Ecochemical studies of interrelationships between epiphytic bacteria and host plants via secondary metabolites

The plant surface, which is representative of the phylloplane and rhizoplane, is a characteristic habitat for microorganisms. In this review, the ecological roles of phytoepiphytic bacteria will be described. The phylloplane and rhizoplane, which are adjacent to the atmosphere and soil sphere respectively, accumulate topically and/or selectively release secondary metabolites that are specific to the plant genera and species which reside within these regions. Some epiphytes have abilities to decarboxylate xenobiotic phenolic acids that have accumulated in the plant tissues and surfaces as a majority of such secondary metabolites. In physicochemically stressed soil, rhizosphere microflora often remedy such microenvironments within the rhizosphere in order to assist in the survival of the host, and some of the microfloral compositions behave as if they were symbionts. Specifically, some Sphingomonas spp., which are frequently isolated from the rhizosphere of acidic soil-tolerant plants in tropical zones, make possible the development of a rhizo-biocomplex. In this review, the possibility of rhizosphere regulation utilizing such a rhizo-biocomplex is discussed.     

Microbial Complexes Occurring on the Apogeotropic Roots and in the Rhizosphere of Cycad Plants

The microbial complexes of soil, the rhizosphere, and the rhizoplane of the apogeotropic (coralloid) roots of cycad plants were comparatively studied. The aseptically prepared homogenates of the surface-sterilized coralloid roots did not contain bacterial microsymbiont, indicating that in the root tissues the symbiosis is a two-component one (plant–cyanobacteria). At the same time, associated bacteria belonging to different taxonomic groups were detected in increasing amounts in the cycad rhizoplane, rhizosphere, and the surrounding soil. The bacterial communities found in the cycad rhizoplane and the surrounding soil were dominated by bacteria from the genus Bacillus. The saprotrophic bacteria and fungi colonizing the cycad rhizosphere and rhizoplane were dominated by microorganisms capable of degrading the plant cell walls. The local degradation of the cell wall was actually observed on the micrographs of the thin sections of cycad roots in the form of channels through which symbiotic cyanobacterial filaments can penetrate into the cortical parenchyma.     

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium–plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.     

The role of plant-associated rhizobacteria in plant growth,biocontrol and abiotic stress management

The rhizosphere is the region around the plant roots where maximum microbial activities occur. In the rhizosphere, microorganisms' beneficial and harmful activities affect plant growth and development. The mutualistic rhizospheric bacteria which improve plant growth and health are known as plant growth-promoting rhizobacteria (PGPR). They are very important due to their ability to help the plant in diverse ways. PGPR such as Pseudomonas, Bacillus, Azospirillum, Azotobacter, Arthrobacter, Achromobacter, Micrococcus, Enterobacter, Rhizobium, Agrobacterium, Pantoea and Serratia are now very well known. Rhizomicrobiome plays critical roles in nutrient acquisition and assimilation, improved soil texture, secreting and modulating extracellular molecules such as hormones, secondary metabolites, antibiotics and various signal compounds, all leading to the enhancement of plant growth and development. The microbes and compounds they secrete constitute valuable biostimulants and play pivotal roles in modulating plant stress responses. In this review, we highlight the rhizobacteria diversity and cutting-edge findings focusing on the role of a PGPR in plant growth and development. We also discussed the role of PGPR in resisting the adverse effects arising from various abiotic (drought, salinity, heat, heavy metals) stresses.     

