a-workflow-for-quantitative-assessment-endophytic-epiphytic-bacterial

A Workflow for the Quantitative Assessment of the Endophytic and Epiphytic Bacterial Microbiomes of the Bark of <em>Populus trichocarpa</em>

Microorganisms colonizing plant surfaces and internal tissues may possess beneficial functions in promoting plant growth and health. However, information ...

Northeast Forestry University

Besides analyzing the composition and dynamics of microbial communities, plant microbiome research aims to understanding the mechanism of plant microbiota assembly and their biological functions. Here, we describe procedures to investigate the role of bacterial interspecies interactions in root microbiome assembly and the beneficial effects of the root microbiota on hosts by using a maize root-associated simplified seven-species (, , , , ,  and ) synthetic bacterial community described in our previous work. Surface-sterilized maize seeds were grown in a gnotobiotic system based on double-tube growth chambers after being soaked in suspensions containing multiple species of bacteria. The dynamics of the composition of the bacterial communities colonized on maize roots were tracked by a culture-dependent method with a selective medium for each of the seven strains. The impact of bacterial interactions on the community assembly was evaluated by monitoring the changes of community structure. The plant-protection effects of the simplified seven-species community were assessed by quantifying (1) the growth of a fungal phytopathogen,  on the surfaces of the seeds and (2) the severity of seedling blight disease the fungus causes, in the presence and absence of the bacterial community. Our protocol will serve as useful guidance for studying plant-microbial community interactions under the laboratory conditions.

Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect.

Plant specialized metabolites modulate root microbiomes.

Major losses of crop yield and quality caused by soil-borne plant diseases have long threatened the ecology and economy of agriculture and forestry. Biological control using beneficial microorganisms has become more popular for management of soil-borne pathogens as an environmentally friendly method for protecting plants. Two major barriers limiting the disease-suppressive functions of biocontrol microbes are inadequate colonization of hosts and inefficient inhibition of soil-borne pathogen growth, due to biotic and abiotic factors acting in complex rhizosphere environments. Use of a consortium of microbial strains with disease inhibitory activity may improve the biocontrol efficacy of the disease-inhibiting microbes. The mechanisms of biological control are not fully understood. In this review, we focus on bacterial and fungal biocontrol agents to summarize the current state of the use of single strain and multi-strain biological control consortia in the management of soil-borne diseases. We discuss potential mechanisms used by microbial components to improve the disease suppressing efficacy. We emphasize the interaction-related factors to be considered when constructing multiple-strain biological control consortia and propose a workflow for assembling them by applying a reductionist synthetic community approach.

Microbial Interactions Within Multiple-Strain Biological Control Agents Impact Soil-Borne Plant Disease.

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell-cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.

Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system.

, a plant parasitic nematode, is the causal agent of pine wilt, a devastating forest tree disease. Essentially, no efficient methods for controlling  and pine wilt disease have yet been developed.  AA4, isolated from the root of maize, has powerful nematocidal activity against  in a new  dye exclusion test. The corrected mortality of the  treated by  AA4 or its cell extract reached 98.3 and 98.6%, respectively. Morphological changes in  treated with a cell extract from strain AA4 suggested that the death of  might be caused by an increased number of vacuoles in non-apoptotic cell death and the damage to tissues of the nematodes. In a greenhouse test, the disease index of the seedlings of Scots pine () treated with the cells of strain AA4 plus  or those treated by AA4 cell extract plus  was 38.2 and 30.3, respectively, was significantly lower than 92.5 in the control plants treated with distilled water and . We created a  gene knockout in strain AA4 by deleting the gene that was putatively encoding the beta-subunit of L-serine dehydratase through Red homologous recombination. The nematocidal and disease-suppressing activities of the knockout strain were remarkably impaired. Finally, we revealed a robust colonization of  seedling needles by  AA4, which is supposed to contribute to the disease-controlling efficacy of strain AA4. Therefore,  AA4 has significant potential to serve as an agent for the biological control of pine wilt disease caused by .

Gene  Is Involved in the Nematocidal Activity of  AA4 Against the Pine Wood Nematode .

