Negative plant‐phyllosphere feedbacks in native Asteraceae hosts – a novel extension of the plant‐soil feedback framework

Over the past 25 years, the plant‐soil feedback (PSF) framework has catalyzed our understanding of how belowground microbiota impact plant fitness and species coexistence. Here, we apply a novel extension of this framework to microbiota associated with aboveground tissues, termed ‘plant‐phyllosphere feedback (PPFs)’. In parallel greenhouse experiments, rhizosphere and phyllosphere microbiota of con‐ and heterospecific hosts from four species were independently manipulated. In a third experiment, we tested the combined effects of soil and phyllosphere feedback under field conditions. We found that three of four species experienced weak negative PSF whereas, in contrast, all four species experienced strong negative PPFs. Field‐based feedback estimates were highly negative for all four species, though variable in magnitude. Our results suggest that phyllosphere microbiota, like rhizosphere microbiota, can potentially mediate plant species coexistence via negative feedbacks. Extension of the PSF framework to the phyllosphere is needed to more fully elucidate plant‐microbiota interactions.     

Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice

The above- and below-ground parts of rice plants create specific habitats for various microorganisms. In this study, we characterized the phyllosphere and rhizosphere microbiota of rice cultivars using a metaproteogenomic approach to get insight into the physiology of the bacteria and archaea that live in association with rice. The metaproteomic datasets gave rise to a total of about 4600 identified proteins and indicated the presence of one-carbon conversion processes in the rhizosphere as well as in the phyllosphere. Proteins involved in methanogenesis and methanotrophy were found in the rhizosphere, whereas methanol-based methylotrophy linked to the genus Methylobacterium dominated within the protein repertoire of the phyllosphere microbiota. Further, physiological traits of differential importance in phyllosphere versus rhizosphere bacteria included transport processes and stress responses, which were more conspicuous in the phyllosphere samples. In contrast, dinitrogenase reductase was exclusively identified in the rhizosphere, despite the presence of nifH genes also in diverse phyllosphere bacteria.     

Resilience of the Natural Phyllosphere Microbiota of the Grapevine to Chemical and Biological Pesticides

The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent (Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards.     

青杨雌雄株叶际微生物群落多样性和结构的差异

【目的】本论文探究了青杨雌雄株的叶际微生物的群落结构差异及其主要环境影响因素。【方法】以河北小五台山的天然青杨林为研究对象,采用基于16S rRNA/ITS1基因的MiSeq高通量测序技术,分析了青杨雌雄株叶际细菌和真菌的群落结构,并耦合分析其与叶片理化性质的相关性。【结果】测序结果表明细菌和真菌的多样性指数ACE、Chao1、Shannon、Simpson在雌雄株间都无显著性差异(P>0.05)。Metastats组间群落显著性差异分析表明,在门水平,青杨雌雄株叶际细菌和真菌都无显著差异。而在属水平,青杨雌雄株的叶际细菌Amnibacterium和Spingomonas及真菌Aureobasidium、Elmerina、Exobasidium、Endoconidioma、Monilinia和Rhodotorula的相对丰度在雌雄株叶际有显著差异(P<0.05)。基于OTUs的菌群分析表明,青杨雌株和雄株的叶际环境上都有其各自的特有菌群,如雌株的特有真菌Pringsheimia(0.15%)和细菌Chitinophaga(0.04%)。RDA冗余分析表明,叶片含水量与青杨叶际真菌的群落结构有显著相关性(P<0.05),而未发现青杨细菌群落结构与测定的叶片理化性质有显著相关。【结论】青杨雌雄株叶际微生物在属水平有显著分异的菌属,且可能受叶片理化性质影响,该结果为揭示雌雄异株植物的叶际微生物差异有重要借鉴意义。     

Summer season and long-term drought increase the richness of bacteria and fungi in the foliar phyllosphere of Quercus ilex in a mixed Mediterranean forest

