Ectomycorrhizal community associated with Cedrus deodara in four urban forests of Nantong in East China.

Ectomycorrhizal (ECM) fungi play fundamental roles in host plant growth and terrestrial ecosystems. Cedrus deodara is cultivated in several regions in China, has high ecological, economic and medicinal value, for its afforestation and providing timber and wood oil. Here, we investigated ECM colonization status of four urban C. deodara forests in Nantong, East China. We also characterized soil spore banks by conducting bioassay experiments using soils collected from these forests. In total, we identified 19 ECM fungal species, of which 13 species were found in mature forests and 9 species were identified in bioassay experiments, with only 3 species shared. Soil pH and available P content had significant effects on species occurrence in both mature trees and bioassay seedlings on local scales. ECM communities clearly (A = 0.391, p = 0.006) separated mature forests from spore banks. Thelephoracae was the richest family we detected associated with C. deodara, while Trichophaea sp. was the most dominant in mature forests, and Wilcoxina sp. was dominant in spore banks. ECM richness affected the growth of bioassay seedlings, especially after inoculation with 2 ECM species, promoting root growth, significantly (F = 3.028, p = 0.050), but it had no effects on shoots (F = 1.778, p = 0.177). No effect of inoculation rate was found on seedlings growth. To conserve this important tree species, the ECM fungi that are associated with it should be considered.

Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor.

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification. The increasing numbers of available genomic sequences of ectomycorrhizal (ECM) fungi enable deeper insights into the characteristics of MT genes in these fungi that form the most important symbiosis with the host trees in forest ecosystems. The aim of this study was to establish a comprehensive, genome-wide inventory of MT genes from the ECM fungus Laccaria bicolor. Eight MT genes in L. bicolor were cloned, and the expression patterns of their transcripts at various developmental stages based on expressed sequence tag (EST) counts were analyzed. The expression levels of four MTs were significantly increased during symbiosis stages. Quantitative real-time PCR (qRT-PCR) analysis revealed that transcripts of LbMT1 were dominant in free-living mycelia and strongly induced by excessive copper (Cu), cadmium (Cd), and hydrogen peroxide (H2O2). To determine whether these eight MTs functioned as metal chelators, we expressed them in the Cu- and Cd-sensitive yeast mutants, cup1∆ and yap1∆, respectively. All LbMT proteins provided similar levels of Cu(II) or Cd(II) tolerance, but did not affect by H2O2. Our findings provide novel data on the evolution and diversification of fungal MT gene duplicates, a valuable resource for understanding the vast array of biological processes in which these proteins are involved.

Diversity and community structure of ectomycorrhizal fungi in Pinus thunbergii coastal forests bordering the Yellow Sea of China.

Ectomycorrhizas play a fundamental role in the function of forest ecosystems, being essential for plant nutrition absorption and soil quality. Many afforestation and reforestation programmes have begun to recover and maintain coastal forests in China, using pine species including Pinus thunbergii. We investigated the ectomycorrhizal colonization status of P. thunbergii in coastal pine forests of the Yellow Sea of China. We identified a total of 53 ectomycorrhizal fungal species in 74 soil samples collected from three sites and found that Thelephoraceae (10 spp.) and Russulaceae (8 spp.) were the most species-rich ectomycorrhizal fungal lineages. Russula sp. 1 was the most abundant species, accounting for 15.3% of the total ectomycorrhizal tips identified. Most of the remaining species were rare. At this small scale, host identity had no significant effect on the ectomycorrhizal fungal community composition (A = 0.036, P = 0.258), but sampling sites did (A = 0.135, P = 0.041). In addition, Na+ and K+ content and soil pH had significant effects on the ectomycorrhizal fungal community. The ectomycorrhizal fungal community associated with different host plants will become an important new direction for research, as ectomycorrhiza may have the potential to improve host capacity to establish in salt-stressed environments. This will provide a theoretical basis and technical support for saline soil reforestation and rehabilitation using pine species with compatible, native ectomycorrhizal fungi in Yellow Sea coastal areas.

