Study on the mechanisms of sporocarp formation of Laccaria japonica colonising the seedlings of Pinus densiflora

概要

In forest ecosystems, ectomycorrhizal (ECM) fungi commonly form a symbiotic relationship with various species of higher plants. ECM fungi gain carbon and other essential substances from host plants, and in return, help host plants take up minerals, water, and metabolites. ECM fungi can also fight against parasites, soil pathogens and interact with other microbes beneficial for plants. Moreover, many ECM fungi can produce sporocarps, which are considered culinary delicacies with high medicinal or nutritional values. However, a number of in-demand and commercially valuable ECM sporocarps such as Tricholoma matsutake and Boletus species (Porcini) cannot be cultivated under the controlled conditions but form in forests.

The environmental factors including temperature, light, soil humidity and nutrient availability as well as interactions with other microbes in the soils, are fundamentally important for growth and sporocarp formation of ECM fungi. The co-occurring fungi and bacteria in the soils may affect the mycorrhizal rates and mycelial growth of the other ECM fungi due to the competition for the infection ability and mycelial growth. Soil nutrient is also an important factor influencing species richness or abundance in fields and sexual reproduction of ECM fungi, thus affecting the sporocarp formation of ECM fungi. However, the impact of co-colonising ECM fungi or soil nutrients on sporocarp formation is still poorly understood. Hence it is important to understand how the detectable changes at the different stages of sporocarp formation of ECM fungi accompanied by profound changes in the genes that regulate the induction, development and maturation of sporocarps.

In this dissertation, the effects of co-colonising ECM fungi (Cenococcum geophilum, Pisolithus sp., and Suillus luteus) and nitrogen (N) application on mycorrhizal colonisation and sporocarp formation of Laccaria japonica colonising Pinus densiflora seedlings were investigated. The genes expressed specifically during the primordial sporocarp induction and formation of L. japonica were also analyzed by using next-generation sequencing.

Effects of co-colonising ECM fungi on sporocarp formation in L. japonica colonising seedlings of P. densiflora
I examined host plant growth, mycorrhizal colonisation and sporocarp formation when roots of P. densiflora were colonised by L. japonica (Lj) and three other ECM fungal species [Cenococcum geophilum (Cg), Pisolithus sp. (PS), and Suillus luteus (Sl)]. Each pot contains four seedlings as follows: one seedling inoculated with Lj + three non-mycorrhizal (NM) seedlings (Lj+NM), one seedling inoculated with Lj + one with other ECM fungus + two NM seedlings (Lj+Cg, Lj+PS, Lj+Sl, respectively) or four NM seedlings for control. Sporocarp numbers were recorded throughout the experimental period. The biomass, photosynthetic rate, and mycorrhizal colonisation rate of the seedlings were also measured at 45 days (before Lj primordia appeared), 62 days (when Lj sporocarps first appeared), and 1 year after seedlings were transplanted. Results indicated that C. geophilum and S. luteus may have negatively impact on mycorrhizal colonisation and sporocarp formation in L. japonica. Sporocarp formation in L. japonica was positively correlated with conspecific mycorrhizal colonisation, but negatively correlated with the biomass of seedlings of P. densiflora. The co-occurring ECM fungi largely competed with L. japonica, resulting in various effects on mycorrhizal colonisation and sporocarp formation in L. japonica. A variety of mechanisms may be involved in the competitive interactions among the different ECM fungal species, including abilities to more rapidly colonise root tips, acquire soil nutrients, or produce antibiotics.

Effects of nitrogen application on sporocarp formation of L. japonica
Two experiments were conducted in this study. Firstly, the effect of N application on the growth of mycelia of L. japonica was investigated in vitro. The fresh cultured mycelium of L. japonica was transferred to 20 ml MMN (modified Melin-Norkrans) agar media containing the different concentrations of N (0, 5, 25, 50, 100 and 200 mg N/L, added as NH4NO3). After incubation for one month, the growth area and dry weight of mycelia of L. japonica were determined. The area of mycelial growth of L. japonica was highest for the treatment of 50 mg N/L and was significantly inhibited when the concentration of N was higher than 100 N mg/L. The dry weight of mycelia also increased with increasing N concentration up to 50 N mg/L. However, the dry weight was constant in the range of N concentration from 50 to 100 mg N/L and then markedly decreased at the treatment of 200 mg N/L. From the results it could be speculated that the high concentration of N may affect the morphology of mycelium of L. japonica and change the degree of compactness of mycelium. Secondly, the effect of N application on mycorrhizal colonisation and sporocarp formation was investigated in pots under a controlled condition. The seedlings of P. densiflora were inoculated with the L. japonica. The 45-day old Lj-mycorrhizal seedlings and non-mycorrhizal (NM) seedlings were transplanted to each pot containing three seedlings as follows: one seedling colonised with Lj and two NM seedlings (Lj+NM); or three NM seedlings for control. The seedlings were watered by the nutrient solutions containing 0, 5, 25, 50, and 100 mg N/L at the interval of 3–4 days, respectively. After 4 months of transplanting, the total amount of N addition in the corresponding N treatments were 0, 3.15, 15.75, 31.50, and 63.00 mg N/pot, respectively. The seedlings were harvested at 45 days (primordium initial appearing), 65 days (sporocarp appearing on the surface of the substrate), and 4 months after transplanting, respectively. The results showed that the sporocarp numbers of L. japonica increased with the N supply from 0 to 100 mg N/L, while the biomass of sporocarp of L. japonica significantly decreased at the highest level of treatment of 100 mg N/L. I examined the mycorrhizal colonisation rates of two formerly NM seedlings. During the experimental period, the mycorrhizal colonisation rate in the formerly NM seedlings significantly increased when the N supply was increased to 25 mg N/L. There was no significant difference of mycorrhizal colonization rate between the treatments 25 and 50 mg N/L. However, the mycorrhizal formation was significantly inhibited in the treatment of 100 mg N/L. The mycorrhizal colonization rate in the treatment of 100 mg N/L was significantly lower than those in the treatments of 25 mg N/L and 50 mg N/L. Mycorrhizal root tip in the formerly NM seedlings was not found in any treatment without N supply after 45 days and 65 days of transplanting. The N supply did not affect the dry weight of NM seedlings in control treatments. However, the dry weight of Lj-colonised seedlings was significantly higher in the treatments of higher N supply (i.e., 50 and 100 mg N/L) after 4 months of transplanting. With the N supply increased to 50 mg N/L, the total N content and C/N ratio of seedlings of P. densiflora significantly increased and decreased, respectively. There was no difference of C/N ratio of seedlings between the treatments of 50 and 100 mg N/L, while the total N content in seedling in the treatment of 100 mg N/L was higher than that in the treatment of 50 mg N/L. A similar trend of change in the total N content and C/N ratio was observed in the sporocarps of L. japonica