We explored the changes in richness, diversity and evenness of epiphytic (on the leaf surface) and endophytic (within leaf tissues) bacteria and fungi in the foliar phyllosphere of Quercus ilex, the dominant tree species of Mediterranean forests. Bacteria and fungi were assessed during ontogenic development of the leaves, from the wet spring to the dry summer season in control plots and in plots subjected to drought conditions mimicking those projected for future decades. Our aim was to monitor succession in microbiota during the colonisation of plant leaves and its response to climate change. Ontogeny and seasonality exerted a strong influence on richness and diversity of the microbial phyllosphere community, which decreased in summer in the whole leaf and increased in summer in the epiphytic phyllosphere. Drought precluded the decrease in whole leaf phyllosphere diversity and increased the rise in the epiphytic phyllosphere. Both whole leaf bacterial and fungal richness decreased with the decrease in physiological activity and productivity of the summer season in control trees. As expected, the richness of epiphytic bacteria and fungi increased in summer after increasing time of colonisation. Under summer dry conditions, there was a positive relationship between TRF (terminal restriction fragments) richness and drought, both for whole leaf and epiphytic phyllosphere, and especially for fungal communities. These results demonstrate that changes in climate are likely to significantly alter microbial abundance and composition of the phyllosphere. Given the diverse functions and large number of phyllospheric microbes, the potential functional implications of such community shifts warrant exploration.     

A Synthetic Community Approach Reveals Plant Genotypes Affecting the Phyllosphere Microbiota

The identity of plant host genetic factors controlling the composition of the plant microbiota and the extent to which plant genes affect associated microbial populations is currently unknown. Here, we use a candidate gene approach to investigate host effects on the phyllosphere community composition and abundance. To reduce the environmental factors that might mask genetic factors, the model plant Arabidopsis thaliana was used in a gnotobiotic system and inoculated with a reduced complexity synthetic bacterial community composed of seven strains representing the most abundant phyla in the phyllosphere. From a panel of 55 plant mutants with alterations in the surface structure, cell wall, defense signaling, secondary metabolism, and pathogen recognition, a small number of single host mutations displayed an altered microbiota composition and/or abundance. Host alleles that resulted in the strongest perturbation of the microbiota relative to the wild-type were lacs2 and pec1. These mutants affect cuticle formation and led to changes in community composition and an increased bacterial abundance relative to the wild-type plants, suggesting that different bacteria can benefit from a modified cuticle to different extents. Moreover, we identified ein2, which is involved in ethylene signaling, as a host factor modulating the community''s composition. Finally, we found that different Arabidopsis accessions exhibited different communities, indicating that plant host genetic factors shape the associated microbiota, thus harboring significant potential for the identification of novel plant factors affecting the microbiota of the communities.     

How does plant sex alter microbiota assembly in dioecious plants?

Plant microbiota can greatly impact plant growth, defense, and health in different environments. Thus, it might be evolutionarily beneficial for plants to be able to control processes related to microbiota assembly. Dioecious plant species display sexual dimorphism in morphology, physiology, and immunity. These differences imply that male and female individuals might differently regulate their microbiota, but the role of sex in microbiota assembly has been largely neglected so far. Here, we introduce the mechanism of how sex controls microbiota in plants analogically to the sex regulation of gut microbiota in animals, in particular in humans. We argue that plant sex imposes selective pressure on filtering and constructing microbiota in the rhizosphere, phyllosphere, and endosphere along the soil–plant continuum. Since male plants are more resistant than female plants to environmental stresses, we suggest that a male host forms more stable and resistant plant microbiota that cooperate more effectively with the host to resist stresses. Male and female plants can distinguish whether a plant is of the same or different sex, and males can alleviate stress-caused damage in females. The impact of a male host on microbiota would protect female plants from unfavorable environments.     

