Cenococcum geophilum impedes cadmium toxicity in Pinus massoniana by modulating nitrogen metabolism.

Nitrogen (N) is of great significance to the absorption, distribution and detoxification of cadmium (Cd). Ectomycorrhizal fungi (EMF) are able to affect the key processes of plant N uptake to resist Cd stress, while the mechanism is still unclear. Therefore, we explored potential strategies of Cenococcum geophilum (C. geophilum) symbiosis to alleviate Cd stress in Pinus massoniana (P. massoniana) from the perspective of plant N metabolism and soil N transformation. The results showed that inoculation of C. geophilum significantly increased the activities of NR, NiR and GS in the shoots and roots of P. massoniana, thereby promoting the assimilation of NO3- and NH4+ into amino acids. Moreover, C. geophilum promoted soil urease and protease activities, but decreased soil NH4+ content, indicating that C. geophilum might increase plant uptake of soil inorganic N. qRT-PCR results showed that C3 symbiosis significantly up-regulated the expression of genes encoding functions involved in NH4+ uptake (AMT3;1), NO3- uptake (NRT2.1, NRT2.4, NRT2.9), as well as Cd resistance (ABCC1 and ABCC2), meanwhile down-regulated the expression of NRT7.3, Cd transporter genes (HMA2 and NRAMP3) in the roots of P. massoniana seedlings. These results demonstrated that C. geophilum was able to alleviate Cd stress by increasing the absorption and assimilation of inorganic N in plants and inhibiting the transport of Cd from roots to shoots, which provided new insights into how EMF improved host resistance to abiotic stress.

Jasmonic acid's impact on Sedum alfredii growth and cadmium tolerance: A physiological and transcriptomic study.

Soil cadmium (Cd) pollution is escalating, necessitating effective remediation strategies. This study investigated the effects of exogenous jasmonic acid (JA) on Sedum alfredii Hance under Cd stress, aiming to enhance its phytoextraction efficiency. Initially, experiments were conducted to assess the impact of various concentrations of JA added to environments with Cd concentrations of 100, 300, and 500 µmol/L. The results determined that a concentration of 1 µmol/L JA was optimal. This concentration effectively mitigated the level of ROS products by enhancing the activity of antioxidant enzymes. Additionally, JA fostered Cd absorption and accumulation, while markedly improving plant biomass and photosynthetic performance. In further experiments, treatment with 1 µmol/L JA under 300 µmol/L Cd stress was performed and transcriptomic analysis unveiled a series of differentially expressed genes (DEGs) instrumental in the JA-mediated Cd stress response. These DEGs encompass not only pathways of JA biosynthesis and signaling but also genes encoding functions that influence antioxidant systems and photosynthesis, alongside genes pertinent to cell wall synthesis, and metal chelation and transport. This study highlights that JA treatment significantly enhances S. alfredii's Cd tolerance and accumulation, offering a promising strategy for plant remediation and deepening our understanding of plant responses to heavy metal stress.

Metal-non-tolerant ecotypes of ectomycorrhizal fungi can protect plants from cadmium pollution.