Mineralogy of the rhizosphere in forest soils of the eastern United States

Chemical and mineralogical studies of forest soils from six sites in the northeastern and southeastern United States indicate that soil in the immediate vicinity of roots and fine root masses may show marked differences in physical characteristics, mineralogy and weathering compared to the bulk of the forest soil. Examination of rhizosphere and rhizoplane soils revealed that mineral grains within these zones are affected mechanically, chemically and mineralogically by the invading root bodies. In SEM/EDS analyses, phyllosilicate grains adjacent to roots commonly aligned with their long axis tangential to the root surface. Numerous mineral grains were also observed for which the edge abutting a root surface was significantly more fractured than the rest of the grain. Both the alignment and fracturing of mineral grains by growing roots may influence pedogenic processes within the rhizosphere by exposing more mineral surface to weathering in the root-zone microenvironment. Chemical interactions between roots and rhizosphere minerals included precipitation of amorphous aluminium oxides, opaline and amorphous silica, and calcium oxalate within the cells of mature roots and possible preferential dissolution of mineral grains adjacent to root bodies. Mineralogical analyses using X-ray diffraction (XRD) techniques indicated that kaolin minerals in some rhizosphere samples had a higher thermal stability than kaolin in the surrounding bulk forest soil. In addition, XRD analyses of clay minerals from one of the southeastern sites showed abundant muscovite in rhizoplane soil adhering to root surfaces whereas both muscovite and degraded mica were present in the immediately surrounding rhizosphere soil. This difference in mineral assemblages may be due to either K-enrichment in rhizoplane soil solutions or the preferential dissolution of biotite at the root-soil interface     

Selective effect of the systemic insecticide Phosphamidon on soil,root-surface and leaf-surface fungi

Results of the effect of Phosphamidon on soil, root- and leaf-surface fungi of Vigna sinensis var. azmerly were generally inconsistent. However, there were some consistent ones. Its effect on soil, root- and leaf-surface fungi persisted till after 40 days. In soil the total count was regularly increased after 20 days by the low and medium doses and after 40 days by the high dose. In the rhizosphere, Fusarium was almost consistently activated by the three doses after 5 days of treatment and by the low dose after 20 days. In the rhizoplane, the total count of fungi was lowered by the three doses after 40 days in the two sowings. Alternaria alternata was toxicated by the three doses after several experimental periods of the two sowings in the phyllosphere and phylloplane. When this insecticide was incorporated with the agar medium, it was of no significant effect on the total count of fungi at the low and medium doses but abnormally increased the total count at the high dose. In the liquid medium, the mycelia of the test fungi were not significantly affected except that of Penicillium corylophilum which was significantly retarded by the three doses and Rhizopus stolonifer which was significantly activated by the high dose.     

Biological control of anthracnose of chilli with rhizosphere and rhizoplane fungal isolates from grasses

The fungal species from rhizosphere and rhizoplane of perennial grasses of the Western Ghats of India were studied for their pathogenicity, antagonism in vitro, substrate and root colonization abilities, rhizosphere competence, growth in different soil pH and inoculum shelf-life. Out of 138 non-pathogenic fungal isolates tested, 85 were antagonistic in vitro to chilli anthracnose pathogen Colletotrichum capsici. Fifteen isolates with >60% inhibition zone to pathogen culture had saprophytic and root and rhizosphere colonization abilities. The sorghum grain inocula of test antagonistic fungi- Fusarium oxysporum, Chaetomium globosum and Trichoderma harzianum had the shelf-life of 90 days at 20?±?2?°C and required optimum soil pH of 6.5. The above fungal isolates when tested for biocontrol of anthracnose disease in greenhouse and field caused reduction in seedling mortality and decreased disease incidence and severity at various plant growth stages and significant reduction in chilli fruit and seed infection. The test antagonistic fungi promoted seedling and mature plant growth and increased fruit and seed yield. Populations of these antagonistic fungi were fairly high in chilli rhizosphere at harvest. The present study indicated that antagonistic fungi from grass rhizosphere and rhizoplane could be used to control anthracnose and promote plant growth, and increase yield of chilli in field.     

西北黄土高原柠条种植区土壤微生物多样性分析

柠条锦鸡儿(Caragana korshinskii)是我国黄土高原区重要的饲用豆科灌木植物。为揭示土壤微生物与柠条种植之间的关系,采用未培养技术提取样品宏基因组DNA,分别构建柠条根表、根际和自然土16SrDNA文库,分析各文库微生物群落的变化。结果显示,随距离柠条根部渐远,微生物数量呈现递减趋势。聚类分析发现,变形杆菌纲是根表土壤区系中的优势微生物种群(70.3%),尤其存在大量α-Proteobacteria类的能诱使植物形成根瘤的根瘤菌和对植物有促生作用的γ-Proteobacteria类微生物;而在根际和自然土中,酸杆菌属(Acidobacteria)和古菌(Archaea)数量较多。柠条根际的多样性指数最高,而根表和自然土微生物类群具有较高的优势度,表现出从根表、根际植物相关微生物到自然土单一简单微生物类群的过渡。说明植物根系和土壤环境与微生物类群具有相互选择性。     