Distinct Phyllosphere Bacterial Communities on Arabidopsis Wax Mutant Leaves

The phyllosphere of plants is inhabited by diverse microorganisms, however, the factors shaping their community composition are not fully elucidated. The plant cuticle represents the initial contact surface between microorganisms and the plant. We thus aimed to investigate whether mutations in the cuticular wax biosynthesis would affect the diversity of the phyllosphere microbiota. A set of four Arabidopsis thaliana eceriferum mutants (cer1, cer6, cer9, cer16) and their respective wild type (Landsberg erecta) were subjected to an outdoor growth period and analysed towards this purpose. The chemical distinctness of the mutant wax phenotypes was confirmed by gas chromatographic measurements. Next generation amplicon pyrosequencing of the bacterial communities showed distinct community patterns. This observation was supported by denaturing gradient gel electrophoresis experiments. Microbial community analyses revealed bacterial phylotypes that were ubiquitously present on all plant lines (termed “core” community) while others were positively or negatively affected by the wax mutant phenotype (termed “plant line-specific“ community). We conclude from this study that plant cuticular wax composition can affect the community composition of phyllosphere bacteria.     

Untangling metabolic and communication networks: interactions of enterics with phytobacteria and their implications in produce safety

Recent outbreaks of vegetable-borne gastrointestinal illnesses across the globe demonstrate that human enteric pathogens can contaminate produce at any stage of production. Interactions of enterics with native plant-associated microbiota influence the microbiological safety of produce by affecting the attachment, persistence and proliferation of human pathogens on plants. Supermarket surveys have revealed that bacteria, but not fungi or mechanical damage, promote the growth of Salmonella enterica on produce. Field and laboratory studies have indicated that some plant pathogenic bacteria and fungi facilitate the entry and internalization of human pathogens in plants. Conversely, some phytobacteria, including those involved in biocontrol of plant diseases, significantly inhibit attachment and plant colonization by non-typhoidal Salmonella and enterovirulent Escherichia coli by producing antibiotics or competing for nutrients in the phyllosphere. In this review, we attempt to elucidate the mechanisms of interactions between human enteric pathogens and plant-associated microbiota, and describe how these interactions affect produce safety.     

Two-way microscale interactions between immigrant bacteria and plant leaf microbiota as revealed by live imaging

The phyllosphere – the aerial parts of plants – is an important microbial habitat that is home to diverse microbial communities. The spatial organization of bacterial cells on leaf surfaces is non-random, and correlates with leaf microscopic features. Yet, the role of microscale interactions between bacterial cells therein is not well understood. Here, we ask how interactions between immigrant bacteria and resident microbiota affect the spatial organization of the combined community. By means of live imaging in a simplified in vitro system, we studied the spatial organization, at the micrometer scale, of the biocontrol agent Pseudomonas fluorescens A506 and the plant pathogen P. syringae B728a when introduced to pear and bean leaf microbiota (the corresponding native plants of these strains). We found significant co-localization of immigrant and resident microbial cells at distances of a few micrometers, for both strains. Interestingly, this co-localization was in part due to preferential attachment of microbiota cells near newly formed P. fluorescens aggregates. Our results indicate that two-way immigrant bacteria – resident microbiota interactions affect the microscale spatial organization of leaf microbiota, and possibly that of other surface-related microbial communities.Subject terms: Microbial ecology, Microbial ecology     

Toward the construction of a technology platform for chemicals production from methanol: d-lactic acid production from methanol by an engineered yeast Pichia pastoris

In this study, the effects of di-n-butyl phthalate (DBP) on the phyllosphere bacterial community of field mustard (Brassica campestris L.) at the five-leaf stage were investigated. The indigenous alpha-diversity of the phyllosphere bacteria was altered after spraying with different concentrations of DBP. Shannon diversity indices were significantly changed on day 5 after treatment at DBP concentrations >?400 mg L^?1 (P?>?0.05). Nevertheless, the difference between treatment and control was not significant on day 9 after DBP treatment (P?>?0.05). Exposure to DBP resulted in a decrease in Proteobacteria and Firmicutes, and an increase in Actinobacteria at all sampling intervals. These changes included significant increases in the relative abundance of Paracoccus and Rhodococcus, and significant decreases in that of Pseudomonas, Exiguobacterium, an unclassified genus of Pseudomonadaceae, and an unclassified genus of Enterobacteriaceae. This study provides new evidence for the possibility of using phyllosphere microbiota to remediate DBP contamination.

