The results of ITS sequence alignment and pure culture of four kinds of Bolete
The mycelia of four bolete species were obtained through tissue isolation and purification. After two months of mycelium culture, the ITS sequences were acquired by amplifying the DNA of the mycelium, followed by BLAST alignment in GenBank. The ITS sequences of strains S1, S2, and R1 (OM846602, OM846601, OL339344) showed 99.69%, 99.32%, and 100% homology with the Chinese specimens of S. bovinus, S. luteus, and R. sinensis [19–21], respectively. The ITS sequence of S3 (OM865366) showed 99.03% homology with the Japanese S. grevillei[22]. The original fruiting bodies were sampled from coniferous and broad-leaved mixed forests in Yantai, China, at Xiaoyuanmiao Forest Farm and Trapped Mountain Forest Farm (Table 1). One representative strain was registered in the China General Microbiological Culture Collection Center (CGMCC No. 23887).
The three Bolete species have similar mycelial morphology. The mycelia are white and dense, primarily comprising creeping mycelium, and secreting brown substances during the later stages of culture (Fig. 1a-c). The mycelia of Retiboletus sinensis are light yellow, mainly consisting of erect mycelium, and also secreting brown substances during the later stages of culture. During the pure culture process, the mycelium was stimulated by temperature and scattered light. It could produce primordium and fruiting bodies independently from symbiotic plants. The primordium of the fruiting body was light yellow and millet grain-shaped, the pileus was light yellow during the early stage of fruiting body differentiation, and the stipe was white with villi (Fig. 1d-f).
Observation of mycorrhizal appearance morphology
The appearance of Bolete mycorrhiza was observed using a stereoscope. The mycorrhiza formed by the combination of four types of mushrooms and black pine (Fig. 2a-d) was predominantly binary branching structure, with a small amount of rod-like structure mycorrhiza. This rod-like structure was likely an unbranched or soon-to-be bifurcated binary branching structure. A small amount of hyphae were seen on the surface of the mycorrhiza, and there was no apparent fungal sheath. Mycorrhizal branch length ranged from 0.6 to 1.23 mm, with a thickness of 0.22 to 0.29 mm.
Observing the mycorrhizal fungi combined with Q. acutissima (Fig. 2e-h), it was found that they were all rod-like structures, and no two-branched structure similar to Q. acutissima mycorrhizal was found. The hyphae on the surface of mycorrhizal were visibly entangled. The length of mycorrhiza ranged from 0.33 to 0.96 mm, with a thickness of 0.05 to 0.18 mm. From an appearance perspective, the rod-shaped mycorrhizal size of Q. acutissima was significantly smaller than that of P. thunbergii, but the mycorrhizal density of Q. acutissima was much higher than that of P. thunbergii (Fig. 3, Fig. 4). The mycorrhizal morphology is not related to the species of boletes, but is determined by the species of symbiotic plants.
The mycorrhizal infection rates of four types of mushrooms (Suillus bovinus, S. luteus, S. grevillei, and Retiboletus sinensis) using liquid inoculation aseptic culture method were as follows: Pinus thunbergii − 49.7%, 54.97%, 52.3%, and 43.4%; Quercus acutissima − 54.83%, 59.6%, 48.43%, and 55.37%.
Hormone contents of Seedling
Compared to the uninoculated Quercus acutissima seedlings, the total IAA content in seedlings inoculated with four types of mushrooms (Suillus bovinus, Suillus luteus, Suillus grevillei, and Retiboletus sinensis) increased by 27.1%, 29.5%, 25.2%, and 30%, respectively(Fig. 10). Similarly, the total ZT content in inoculated seedlings increased by 25.4%, 34.8%, 34.6%, and 36.7%, respectively. Among various plant hormones, GA content exhibited the most significant change, with total GA content increasing by 86.3%, 95.1%, 95.7%, and 83.1%, respectively. In contrast to the changes in IAA, ZT, and GA, ABA content in inoculated seedlings was much lower than that in control seedlings, with total ABA content in roots, stems, and leaves decreasing by 22.7%, 23.3%, 23.1%, and 26.8%, respectively.