Comparative transcriptomic analysis reveals novel insights into the response to Cr(VI) exposure in Cr(VI) tolerant ectomycorrhizal fungi Pisolithus sp. 1 LS-2017.

Chromium (Cr) is one of the most toxic heavy metals and a health hazard to millions of people worldwide. Ectomycorrhizal (ECM) fungi can assist plants in phytoremediation of heavy metal contaminated soil. Cr tolerance differs among ECM fungal varieties, but the underlying molecular mechanisms of Cr tolerance in ECM fungi are not clear. This study identified, analysed and compared the Cr(VI)-induced transcriptional changes between Cr(VI)-tolerant strain (Pisolithus sp. 1 LS-2017) and Cr(VI)-sensitive strain (Pisolithus sp. 2 LS-2017) by de novo transcriptomic analysis. The results showed that 93,642 assembled unique transcripts representing the 22,353 (46.76%) unigenes matched the proteins we have known in the Nr database and 47,801 unigenes were got from the Pisolithus spp. For DEGs between the control and 10 mg/L Cr(VI) treatment, cyanoamino acid metabolic, type I diabetes mellitus metabolism, nitrogen metabolism and beta-Alanine metabolism pathways were significantly enriched (p < 0.05) in Pisolithus sp. 1 LS-2017. Two nitrate reductase family genes (nidD, niiA) provide Cr(VI) tolerance for Pisolithus sp. 1 LS-2017 by regulating Cr(VI) reduction. In addition, NO produced by nidD, niiA regulated denitrification can alleviate Cr(VI) induced oxidative stress. In Pisolithus sp. 2 LS-2017, the alcC, aldA and lcf2 gene may alleviate Cr(VI) induced oxidative stress by protecting SH groups and increasing secondary metabolism, reducing detoxify aldehydes to carboxylic acids and producing LCPUFAs respectively; .T gene regulate Cr(VI) induced wound healing by pigmentation and stability of melanin in spore; MKP2 gene accelerate Cr(VI) induced cell death and gpmA gene regulated Cr(VI) induced energy emergency.

Hydrogen ions and organic acids secreted by ectomycorrhizal fungi, Pisolithus sp1, are involved in the efficient removal of hexavalent chromium from waste water.

Pisolithus sp1 is an ectomycorrhizal (ECM) fungi that was chosen during a screening test of six strains of ECM fungi due to its ability to tolerate and remove hexavalent chromium (Cr(VI)). The physiological responses of Pisolithus sp1 to Cr(VI) exposure, the relationship between Pisolithus sp1 and exogenously added organic acids (EAOAs) or Na3VO4 (H+-ATPase inhibitor) and the ability of Pisolithus sp1 to reduce Cr(VI) in liquid culture were also investigated. Hydrogen ions (H+), which were produced directly by Pisolithus sp1, reduced the pH of the medium and played an important role in Cr(VI) reduction; however, Na3VO4 significantly inhibited this process and resulted in a decrease in the Cr(VI) reduction rates. Organic acids were secreted after the reduction in Cr(VI) by Pisolithus sp1, and EAOAs did not significantly affect Cr(VI) reduction; those results revealed the secondary role of organic acids in Cr(VI) reduction. The Cr(VI) removal rate of Pisolithus sp1 approached 99% after Cr(VI) treatment for 12 days. Overall, 75% of the Cr(VI) removal was due to extracellular reduction and 24% was due to adsorption. The results of this study provide a strong basis for using Cr(VI)-tolerant and Cr(VI)-reducing fungi, as well as ectomycorrhiza, in the remediation of Cr(VI)-contaminated sites.

Soil spore bank communities of ectomycorrhizal fungi in endangered Chinese Douglas-fir forests.