     

Fertilization alters protistan consumers and parasites in crop-associated microbiomes

Crop plants carry an enormous diversity of microbiota that provide massive benefits to hosts. Protists, as the main microbial consumers and a pivotal driver of biogeochemical cycling processes, remain largely understudied in the plant microbiome. Here, we characterized the diversity and composition of protists in sorghum leaf phyllosphere, and rhizosphere and bulk soils, collected from an 8-year field experiment with multiple fertilization regimes. Phyllosphere was an important habitat for protists, dominated by Rhizaria, Alveolata and Amoebozoa. Rhizosphere and bulk soils had a significantly higher diversity of protists than the phyllosphere, and the protistan community structure significantly differed among the three plant–soil compartments. Fertilization significantly altered specific functional groups of protistan consumers and parasites. Variation partitioning models revealed that soil properties, bacteria and fungi predicted a significant proportion of the variation in the protistan communities. Changes in protists may in turn significantly alter the compositions of bacterial and fungal communities from the top-down control in food webs. Altogether, we provide novel evidence that fertilization significantly affects the functional groups of protistan consumers and parasites in crop-associated microbiomes, which have implications for the potential changes in their ecological functions under intensive agricultural managements.     

Microbial and Functional Diversity within the Phyllosphere of Espeletia Species in an Andean High-Mountain Ecosystem

Microbial populations residing in close contact with plants can be found in the rhizosphere, in the phyllosphere as epiphytes on the surface, or inside plants as endophytes. Here, we analyzed the microbiota associated with Espeletia plants, endemic to the Páramo environment of the Andes Mountains and a unique model for studying microbial populations and their adaptations to the adverse conditions of high-mountain neotropical ecosystems. Communities were analyzed using samples from the rhizosphere, necromass, and young and mature leaves, the last two analyzed separately as endophytes and epiphytes. The taxonomic composition determined by performing sequencing of the V5-V6 region of the 16S rRNA gene indicated differences among populations of the leaf phyllosphere, the necromass, and the rhizosphere, with predominance of some phyla but only few shared operational taxonomic units (OTUs). Functional profiles predicted on the basis of taxonomic affiliations differed from those obtained by GeoChip microarray analysis, which separated community functional capacities based on plant microenvironment. The identified metabolic pathways provided insight regarding microbial strategies for colonization and survival in these ecosystems. This study of novel plant phyllosphere microbiomes and their putative functional ecology is also the first step for future bioprospecting studies in search of enzymes, compounds, or microorganisms relevant to industry or for remediation efforts.     