Rhizosphere microorganisms are essential parts in maintaining soil ecological functions. Reforestation using genetically modified trees might have great potential to enhance tree production in biotic and abiotic stress, however, their long-term impact on rhizosphere microorganisms is scant. In this study, we studied soil enzyme activities and composition of rhizosphere microorganisms in 2-year-old transgenic  overexpression (OE), repressed expression (RE) and wild-type (WT) poplar (). The root exudates of  transgenic poplar () were analyzed by liquid chromatography-mass spectrometry (LC-MS). The results showed that there were significant difference for soil sucrase, urease, catalase, neutral protease and cellulase between the transgenic and WT lines at different growth periods. Alpha diversity analysis showed that bacterial community abundance and diversity for RE lines were significantly lower than WT ( < 0.05), while RE lines for fungi were significantly higher than WT lines. At the genus level,  was the dominant group of rhizosphere bacterial community, and the relative abundance for RE was significantly higher than WT.  was the dominant group for fungi community.  for RE was significantly higher than WT and OE. Main metabolite contents of (S)-ACPA, geniposidic acid, agnuside, hydroquinone and pyranocoumarins were significantly different among transgenic lines. These results suggest that transgenic activities have effects on root exudates, rhizosphere soil enzyme activities and soil microbial community composition, but long term effects need to be further investigated.

Impact of  transgenic poplar on microbial community and soil enzyme activity in rhizosphere soil.

To understand the effect of seasonal variations on soil microbial communities in a forested wetland ecotone, here, we investigated the dynamics of the diversities and functions of both soil bacterial and fungal communities inhabiting three wetland types (forested wetland, shrub wetland and herbaceous vegetation wetland) from forest-wetland ecotone of northern Xiaoxing'an Mountains spanning different seasons. β-diversity of soil microbial communities varied significantly among different vegetation types (-, , , and  wetlands). We totally detected 34 fungal and 14 bacterial indicator taxa among distinctive groups by using Linear discriminant analysis effect size (LEfSe) analysis, and identified nine network hubs as the most important nodes detected in whole fungi, bacteria, and fungi-bacteria networks. At the vegetation type-level, bacterial and fungal microbiome living in  wetland soil possessed fewer positive interactions and lower modularity than those in other types of wetland soil. Furthermore, we also discovered that ectomycorrhizal fungi were dominant in the fungal microbiota existing in forested and shrub wetland soils, whereas arbuscular mycorrhizal fungi were predominated in those residing in herbaceous vegetation wetland soil. The distribution of the predicted bacterial functional enzymes also obviously varied among different vegetation-types. In addition, the correlation analysis further revealed that the key fungal network modules were significantly affected by the contents of total N and soil water-soluble K, whereas most of the bacterial network modules were remarkably positively driven by the contents of total N, soil water-soluble K, Mg and Na. Our study suggested that vegetation type are substantive factors controlling the diversity, composition and functional group of soil microbiomes from forest-wetland ecotone of northern Xiaoxing'an Mountains.

The characteristics of soil microbial co-occurrence networks across a high-latitude forested wetland ecotone in China.

The continued large consumption of maize makes it one of the most important food crops worldwide. However, the yield and quality of maize are greatly affected by global warming, and mycotoxin pollution keeps increasing. The effect of environmental factors, especially rhizosphere microorganisms, on mycotoxin pollution of maize is not completely clear, so we carried out relevant studies. In this study, we found that microbial communities inhabiting the maize rhizosphere, which consists of soil particles firmly attached to roots, as well as the soil, have a significant influence on the aflatoxin pollution of maize. The ecoregion and soil properties also had considerable effects on the microbial structure and diversity. The bacterial communities from the rhizosphere soil were profiled using a high-throughput next-generation sequencing method. The ecoregion and soil properties had considerable effects on the microbial structure and diversity. A comparison of the aflatoxin high concentration group with the low concentration group found that bacteria of the phylum  and order  were significantly more abundant in the high concentration samples. Furthermore, these bacteria were significantly correlated with aflatoxin contamination and could aggravate its contamination of maize. The results of these analyses showed that seeding location could cause significant shifts in the root microbiota of maize, and the bacteria enriched in high aflatoxin contamination area soils should attract special concern. These findings will support strategies for improving maize yield and aflatoxin contamination control.

