Identification of non-canonical antagonistic bacteria via interspecies contact-dependent killing.

BACKGROUND: Canonical biocontrol bacteria were considered to inhibit pathogenic bacteria mainly by secreting antibiotic metabolites or enzymes. Recent studies revealed that some biocontrol bacteria can inhibit pathogenic bacteria through contact-dependent killing (CDK) mediated by contact-dependent secretion systems. The CDK was independent of antibiotic metabolites and often ignored in normal biocontrol activity assay. RESULTS: In this study, we aimed to use a pathogen enrichment strategy to isolate non-canonical bacteria with CDK ability. Rhizosphere soil samples from Chinese cabbage showing soft rot symptom were collected and Pectobacterium carotovorum subsp. carotovorum (Pcc), the pathogen of cabbage soft rot, were added into these samples to enrich bacteria which attached on Pcc cells. By co-culture with Pcc, four bacteria strains (named as PcE1, PcE8, PcE12 and PcE13) showing antibacterial activity were isolated from Chinese cabbage rhizosphere. These four bacteria strains showed CDK abilities to different pathogenic bacteria of horticultural plants. Among them, PcE1 was identified as Chryseobacterium cucumeris. Genome sequencing showed that PcE1 genome encoded a type VI secretion system (T6SS) gene cluster. By heterologous expression, four predicted T6SS effectors of PcE1 showed antibacterial activity to Escherichia coli. CONCLUSION: Overall, this study isolated four bacteria strains with CDK activity to various horticultural plant pathogens, and revealed possible involvement of T6SS of Chryseobacterium cucumeris in antibacterial activity. These results provide valuable insight for potential application of CDK activity in biocontrol bacteria. © 2024 Society of Chemical Industry.

Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome.

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae, from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity in vitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.

The role of tumor-associated macrophages in the progression, prognosis and treatment of endometrial cancer.

Tumor-associated macrophages (TAMs) are the main immune cells in the tumor microenvironment (TME) of endometrial cancer (EC). TAMs recruitment and polarization in EC is regulated by the TME of EC, culminating in a predominantly M2-like macrophage infiltration. TAMs promote lymphatic angiogenesis through cytokine secretion, aid immune escape of EC cells by synergizing with other immune cells, and contribute to the development of EC through secretion of exosomes so as to promoting EC development. EC is a hormone- and metabolism-dependent cancer, and TAMs promote EC through interactions on estrogen receptor (ER) and metabolic factors such as the metabolism of glucose, lipids, and amino acids. In addition, we have explored the predictive significance of some TAM-related indicators for EC prognosis, and TAMs show remarkable promise as a target for EC immunotherapy.

Assembly of an active microbial consortium by engineering compatible combinations containing foreign and native biocontrol bacteria of kiwifruit.

Assembling functional bacterial biocontrol consortia is expected to expand the scope and efficiency of biocontrol agents. Generally, bacterial interspecies interactions lead to incompatibility events, as bacteria can produce antibacterial compounds and/or assemble contact-dependent killing (CDK) devices. Here, we aimed to assemble a bacterial consortium comprising Lysobacter enzymogenes OH11 and Bacillus safensis ZK-1 for the synergistic control of bacterial and fungal diseases of kiwifruit. ZK-1, a native kiwifruit biocontrol bacterium, is effective against Pseudomonas syringae pv. actinidiae (Psa) that causes bacterial kiwifruit canker, but has weak antifungal activity. OH11 is a foreign kiwifruit biocontrol agent with strong antifungal activity. While OH11 was unable to produce anti-Gram-negative metabolites, this strain could utilize type IV secretion system as an antibacterial CDK weapon. We first observed that OH11 could inhibit growth of ZK-1 by generating diffusible anti-Gram-positive antibiotic WAP-8294A2, whereas ZK-1 failed to generate diffusible antibacterial compound to inhibit growth of OH11. To disrupt this interspecies incompatibility, we generated a transgenic OH11-derived strain, OH11W, by deleting the WAP-8294A2 biosynthetic gene and found that OH11W did not kill ZK-1. We further observed that when OH11W and ZK-1 were co-inoculated on agar plates, no CDK effect was observed between them, whereas co-culture of OH11W or ZK-1 with Psa on agar plates resulted in Psa killing, suggesting L. enzymogenes and B. safensis assemble antibacterial CDK weapons against bacterial pathogens, and these CDK weapons did not affect the compatibility between OH11W and ZK-1. Based on these findings, we assembled an OH11W/ZK-1 dependent consortium that was shown to be functional in controlling bacterial canker and several representative fungal diseases of kiwifruit.

