Phyllosphere eukaryotic microalgal communities in rainforests:Drivers and diversity

Phyllosphere algae are common in tropical rainforests,forming visible biofilms or spots on plant leaf surfaces.However,knowledge of phyllosphere algal diversity and the environmental factors that drive that diversity is limited.The aim of this study is to identify the environmental factors that drive phyllosphere algal community composition and diversity in rainforests.For this purpose,we used single molecule real-time sequencing of full-length 18S rDNA to characterize the composition of phyllosphere microalgal communities growing on four host tree species(Ficus tikoua,Caryota mitis,Arenga pinnata,and Musa acuminata) common to three types of forest over four months at the Xishuangbanna Tropical Botanical Garden,Yunnan Province,China.Environmental 18S rDNA sequences revealed that the green algae orders Watanabeales and Trentepohliales were dominant in almost all algal communities and that phyllosphere algal species richness and biomass were lower in planted forest than in primeval and reserve rainforest.In addition,algal community composition differed significantly between planted forest and primeval rainforest.We also found that algal communities were affected by soluble reactive phosphorous,total nitrogen,and ammonium contents.Our findings indicate that algal community structure is significantly related to forest type and host tree species.Furthermore,this study is the first to identify environmental factors that affect phyllosphere algal communities,significantly contributing to future taxonomic research,especially for the green algae orders Watanabeales and Trentepohliales.This research also serves as an important reference for molecular diversity analysis of algae in other specific habitats,such as epiphytic algae and soil algae.

Effect of cypermethrin insecticide on the microbial community in cucumber phyllosphere

Cucumber (Cucumis sativus) is one of the most widely used vegetable in the world,and different pesticides have been extensively used for controlling the insects and disease pathogens of this plant.However,little is known about how the pesticides affect the microbial community in cucumber phyllosphere.This study was the first attempt to assess the impact of pyrethroid insecticide cyperemethrin on the microbial communities of cucumber phyllosphere using biochemical and genetic approaches.Phospholipid fatty ac...

Assessing the impact of fungicide enostroburin application on bacterial community in wheat phyllosphere

Fungicides have been used extensively for controlling fungal pathogens of plants. However, little is known regarding the effects that fungicides upon the indigenous bacterial communities within the plant phyllosphere. The aims of this study were to assess the impact of fungicide enostroburin upon bacterial communities in wheat phyllosphere. Culture-independent methodologies of 16S rDNA clone library and 16S rDNA directed polymerase chain reaction with denaturing gradient gel electrophoresis （PCR-DGGE） were used for monitoring the change of bacterial community. The 16S rDNA clone library and PCR-DGGE analysis both confirmed the microbial community of wheat plant phyllosphere were predominantly of the γ-Proteobacteria phyla. Results from PCR-DGGE analysis indicated a significant change in bacterial community structure within the phyllosphere following fungicide enostroburin application. Bands sequenced within control cultures were predominantly of Pseudomonas genus, but those bands sequenced in the treated samples were predominantly strains of Pantoea genus and Pseudomonas genus. Of interest was the appearance of two DGGE bands following fungicide treatment, one of which had sequence similarities （98%） to Pantoea sp. which might be a competitor of plant pathogens. This study revealed the wheat phyllosphere bacterial community composition and a shift in the bacterial community following fungicide enostroburin application.

Screening of Antagonistic Bacteria from Phyllosphere towards Tobacco Brown Spot Fungus Alternaria alternata

The possibility of employing antagonistic bacteria for the control of tobacco brown spot was studied. Approximately 136 strains of bacteria were isolated from phyllospheres of tobacco and 9 of these possessed high levels of antagonistic properties, They significantly reduced brown spot in detached tobacco leaves when artificially inoculated with Alternaria alternata. Culture filtrate of the most effective bacterial isolate which designated as Tpb88 was shown to be very efficient in inhibiting mycelial growth of A alternata in dual cultures. Culture filtrate of Tpb88 inhibited germination and germ tube elongation of A alternata. The results showed that the culture filtrate directly inhibited spore germination of A alternata, especially during the first hours of the paired cultivation. The rate of antagonistic activity of culture filtrate of Tpb88 depended on its concentration in the mixture. The greatest inhibition of spore germination was ob served at the highest concentration of filtrate （filtrate to fungal spores inocula mixed in proportion 1 ： 50）. These suggest that the hypothetic mechanism of Tpb88 against tobacco brown spot is to produce antagonistic substances.