健康与患病刺梨植株可培养叶际真菌菌群差异比较

本研究比较了健康与患叶斑病刺梨植株叶际真菌群落特征差异,以期探索病原菌的潜在来源,为人工构建具拮抗功能群落和刺梨叶斑病的生物防控提供参考。通过可培养方法对不同健康状况的刺梨叶际真菌进行分离培养,结合形态学和分子系统学对菌株进行综合鉴定;利用FUNGuild平台对真菌进行功能注释;结合根际、根部真菌作拆分网络分析探索病原菌的潜在来源。本研究结果表明：1）刺梨叶际真菌具有丰富的多样性。从刺梨叶际8个样品中共分离到真菌266株,其隶属于3门、6纲、13目、30科、46属中的61个种。其中,健康植株叶际内生真菌（LHE）包括8属10种27株,附生真菌（LHS）包括33属37种77株。患病叶际内生真菌（LDE）分离到7属10种38株;附生真菌（LDS）分离到31属35种124株。2）不同样品的真菌优势属和特有类群有差异。不同健康状况下叶际附生真菌的优势属均为拟盘多毛孢属Pestalotiopsis,但二者的相对多度存在差异,LHS为11.49%,LDS为32.26%;内生真菌优势属二者均为链格孢属Alternaria,但相对多度各异,LHE为33.33%,LDE为63.16%。其中,LHE特有类群为盘长孢状刺盘孢 Colletotrichum gloeosporioides和果生刺盘孢Colletotrichum fructicola等8种;LDE特有类群为茄链格孢 Alternaria solani和Didymella sinensis 等8种;LHS特有类群是草酸青霉Penicillium oxalicum和Peniophora crassitunicata等21种;LDS特有类群是尖孢镰刀菌Fusarium oxysporum、赭绿青霉Penicillium ochrochloron和易脆毛霉Mucor fragilis等19种。3）不同样品叶际真菌功能不同。经FUNGuild解析表明,LHS、LHE和LDE的叶际真菌功能群主要以腐生型为主,LDS则主要以植物病原菌群为主。本研究结果初步揭示刺梨植株健康与患叶斑病叶际间真菌多样性、群落组成及营养功能群存在差异,植株健康状况与其真菌群落特征密切相关;叶斑病病原菌主要源于刺梨叶际的附生微生物群。     

不同生境中桂花和夹竹桃叶际细菌的群落结构

植物叶际微生物多样性是目前植物-微生物关系研究的热点之一,但影响叶际微生物群落结构的主要因素目前还存在很大争议.本研究以生长在3处生境的桂花和夹竹桃为对象,基于高通量测序技术,分析2种植物叶际细菌的群落结构,探讨影响植物叶际细菌群落结构的主要因素.结果表明:来自3处生境的2种植物叶际细菌多样性无显著差异,构成叶际细菌群落的优势门主要包括放线菌门、拟杆菌门、衣原体门、蓝细菌门、厚壁菌门和变形菌门,优势属主要包括甲基杆菌属、鞘氨醇单胞菌属、薄层杆属、Polaromonas和无毛螺旋体属.植物种类、生境及二者的交互作用均能显著影响叶际细菌群落结构,其中生境的影响最大.     

Isolation and characterization of cultivable fermentative bacteria from the intestine of two edible snails, Helixpomatia and Cornu aspersum (Gastropoda: Pulmonata)

The intestinal microbiota of the edible snails Cornu aspersum fSyn: H. aspersa), and Helix pomatia were investigated by culture-based methods, 16S rRNA sequence analyses and phenotypic characterisations. The study was carried out on aestivating snails and two populations of H. pomatia were considered. The cultivable bacteria dominated in the distal part of the intestine, with up to 5.10(9) CFU g -1, but the Swedish H. pomatia appeared significantly less colonised, suggesting a higher sensitivity of its microbiota to climatic change. All the strains, but one, shared >/= 97% sequence identity with reference strains. They were arranged into two taxa: the Gamma Proteobacteria with Buttiauxella, Citrobacter, Enterobacter, Kluyvera, Obesumbacterium, Raoultella and the Firmicutes with Enterococcus, Lactococcus, and Clostridium. According to the literature, these genera are mostly assigned to enteric environments or to phyllosphere, data in favour of culturing snails in contact with soil and plants. None of the strains were able to digest filter paper, Avicel cellulose or carboxymethyl cellulose (CMC). Acetogens and methanogenic archaea were not cultivated, so the fate of hydrogen remains questionable. This microbiota could play important roles in the digestive process (fermentation) and the energy supply of the snail (L-lactate, acetate). The choice of cereals and plants by snail farmers should take into account the fermentative abilities of the intestinal microbiota.     

