activity profiles of partial test strains on PDA and VA-PDA media. A, B, C and D represent different types of characteristic curve vp1, vp2, vp3 and vp4.

The laccase activity variation curve of the test strains after growing in test tubes of sawdust-based substrate for different days was shown in Supplementary Fig. S4, which can be divided into 4 different profiles of characteristic types. Among them, the three strains in Fig. 4A (sp1) typically showed that the laccase had the highest activity when the strains firstly contacted with the sawdust-based substrate. With the increase of the cultivation days, the enzyme activity gradually decreased until it was almost impossible to be detected. Characteristic curves of some strains in second profile (sp2) (Fig. 4B) were similar to the first kind of profile in the early stage of culture, but laccase activity suddenly increased significantly after 32 d of cultivation. Laccase activity of some strains increased continuously during the culture period. The third profile (sp3) showed that the laccase activity was low in the early and late culture and relatively high in the middle of the culture (Fig. 4C). Laccase activity of strains in the last profile (sp4) was very low (less than 1 U/mg) during all culture stages (Fig. 4D).

Fig. 4. The laccase activity profiles of partial test strains on sawdust substrates. A, B, C and D represent different types of characteristic curve sp1, sp2, sp3 and sp4.

Laccase activity profiles of all strains on three different culture media were clustered according to the activity characteristic curve (Table 1). On non-induced PDA media, the laccase activity profiles of 67% strains with fruiting ability was pp1 type, and 33% strains were pp2 type. The laccase activity profiles of 22% strains without fruiting ability was pp1 type, 48% strains were pp2 type and 30% strains were pp3 type. On VA-PDA media, 56% strains with fruiting ability had vp1 profiles, and 44% strains had vp2 profiles. The distribution proportions of vp1, vp2, vp3, vp4 profiles of strains without fruiting ability were 19%, 33%, 22% and 26% respectively. On sawdust-based substrates, the distribution proportions of sp1, sp2, sp4 profiles of strains with fruiting ability were 38%, 12% and 50% respectively. The distribution proportions of sp1, sp2, sp3, sp4 profiles of strains without fruiting ability were 25%, 17%, 25% and 33% respectively.

Correlation analysis results between laccase activity of mycelium cultured for different days on different media and fruiting ability were shown in Table 2. Only the fruiting ability had a significant positive correlation with the laccase activity on non-induced PDA medium in the culture period of 4-8 d. According to the results of laccase activity profiles on non-induced PDA medium, in 4-8 d, with the increase of culture days, the laccase activity of most strains showed an overall growth trend. On the 8th d, the difference of laccase activity among strains was the most significant.

According to the above experimental results, the laccase activity of mycelium cultured in non-induced medium for 8 d could be used as an early screening index to judge whether it had fruiting ability at the later stage.

127 strains of the five newly constructed hybrid populations were used to detect laccase activity of mycelium cultured on non-induced PDA medium for 8 d and fruiting ability (Supplementary Table S1). Correlation analysis between fruiting ability and the laccase activity was performed (Table 3). The results showed that there was an extremely significant positive correlation between laccase activity and fruiting ability. However, there were great differences among different hybrid populations. The correlation coefficient of the “SZ” population was as high as 0.755, but there was no correlation between laccase activity and fruiting ability in “L86” and “L62” populations. The results showed that the appropriate threshold of laccase activity should be selected when early screening method was used.

When different laccase activity values were used as thresholds, filtering rate, the fruiting rate of retained strains, error-filtering rate and error-filtering rate of fruiting strains of each population were shown in Table 4. The average filtering rate increased from 42.15% to 79.86% with the increase of laccase activity threshold from 1 to 8. The fruiting rate of the retained strains increased from 32.65% to 58.00%. The error-filtering rate only increased from 14.14% to 17.13%. The error-filtering rate of fruiting strains increased from 25.52% to 54.54%. Therefore, an excessively high screening threshold will lose more than half of strains with fruiting ability, but it can raise more than 2 times efficiency of cultivation and screening, and the number of constructed breeding populations could greatly increase.

The artificial substitute cultivation of L. edodes requires a long process from the mycelial growth to the fruit body formation, and some special changes relative to other edible fungi occur in it, such as the cultivation of L. edodes relies heavily on sawdust substrates. In mycelial growth stage, the mycelial mat will be formed on the surface of the artificial log, which is used to breed the primordia and form fruit body in the later stage. In addition, the mycelial mat will also change color in the later stage of culture, forming a brown protective film for moisture retention, resistance to other microorganisms and adverse conditions. In these relatively special processes, laccase seems to be able to play a key role in it, such as the degradation of lignin, the formation of the brown film, and the occurrence and development of fruit body (Leatham & Stahmann, 1981; Vetchinkina, Gorshkov, Ageeva, Gogolev, & Nikitina, 2015).

At present, there are many methods to determine fungal laccase activity. In 1983, Badiani et al. established high performance liquid chromatography (HPLC) method to detect laccase activity. This method has the characteristics of high accuracy, short time and high cost (Badiania, Felicia, Luna, & Artemia, 1983). In 1995, Srinivasan et al. established a method for the determination of enzyme activity by plate using ABTS as the substrate, which could only qualitatively analyze the enzyme activity (Srinivasan, D'Souza, Boominathan, & Reddy, 1995). Spectrophotometric method is the most popular methods to determine laccase activity because of its simple operation, low cost and short time consuming (Hatvani & Mécs, 2001; Hublik & Schinner, 2000; Meza, Auria, Lomascolo, Sigoillot, & Casalot, 2007; Murugesan, Nam, Kim, & Chang, 2007), but the original method is not suitable for the detection of large quantities of strains. A large number of hybrid progenies strains needs to be tested for laccase activity, this study first developed a method for laccase detection based on the agar diffusion. The prime laccase detection method was innovated and improved, and the optimal diffusion and reaction time was 8 hours, and stable results were obtained in 163 strains tested in this research. Compared with other common detection methods, this method is simpler and faster, and can be applied to the detection of laccase activity in large quantities of strains.