Microbial complexes from apogeotropic roots and from rhizosphere of cycad plants

The microbial complexes of soil, the rhizosphere, and the rhizoplane of the apogeotropic (coralloid) roots of cycad plants were comparatively studied. The aseptically prepared homogenates of the surface-sterilized coralloid roots did not contain bacterial microsymbiont, indicating that it was absent in the root tissues. At the same time, associated bacteria belonging to different taxonomic groups were detected in increasing amounts in the cycad rhizoplane, rhizosphere, and the surrounding soil. The bacterial communities found in the cycad rhizoplane and the surrounding soil were dominated by bacteria from the genus Bacillus. The saprotrophic bacteria and fungi colonizing the cycad rhizosphere and rhizoplane were dominated by microorganisms capable of degrading the plant cell walls. The local degradation of the cell wall was actually observed on the micrographs of the thin sections of cycad roots in the form of channels, through which symbiotic cyanobacterial filaments can penetrate into the cortical parenchyma.     

Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Artificial induction of the apple replant problem byPenicillium claviforme inoculation

Inoculation of apple seedling roots withPenicillium claviforme reduced plant growth and resulted in morphological changes of roots resembling those found in seedlings growing in ‘apple replant problem’ soil (apple-sick soil). The introduced fungus persisted in the rhizosphere throughout the 30-month test period. The numbers of colony-forming units (CFU) ofPenicillium claviforme, as well as the ‘total’ number of CFU of micromycetes, were higher in the rhizoplane of the inoculated seedling in comparison with uninoculated plants. The numbers of CFU of phytotoxic micromycetes in the rhizosphere soil of inoculated seedlings were also proportionally higher. Apple tree growth was also reduced when seedlings were inoculated with the flucrescent bacteriumPseudomonas putida; however, no morphological changes were observed in the roots. Both micro-organisms introduced into the apple seedling rhizosphere caused changes in the microbial community. Inoculation withPenicillium claviforme andPseudomonas putida caused a decrease in the number of mycolytic bacteria in the rhizoplane of apple seedlings.     

Frankia populations in soils under different tree species—with special emphasis on soils under Betula pendula

The major source of substrates for microbial activity in the ectorhizosphere and on the rhizoplane are rhizodeposition products. They are composed of exudates, lysates, mucilage, secretions and dead cell material, as well as gases including respiratory CO₂. Depending on plant species, age and environmental conditions, these can account for up to 40% (or more) of the dry matter produced by plants. The microbial populations colonizing the endorhizosphere, including mycorrhizae, pathogens and symbiotic N₂-fixers have greater access to the total pool of carbon including that recently derived from photosynthesis. Utilization of rhizodeposition products induces at least a transient increase in soil biomass but a sustained increase depends on the state of the native soil biomass, the flow of other metabolites from the soil to the rhizosphere and the water relations of the soil. In addition, the phenomena of oligotrophy, cryptic growth, plasmolysis, dormancy and arrested metabolism can all influence the longevity of rhizosphere organisms. With this background, microbial growth in the rhizosphere will be discussed.     

PYRENE EFFECTS ON RHIZOPLANE BACTERIAL COMMUNITIES

Certain plant species promote biodegradation of polycyclic aromatic hydrocarbons (PAHs), but few studies have examined the microbial populations that are associated with the rhizoplane of these plants. In this study, the bacterial composition of the rhizoplane were characterized for four plant species during in soils with different histories of exposure to PAH and in the presence or absence of a pyrene spike at 100 mg kg^?1 pyrene. Three of the plant species including Andropogon gerrardii, Panicum coloratum and Melilotus officinalis were known to stimulate PAH degradation. Wheat (Triticum aestivum) was used as a reference species. Results showed that after 90 days, approximately 45% of the pyrene spike disappeared from soil without plants. In contrast, cultivation of plants resulted in 95% disappearance of pyrene. There were no significant differences in the extent of pyrene disappearance for different plants. In all cases, 16S rRNA gene profiles of the rhizoplane were less complex in the pyrene-spiked soils, suggesting that richness and evenness of the predominant bacteria were reduced. Our results show that pyrene contamination results in significant shifts in the composition of rhizosphere bacterial communities that are still further influenced by the plant species and prior exposure history to PAH contamination.     