Host plant traits play a crucial role in shaping the composition of epiphytic microbiota in the arid desert,Northwest China

Phyllosphere microorganisms are a crucial component of environmental microorganisms,highly influenced by host characteristics,and play a significant role in plant health and productivity.Nonetheless,the impact of host characteristics on shaping phyllosphere microbial communities of plants with different life forms remains ambiguous.Utilizing high-throughput sequencing technology,this study analyzed the diversity and community composition of phyllosphere epiphytic microorganisms(e.g.,bacteria and fungi)of various plant life forms in the hinterland of the Gurbantunggut Desert,Northwest China.Functional annotation of prokaryotic taxa(FAPROTAX)and fungi function guild(FUNGuild)were employed to assess the ecological functions of microorganisms and to investigate the role of stochastic and deterministic processes in shaping phyllosphere microbial communities.Result showed a diverse array of phyllosphere epiphytic microorganisms in the desert plants,with Proteobacteria,Cyanobacteria,and Actinobacteriota dominating bacterial community,while Ascomycota and Basidiomycota were prevalent in fungal community.Comparison across different plant life forms highlighted distinct microbial communities,indicating strong filtering effects by plant characteristics.FAPROTAX prediction identified intracellular parasites(accounting for 27.44%of bacterial community abundance),chemoheterotrophy(10.12%),and phototrophy(17.41%)as the main functions of epiphytic bacteria on leaves of different life form plants.FUNGuild prediction indicated that phyllosphere epiphytic fungi primarily served as Saprotrophs(81.77%),Pathotrophs(17.41%),and Symbiotrophs(0.82%).Co-occurrence network analysis demonstrated a predominance of positive correlations among different microbial taxa.Raup-Crick dissimilarity index analysis revealed that deterministic processes predominantly influenced phyllosphere bacterial and fungal community assembly.Variance partitioning analysis and random forest modeling suggested that plant leaf functional traits significantly impacted both bacterial and fungal community composition,with fungal community composition showing a closer association with leaf nutrients and physiology compared with bacterial community composition.The distinct responses of bacterial and fungal communities to plant traits were attributed to the differing properties of bacteria and fungi,such as bacteria having higher potential dispersal rates and broader ecological niches than fungi.Overall,the results indicate that phyllosphere bacterial and fungal communities undergo similar community assembly processes,with fungi being more influenced by plant characteristics than bacteria.These findings offer novel insights into the ecology of phyllosphere microbial communities of desert plants.

Phyllosphere microbiota:Community dynamics and its interaction with plant hosts

Plants are colonized by various microorganisms in natural environments.While many studies have demonstrated key roles of the rhizosphere microbiota in regulating biological processes such as nutrient acquisition and resistance against abiotic and biotic challenges,less is known about the role of the phyllosphere microbiota and how it is established and maintained.This review provides an update on current understanding of phyllosphere community assembly and the mechanisms by which plants and microbes establish the phyllosphere microbiota for plant health.