Effect of rhizosphere microorganisms on aflatoxin contamination of maize.

By ecologically interacting with various biotic and abiotic agents acting in soil ecosystems, highly diverse soil microorganisms establish complex and stable assemblages and survive in a community context in natural settings. Besides facilitating soil microbiome to maintain great levels of population homeostasis, such microbial interactions drive soil microbes to function as the major engine of terrestrial biogeochemical cycling. It is verified that the regulative effect of microbe-microbe interplay plays an instrumental role in microbial-mediated promotion of soil health, including bioremediation of soil pollutants and biocontrol of soil-borne phytopathogens, which is considered an environmentally friendly strategy for ensuring the healthy condition of soils. Specifically, in microbial consortia, it has been proven that microorganism-microorganism interactions are involved in enhancing the soil health-promoting effectiveness (i.e., efficacies of pollution reduction and disease inhibition) of the beneficial microbes, here defined as soil health-promoting agents. These microbial interactions can positively regulate the soil health-enhancing effect by supporting those soil health-promoting agents utilized in combination, as multi-strain soil health-promoting agents, to overcome three main obstacles: inadequate soil colonization, insufficient soil contaminant eradication and inefficient soil-borne pathogen suppression, all of which can restrict their probiotic functionality. Yet the mechanisms underlying such beneficial interaction-related adjustments and how to efficiently assemble soil health-enhancing consortia with the guidance of microbe-microbe communications remain incompletely understood. In this review, we focus on bacterial and fungal soil health-promoting agents to summarize current research progress on the utilization of multi-strain soil health-promoting agents in the control of soil pollution and soil-borne plant diseases. We discuss potential microbial interaction-relevant mechanisms deployed by the probiotic microorganisms to upgrade their functions in managing soil health. We emphasize the interplay-related factors that should be taken into account when building soil health-promoting consortia, and propose a workflow for assembling them by employing a reductionist synthetic community approach.

Microbial interactions within beneficial consortia promote soil health.

As an ephemeral and oligotrophic environment, the phyllosphere harbors many highly diverse microorganisms. Importantly, it is known that their colonization of plant leaf surfaces is considerably influenced by a few abiotic factors related to climatic conditions. Yet how the dynamics of phyllosphere bacterial community assembly are shaped by detailed climatological elements, such as various bioclimatic variables, remains poorly understood.

Differentiated responses of the phyllosphere bacterial community of the yellowhorn tree to precipitation and temperature regimes across Northern China.

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in  to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial  and the phytopathogen . We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between  and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

Article	Year
Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect. Bio-protocol\| PubMed ID: 30123815
Plant specialized metabolites modulate root microbiomes. Science China. Life sciences\| PubMed ID: 31290099
Microbial Interactions Within Multiple-Strain Biological Control Agents Impact Soil-Borne Plant Disease. Frontiers in microbiology\| PubMed ID: 33162962
Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system. Environmental microbiology\| PubMed ID: 34227200	2021
Gene Is Involved in the Nematocidal Activity of AA4 Against the Pine Wood Nematode . Frontiers in microbiology\| PubMed ID: 35602027	2022
Impact of transgenic poplar on microbial community and soil enzyme activity in rhizosphere soil. Frontiers in bioengineering and biotechnology\| PubMed ID: 35942008	2022
The characteristics of soil microbial co-occurrence networks across a high-latitude forested wetland ecotone in China. Frontiers in microbiology\| PubMed ID: 37025633	2023
Effect of rhizosphere microorganisms on aflatoxin contamination of maize. Heliyon\| PubMed ID: 37215779	2023
Microbial interactions within beneficial consortia promote soil health. The Science of the total environment\| PubMed ID: 37499809	2023
Differentiated responses of the phyllosphere bacterial community of the yellowhorn tree to precipitation and temperature regimes across Northern China. Frontiers in plant science\| PubMed ID: 37954985	2023
Identification of atypical T4SS effector proteins mediating bacterial defense. mLife\| PubMed ID: 38817810	2023

Ben Niu

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