The ratio of total GA to total ABA (GA/ABA) in mycorrhizal seedlings was significantly higher than that in sterile root seedlings (p < 0.05). The GA/ABA ratio for sterile root seedlings was 0.31, whereas the GA/ABA ratios for mycorrhizal seedlings of Suillus bovinus, Suillus luteus, Suillus grevillei, and Retiboletus sinensis were 0.74, 0.78, 0.78, and 0.76, respectively, with no significant difference. The IAA/ABA ratios for mycorrhizal seedlings were 5.50, 5.64, 5.44, and 5.98, respectively, which were significantly higher than the 3.34 ratio observed in sterile seedlings. The ZT/ABA ratios for mycorrhizal seedlings were 4.55, 4.93, 4.91, and 5.24, respectively, which were significantly higher than the 2.81 ratio observed in sterile seedlings. The IAA/GA ratios for mycorrhizal seedlings were 7.46, 7.36, 6.99, and 7.83, respectively, which were significantly lower than the 10.95 ratio observed in aseptic seedlings. The IAA/ZT ratios for mycorrhizal seedlings were 1.21, 1.44, 1.11, and 1.14, with no significant difference compared to the 1.19 ratio observed in sterile root seedlings.
The contents of IAA, ZT and GA in Pinus thunbergii seedlings inoculated with 4 kinds of bolete increased, and the content of ABA was inhibited(Fig. 11). Compared with CK, the total content of IAA in seedlings inoculated with four kinds of bolete increased by 28.7%, 30.8%, 29.8% and 33% respectively. The total ZT content increased by 28.1%, 28.05%, 38.5% and 42.5%, respectively. The total GA content increased by 98.1%, 90%, 102.8% and 102%, respectively. On the contrary, the ABA content of Pinus thunbergii seedlings inoculated with four kinds of boletes was significantly lower than that of the control group, and the total content was reduced by 24.1%, 26.9%, 22.6% and 29%, respectively.
The ratio of total GA to total ABA ( GA/ABA ) in mycorrhizal seedlings was significantly higher than that in sterile root seedlings (p < 0.05). The ratio of GA / ABA in sterile root seedlings was 0.14, and the ratio of GA/ABA in mycorrhizal seedlings of Suillus bovinus, Suillus luteus, Suillus grevillei, Retiboletus sinensis was 0.38, and there was no significant difference. The IAA/ABA of mycorrhizal seedlings were 3.43,3.62,3.39,3.53, respectively, which were significantly higher than 2.02 of aseptic seedlings. The ZT/ABA of mycorrhizal seedlings were 2.56,2.66,2.55 and 2.84, respectively, which were significantly higher than 1.52 of sterile seedlings. The IAA/GA of mycorrhizal seedlings were 8.42,9.52,8.79 and 8.31, respectively, which were significantly lower than 15.96 of aseptic seedlings. The IAA/ZT of mycorrhizal seedlings were 1.34,1.36,1.32,1.24, and the sterile root seedlings were 1.33, with no significant difference.
Bolete mushrooms have a growth-promoting effect on plant seedlings (Quercus acutissima and Pinus thunbergii). After inoculation with bolete mushrooms, the seedlings' growth hormone levels undergo significant changes.
After inoculation with bolete mushrooms, the seedlings' indoleacetic acid (IAA), zeatin (ZT), and gibberellic acid (GA) levels generally increase, while abscisic acid (ABA) levels decrease. This suggests that bolete mushrooms form a symbiotic relationship with the plant seedlings, promoting plant growth. The seedlings' GA/ABA, IAA/ABA, and ZT/ABA ratios significantly increase. This implies that the ratio of growth-promoting hormones (such as GA, IAA, and ZT) to growth-inhibiting hormones (such as ABA) increases, potentially enhancing the growth rate of plant seedlings.
For mycorrhizal seedlings, the IAA/GA ratio significantly decreases, which may help regulate plant growth and maintain a balance between growth rate and structural development. However, there is no significant difference in the IAA/ZT ratio between the two types of seedlings, indicating that the relative proportion between growth hormone and cytokinin remains relatively stable.