Different strains of L. edodes, especially new strains obtained through hybridization of spore monokaryons, may have different phenotypes in the traits of the above-mentioned hybrids due to meiosis recombination and exchange of basidiospores. The proportion of strains with fruiting ability (The primordia can be formed in the later cultivation process, indicating that it has fruiting ability, otherwise it does not have fruiting ability) in the hybrid progenies of L. edodes is low, which adds great difficulty to the breeding work. The numerous quantities and types of laccase and their different expression and secretion methods also determine its extensive role in L. edodes. In this study, the laccase activity profiles of strains with and without fruiting ability cultured on different media for different days were constructed, it was found the laccase activity profiles on different media have certain characteristics. On PDA medium with vanillic acid as an inducer, only a few strains induced higher laccase production than that of non-induced PDA medium. Many strains did not show an increase in laccase activity, even show an inhibited production of laccase. Vanillic acid, as a laccase inducer, induces a variety of edible fungi, but has not been reported in L. edodes (Galhaup & Haltrich, 2001; Hou, Zhou, Jing, Du, & Yan, 2004; Piscitelli et al., 2011; Souza, Tychanowicz, Souza, & Peralta, 2004). The findings of this study indicate that an inducer substrate will also show different effects on different strains of the same species. Sequence comparison and transcription analysis of several fungi demonstrated that laccases were encoded by multigene families. The laccase genes not only vary between different species, but also between different varieties (Madhavan, Krause, Jung, & Kothe, 2014; Saparrat, Balatti, Martínez, & Jurado, 2010; Li et al., 2016; Sakamoto et al., 2015). Our results also showed that the enzyme activities of different L. edodes varieties were different under the same culture conditions. Some strains have high activity on agar medium but low activity on sawdust-based medium. This may be due to different laccase genes in different varieties suggest the differences in their physicochemical and regulatory mechanisms, especially their physiological roles in response to nutrition change and environmental stress (Hua et al., 2018; Wong, Cheung, Au, & Kwan, 2013). Laccases in L. edodes respond to stresses such as heat and carbon sources (Cai, Gong, Wei, Yue, & Bian, 2017; Wang et al., 2018). The analysis results of 14 laccase genes in the draft genome sequence of L. edodes strain w1-26 also showed that all laccase genes were significantly regulated by varying carbon source materials. The expression of only two laccase genes was induced by sodium-lignosulphonate and the expression of most laccase genes was specifically upregulated in glucose medium (Yan, Xu, Bian, Li, & Zhou, 2019). Some strains without fruiting ability could be eliminated according to the type of profile. Among the 36 strains of ‘SH’ hybrid progenies, according to the profile characteristics, the proportion of strains without fruiting ability that could be significantly eliminated on non-induced PDA, VA-PDA and sawdust-based substrates accounted for 30%, 48% and 25% of strains without fruiting ability respectively. Previous studies have also reported that the laccase activity in the vegetative growth stage was closely related to the time when L. edodes entered the reproductive stage. The greater the enzyme activity, the earlier the brown film formation and primordium initiation (Zhang, 2016). This indicated that the laccase activity of mycelium could be used as a screening index for the fruiting ability of strains in the later stage. Through correlation analysis between laccase activity of mycelium cultured for different days on different media and fruiting ability, we found that the fruiting ability had a significant positive correlation with the laccase activity on non-induced PDA medium in the culture period of 4-8 d. On the 8th d, the difference of laccase activity among strains was the most significant.

From the verification results of five newly constructed hybrid populations, we found correlation degree between laccase activity and fruiting ability in different breeding populations was different, the corresponding screening efficiency was different. In addition, the laccase activity threshold used for screening would also cause certain degrees of error-filtering. An excessively high screening threshold will lose more than half of the fruiting strains, but it can raise more than 2 times efficiency of cultivation and screening. The selection of enzyme activity threshold should be based on the size of the constructed population and the corresponding screening capability.

Based on the above results, a schematic diagram of early screening method for fruiting ability of L. edodes hybrid progenies was drawn (Fig. 5). This screening method could improve the breeding efficiency, not only save the economic costs, but also speed up the breeding process of L. edodes.

Fig. 5. Schematic diagram of early screening method for fruiting ability of Lentinula edodes hybrid progenies.

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

The Supplementary Fig. S1 showed the plates before and after detection. Supplementary Fig. S2 showed the characteristic curves of laccase activity changes of 36 strains on non-induced PDA plates with increasing culture days. Supplementary Fig. S3 showed four different profiles of the laccase activities variation curves of all the test strains. The laccase activity variation curve of the test strains after growing in test tubes of sawdust-based substrate for different days was shown in Supplementary Fig. S4. Supplementary Table S1 showed laccase activity on non-induced PDA and fruiting ability of 127 strains of the five newly constructed hybrid populations.

This work was financially supported by National key Research and development plan (2019YFD1001905 - 31), China Agriculture Research System (CARS20), SAAS Program for Excellent Research Team.

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