Film mulching reduces antibiotic resistance genes in the phyllosphere of lettuce

Phyllosphere is an important reservoir of antibiotic resistance genes(ARGs),but the transfer mechanism of ARGs from soil and air to phyllosphere remains unclear.This study demonstrated that soil-air-phyllosphere was the dominant ARG transfer pathway,and blocking it by film mulching can reduce typical phyllosphere ARGs in lettuce by 80.7%-98.7%(89.5%on average).To further eliminate phyllosphere ARGs in lettuce grown with film mulching,the internal soil-endosphere-phyllosphere transfer pathway deserves more attention.We analyzed the ARG hosts and the resistome in lettuce rhizosphere and phyllosphere with film mulching via hybrid Illumina-Nanopore sequencing.Pseudomonas sp.7SR1 was more abundant than other ARG hosts,accounting for 1.0%and 47.1%of the total bacteria in rhizosphere and phyllosphere,respectively.The species has flagella that can promote mobility and can excrete extracellular polymeric substances and/or surfactant-like microbial products,which benefits its colonization in the phyllosphere.Impeding the migration of Pseudomonas sp.7SR1 via the soil-endosphere-phyllosphere pathway would be effective to further reduce ARGs in phyllosphere.Multidrug resistant genes were predominant in phyllosphere(40.3%of the total),and 87.6%of the phyllosphere ARGs were located on chromosomes,indicating relatively low horizontal gene transfer(HGT)potentials.This study provides insights into the transfer mechanism,hosts,and control strategies of phyllosphere ARGs in typical plants.

Leaf metabolic influence of glyphosate and nanotubes on the Arabidopsis thaliana phyllosphere

Chemical exposure can indirectly affect leaf microbiota communities,but the mechanism driving this phenomenon remains largely unknown.Results revealed that the co-exposure of glyphosate and multi-carbon nanotubes(CNTs)caused a synergistic inhibitory effect on the growth and metabolism of Arabidopsis thaliana shoots.However,only a slight inhibitory effect was induced by nanotubes or glyphosate alone at the tested concentrations.Several intermediate metabolites of nitrogen metabolism and fatty acid synthesis pathways were upregulated under the combined treatment,which increased the amount of energy required to alleviate the disruption caused by the combined treatment.Additionally,compared with the two individual treatments,the glyphosate/nanotube combination treatment induced greater fluctuations in the phyllosphere bacterial community members with low abundance(relative abundance(RA)<1%)at both the family and genus levels,and among these bacteria some plant growth promotion and nutrient supplement related bacteria were markable increased.Strikingly,strong correlations between phyllosphere bacterial diversity and metabolites suggested a potential role of leaf metabolism,particularly nitrogen and carbohydrate metabolism,in restricting the range of leaf microbial taxa.These correlations between phyllosphere bacterial diversity and leaf metabolism will improve our understanding of plant-microbe interactions and the extent of their drivers of variation and the underlying causes of variability in bacterial community composition.

Profile of the culturable microbiome capable of producing acyl-homoserine lactone in the tobacco phyllosphere

Bacterial populations coexisting in the phyllosphere niche have important effects on plant health. Quorum sensing （QS） allows bacteria to communicate via diffusible signal molecules, but QS-dependent behaviors in phyllosphere bacterial populations are poorly understood. We investigate the dense and diverse N-acyl-homoserine lactone （AHL）-producing phyllosphere bacteria living on tobacco leaf surfaces via a culture-dependent method and 16S rRNA gene sequencing. Our results indicated that approximately 7.9%-11.7% of the culturable leaf-associated bacteria have the ability to produce AHL based on the assays using whole-cell biosensors. Sequencing of the 16S rRNA gene assigned the AHL-producing strains to two phylogenetic groups, with Gammaproteobacteria （93%） as the predominant group, followed by Alphaproteobacteria. All of the AHL-producing Alphaproteobacteria were affiliated with the genus Rhizobium, whereas the AHL-producing bacteria belonging to the Gammaproteobacteria mainly fell within the genera Pseudomonas, Acinetobacter, Citrobacter, Enterobacter, Pantoea and Serratia. The bioassays of supernatant extracts revealed that a portion of the strains have a remarkable AHL profile for AHL induction activity using the two different biosensors, and one compound in the active extract of a representative isolate, NTL223, corresponded to 3-oxo-hexanoyl-homoserine lactone. A large population size and diversity of bacteria capable of AHL-driven QS were found to cohabit on leaves, implying that cross-communication based AHL-type QS may be common in the phyllosphere. Furthermore, this study provides a general snapshot of a potential valuable application of AHL-producing bacteria inhabiting leaves for their presumable ecological roles in the phyllosphere.