The role of root exudates and allelochemicals in the rhizosphere

Plant roots serve a multitude of functions in the plant including anchorage, provision of nutrients and water, and production of exudates with growth regulatory properties. The root–soil interface, or rhizosphere, is the site of greatest activity within the soil matrix. Within this matrix, roots affect soil structure, aeration and biological activity as they are the major source of organic inputs into the rhizosphere, and are also responsible for depletion of large supplies of inorganic compounds. Roots are very complicated morphologically and physiologically, and their metabolites are often released in large quantities into the soil rhizosphere from living root hairs or fibrous root systems. Root exudates containing root-specific metabolites have critical ecological impacts on soil macro and microbiota as well as on the whole plant itself. Through the exudation of a wide variety of compounds, roots impact the soil microbial community in their immediate vicinity, influence resistance to pests, support beneficial symbioses, alter the chemical and physical properties of the soil, and inhibit the growth of competing plant species. In this review, we outline recent research on root exudation and the role of allelochemicals in the rhizosphere by studying the case of three plants that have been shown to produce allelopathic root exudates: black walnut, wheat and sorghum     

刺槐林魔芋健康高产的土壤微生态机制

通过对刺槐林和农田魔芋健株根区、根表及根外土壤微生物区系及养分含量比较,探索刺槐林魔芋健康高产的土壤微生态机制。结果表明:(1)刺槐林魔芋根外和根表土壤细菌数量分别较农田增加11.8%和588.9%,根区土壤真菌数量较农田显著减少74.4%。(2)刺槐林魔芋根区、根表及根外土壤中的有益优势微生物数量及其比例远高于农田魔芋,有害微生物数量远低于农田魔芋;在刺槐林魔芋根区、根表及根外土壤中,3种优势细菌为放射型根瘤菌(Rhizobium radiobacter)、苏云金芽孢杆菌(Bacillus thuringiensis)及摩氏假单胞菌(Pseudomonas mosselii),其中,根表土壤中放射型根瘤菌及苏云金芽孢杆菌数量分别为农田的25.7倍及13.0倍;2种优势真菌为黒附球菌(Epicoccum nigrum)和疣孢青霉(Penicillium verruculosum),1种优势放线菌为绿淀粉酶链霉菌(Streptomyces viridodiastaticus),其中刺槐林魔芋根表和根外土壤中黒附球菌数量分别较农田高159.2%和120.3%;大量存在于刺槐林下魔芋根外土壤中的疣孢青霉、以及根区、根表、根外土壤中的绿淀粉酶链霉菌在农田魔芋相应部位均未检出。(3)刺槐林下魔芋根外、根区土壤有机质含量分别较农田显著增加167.6%、39.6%,但速效P、K含量较农田分别显著降低85.6%~91.3%、12.4%~13.0%。研究认为,刺槐林魔芋健康高产与其根区、根表及根外土壤中特有的有益优势微生物数量多、有害微生物数量少以及土壤有机质含量高密切相关。     

