Breeding of a high-yield strain for commercial cultivation by crossing Pholiota adiposa and P. limonella

Pholiota adiposa is an edible mushroom with excellent nutritional and medicinal properties. However, fruiting body yields are low, and the commercial cultivation potential of this fungus is limited. In the present study, 279 crossbred strains were obtained by mono-mono crossing of monokaryotic strains derived from P. adiposa HS5 and P. limonella HS4. Ligninolytic enzymes and mycelial growth rate were used as markers to screen the crossbred strains, and 18 were selected for further analysis. Crossbred strain A10B4 displayed the highest yield, i.e., 165.91 ± 12.56 g per bag, which was 31.34 g and 74.48 g more than that of strains HS5 and HS4, respectively. The mycelial colonization time of A10B4 was 25.18 ± 1.33 days, which was 5.64 days shorter than that of HS5. A10B4 was characterized by inter-simple sequence repeat molecular markers and antagonism tests. Differences in PCR products from parental and crossbred strains were observed. Therefore, the newly developed hybrid strain A10B4, named P. adiposa-limonella HS54 and having a high yield and desirable traits, might be suitable for commercial cultivation.


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Pholiota adiposa (Batsch) P. Kumm. is an edible mushroom that is widely distributed on dead 31 poplars, willows, or birches in forested areas in China [1,2]. It is also a lignin-degrading owing to its delicious taste and beneficial properties [7,8].

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Mushroom breeding involves various methods, but crossbreeding is considered the most 38 efficient way to develop new, good quality strains of edible mushrooms [9][10][11]. Intra-species 39 crossbreeding has been reported in Sparassis latifolia, Pleurotus tuoliensis, P. eryngii species etc Antagonistic activity tests

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Antagonistic activity assays were performed as previously described, with minor modifications (Xiang et al., 2016). Briefly, two parental strains and the putative hybrids were co-cultured at 25°C 93 in 9-cm PDA plates, with every two mycelial fragments placed 2 cm apart. Somatic incompatibility 94 reactions were observed after incubation for 10 days.

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Fruiting and cultivation methods

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18% sawdust, 15% wheat bran, 5% corn flour, 1% gypsum, and 1% lime and placed in 98 polypropylene bags (17 cm × 33 cm × 0.04 cm) at a packing density of 1,000 g of substrate per 99 bag. The bags were autoclaved at 121°C for 120 min. The inoculated bags were then maintained 100 in a spawn running room at 23-25°C and 50-60 relative humidity under dark conditions. After a 101 complete spawn run, mycelial differentiation was induced by stimulation at 0-5°C for 3-5 days.

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The bags were then transferred to a fruiting chamber that was maintained at 18 ± 2°C and a All data were statistically analyzed using SPSS PASW Statistics software version 18. All data 119 were obtained in triplicate, and differences were determined by Duncan's multiple range testing.

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One hundred and twenty-six and 103 monokaryons were isolated from P. adiposa HS5 and 124 P. limonella HS4, respectively. Monokaryons were selected for further study based on analysis of 125 mycelial growth rates and their abilities to decolorization of RBBR (Fig 1)

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Fruiting body production from crossbred strains

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The parental and 18 crossbred strains were cultured, and fresh weight, and mycelial 146 colonization time were compared between the strains (Table 4). Among the 18 crossbred strains,

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A20B3, A4B6, A16B3, and A16B6 showed abnormal fruiting bodies, and A5B1, A20B12, A6B6, 148 and A10B12 did not form fruiting bodies at all. The weight of the fresh fruiting body of parental 149 strain HS5 in one bag was 134.57 ± 5.45g, which was 43.14 g more than that of HS4 (Table 4).

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The BEs of HS5 and HS4 were 40.78 ± 1.65% and 27.71 ± 1.59%, respectively, in the first flush 151 (Table 4). Two crossbred strains (A10B4 and A14B4) were more productive than parental strain 152 HS5 (Fig 2), and five crossbred strains were more productive than parental strain HS4. The most 153 productive strain was A10B4, but the difference in productivity between A10B4 and A14B4 was 154 not significant. The mycelial colonization time of HS5 was 30.82 ± 0.98days, which was 6.1 days 155 longer than HS4 ( DNA from all strains (Fig 3). The size of the polymorphic fragments obtained ranged from 200 bp 163 to 5000 bp, and differences were observed in the number of bands obtained (Fig 3).