Enantioselective effects of imazethapyr residues on Arabidopsis thaliana metabolic profile and phyllosphere microbial communities

Imazethapyr(IM)is a widely used acetolactate synthase-inhibiting chiral herbicide.It has long-term residuals that may be absorbed by the human body through the edible parts of plants,such as vegetable leaves or fruits.Here,we selected a model plant,Arabidopsis thaliana,to determine the effects of R-IM and S-IM on its leaf structure,photosynthetic efficiency,and metabolites,as well as the structures of microorganisms in the phyllosphere,after 7 days of exposure.Our results indicated enantiomeric differences in plant growth between R-IM and S-IM;133μg/kg R-IM showed heavier inhibition of photosynthetic efficiency and greater changes to subcellular structure than S-IM.R-IM and S-IM also had different effects on metabolism and leaf microorganisms.S-IM mainly increased lipid compounds and decreased amino acids,while R-IM increased sugar accumulation.The relative abundance of Moraxellaceae human pathogenic bacteria was increased by R-IM treatment,indicating that R-IM treatment may increase leaf surface pathogenic bacteria.Our research provides a new perspective for evaluating the harmfulness of pesticide residues in soil,phyllosphere microbiome changes via the regulation of plant metabolism,and induced pathogenic bacterial accumulation risks.

Diversity of Endophytic Fungi in Banana Cultivars of Assam India

Endophytic fungal isolates(139 no.)were obtained from 143(62 roots,18 fruits and 54 leaves)samples of 15 dif­ferent varieties of banana collected from 10 sites in Assam,India during 2018-2019.Overall isolation frequency from surface-sterilized tissue ranged from 10%-80%(as per site)and 6%-70%(as per variety of banana).All isolates were segregated into 40 different types on the basis of macromorphological and micro morphological characteristics.Forty different fungal taxa were isolated belonging to 14 genera including Absidia,Arthrinium,Aspergillus,Bipolaris,Cla­dosporium,Curvularia,Dendrophion,Fusarium,Humicola,Mortierella,Mucor,Penicillium,Paecilomyces,Verticilli­um and one mycelium sterile.Among them,Cladosporium cladosporioidies and Paecilomyces sp.frequently occurred in most of the sites surveyed whereas Cladospoirum cladosporioides and Aspergillus sp.8,Fusarium graminseram were most frequently isolated from different varieties.However,all sites differed in their fungal diversity.Banana samples from Narigoan and Jorhat have been found with maximum fungal species followed by marigoan samples so as to Banana varieties Amrit Sagar endowed 27 no.of fungi followed by Jehaji and Honda which were associated with a maximum 14 fungal sp.Isolation frequency and relative abundance of Cladosporium cladosporiodes(80%,4.6),Paecilomyces farinosus(80%,4.6)followed by Penicillium ruburm,Aspergillus sp.8&9(70%,4.02)were recorded as maximum comparatively in different sites.However,Aspergillus sp.8,Mortieralla sp.and Pacilomyces farinosus are isolated frequently from different banana varieties(73.33%,4.93).

Production of a newly isolated Paenibacillus polymyxa biocontrol agent using monosodium glutamate wastewater and potato wastewater

A phyllosphere bacterial strain EBL-06 was isolated from wheat leaves. The morphology, cultural characteristics, phospholipid fatty acids, physiological and antagonistic fungus activities of this strain were investigated. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the relevant bacteria. The results showed that the isolate EBL-06 was a strain of Paenibacillus polymyxa; this strain performed a high level of antagonistic fungus activity toward a broad spectrum of phytopathogens, such as Botrytis cinerea, Cladosporium cucumerinum, Fusarium spp. The isolate EBL-06 can grow well using monosodium glutamate wastewater （MGW） and potato wastewater （PW） as culture medium. The maximum yield of 6.5 × 10^9 CFU/mL of the isolate EBL-06 anti-fungus biocontrol agent was reached in 15 hr cultivation at 28℃, pH 6.0-7.5 using the mixture of MGW and PW （1：9）.