The application of mycorrhizal fungi as a bioaugmentation technology for phytoremediation of heavy metal (HM) contaminated soil has attracted widespread attention. In order to explore whether the adaptation of Pinus massoniana (P. massoniana) to metal polluted soil depends on the metal adaptation potential of their associated ectomycorrhizal fungi (ECMF), we evaluated the cadmium (Cd) tolerance of 10 ecotypes of Cenococcum geophilum (C. geophilum) through a membership function method, and P. massoniana seedlings were not (NM) or inoculated by Cd non-tolerant type (JaCg144), low-tolerant (JaCg32, JaCg151) and high-tolerant (JaCg205) isolates of C. geophilum were exposed to 0 and 100 mg·kg-1 for 3 months. The result showed that, each ecotype of C. geophilum significantly promoted the growth, photosynthesis and chlorophyll content, proline (Pro) content and the activity of peroxidase (POD) of P. massoniana seedlings, and decreased malonaldehyde (MDA) content and catalase (CAT) and superoxide dismutase (SOD) activity. The comprehensive evaluation D value of the tolerance to Cd stress showed that the order of the displaced Cd resistance of the four ecotypic mycorrhizal P. massoniana was: JaCg144 > JaCg151 > JaCg32 > JaCg205. Pearson correlation analysis showed that the Sig. value of the comprehensive evaluation (D) values of the strains and mycorrhizal seedlings was 0.077 > 0.05, indicating that the Cd tolerance of the the C. geophilum isolates did not affect its regulatory effect on the Cd tolerance of the host plant. JaCg144 and JaCg151 which are non-tolerant and low-tolerant ecotype significantly increased the Cd content in the shoots and roots by about 136.64-181.75% and 153.75-162.35%, indicating that JaCg144 and JaCg151 were able to effectively increase the enrichment of Cd from the soil to the root. Transcriptome results confirmed that C. geophilum increased the P. massoniana tolerance to Cd stress through promoting antioxidant enzyme activity, photosynthesis, and lipid and carbohydrate synthesis metabolism. The present study suggests that mental-non-tolerant ecotypes of ECMF can protect plants from Cd pollution, providing more feasible strategies for ectomycorrhizal-assisted phytoremediation.

The Ectomycorrhizal Fungi and Soil Bacterial Communities of the Five Typical Tree Species in the Junzifeng National Nature Reserve, Southeast China.

To explore the contribution of microorganisms to forest ecosystem function, we studied the ectomycorrhizal (ECM) fungal and soil bacterial community of the five typical tree species (Pinus massoniana, PM; Castanopsis carlesii, CC; Castanopsis eyrei, CE; Castanopsis fargesii, CF; and Keteleeria cyclolepis, KC) at the Junzifeng National Nature Reserve. The results indicated that the ECM fungal and soil bacterial diversity of CC and CF was similar, and the diversity rates of CC and CF were higher than those of PM, CE, and KC. Cenococcum geophilum and unclassified_Cortinariaceae II were the most prevalent occurring ECM fungi species in the five typical tree species, followed by unclassified_Cortinariaceae I and Lactarius atrofuscus. In bacteria, the dominant bacterial genera were Acidothermus, Bradyrhizobium, Acidibacter, Candidatus_Solibacter, Candidatus_Koribacter, Roseiarcus, and Bryobacter. EMF fungi and soil bacteria were correlated with edaphic factors, especially the soil pH, TP, and TK, caused by stand development. The results show that the community characteristics of ECM fungi and bacteria in the typical tree species of the Junzifeng National Nature Reserve reflect the critical role of soil microorganisms in stabilizing forest ecosystems.

Increased Tolerance of Massion's pine to Multiple-Toxic-Metal Stress Mediated by Ectomycorrhizal Fungi.

Pinus massoniana (Massion's pine), a pioneer tree species, exhibits restoration potential in polluted mining areas. However, the physiological and molecular mechanisms of ectomycorrhizal (ECM) fungi in Massion's pine adaptability to multiple-toxic-metal stress are still unclear. Hence, Massion's pine seedlings inoculated with two strains of C. geophilum, which were screened and isolated from a polluted mine area, were cultivated in mine soil for 90 days to investigate the roles of EMF in mediating toxic metal tolerance in host plants. The results showed that compared with the non-inoculation control, C. geophilum (CG1 and CG2) significantly promoted the biomass, root morphology, element absorption, photosynthetic characteristics, antioxidant enzyme activities (CAT, POD, and SOD), and proline content of Massion's pine seedlings in mine soil. C. geophilum increased the concentrations of Cr, Cd, Pb, and Mn in the roots of Massion's pine seedlings, with CG1 significantly increasing the concentrations of Pb and Mn by 246% and 162% and CG2 significantly increasing the concentrations of Cr and Pb by 102% and 78%. In contrast, C. geophilum reduced the concentrations of Cr, Cd, Pb, and Mn in the shoots by 14%, 33%, 27%, and 14% on average, respectively. In addition, C. geophilum significantly reduced the transfer factor (TF) of Cr, Cd, Pb, and Mn by 32-58%, 17-26%, 68-75%, and 18-64%, respectively, and the bio-concentration factor (BF) of Cd by 39-71%. Comparative transcriptomic analysis demonstrated that the differently expressed genes (DEGs) were mainly encoding functions involved in "transmembrane transport", "ion transport", "oxidation reduction process", "oxidative phosphorylation", "carbon metabolism", "glycolysis/gluconeogenesis", "photosynthesis", and "biosynthesis of amino acids." These results indicate that C. geophilum is able to mitigate toxic metals stress by promoting nutrient uptake, photosynthesis, and plant growth, thereby modulating the antioxidant system to reduce oxidative stress and reducing the transport and enrichment of toxic metals from the root to the shoot of Massion's pine seedlings.

