High-elevation-induced decrease in soil pH weakens ecosystem multifunctionality by influencing soil microbiomes.

Plant environmental stress response has become a global research hotspot, yet there is a lack of clear understanding regarding the mechanisms that maintain microbial diversity and their ecosystem services under environmental stress. In our research, we examined the effects of moderate elevation on the rhizosphere soil characteristics, microbial community composition, and ecosystem multifunctionality (EMF) within agricultural systems. Our findings revealed a notable negative correlation between EMF and elevation, indicating a decline in multifunctionality at higher elevations. Additionally, our analysis across bacterial and protistan communities showed a general decrease in microbial richness with increasing elevation. Using random forest models, pH was identified as the key environmental stressor influencing microbial communities. Furthermore, we found that microbial community diversity is negatively correlated with stability by mediating complexity. Interestingly, while pH was found to affect the complexity within bacterial networks, it did not significantly impact the ecosystem stability along the elevation gradients. Using a Binary-State Speciation and Extinction (BiSSE) model to explore the evolutionary dynamics, we found that Generalists had higher speciation rates and lower extinction rates compared to specialists, resulting in a skewed distribution towards higher net diversification for generalists under increasing environmental stress. Moreover, structural equation modeling (SEM) analysis highlighted a negative correlation between environmental stress and community diversity, but showed a positive correlation between environmental stress and degree of cooperation & competition. These interactions under environmental stress indirectly increased community stability and decreased multifunctionality. Our comprehensive study offers valuable insights into the intricate relationship among environmental factors, microbial communities, and ecosystem functions, especially in the context of varying elevation gradients. These findings contribute significantly to our understanding of how environmental stressors affect microbial diversity and ecosystem services, providing a foundation for future ecological research and management strategies in similar contexts.

Soil microbiota plays a key regulatory role in the outbreak of tobacco root rot.

Introduction: Root rot caused by the fungal pathogen Fusarium sp. poses significant challenges to tobacco cultivation in China, leading to major economic setbacks. The interplay between this pathogen and the wider soil microbial community remains poorly understood. Methods: High-throughput sequencing technology was utilized to evaluate soil prokaryotic, fungal, and protistan communities. We compared microbial communities in infected soils to those in healthy soils from the same field. Additionally, the influence of pH on the microbial communities was assessed. Results: Infected soils displayed elevated levels of soil nutrients but diminished observed richness across prokaryotic, fungal, and protistan groups. The pathogenic fungi Fusarium solani f sp. eumartii's abundance was notably increased in infected soils. Infection with F. solani significantly altered the soil's microbial community structure and interactions, manifested as a decrease in network scale and the number of keystone species. An evaluation of prokaryotes' role in F. solani's invasion revealed an increased number of connecting nodes in infected soils. Additionally, relationships between predatory protists and fungi were augmented, whereas predation on F. solani declined. Discussion: The study underscores the significance of comprehending the interactions among soil microorganisms and brings to light the susceptibility of soil microbial communities to pathogen invasion. It offers insights into the multifaceted relationships and potential vulnerabilities within the soil ecosystem in the context of Fusarium sp. invasion.

First report of Fusarium cugenangense causing root rot of tea plants (Camellia sinensis) in China.

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 µm to 41.2 µm × 4.2 µm to 5.4 µm (mean= 31.2 µm × 4.8 µm, n=30). The microconidia were measured as 6.7 µm to 12.8 µm × 2.4 µm to 4.9 µm (mean= 10.1 µm × 3.3 µm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7µm (mean= 7.7µm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.

Soil microbial community assembly and stability are associated with potato (Solanum tuberosum L.) fitness under continuous cropping regime.