Effects of imazethapyr spraying on plant growth and leaf surface microbial communities in Arabidopsis thaliana

Imazethapyr (IM) is an acetolactate synthase (ALS)-inhibiting herbicide that has been widely used in recent years.However,IM spraying can lead to the accumulation of herbicide residues in leaves.Here,we determined the effects of IM spraying on the plant growth and leaf surface microbial communities of Arabidopsis thaliana after 7 and 14 days of exposure.The results suggested that IM spraying inhibited plant growth.Fresh weight decreased to 48% and 26% of the control value after 7 and 14 days,respectively,of 0.035 kg/ha IM exposure.In addition,anthocyanin content increased 9.2-fold and 37.2-fold relative to the control content after 7 and 14 days of treatment,respectively.Furthermore,IM spraying destroyed the cell structures of the leaves,as evidenced by increases in the number of starch granules and the stomatal closure rate.Reductions in photosynthetic efficiency and antioxidant enzyme activity were observed after IM spraying,especially after 14 days of exposure.The diversity and evenness of the leaf microbiota were not affected by IM treatment,but the composition of community structure at the genus level was altered by IM spraying.Imazethapyr application increased the abundance of Pseudomonas,a genus that includes species pathogenic to plants and humans,indicating that IM potentially increased the abundance of pathogenic bacteria on leaves.Our findings increase our understanding of the relationships between herbicide application and the microbial community structures on plant leaves,and they provide a new perspective for studying the ecological safety of herbicide usage.

Investigating the bacterial community and amoebae population in rural domestic wastewater reclamation for irrigation

Reclamation of domestic wastewater for agricultural irrigation is viewed as a sustainable option to create an alternative water source and address water scarcity. Free-living amoebae（FLA）, which are amphizoic protozoa, are widely distributed in various environmental sources. The FLA could cause considerable environmental and health risks. However, little information is available on the risk of these protozoa. In this study, we evaluated the feasibility using rural domestic wastewater for agricultural irrigation, and analyzed dynamic changes of the microbial community structure and FLA populations in raw and treated wastewater, as well as the phyllosphere and rhizosphere of lettuce production sites that were irrigated with different water sources. The bacterial community dynamics were analyzed by terminal restriction fragment length polymorphism（T-RFLP）. The bacterial community structures in the influent were similar to that in the effluent, while in some cases relative abundances varied significantly. The populations of Acanthamoeba spp. and Hartmannella vermiformis in the anaerobically treated wastewater were significantly higher than in the raw wastewater. The vegetables could harbor diverse amoebae, and the abundances of Acanthamoeba spp. and H. vermiformis in the rhizosphere were significantly higher than in the phyllosphere. Accordingly, our studies show insight into the distribution and dissemination of amoebae in wastewater treatment and irrigation practices.