Exogenous silicon promotes cadmium (Cd) accumulation in Sedum alfredii Hance by enhancing Cd uptake and alleviating Cd toxicity.

Soil Cadmium (Cd) pollution has become a serious environmental problem. Silicon (Si) plays key roles in alleviating Cd toxicity in plants. However, the effects of Si on mitigation of Cd toxicity and accumulation of Cd by hyperaccumulators are largely unknown. This study was conducted to investigate the effect of Si on Cd accumulation and the physiological characteristics of Cd hyperaccumulator Sedum alfredii Hance under Cd stress. Results showed that, exogenous Si application promoted the biomass, Cd translocation and concentration of S. alfredii, with an increased rate of 21.74-52.17% for shoot biomass, and 412.39-621.00% for Cd accumulation. Moreover, Si alleviated Cd toxicity by: (i) increasing chlorophyll contents, (ii) improving antioxidant enzymes, (iii) enhancing cell wall components (lignin, cellulose, hemicellulose and pectin), (iv) raising the secretion of organic acids (oxalic acid, tartaric acid and L-malic acid). The RT-PCR analysis of genes that involved in Cd detoxification showed that the expression of SaNramp3, SaNramp6, SaHMA2 and SaHMA4 in roots were significantly decreased by 11.46-28.23%, 6.61-65.19%, 38.47-80.87%, 44.80-69.85% and 33.96-71.70% in the Si treatments, while Si significantly increased the expression of SaCAD. This study expanded understanding on the role of Si in phytoextraction and provided a feasible strategy for assisting phytoextraction Cd by S. alfredii. In summary, Si facilitated the Cd phytoextraction of S. alfredii by promoting plant growth and enhancing the resistance of plants to Cd.

Effects of High Temperature-Triggered Transcriptomics on the Physiological Adaptability of Cenococcum geophilum, an Ectomycorrhizal Fungus.

High temperature stress caused by global warming presents a challenge to the healthy development of forestry. Cenococcum geophilum is a common ectomycorrhizal fungus (ECMF) in the forest system and has become an important fungus resource with application potential in forest vegetation restoration. In this study, three sensitive isolates of C. geophilum (ChCg01, JaCg144 and JaCg202) and three tolerant isolates of C. geophilum (ACg07, ChCg28 and ChCg100) were used to analyze the physiological and molecular responses to high temperature. The results showed that high temperature had a significant negative effect on the growth of sensitive isolates while promoting the growth of tolerant isolates. The antioxidative enzymes activity of C. geophilum isolates increased under high temperature stress, and the SOD activity of tolerant isolates (A07Cg and ChCg100) was higher than that of sensitive isolates (ChCg01 and JaCg202) significantly. The tolerant isolates secreted more succinate, while the sensitive isolates secreted more oxalic acid under high temperature stress. Comparative transcriptomic analysis showed that differentially expressed genes (DEGs) of six C. geophilum isolates were significantly enriched in "antioxidant" GO entry in the molecular. In addition, the "ABC transporters" pathway and the "glyoxylate and dicarboxylic acid metabolic" were shared in the three tolerant isolates and the three sensitive isolates, respectively. These results were further verified by RT-qPCR analysis. In conclusion, our findings suggest that C. geophilum can affect the organic acid secretion and increase antioxidant enzyme activity in response to high temperature by upregulating related genes.

