Search

Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200 mg/L) on the growth of Laccaria japonica mycelia in vitro for 1 month. The results showed that L. japonica mycelial biomass was highest in the 50 mg/L treatment and was significantly inhibited at N concentrations higher than 200 mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100 mg N/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45 days (first appearance of primordia), 65 days (sporocarp appearance on the substrate surface), and 4 months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4 months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50 mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.

Ectomycorrhizal (ECM) fungi improve the host plant's tolerance to abiotic and biotic stresses. Cenococcum geophilum (Cg) is among the most common ECM fungi worldwide and often grows in saline environments. However, the physiological and molecular mechanisms of salt tolerance in this fungus are largely unknown. In the present study, 12 isolates collected from different ecogeographic regions were used to investigate the mechanism of salt tolerance of Cg. The isolates were classified into four groups (salt-sensitive, moderately salt-tolerant, salt-tolerant, and halophilic) based on their in vitro mycelial growth under 0, 50, 125, 250, and 500 mM NaCl concentrations. Hence, the Na, Ca, P, and K concentrations of mycelia and the pH of the culture solution were determined. Compared with salt-tolerant isolates, treatment with 250 mM NaCl significantly increased the sodium concentration and decreased the potassium concentration of salt-sensitive isolates. RNA-sequencing and qRT-PCR analysis were conducted to identify differentially expressed genes (DEGs) involved in transmembrane transport and oxidoreductase activity pathways. The hydrogen peroxide concentration and activities of peroxidase and superoxide dismutase in mycelia were determined, and the accumulation and scavenging of reactive oxygen species in the salt-sensitive isolates were more active than those in the salt-tolerant isolates. The results supply functional validations to RNA-seq and qRT-PCR analysis. This study provides novel insights into the salt-stress response of Cg isolates and provides a foundation for elucidation of the salt-tolerance mechanism of ECM fungi.

Sugi (Japanese cedar

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

Pollen allergy caused by sugi (Japanese cedar, Cryptomeria japonica) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of MS1 itself, selecting individuals with ms1-1 and ms1-2 male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of MS1 male-sterile sugi seedlings.

Forest trees are colonised by different species of ectomycorrhizal (ECM) fungi that interact competitively or mutualistically with one another. Most ECM fungi can produce sporocarps. To date, the effects of co-colonising fungal species on sporocarp formation in ECM fungi remain unknown. In this study, we examined host plant growth, mycorrhizal colonisation, and sporocarp formation when roots of Pinus densiflora are colonised by Laccaria japonica and three other ECM fungal species (Cenococcum geophilum, Pisolithus sp., and Suillus luteus). Sporocarp numbers were recorded throughout the experimental period. The biomass, photosynthetic rate, and mycorrhizal colonisation rate of the seedlings were also measured at 45days, 62days, and 1year after seedlings were transplanted. Results indicated that C. geophilum and S. luteus may negatively impact 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. These mechanisms need to be confirmed in further studies.

Flowering cherry is an extremely renowned ornamental tree, consisting of a variety of species and cultivars. Because cherry species have no strict barriers for interspecific hybridization before fertilization, identification of the gene underlying post-zygotic hybrid inviability will help breeders identify specimens for breeding and help us understand speciation mechanisms. In this study, we mapped the genetic linkages and physical genome sequences for a presumed hybrid inviability locus (HIs-1) that we observed in the seedlings crossed between Cerasus × yedoensis 'Somei-yoshino' and its wild relative C. spachiana. By the surveying linkage maps of 'Somei-yoshino' and C. spachiana, we identified correlation with seedling inviability only in linkage group 4 (LG4) of the 'Somei-yoshino' map. When we produced a finer-scaled map of HIs-1 in LG4, we found that HIs-1 is located between two microsatellite (SSR) markers with a 3.8 cM span. Using eight SSR markers based on peach genome sequences, we further refined the candidate region for HIs-1. This region was located between Pp04C001 and Pp04C007 markers, spanning 240 Kb of the peach genome, in which 45 transcribed genes had been estimated. From these candidate genes, it will be feasible to identify molecular mechanisms involved in cherry hybrid inviability.