Reanalysis of single-cell data reveals macrophage subsets associated with the immunotherapy response and prognosis of patients with endometrial cancer.

Endometrial cancer (EC) is an aggressive gynecological malignancy with an increased incidence rate. The immune landscape crucially affects immunotherapy efficacy and prognosis in EC patients. Here, we characterized the distinct tumor microenvironment signatures of EC tumors by analyzing single-cell RNA sequencing data from Gene Expression Omnibus and bulk RNA sequencing data from The Cancer Genome Atlas, which were compared with normal endometrium. Three macrophage subsets were identified, and two of them showed tissue-specific distribution. One of the macrophage subsets was dominant in macrophages derived from EC and exhibited characteristic behaviors such as promoting tumor growth and metastasis. One of the other macrophage subsets was mainly found in normal endometrium and served functions related to antigen presentation. We also identified a macrophage subset that was found in both EC and normal endometrial tissue. However, the pathway and cellular cross-talk of this subset were completely different based on the respective origin, suggesting a tumor-related differentiation mechanism of macrophages. Additionally, the tumor-enriched macrophage subset was found to predict immunotherapy responses in EC. Notably, we selected six genes from macrophage subset markers that could predict the survival of EC patients, SCL8A1, TXN, ANXA5, CST3, CD74 and NANS, and constructed a prognostic signature. To verify the signature, we identified immunohistochemistry for the tumor samples of 83 EC patients based on the selected genes and further followed up with the survival of the patients. Our results provide strong evidence that the signature can effectively predict the prognosis of EC patients.

Evaluation of immunotherapy efficacy in gynecologic cancer.

Various immunotherapies have demonstrated remarkable success over the past few decades, and have been approved for the treatment of different cancer types. However, patient responses to immunotherapy are variable, and approximately 50% of cases are refractory to these agents. Tumor biomarker-based stratification of cases may therefore help identify subpopulations that are sensitive/resistant to immunotherapy; it may also improve prediction of response in various cancers including gynecologic cancer. These biomarkers include the tumor mutational burden, microsatellite instability, mismatch repair deficiency, T cell-inflamed gene expression profile, programmed cell death protein 1 ligand 1, tumor-infiltrating lymphocytes, and numerous other genomic alterations. Future directions in the treatment of gynecologic cancer include the utilization of these biomarkers to select ideal candidates. This review focused on recent advances in the predictive ability of molecular biomarkers in patients with gynecologic cancer who undergo immunotherapy. The most recent developments in combined immunotherapy and targeted therapy strategies and novel immune interventions against gynecologic cancers have also been discussed.

Effects and Mechanisms of Copper Oxide Nanoparticles with Regard to Arsenic Availability in Soil-Rice Systems: Adsorption Behavior and Microbial Response.

Copper oxide nanoparticles (CuO NPs) are widely used as fungicides in agriculture. Arsenic (As) is a ubiquitous contaminant in paddy soil. The present study was focused on the adsorption behavior of CuO NPs with regard to As as well as the characteristics of the microbial community changes in As-contaminated soil-rice systems in response to CuO NPs. The study found that CuO NPs could be a temporary sink of As in soil; a high dose of CuO NPs promoted the release of As from crystalline iron oxide, which increased the As content in the liquid phase. The study also found that the As bioavailability changed significantly when the dose of CuO NPs was higher than 50 mg kg-1 in the soil-rice system. The addition of 100 mg kg-1 CuO NPs increased the microbial diversity and the abundance of genes involved in As cycling, decreased the abundance of Fe(III)-reducing bacteria and sulfate-reducing genes, and decreased As accumulation in grains. Treatment with 500 mg kg-1 CuO NPs increased the abundance of Fe(III)-reducing bacteria and sulfate-reducing genes, decreased Fe plaques, and increased As accumulation in rice. The adverse effects of CuO NPs on crops and associated risks need to be considered carefully.

Colonization of Penicillium oxalicum SL2 in Pb-contaminated paddy soil and its immobilization effect on soil Pb.