高寒特境中甸黄芪植物根际微生物物种多样性及其抗生物膜活性成分

滇西北高寒地区分布着丰富的黄芪属植物资源，该属植物“根际效应”明显，其根际微生物极具抗菌药用资源研究价值。【目的】认知滇西北高寒特境中甸黄芪根际微生物的物种多样性，探究其可培养菌株次生代谢产物的化学多样性及抗菌、抗生物膜活性。【方法】采用宏基因组和微生物纯培养方法对中甸黄芪植物根际微生物进行物种多样性分析，同时采用高效液相色谱（high performance liquid chromatography，HPLC）、超高效液相色谱-质谱联用法（ultra-performance liquid chromatography-mass spectrometry，UPLC-MS）结合“微量肉汤稀释法” “孔板法”等多级联合筛选策略综合评估可培养菌株的抗菌活性药源研究价值。【结果】对中甸黄芪根际土壤样本的微生物分类操作单元（operational taxonomic units，OTU）进行分类注释，得到22门54纲105目187科316属856种微生物，其中优势菌群为慢生根瘤菌属。纯培养共获得27属54种95株可培养菌株，包括20属33种54株细菌和7属21种41株真菌，优势属分别为芽孢杆菌属和青霉属。其中，1株细菌Pseudomonas tolaasii ZTB4和3株真菌Aspergillus tabacinus ZNF17、Lecanicillium aphanocladii ZNF15、Umbelopsis nana ZTF31的次生代谢产物具有广谱抗菌活性。同时，菌株ZTB4和ZNF17的次生代谢产物也显示出优秀的抗耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）生物膜活性，并已验证这2株菌株的主要活性成分分别为环脂肽类与黄酮类。【结论】中甸黄芪植物根际微生物物种多样性较为丰富，其可培养菌株次生代谢产物有较好的化学多样性和抗菌、抗生物膜活性。研究结果为我国特境特色微生物药用资源的开发利用提供理论依据。     

Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Antibiotic-resistantArthrobacter sp. andPseudomonas sp. responses in the rhizosphere of blue grama after herbage removal

Summary Streptomycin-resistant Pseudomonas and Arthrobacter were isolated from semi-arid grassland soil and their relative responses in the rhizosphere of blue grama (Bouteloua gracilis) subjected to herbage removal were evaluated. Using plants grown in normal soil, the two bacteria showed differential responses to herbage removal, which were most marked in the rhizoplane, where the Pseudomonas showed a two-log unit increase over a 60 hour period, while Arthrobacter, in contrast, exhibited a one-log unit decrease in viable counts for at least 48 hours after defoliation, responses which are similar to those observed in root exudate medium experiments by earlier workers. These results suggest that the rhizoplane may be a critical environment for interaction of these two types of microorganisms, and that sequential responses of the root-associated soil microorganisms may occur after herbage removal from this important rangeland plant. These responses are most likely associated with increased exudate release following herbage removal, which has been best documented using blue grama grown under sterile conditions.     

Soil mycoflora in tomato fields

Density and species richness of fungal communities in soils ofFusarium infested and non-infested tomato-growing localities were studied by comparison of rhizoplanes, rhizospheres, and root-free soils. The rhizosphere soils harbored the highest counts of fungi, followed by root-free soil and rhizoplanes in both localities. Species richness was high in the rhizosphere and root-free soil but distinctly low in the rhizoplane. The population density of the zhizosphere and the rhizoplane showed a significant difference between infested and non-infested localities.     

Nanoparticle‐based measurements of pH and O2 dynamics in the rhizosphere of Zostera marina L.: effects of temperature elevation and light‐dark transitions

Seagrasses can modulate the geochemical conditions in their immediate rhizosphere through the release of chemical compounds from their below‐ground tissue. This is a vital chemical defence mechanism, whereby the plants detoxify the surrounding sediment. Using novel nanoparticle‐based optical O₂ and pH sensors incorporated in reduced and transparent artificial sediment, we investigated the spatio‐temporal dynamics of pH and O₂ within the entire rhizosphere of Zostera marina L. during experimental manipulations of light and temperature. We combined such measurements with O₂ microsensor measurements of the photosynthetic productivity and respiration of seagrass leaves. We found pronounced pH and O₂ microheterogeneity within the immediate rhizosphere of Z. marina, with higher below‐ground tissue oxidation capability and rhizoplane pH levels during both light exposure of the leaf canopy and elevated temperature, where the temperature‐mediated stimuli of biogeochemical processes seemed to predominate. Low rhizosphere pH microenvironments appeared to correlate with plant‐derived oxic microzones stimulating local sulphide oxidation and thus driving local proton generation, although the rhizoplane pH levels generally where much higher than the bulk sediment pH. Our data show that Z. marina can actively alter its rhizosphere pH microenvironment alleviating the local H₂S toxicity and enhancing nutrient availability in the adjacent sediment via geochemical speciation shift.