The number and the dry weight of the sporocarps were significantly positively correlated with total increased of dry weight, the net photosynthetic rate and total N content of P. densiflora seedlings at 4 months post-transplant, respectively. There was a similar relationship between the dry weight of sporocarp and the mycorrhizal rates. The number and the dry weight of the sporocarps were significantly negatively correlated with C/N ratio of P. densiflora seedlings at 4 months post-transplant.

These results indicated that the N concentrations affecting the growth of the mycelia in vitro and the sporocarp formation of L. japonica in the pot experiment were similar. The improvement of growth and mycorrhizal rates of seedlings of P. densiflora by N supply under the condition of low-N concentrations were responsible for the enhancement of the sporocarp formation of L. japonica.

Comparative transcriptome analysis of genes expressed specifically during the sporocarp formation of L. japonica
To investigate the molecular mechanisms of primordium and sporocarp formation of L. japonica, transcriptome sequencing and comparative analysis of mycelia were conducted in this study. RNA samples were extracted from the materials of free-living mycelial stage (FM), the stage of the mycelia in the Ljmycorrhizal roots before primordia appearing (BP) and the stage of mycelia in the Lj-mycorrhizal roots after primordia initial appearing (PI), and used for sequencing and data analysis. Results showed that 2053 genes were differentially expressed genes (DEGs). Among these genes, 652 and 755 of DEGs were upregulated, and 584 and 902 of DEGs were down-regulated in BP and PI compared to FM, respectively. Comparing with PI, 230 and 76 of DEGs were up-regulated and down-regulated in BP. In addition, 88 of DEGs were co-expressed in FM, BP and PI, of which 66 and 22 of DEGs were up-regulated in BP and PI samples, respectively

The functional annotation and enrichment analyses of DEGs showed that one gene encoding aquaporin related protein and five genes encoding heat shock proteins had strongly higher expression levels in BP stage than those in PI stage. Two genes involved in the pathways of fatty acid metabolism and many genes related to mitochondrial proteins had higher expression levels in PI stage than those in BP stage. In addition, the DEGs were homologous to genes encoding mitochondrial ADP/ATP carrier proteins and ubiquitinrelated proteins. Meanwhile, the genes participating in nitrogen metabolism also had high expression levels in the BP and PI stage. Some DEGs were selected for quantitative real-time reverse transcription PCR (qRTPCR) to verify the results of transcriptome analysis, and the trend analysis was consistent with the data of transcriptome sequencing. These results suggest that the sporocarp formation of L. japonica is a complicated process, and a series of genes encoding aquaporin related proteins, fatty acid synthase and heat shock proteins may participate in the sporocarp formation of L. japonica

In the present study, from the analysis of factors influencing sporocarp formation of ECM fungus, L. japonica, it can be concluded that the ability to colonise root tips, acquire soil nutrients, or produce antibiotics is one of the key factors determining the sporocarp formation and production of ECM fungus when there are several ECM fungi co-occurring in the field. The N concentration (50 mg N/L) that is favorable to the growth of mycelia in vitro, and the sporocarp formation of L. japonica in the pot experiment is similar. In the soils which lack N nutrient, the suitable N supply can enhance the sporocarp formation of L. japonica, but the sporocarp formation can be inhibited by oversupply of nitrogen. From the comparative transcriptome analysis, a series of genes encoding aquaporin related proteins, fatty acid synthase and heat shock proteins may underlie the sporocarp formation of L. japonica. These results are useful for artificial cultivation of sporocarps of edible ECM fungi.