Penicillium oxalicum SL2 (SL2) is a previously screened Pb-tolerant fungus that can promote crops growth. The relationship between SL2 colonization and Pb immobilization was studied to provide a theoretical basis for microbial remediation of Pb-contaminated paddy soil. In this study, green fluorescent protein (GFP) labeled SL2 was inoculated into different Pb-contaminated paddy soils (S1-S6). The Pb extracted from the soil by HNO3, EDTA and CaCl2 were used to characterize the available Pb. The results showed that the colonization of SL2 was divided into lag phase (0-7 days), growth phase (7-30 days), and mortality phase (30-90 days). SL2 colonized well in sandy soils rich in clay and total phosphorus with initial pH of 4.5-7.0. In addition, SL2 increased soil pH and decreased soil Eh, which was beneficial to immobilize Pb. In different soils, the highest percentages of CaCl2-Pb, EDTA-Pb, and HNO3-Pb immobilized by SL2 were 34.34%-40.53%, 17.05%-20.11%, and 7.39%-15.62%, respectively. Pearson correlation analysis showed that the percentages of CaCl2-Pb and EDTA-Pb immobilized by SL2 were significantly positively correlated with the number of SL2 during the growth phase. SL2 mainly immobilized Pb in the growth phase and a higher peak number of SL2 was beneficial to the immobilization of Pb.

Regulatory Mechanism of Copper Oxide Nanoparticles on Uptake of Different Species of Arsenic in Rice.

Copper oxide nanoparticles (CuO NPs) are widely used as a fungicide in agriculture. The application of CuO NPs in agriculture affects the growth of rice and metal accumulation in rice. However, the mechanism of CuO NPs on arsenic (As) accumulation in rice remains unclear. In this study, a hydroponic culture was produced to investigate the mechanism of the effect of 50 and 100 mg L-1 CuO NPs on As accumulation in rice. Our results showed that CuO NPs decreased As(III/V) accumulation in the roots and shoots by adsorbing As(III/V), oxidizing of As(III) on the surface, and thickening the root cell wall. The addition of CuO NPs regulated the expression of the OsNIP1;1, OsHAC1;1, and OsHAC4 genes, which decreased As(III) transport and promoted As(V) reduction in the roots. Moreover, when CuO NPs were co-exposed to As, a negative correlation between the concentration of Cu and As in rice was also found in our study. However, CuO NPs significantly increased Cu accumulation in rice and constrained the rice growth. In conclusion, CuO NPs might be a promising way to decrease As accumulation in rice, but the negative effects such as growth inhibition should be further considered. Therefore, the application of CuO NPs in rice plants should take a more restrained approach.

Water management of alternate wetting and drying combined with phosphate application reduced lead and arsenic accumulation in rice.

Lead (Pb) and arsenic (As) exist in soil with different ionic forms, and it is difficult to immobilize simultaneously Pb and As in soil. The objective of this study is to determine the effects of water management including flooding (FL), alternate wetting and drying (AWD) and dry farming (DF) combined with addition of phosphate (P) on the accumulation of Pb and As in rice. Our results showed that Pb accumulated in root during vegetative stage, and most of As in root was transported to the above ground parts during the reproductive stage. Pb was evenly distributed in grains, and As was mostly accumulated in bran and aleurone layer. Water management had a reverse effect on the accumulation of Pb and As in rice. However, the effects of P on arid soil environment and Pb, As accumulation in rice were stronger than that in flooded soil. Application of P under AWD treatment could maintain a similar quantity of Fe plaque with flooding, decrease the availability of Pb in rhizosphere soil, reduce Pb and As accumulation in root, and result in the reduction of Pb and As accumulation in grains by 86% and 66% respectively. Besides, our study also found that flooding or AWD during vegetative stage facilitated the formation of iron plaque. In conclusion, AWD combined with P application could maintain a relatively lower concentrations of Pb and As in grains.

Controlling Factors and Prediction of Lead Uptake and Accumulation in Various Soil-Pepper Systems.

Lead (Pb) is a typical toxic heavy metal element in soils and plants, which has a potential threat to human health through the food chain. Uptake of Pb in the soil-vegetable system has attracted broad attention, whereas reports on the main controlling factors of Pb uptake and accumulation in different soil-vegetable systems are limited. The effect of soil properties on Pb uptake and accumulation in pepper (Capsicum annuum L.) was studied by a pot experiment with 16 typical soils in China. The results showed that the Pb bioavailability was lower in alkaline soils, and that soil cation exchange capacity (CEC), CaCO3 , and total phosphorus contents might influence the uptake and transfer of Pb by peppers. Soil pH and CEC were the most significant factors affecting Pb accumulation in pepper fruits. Soil pH was negatively correlated with Pb uptake and accumulation due to its influence on Pb mobility and bioavailability. The accumulation of Pb decreased as soil CEC increased, which might inhibit the absorption and transfer of Pb in peppers. The multiple linear regression function based on soil Pb content, pH, and CEC could provide enough information for a good prediction of the accumulation of Pb in soil-pepper systems (R2 = 0.733). The results are in favor of developing a Pb threshold for vegetables in agricultural soils in China, thus improving the food safety of crops. Environ Toxicol Chem 2021;40:1443-1451. © 2021 SETAC.