Continuous cropping obstacles caused by the over-cultivation of a single crop trigger soil degradation, yield reduction and the occurrence of plant disease. However, the relationships among stability, complexity and the assembly process of soil microbial community with continuous cropping obstacles remains unclear. In this study, molecular ecological networks analysis (MENs) and inter-domain ecological networks analysis (IDENs), and a new index named cohesion tools were used to calculate the stability and complexity of soil microbial communities from eight potato cultivars grown under a continuous cropping regime by using the high-throughput sequencing data. The results showed that the stability (i.e., robustness index) of the bacterial and fungal communities for cultivar ZS5 was significantly higher, and that the complexity (i.e., cohesion values) was also significantly higher in the bacterial, fungal and inter-domain communities (i.e., bacterial-fungal community) of cultivar ZS5 than other cultivars. Network analysis also revealed that Actinobacteria and Ascomycota were the dominant phyla within intra-domain networks of continuous cropping potato soil communities, while the phyla Proteobacteria and Ascomycota dominated the correlation of the bacterial-fungal network. Infer community assembly mechanism by phylogenetic-bin-based null model analysis (iCAMP) tools were used to calculate the soil bacterial and fungal communities' assembly processes of the eight potato cultivars under continuous cropping regime, and the results showed that the bacterial community was mainly dominated by deterministic processes (64.19% - 81.31%) while the fungal community was mainly dominated by stochastic processes (78.28% - 98.99%), indicating that the continuous-cropping regime mainly influenced the potato soil bacterial community assembly process. Moreover, cultivar ZS5 possessed a relatively lower homogeneous selection, and a higher TP, TN, AP and yield than other cultivars. Our results indicated that the soil microbial network stability and complexity, and community assemble might be associated with yield and soil properties, which would be helpful in the study for resistance to potato continuous cropping obstacles.

iNAP: An integrated network analysis pipeline for microbiome studies.

Integrated network analysis pipeline (iNAP) is an online analysis pipeline for generating and analyzing comprehensive ecological networks in microbiome studies. It is implemented in two sections, that is, network construction and network analysis, and integrates many open-access tools. Network construction contains multiple feasible alternatives, including correlation-based approaches (Pearson's correlation and Spearman's rank correlation along with random matrix theory, and sparse correlations for compositional data) and conditional dependence-based methods (extended local similarity analysis and sparse inverse covariance estimation for ecological association inference), while network analysis provides topological structures at different levels and the potential effects of environmental factors on network structures. Considering the full workflow, from microbiome data set to network result, iNAP contains the molecular ecological network analysis pipeline and interdomain ecological network analysis pipeline (IDENAP), which correspond to the intradomain and interdomain associations of microbial species at multiple taxonomic levels. Here, we describe the detailed workflow by taking IDENAP as an example and show the comprehensive steps to assist researchers to conduct the relevant analyses using their own data sets. Afterwards, some auxiliary tools facilitating the pipeline are introduced to effectively aid in the switch from local analysis to online operations. Therefore, iNAP, as an easy-to-use platform that provides multiple network-associated tools and approaches, can enable researchers to better understand the organization of microbial communities. iNAP is available at http://mem.rcees.ac.cn:8081 with free registration.

Fungi-Bacteria Associations in Wilt Diseased Rhizosphere and Endosphere by Interdomain Ecological Network Analysis.

In the plant rhizosphere and endosphere, some fungal and bacterial species regularly co-exist, however, our knowledge about their co-existence patterns is quite limited, especially during invasion by bacterial wilt pathogens. In this study, the fungal communities from soil to endophytic compartments were surveyed during an outbreak of tobacco wilt disease caused by Ralstonia solanacearum. It was found that the stem endophytic fungal community was significantly altered by pathogen invasion in terms of community diversity, structure, and composition. The associations among fungal species in the rhizosphere and endosphere infected by R. solanacearum showed more complex network structures than those of healthy plants. By integrating the bacterial dataset, associations between fungi and bacteria were inferred by Inter-Domain Ecological Network (IDEN) approach. It also revealed that infected samples, including both the rhizosphere and endosphere, had more complex interdomain networks than the corresponding healthy samples. Additionally, the bacterial wilt pathogenic Ralstonia members were identified as the keystone genus within the IDENs of both root and stem endophytic compartments. Ralstonia members was negatively correlated with the fungal genera Phoma, Gibberella, and Alternaria in infected roots, as well as Phoma, Gibberella, and Diaporthe in infected stems. This suggested that those endophytic fungi may play an important role in resisting the invasion of R. solanacearum.

Network analysis infers the wilt pathogen invasion associated with non-detrimental bacteria.