叶际微生物及其与外界互作的研究进展

叶际微生物及其生存环境共同形成了一个复杂的生态系统。建立在纯种分离和纯培养技术基础之上的传统研究方法只能了解其中部分叶际微生物,但对物种组成、种群结构和生态学作用等方面的认识都比较片面。近年来随着分子生物学和生物信息学的进步,人们对叶际微生物总群落的分析逐渐揭示了叶际微生物组成的多样性及其特点,以及与外界互相作用的复杂性。研究表明,植物种类、地理位置和季节差异等都不同程度地影响着叶际微生物群落的构成。本文综述了近年来国内外叶际微生物群落结构组成及其与外界互作方面的研究进展,有利于加深对叶际微生物的了解,也有助于深入理解叶际微生物与植物生长和植物病虫害防治的关联关系。     

Colonization of Japanese beech leaves by phyllosphere fungi

To examine the colonization of living leaves from buds and twigs by phyllosphere fungi of Japanese beech, the mycobiota were investigated on buds and twigs and on leaves covered with well-ventilated bags before budbreak. Ten phyllosphere fungi were isolated from rolled-up leaves within buds, bud scales, and twigs. However, frequencies of phyllosphere fungi on expanded leaves were reduced markedly when the buds were covered with well-ventilated bags before budbreak compared to the leaves that were not covered. This observation suggests that invasion of the fungi to the leaves from buds and twigs may be possible but is not the main route. Horizontal transmission may be common in endophytes and epiphytes of beech leaves. Phyllosphere mycobiota were then compared between sun and shade leaves. Of 13 species recorded as phyllosphere fungi, the frequencies of 2 species were lower and those of 3 species were higher in sun leaves than in shade leaves. Frequencies of the other 8 phyllosphere species were not different between sun and shade leaves. This result indicates that the colonization of leaves by some phyllosphere fungi was affected by the microenvironmental conditions on leaf surfaces.     

温室黄瓜叶部微生物区系

采用稀释分离法和消毒叶片研磨液培养法对温室黄瓜叶围和内生微生物进行了分离,共分离到248个菌株,初步鉴定出13个属的叶围真菌,其中链格孢属(Alternaria)和青霉属(Penicillium)真菌为优势类群;鉴定出4个属的内生真菌,其中曲霉属(Aspergillus)真菌为优势类群;10个属的叶围细菌,其中芽孢杆菌属(Bacillus)和黄单胞菌属(Xanthomonas)细菌为优势类群;6个属的内生细菌,其中芽孢杆菌属和假单胞菌属(Pseudomonas)细菌为优势类群;6个属的叶围酵母菌,其中隐球酵母属(Cryptococcus)为优势类群;已鉴定出2个属的叶围放线菌,分别为链霉菌属(Streptomyces)和小多孢菌属(Micropolpspora).未分离到内生酵母菌和放线菌.     

Phyllosphere Bacterial Community of Floating Macrophytes in Paddy Soil Environments as Revealed by Illumina High-Throughput Sequencing

The phyllosphere of floating macrophytes in paddy soil ecosystems, a unique habitat, may support large microbial communities but remains largely unknown. We took Wolffia australiana as a representative floating plant and investigated its phyllosphere bacterial community and the underlying driving forces of community modulation in paddy soil ecosystems using Illumina HiSeq 2000 platform-based 16S rRNA gene sequence analysis. The results showed that the phyllosphere of W. australiana harbored considerably rich communities of bacteria, with Proteobacteria and Bacteroidetes as the predominant phyla. The core microbiome in the phyllosphere contained genera such as Acidovorax, Asticcacaulis, Methylibium, and Methylophilus. Complexity of the phyllosphere bacterial communities in terms of class number and α-diversity was reduced compared to those in corresponding water and soil. Furthermore, the bacterial communities exhibited structures significantly different from those in water and soil. These findings and the following redundancy analysis (RDA) suggest that species sorting played an important role in the recruitment of bacterial species in the phyllosphere. The compositional structures of the phyllosphere bacterial communities were modulated predominantly by water physicochemical properties, while the initial soil bacterial communities had limited impact. Taken together, the findings from this study reveal the diversity and uniqueness of the phyllosphere bacterial communities associated with the floating macrophytes in paddy soil environments.