Comparative Physiological and Transcriptome Analysis Provide Insights into the Response of Cenococcum geophilum, an Ectomycorrhizal Fungus to Cadmium Stress.

Cadmium (Cd) displays strong toxicity, high mobility, and cannot be degraded, which poses a serious threat to the environment. Cenococcum geophilum (C. geophilum) is one of the most common ectomycorrhizal fungi (ECMF) in the natural environment. In this study, three Cd sensitive and three Cd tolerant strains of C. geophilum were used to analyze the physiological and molecular responses to Cd exposure. The results showed that Cd inhibited the growth of all strains of C. geophilum but had a less toxic effect on the tolerant strains, which may be correlated to a lower content of Cd and higher activity of antioxidant enzymes in the mycelia of tolerant strains. Comparative transcriptomic analysis was used to identify differentially expressed genes (DEGs) of four selected C. geophilum strains after 2 mg/L Cd treatment. The results showed that the defense response of C. geophilum strain to Cd may be closely related to the differential expression of functional genes involved in cell membrane ion transport, macromolecular compound metabolism, and redox pathways. The results were further confirmed by RT-qPCR analysis. Collectively, this study provides useful information for elucidation of the Cd tolerance mechanism of ECMF.

The Transcriptional Responses of Ectomycorrhizal Fungus, Cenococcum geophilum, to Drought Stress.

With global warming, drought has become one of the major environmental pressures that threaten the development of global agricultural and forestry production. Cenococcum geophilum (C. geophilum) is one of the most common ectomycorrhizal fungi in nature, which can form mycorrhiza with a large variety of host trees of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. In this study, six C. geophilum strains with different drought tolerance were selected to analyze their molecular responses to drought stress with treatment of 10% polyethylene glycol. Our results showed that drought-sensitive strains absorbed Na and K ions to regulate osmotic pressure and up-regulated peroxisome pathway genes to promote the activity of antioxidant enzymes to alleviate drought stress. However, drought-tolerant strains responded to drought stress by up-regulating the functional genes involved in the ubiquinone and other terpenoid-quinone biosynthesis and sphingolipid metabolism pathways. The results provided a foundation for studying the mechanism of C. geophilum response to drought stress.

Deciphering Rhizosphere Microbiome Assembly of Castanea henryi in Plantation and Natural Forest.

Based on the importance and sensitivity of microbial communities to changes in the forest ecosystem, soil microorganisms can be used to indicate the health of the forest system. The metagenome sequencing was used to analyze the changes of microbial communities between natural and plantation Castanea henryi forests for understanding the effect of forest types on soil microbial communities. Our result showed the soil microbial diversity and richness were higher in the natural forests than in the plantation. Proteobacteria, Actinobacteria, and Acidobacteria are the dominant categories in the C. henryi rhizosphere, and Proteobacteria and Actinobacteria were significantly enriched in the natural forest while Acidobacteria was significantly enriched in the plantation. Meanwhile, the functional gene diversity and the abundance of functions in the natural forest were higher than that of the plantation. Furthermore, we found that the microbial network in the natural forests had more complex than in the plantation. We also emphasized the low-abundance taxa may play an important role in the network structure. These results clearly showed that microbial communities, in response to different forest types, provide valuable information to manipulate microbiomes to improve soil conditions of plantation.

Overexpression of PtrMYB121 Positively Regulates the Formation of Secondary Cell Wall in Arabidopsis thaliana.