The microbiota colonizing the root endophytic compartment and surrounding rhizosphere soils contribute to plant growth and health. However, the key members of plant soil and endophytic microbial communities involved in inhibiting or assisting pathogen invasion remain elusive. By utilizing 16S high-throughput sequencing and a molecular ecological network (MEN) approach, we systematically studied the interactions within bacterial communities in plant endophytic compartments (stem and root) and the surrounding soil (bulk and rhizosphere) during bacterial wilt invasion. The endophytic communities were found to be strongly influenced by pathogen invasion according to analysis of microbial diversity and community structure and composition. Endophytic communities of the infected plants were primarily derived from soil communities, as assessed by the SourceTracker program, but with rare migration from soil communities to endophytic communities observed in healthy plants. Soil and endophytic microbiomes from infected plants showed modular topology and greater complexity in network analysis, and a higher number of interactions than those in healthy plants. Furthermore, interactions among microbial members revealed that pathogenic Ralstonia members were positively correlated with several bacterial genera, including Delftia, Stenotrophomonas, Bacillus, Clostridium XlVa, Fontibacillus, Acidovorax, Herminiimonas, and three unclassified bacterial genera, in infected plant roots. Our findings indicated that the pathogen invasion in the rhizosphere and endophytic compartments may be highly associated with bacteria that are normally not detrimental, and sometimes even beneficial, to plants.

Inhibition of migration and induction of apoptosis in LoVo human colon cancer cells by polysaccharides from Ganoderma lucidum.

Ganoderma lucidum polysaccharides (GLPs), which were purified from the medicinal herb G. lucidum followed by ethanol precipitation, protein depletion using the Sevage assay, purification using DEAE­cellulose (DE-52), dialysis and the use of ultrafiltration membranes, are used as an ingredient in traditional anticancer treatments in China. The aim of the current study was to evaluate the anticancer effects and investigate the underlying molecular mechanisms of GLPs on LoVo human colon cancer cells. The results demonstrated that the GLP­mediated anticancer effect in LoVo cells was characterized by cytotoxicity, migration inhibition, enhanced DNA fragmentation, morphological alterations and increased lactate dehydrogenase release. Furthermore, the activation of caspases­3, ­8 and ­9 was involved in GLP­stimulated apoptosis. Additionally, treatment with GLPs promoted the expression of Fas and caspase­3 proteins, whilst reducing the expression of cleaved poly(ADP­ribose) polymerase. These data indicate that GLPs demonstrate potential antitumor activity in human colon cancer cells, predominantly through the inhibition of migration and induction of apoptosis. Furthermore, activation of the Fas/caspase-dependent apoptosis pathway is involved in the cytotoxicity of GLPs.

Ganoderma lucidum polysaccharides target a Fas/caspase dependent pathway to induce apoptosis in human colon cancer cells.

Ganoderma lucidum polysaccharides (GLP) extracted from Ganoderma lucidum have been shown to induce cell death in some kinds of cancer cells. This study investigated the cytotoxic and apoptotic effect of GLP on HCT-116 human colon cancer cells and the molecular mechanisms involved. Cell proliferation, cell migration, lactate dehydrogenase (LDH) levels and intracellular free calcium levels ([Ca(2+)]i) were determined by MTT, wound-healing, LDH release and fluorescence assays, respectively. Cell apoptosis was observed by scanning and transmission electron microscopy. For the mechanism studies, caspase-8 activation, and Fas and caspase-3 expression were evaluated. Treatment of HCT-116 cells with various concentrations of GLP (0.625-5 mg/mL) resulted in a significant decrease in cell viability (P< 0.01). This study showed that the antitumor activity of GLP was related to cell migration inhibition, cell morphology changes, intracellular Ca(2+) elevation and LDH release. Also, increase in the levels of caspase-8 activity was involved in GLP-induced apoptosis. Western blotting indicated that Fas and caspase-3 protein expression was up-regulated after exposure to GLP. This investigation demonstrated for the first time that GLP shows prominent anticancer activities against the HCT-116 human colon cancer cell line through triggering intracellular calcium release and the death receptor pathway.

Chemical characterization and antitumor activities of polysaccharide extracted from Ganoderma lucidum.

Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced cell viability on HCT-116 cells in a time- and dose-dependent manner, which in turn, induced cell apoptosis. The observed apoptosis was characterized by morphological changes, DNA fragmentation, mitochondrial membrane potential decrease, S phase population increase, and caspase-3 and -9 activation. Furthermore, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 led to a dramatic decrease of the GLP-induced apoptosis. Western blot analysis unveiled that GLP up-regulated the expression of Bax/Bcl-2, caspase-3 and poly (ADP-ribose) polymerase (PARP). These results demonstrate that apoptosis stimulated by GLP in human colorectal cancer cells is associated with activation of mitochondrial and mitogen-activated protein kinase (MAPK) pathways.