Unlocking the bacterial contact-dependent antibacterial activity to engineer a biocontrol alliance of two species from natural incompatibility to artificial compatibility.

Plant growth-promoting rhizobacteria (PGPR) contain various biocontrol bacteria with broad-spectrum antimicrobial activity, and their single species has been extensively applied to control crop diseases. The development of complex biocontrol community by mixing two or more PGPR members together is a promising strategy to enlarge the efficacy and scope of biocontrol. However, an effective method to assess the natural compatibility of PGPR members has not yet been established to date. Here, we developed such a tool by using the bacterial contact-dependent antibacterial activity (CDAA) as a probe. We showed that the CDAA events are common in two-species interactions in the four selected representative PGPRs, represented by the incompatible interaction of Lysobacter enzymogenes strain OH11 (OH11) and Lysobacter antibioticus strain OH13 (OH13). We further showed that the CDAA between OH11 and OH13 is jointly controlled by a contact-dependent killing device, called the type IV secretion system (T4SS). By deleting the respective T4SS synthesis genes, the T4SS in both strains was co-inactivated and this step unlocked  their natural CDAA, resulting in an engineered, compatible mutant alliance that co-displayed antibacterial and antifungal activity. Therefore, this study reveals that releasing bacterial CDAA is effective to rationally engineer the biocontrol community.

Antifungal activity of hypocrellin compounds and their synergistic effects with antimicrobial agents against Candida albicans.

Candida albicans is a common human fungal pathogen. The previous study revealed that quinone compounds showed antimicrobial activity against C. albicans by inhibiting cell growth. However, it was unclear whether quinones have other antifungal effects against C. albicans in addition to fungicidal effects. In this study, we assessed the inhibitory activity of a total of 25 quinone compounds against C. albicans morphological transition, which is essential for the pathogenicity of C. albicans. Several quinones exhibited strong inhibition of mycelium formation by C. albicans SC5314. Three leading compounds, namely hypocrellins A, B and C, also exhibited marked attenuation of C. albicans SC5314 virulence in both human cell lines and mouse infection models. These three compounds significantly suppressed the proliferation of C. albicans SC5314 cells in a mouse mucosal infection model. Intriguingly, hypocrellins not only attenuated the cytotoxicity of a nystatin-resistant C. albicans strain but also showed excellent synergistic effects with antifungal agents against both wild-type C. albicans SC5314 and the drug-resistant mutant strains. In addition, hypocrellins A, B and C interfered with the biological functions and virulence of various clinical Candida species, suggesting the promising potential of these compounds for development as new therapeutic agents against infections caused by Candida pathogens.

Toxicity of different forms of antimony to rice plants: Effects on reactive oxidative species production, antioxidative systems, and uptake of essential elements.

Antimonite [Sb(III)] and antimonate [Sb(V)] are known to have different toxicity to plants, but the corresponding mechanisms are not fully understood. This study was conducted to investigate reactive oxygen species (ROS), antioxidant systems, and levels of certain essential elements in response to exposure to Sb(III) and Sb(V). Results showed that exposure to Sb(V) caused oxidative stress in a rice plant (Yangdao No.6). Sb(III) was shown to be more toxic than Sb(V) as judged from a lower shoot biomass, a higher loss of essential elements, and higher production of superoxide anion free radicals (O2-). The toxicity of Sb(III) might partially be due to the disturbance of the O2- dismutation reaction, which resulted in root cell membrane damage under exposure to 20 mg L-1 Sb(III). Sb(V) stimulated the shoot fresh weight and the shoot uptake of many essential elements. Moreover, Sb(V) and Sb(III) both stimulated the accumulation of calcium in the shoots and roots, and calcium was found to significantly correlate with the concentrations of many essential elements and with some parameters correlated to antioxidant systems, suggesting a Ca-induced regulatory mechanism. The activity of glutathione peroxidase was significantly enhanced by Sb(V) and Sb(III), suggesting a role in scavenging hydrogen peroxide. Catalase was activated by exposure to 20 mg L-1 Sb(III) in the roots and by exposure to 20 mg L-1 Sb(V) both in the shoots and roots. However, peroxidase was activated by exposure to 5 mg L-1 Sb(III) in the shoots and by exposure to 5 mg L-1 Sb(V) in the roots. This study, for the first time, showed the differences between Sb(V) and Sb(III) toxicity when looking at the antioxidant response and essential element uptake.