Chinese Douglas-fir (Pseudotsuga sinensis) is an endangered Pinaceae species found in several isolated regions of China. Although soil spore banks of ectomycorrhizal (ECM) fungi can play an important role in seedling establishment after disturbance, such as in the well-known North American relative (Pseudotsuga menziesii), we have no information about soil spore bank communities in relict forests of Chinese Douglas-fir. We conducted bioassays of 73 soil samples collected from three Chinese Douglas-fir forests, using North American Douglas-fir as bait seedlings, and identified 19 species of ECM fungi. The observed spore bank communities were significantly different from those found in ECM fungi on the roots of resident trees at the same sites (p = 0.02). The levels of potassium (K), nitrogen (N), organic matter, and the pH of soil were the dominant factors shaping spore bank community structure. A new Rhizopogon species was the most dominant species in the spore banks. Specifically, at a site on Sanqing Mountain, 22 of the 57 surviving bioassay seedlings (representing 21 of the 23 soil samples) were colonized by this species. ECM fungal richness significantly affected the growth of bioassay seedlings (R 2 = 0.20, p = 0.007). Growth was significantly improved in seedlings colonized by Rhizopogon or Meliniomyces species compared with uncolonized seedlings. Considering its specificity to Chinese Douglas-fir, predominance in the soil spore banks, and positive effect on host growth, this new Rhizopogon species could play critical roles in seedling establishment and forest regeneration of endangered Chinese Douglas-fir.

Effects of Pisolithus tinctorius and Cenococcum geophilum inoculation on pine in copper-contaminated soil to enhance phytoremediation.

We used Pisolithus tinctorius and Cenococcum geophilum to determine the copper (Cu) resistance of ectomycorrhizal (ECM) fungi and their potential for improving phytoremediation of Cu-contaminated soil by Chinese red pine (Pinus tabulaeformis). The results showed that nutrient accumulation in C. geophilum mycelium was significantly lower under higher Cu concentrations in the soil, which was not observed in P. tinctorius. Meanwhile, P. tinctorius exhibited greater Cu tolerance than C. geophilum. Inoculation with ECM fungi significantly improved the growth of pine shoots planted in polluted soil in pot experiments (p < 0.01). The total accumulated Cu in pine seedlings planted in Cu-contaminated soil increased by 72.8% and 113.3% when inoculated with P. tinctorius and C. geophilum, respectively, indicating that ECM fungi may help their host to phytoextract heavy metals. Furthermore, the majority of the total absorbed metals remained in the roots, confirming the ability of ECM fungi to promote heavy metal phytostabilization. There were no differences between the effects of the two fungi in helping the host stabilize and absorb Cu, even though they have different Cu tolerances. Inoculation with ECM fungi can benefit plant establishment in polluted environments and assist plants with phytoremediating heavy-metal-contaminated soils.

Growth and photosynthetic responses of ectomycorrhizal pine seedlings exposed to elevated Cu in soils.

It is still controversial whether ectomycorrhizal (ECM) mycelia filter out toxic metals in nutrient absorption of host trees. In this study, pine (Pinus densiflora) seedlings colonized by Cu-sensitive and Cu-tolerant ECM species were exposed to a wide spectrum of soil Cu concentrations to investigate functions of ECM fungi under Cu stress. The photosynthetic rates of intact needles were monitored in situ periodically. The biomass and elements of plants were also measured after harvest. The ameliorating effect of ECM infection on host plants exposed to toxic stress was metal concentration specific. Under lower-level Cu stress, ECM fungi increased seedling performance, while ECM seedlings accumulated more Cu than nonmycorrhizal (NM) seedlings. Under higher-level Cu stress, photosynthesis decreased well before visible symptoms of Cu toxicity appeared. The reduced photosynthesis and biomass in ECM seedlings compared to NM seedlings under higher Cu conditions were also accompanied by lower phosphorus in needles. There was no marked difference between the two fungal species. Our results indicate that the two ECM fungi studied in our system may not have an ability to selectively eliminate Cu in nutrient absorption and may not act as effective barriers that decrease toxic metal uptake into host plants.