MYB transcription factors have a wide range of functions in plant growth, hormone signaling, salt, and drought tolerances. In this study, two homologous transcription factors, PtrMYB55 and PtrMYB121, were isolated and their functions were elucidated. Tissue expression analysis revealed that PtrMYB55 and PtrMYB121 had a similar expression pattern, which had the highest expression in stems. Their expression continuously increased with the growth of poplar, and the expression of PtrMYB121 was significantly upregulated in the process. The full length of PtrMYB121 was 1395 bp, and encoded protein contained 464 amino acids including conserved R2 and R3 MYB domains. We overexpressed PtrMYB121 in Arabidopsis thaliana, and the transgenic lines had the wider xylem as compared with wild-type Arabidopsis. The contents of cellulose and lignin were obviously higher than those in wild-type materials, but there was no significant change in hemicellulose. Quantitative real-time PCR demonstrated that the key enzyme genes regulating the synthesis of lignin and cellulose were significantly upregulated in the transgenic lines. Furthermore, the effector-reporter experiment confirmed that PtrMYB121 bound directly to the promoters of genes relating to the synthesis of lignin and cellulose. These results suggest that PtrMYB121 may positively regulate the formation of secondary cell wall by promoting the synthesis of lignin and cellulose.

Variations of Escherichia coli O157:H7 Survival in Purple Soils.

Escherichia coli O157:H7 is a well-recognized cause of human illness. Survival of Escherichia coli O157:H7 in five purple soils from Sichuan Province was investigated. The dynamics of E. coli O157:H7 survival in purple soils were described by the Weibull model. Results showed that this model is suitable to fit survival curves of E. coli O157:H7 in purple soils, with the calculated td value (survival time needed to reach the detection limit of 100 CFU·g-1) ranging from 2.99 days to 26.36 days. The longest survival time of E. coli O157:H7 was observed in neutral purple soils (24.49 days), followed by alkalescent purple soil (18.62 days) and acid purple soil (3.48 days). The redundancy analysis (RDA) revealed that td values were significantly enhanced by soil nutrition (total organic carbon (OC), total nitrogen (TN), available potassium (AK) and the ratio of humic acid to fulvic acid (Ha/Fa)), but were significantly suppressed by iron and aluminum oxide.

Distance-dependent varieties of microbial community structure and metabolic functions in the rhizosphere of Sedum alfredii Hance during phytoextraction of a cadmium-contaminated soil.

The recovery of microbial community and activities is crucial to the remediation of contaminated soils. Distance-dependent variations of microbial community composition and metabolic characteristics in the rhizospheric soil of hyperaccumulator during phytoextraction are poorly understood. A 12-month phytoextraction experiment with Sedum alfredii in a Cd-contaminated soil was conducted. A pre-stratified rhizobox was used for separating sub-layer rhizospheric (0-2, 2-4, 4-6, 6-8, 8-10 mm from the root mat)/bulk soils. Soil microbial structure and function were analyzed by phospholipid fatty acid (PLFA) and MicroResp™ methods. The concentrations of total and specified PLFA biomarkers and the utilization rates for the 14 substrates (organic carbon) in the 0-2-mm sub-layer rhizospheric soil were significantly increased, as well as decreased with the increase in the distance from the root mat. Microbial structure measured by the ratios of different groups of PLFAs such as fungal/bacterial, monounsaturated/saturated, ratios of Gram-positive to Gram-negative (GP/GN) bacterial, and cyclopropyl/monoenoic precursors and 19:0 cyclo/18:1ω7c were significantly changed in the 0-2-mm soil. The PLFA contents and substrate utilization rates were negatively correlated with pH and total, acid-soluble, and reducible fractions of Cd, while positively correlated with labile carbon. The dynamics of microbial community were likely due to root exudates and Cd uptake by S. alfredii. This study revealed the stimulations and gradient changes of rhizosphere microbial community through phytoextraction, as reduced Cd concentration, pH, and increased labile carbons are due to the microbial community responses.

Classification and identification of metal-accumulating plant species by cluster analysis.