The interaction between particulate organic matter and copper, zinc in paddy soil.

Particulate organic matter (POM) acts as a metals sink in soil, but only a few studies focused on the interaction of POM and heavy metals in paddy soil. The aim of this study is to investigate the interaction between POM and Copper (Cu)/Zinc (Zn). Two levels of Cu (100, 400 mg kg-1) and Zn (250, 500 mg kg-1) were used in a soil culture experiment. Our results showed that POM was porous structure and varied in size. Hydroxyl and carboxyl involved in POM adsorption of Cu and Zn. Rhizosphere effects roughen the surface of POM and enhanced the capacity of POM on heavy metals absorption. Cu-humic (26.2-33.9%) and Cu-citrate (38.5-42.4%) were dominated in POM, and Cu-goethite (41.7-57.7%), Cu-sulphide (6.6-27.6%) was dominated in soil. Rhizosphere effects decreased the proportion of organic-bond Cu along with the increasing the proportion of Cu-sulphide in POM. Addition of Cu and Zn inhibited the degradation of POM but rhizosphere effects promoted. Carbon content was increased in POM by heavy metal and rhizosphere effects. Our findings indicated that POM tended to retain the heavy metals in soil and heavy metals inhibited the degradation of POM, however, rhizosphere effects decreased the stability of POM-metals interactions.

Root application of selenite can simultaneously reduce arsenic and cadmium accumulation and maintain grain yields, but show negative effects on the grain quality of paddy rice.

Most current technologies can hardly simultaneously reduce the accumulation of arsenic (As) and cadmium (Cd) in crops. In this study, root application of selenite [Se (IV)] and selenate [Se (VI)] was used to assess their abilities to reduce the accumulation of As and Cd, and maintain the yields and quality of rice grains. The results show that Se (IV) showed a weaker ability than Se (VI) to maintain the grain contents of many essential elements, but a stronger ability to decrease As and Cd contents in rice grains, and maintain the yields, photosynthesis rate and stomatal conductance, and increase the grain contents of several amino acids (AAs), total Se, selenomethionine (SeMet) and selenocysteine (SeCys). The best outcomes resulted at a relatively high application of 5 mg kg-1 Se (IV), reflecting in the highest total Se, SeCys and SeMet content (14.95, 118.70 and 864.73 µg kg-1, respectively) in the grains, highest grain yield, and lowest grain As and Cd content (0.36 and 0.07 mg kg-1, respectively). In addition, the application of 1-5 mg kg-1 Se (IV) seemed to facilitate the formation of SeMet in the grains, but most inorganic Se in the grains were transformed into SeCys and SeMet under Se (VI) treatments. This study provides a new idea to resolve the problems of high accumulation of As and Cd in rice grains and insufficiency of Se intake in China.

Efficiency evaluation for remediating paddy soil contaminated with cadmium and arsenic using water management, variety screening and foliage dressing technologies.

Paddy soils in many regions of China have been seriously polluted by multiple heavy metals or metalloids, such as arsenic (As), cadmium (Cd) and lead (Pb). In order to ensure the safety of food and take full advantage of the limited farmland resources of China, exploring an effective technology to repair contaminated soils is urgent and necessary. In this study, three technologies were employed, including variety screening, water management and foliage dressing, to assess their abilities to reduce the accumulation of Cd and As in the grains of different rice varieties, and meanwhile monitor the related yields. The results of variety screening under insufficient field drying condition showed that the As and Cd contents in the grains of only four varieties [Fengliangyouxiang 1 (P6), Zhongzheyou 8 (P7), Guangliangyou 1128 (P10), Y-liangyou 696 (P11)] did not exceed their individual national standard. P6 gained a relatively high grain yield but accumulated less As and Cd in the grains despite of the relatively high As and Cd concentrations in the rhizosphere soil. However, long-playing field drying in water management trial significantly increased Cd but decreased As content in the grains of all tested three varieties including P6, suggesting an important role of water supply in controlling the accumulation of grain As and Cd. Selenium (Se) showed a stronger ability than silicon (Si) to reduce As and Cd accumulation in the grains of Fengliangyou 4 (P2) and Teyou 524 (P13), and keep the yields. The results of this study suggest that combined application of water management and foliage dressing may be an efficient way to control As and Cd accumulation in the grains of paddy rice exposing to As- and Cd-contaminated soils.