Flavonoids, which modulate plant resistance to various stresses, can be induced by high light. B-box (BBX) transcription factors (TFs) play crucial roles in the transcriptional regulation of flavonoids biosynthesis, but limited information is available on the association of BBX proteins with high light. We present a detailed overview of 45 Populus trichocarpa BBX TFs. Phylogenetic relationships, gene structure, tissue-specific expression patterns and expression profiles were determined under 10 stress or phytohormone treatments to screen candidate BBX proteins associated with the flavonoid pathway. Sixteen candidate genes were identified, of which five were expressed predominantly in young leaves and roots, and BBX23 showed the most distinct response to high light. Overexpression of BBX23 in poplar activated expression of MYB TFs and structural genes in the flavonoid pathway, thereby promoting the accumulation of proanthocyanidins and anthocyanins. CRISPR/Cas9-generated knockout of BBX23 resulted in the opposite trend. Furthermore, the phenotype induced by BBX23 overexpression was enhanced under exposure to high light. BBX23 was capable of binding directly to the promoters of proanthocyanidin- and anthocyanin-specific genes, and its interaction with HY5 enhanced activation activity. We identified novel regulators of flavonoid biosynthesis in poplar, thereby enhancing our general understanding of the transcriptional regulatory mechanisms involved.

Many wild populations of Chamaecyparis pisifera (Cupressaceae) are recently fragmented and surrounded by coniferous plantations. To distinguish the species composition of pollens that are dispersed to female flowers of C. pisifera, genetic markers (Chaps-trnL01 and Chaps-trnL02) were developed based on the differences in trnL (UAA) intron region of chloroplast DNA. This region in C. pisifera contains the site of the Cla I restriction enzyme and allows the distinction of C. pisifera from other Cupressaceae species after digestion of the products with the enzyme resulting in the development of cleaved amplified polymorphic sequence (CAPS) markers. The availability of markers was determined by single-pollen genotyping. Amplification success rates of 75.0, 58.3, and 100% were obtained for the pollens of C. pisifera, Chamaecyparis obtusa, and Cryptomeria japonica, respectively. Then, the markers and genotyping methods were applied to airborne pollen grains sampled from a remnant natural stand of C. pisifera. Over 90% of the pollen grains were identified as belonging to C. obtusa. Thus, we concluded that abundant heterospecific pollen dispersal was present during the flowering periods of C. pisifera. These results suggest that the remnant C. pisifera population might suffer reproductive interference due to the lower chances to be pollinated with conspecific pollens.

Timing of acorn development and abortion is an important maternal strategy in oak sexual reproduction. To understand the significance of acorn abortion in Quercus serrata, artificial pollination of different mating patterns (outcross, self-, and nonpollination) was performed, and the timing of abortion and the size and internal development of acorns from each mating type were investigated. Acorns were aborted similarly in every pollination treatment until 80 days after pollination. Almost all self- and nonpollinated acorns were rapidly aborted during the period 80–120 days after pollination. During that period, differences in internal fruit development between outcross-pollinated and unsuitably pollinated (self- and nonpolinated) acorns were observed. In addition, cotyledon development and a rapid increase in fruit size were observed in the acorns gained by outcross pollination. The correspondence between the timing of abortion and the timing of rapid size growth and the development of storage organs suggests that this acorn-maintenance strategy may be the consequence of a maternal adaptation that allows better reproductive success under resource limitation.

Cerasus × yedoensis ‘Somei-yoshino’ (2n = 16), one of the most popular cultivars of Japanese flowering cherry, is considered a hybrid between Cerasus spachiana and Cerasus speciosa. In the present study, we observed segregation of the trait for seedling growth in F 1 progenies of Somei-yoshino. In all three families crossed with ancestral wild species, C. spachiana, approximately half of the seedlings showed growth failure. To identify loci involved in seedling inviability, we constructed a genetic linkage map of Somei-yoshino. Both normal growth (N = 77) and growth failure seedlings (N = 101) of an F 1 progenies crossed between Somei-yoshino (CY) and C. spachiana (E750) were used for map construction. In accordance to pseudo-testcross strategy, female (CY) and male (E750) linkage maps were constructed. The CY map consists of eight linkage groups with 59 simple sequence repeat (SSR) and 17 amplified fragment length polymorphism (AFLP) markers. The map length is 574.9 cM. In contrast, the E750 map reached 196.8 cM and included 17 SSRs. Subsequently, we explored positions of loci associated to seedling inviability along with linkage maps. Results of segregation distortion of markers, association analysis, and interval mapping consistently showed that an inviability gene was located between two SSR markers, EMPaS13 and BPPCT005 in the fourth linkage group (LG4) of the Somei-yoshino map with 99.6 % explanations for the inviability trait but not present in LG4 of the E750 map. From the genotypes and expressions of seedling inviability, we named the locus Hybrid Inviability of seedlings 1 (HIs1). Finally, HIs1 was mapped to LG4 as Mendelian trait loci and was completely linked with an SSR, EMPaS13. Although the mechanisms of seedling inviability cannot be explained by only one locus model of HIs1, one of the candidate genes involved in hybrid incompatibility between Somei-yoshino and C. spachiana is certainly located on LG4 of the CY map.