Plant-associated fungal communities in the light of meta’omics

Approaches for the cultivation-independent analysis of microbial communities are summarized as meta’omics,which predominantly includes metagenomic,-transcriptomic,-proteomic and-metabolomic studies.These have shown that endophytic,root-associated and soil fungal communities are strongly shaped by associated plant species.The impact of plant identity on the composition of its litterssociated fungal community remains to be disentangled from the impact of litter chemistry.The composition of the plant community also shapes the fungal community.Most strikingly,adjacent plant species may share mycorrhizal symbionts even if the plants usually have different types of mycorrhizal fungi associated with them(ectomycorrhizal,ericoid and arbuscular mycorrhizal fungi).Environmental parameters weakly explain fungal community composition globally,and their effect is inconsistent at local and regional scales.Decrease in similarity among communities with increasing distance(i.e.distance decay)has been reported from local to global scales.This pattern is only exceptionally caused by spatial dispersal limitation of fungal propagules,but mostly due to the inability of the fungi to establish at the particular locality(i.e.environmental filtering or competitive exclusion).Fungal communities usually undergo pronounced seasonal changes and also differ between consecutive years.This indicates that development of the communities is usually not solely cyclic.Meta’omic studies challenge the classical view of plant litter decomposition.They show that mycorrhizal and(previously)endophytic fungi may be involved in plant litter decomposition and only partly support the idea of a succession from an Ascomycota to a Basidiomycota-dominated community.Furthermore,vertical separation of saprotrophic and mycorrhizal species in soil and sequential degradation from easily accessible to‘recalcitrant’plant compounds,such as lignin,can probably not be generalized.The current models of litter decomposition may therefore have to be eventually refined for certain ecosystems and environmental conditions.To gain deeper insights into fungal ecology,a meta’omic study design is outlined which focuses on environmental processes,because fungal communities are usually taxonomically diverse,but functionally redundant.This approach would initially identify dynamics of chemical shifts in the host and/or substrate by metametabolomics.Detected shifts would be subsequently linked to microbial activity by correlation with metatranscriptomic and/or metaproteomic data.A holistic trait-based approach might finally identify factors shaping taxonomic composition in communities against the dynamics of the environmental process(es)they are involved in.

OPPORTUNITIES AND APPROACHES FOR MANIPULATING SOIL-PLANT MICROBIOMES FOR EFFECTIVE CROP NITROGEN USE IN AGROECOSYSTEMS

Soil microbiomes drive the biogeochemical cycling of nitrogen and regulate soil N supply and loss,thus,pivotal nitrogen use efficiency(NUE).Meanwhile,there is an increasing awareness that plant associated microbiomes and soil food web interactions is vital for modulating crop productivity and N uptake.The rapid advances in modern omics-based techniques and biotechnologies make it possible to manipulate soil-plant microbiomes for improving NUE and reducing N environmental impacts.This paper summarizes current progress in research on regulating soil microbial N cycle processes for NUE improvement,plant-microbe interactions benefiting plant N uptake,and the importance of soil microbiomes in promoting soil health and crop productivity.We also proposes a potential holistic(rhizosphere-root-phyllosphere)microbe-based approach to improve NUE and reduce dependence on mineral N fertilizer in agroecosystems,toward nature-based solution for nutrient management in intensive cropping systems.

Immigrant Pantoea agglomerans embedded within indigenous microbial aggregates: A novel spatial distribution of epiphytic bacteria

Immigrant bacteria located on leaf surfaces are important to the health of plants as well as to people who consume fresh fruits and vegetables. However, the spatial distribution and organization of these immigrant bacteria on leaf surfaces are still poorly understood. To examine the spatial organization of these strains, two bacterial strains on tobacco leaves： （1） an indigenous strain, Pseudomonas stutzeri Nov. Y2011 labeled with green fluorescent protein, and （2） an immigrant strain Pantoea agglomerans labeled with cyan fluorescent protein isolated from pear, were studied. Under moist conditions, P. agglomerans cells quickly disappeared from direct observation by laser- scanning confocal microscopy, although elution results indicated that large amounts of live cells were still present on the leaves. Following exposure to desiccation stress, particles of cyan fluorescent protein-labeled P. agglomerans were visible within cracked aggregates of P. stutzeri Nov. Y2011. Detailed observation of sectioned aggregates showed that colonies of immigrant P. agglomerans were embedded within aggregates of P. stutzeri Nov. Y2011. Furthermore, carbon-resource partitioning studies suggested that these two species could coexist without significant nutritional competition. This is the first observation of an immigrant bacterium embedding within aggregates of indigenous bacteria on leaves to evade harsh conditions in the phyllosphere.