Identification and classification of metal-accumulating plant species is essential for phytoextraction. Cluster analysis is used for classifying individuals based on measured characteristics. In this study, classification of plant species for metal accumulation was conducted using cluster analysis based on a practical survey. Forty plant samples belonging to 21 species were collected from an ancient silver-mining site. Five groups such as hyperaccumulator, potential hyperaccumulator, accumulator, potential accumulator, and normal accumulating plant were graded. For Cd accumulation, the ancient silver-mining ecotype of Sedum alfredii was treated as a Cd hyperaccumulator, and the others were normal Cd-accumulating plants. For Zn accumulation, S. alfredii was considered as a potential Zn hyperaccumulator, Conyza canadensis and Artemisia lavandulaefolia were Zn accumulators, and the others were normal Zn-accumulating plants. For Pb accumulation, S. alfredii and Elatostema lineolatum were potential Pb hyperaccumulators, Rubus hunanensis, Ajuga decumbens, and Erigeron annuus were Pb accumulators, C. canadensis and A. lavandulaefolia were potential Pb accumulators, and the others were normal Pb-accumulating plants. Plant species with the potential for phytoextraction were identified such as S. alfredii for Cd and Zn, C. canadensis and A. lavandulaefolia for Zn and Pb, and E. lineolatum, R. hunanensis, A. decumbens, and E. annuus for Pb. Cluster analysis is effective in the classification of plant species for metal accumulation and identification of potential species for phytoextraction.

Survival of Escherichia coli O157:H7 in soils under different land use types.

Laboratory studies on Escherichia coli O157:H7 survival in soils from four different land use types: forest, tea plantation, bamboo grove, and vegetable garden were investigated at 25 ± 1 °C with the field capacity (soil water content at -33 kPa). Results showed that E. coli O157:H7 declined quickly in the test soils, but its survival dynamics varied in the soils under different land use types. The survival time needed to reach the detection limit (t d) in the test soils ranged from 2.1 to 3.6 days, with slightly longer t d values being observed in soils from the bamboo grove. Stepwise multiple regression analysis revealed that the t d values were shorter in sandy, lower pH, and lower organic carbon content soils. Different E. coli O157:H7 survival time in the soils under different land uses suggests that it is important to adapt proper management practices for reducing the potential risks of pathogen contamination when diary manure is applied to agricultural land.

Survival of Escherichia coli O157:H7 in soils from Jiangsu Province, China.

Escherichia coli O157:H7 (E. coli O157:H7) is recognized as a hazardous microorganism in the environment and for public health. The E. coli O157:H7 survival dynamics were investigated in 12 representative soils from Jiangsu Province, where the largest E. coli O157:H7 infection in China occurred. It was observed that E. coli O157:H7 declined rapidly in acidic soils (pH, 4.57 - 5.14) but slowly in neutral soils (pH, 6.51 - 7.39). The survival dynamics were well described by the Weibull model, with the calculated td value (survival time of the culturable E. coli O157:H7 needed to reach the detection limit of 100 CFU g(-1)) from 4.57 days in an acidic soil (pH, 4.57) to 34.34 days in a neutral soil (pH, 6.77). Stepwise multiple regression analysis indicated that soil pH and soil organic carbon favored E. coli O157:H7 survival, while a high initial ratio of Gram-negative bacteria phospholipid fatty acids (PLFAs) to Gram-positive bacteria PLFAs, and high content of exchangeable potassium inhibited E. coli O157:H7 survival. Principal component analysis clearly showed that the survival profiles in soils with high pH were different from those with low pH.

C3v-symmetrical tribenzotriquinacenes extended by six C1-functional groups and the first triquinacene-based tris(dithiametacyclophanes).

The first examples of novel bowl-shaped tribenzotriquinacenes (TBTQs) bearing three dithiametacyclophane units within their arene peripheries are reported. The synthesis is based on a C3v-symmetrical hexakis(chloromethyl)tribenzotriquinacene as the key intermediate and yields the inter-ring metacyclophane-type macrocyclization instead of the intra-ring orthocyclophane-type cyclocondensation. Multiple nucleophilic substitution of the same key intermediate leads to a number of other new 6-fold functionalized tribenzotriquinacenes, some of which may be of interest as readily accessible building blocks for the construction of novel bowl-shaped organic networks. The molecular structures of the novel tris(dithiametacyclophanes) and of the hexakis(chloromethyl)- and hexakis(hydroxymethyl)tribenzo-triquinacenes have been determined by X-ray analysis and interesting host/guest aggregation and torsional effects in the solid state are discussed.