David E. Crowley

Soil and plant specific effects on bacterial community composition in the rhizosphere

Eubacterial community structures in the plant rhizosphere were examined with respect to plant species, soil type, and root zone location. Three plant species (chickpea, rape and Sudan grass) were grown in intact cores of three California soils (a sandy soil, a sandy loam, and a clay) and were provided with a complete fertilizer solution with or without nitrogen supplied as ammonium nitrate. After 7.5 weeks, the plants were harvested and DNA was extracted from soil adhering to the root tips and from mature root zones at the sites of lateral root emergence. Eubacterial community structures were examined by PCR-DGGE of 16S rDNA to determine the relative abundance and species diversity. While both soil type and nitrogen fertilization affected plant growth, canonical correspondence analyses showed that nitrogen had no significant effect on eubacterial community structures. Eubacterial species diversity was higher in the mature root zones than at the root tips in the sandy soil and the clay but not in the loamy sand. Monte Carlo permutation tests indicated that plant species, root zone and soil type as well as the interactions between these variables had significant effects on community structure. The bacterial rhizosphere community of chickpea was influenced primarily by soil type, whereas root zone was less important. In contrast to chickpea, the community in the rhizosphere of rape and Sudan grass was more affected by the root zone than the soil type. In the sandy soil and the loamy sand, the eubacterial rhizosphere community structure was more affected by the root zone than the plant species and the three plant species had distinct communities. In the clay however, the root zone was less important than the plant species and the rhizosphere communities of chickpea differed from those of rape and Sudan grass. It is concluded that the bacterial community composition in the rhizosphere is affected by a complex interaction between soil type, plant species and root zone location.

Rhizosphere Microbial Community Structure in Relation to Root Location and Plant Iron Nutritional Status

ABSTRACT Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status.

Development of specific rhizosphere bacterial communities in relation to plant species, nutrition and soil type

Rhizosphere microbial communities are important for plant nutrition and plant health. Using the culture-independent method of PCR-DGGE of 16S rDNA for community analyses, we conducted several experiments to investigate the importance of pH, soil type, soil amendment, nutritional status of the plant, plant species and plant age on the structure of the bacterial community in the rhizosphere. At the same time, we assessed the spatial variability of bacterial communities in different root zone locations. Our results showed that the bacterial community structure is influenced by soil pH and type of P fertilization. In a short-term experiment (15–22 days) with cucumber and barley growing in a N deficient or a P deficient soil, the bacterial community structure in the rhizosphere was affected by soil type and fertilization but not by plant species. In a 7.5-week experiment with three plant species (chickpea, canola, Sudan grass) growing in three different soils (a sand, a loam and a clay), the complex interactions between soil and plant effects on the rhizosphere community were apparent. In the sand and the loam, the three plant species had distinct rhizosphere communities while in the clay soil the rhizosphere community structures of canola and Sudan grass were similar and differed from those of chickpea. In all soils, the rhizosphere community structures of the root tip were different from those in the mature root zone. In white lupin, the bacterial community structure of the non-cluster roots differed from those of the cluster roots. As plants matured, different cluster root age classes (young, mature, old) had distinct rhizosphere communities. We conclude that many different factors will contribute to shaping the species composition in the rhizosphere, but that the plant itself exerts a highly selective effect that is at least as great as that of the soil. Root exudate amount and composition are the key drivers for the differences in community structure observed in this study.

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97–100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

Agronomic approaches for improving the micronutrient density in edible portions of field crops

Abstract Increased micronutrient density in grain destined for human consumption may alleviate micronutrient deficiencies (Fe, Zn, Cu, and I) in human populations around the world. The review of literature indicates that fertilization with inorganic and organic forms of micronutrients has a potential to increase their concentrations in grain. The most effective fertilization could be via soil (for Zn and, to some extent, Cu), foliarly (for Fe) and by adding fertilizers to the irrigation water (for iodine). Care should be exercised not to overfertilize crops with micronutrients because of consequent toxicity and losses in quality and quantity of grain yield. Effectiveness of various agricultural measures in increasing micronutrient density depends on soil type, crop, cultivar, rotation, and environmental and other factors, thus necessitating development of a specific set of measures for individual regions. Agricultural measures would need to be supplemented with appropriate changes in the milling technology to ascertain that increased micronutrient concentrations in some grain parts are passed into the food chain.

Secondary plant metabolites in phytoremediation and biotransformation

For millennia, secondary plant metabolites have antagonized microorganisms, insects and humans alike, ultimately generating a complex and dynamic mixture of facultative and obligate interactions from symbioses to pathogenicity. Secondary plant metabolites have an important role in developing the myriad of organic pollutant-degrading enzymes found in nature. The link between secondary plant metabolites and enzymatic diversity has yet to be exploited, with potential applications in fields as varied as pest management, bioremediation and fine chemical production.

MECHANISMS AND PRACTICAL CONSIDERATIONS INVOLVED IN PLANT GROWTH PROMOTION BY RHIZOBACTERIA

Rhizobacteria are capable of stimulating plant growth through a variety of mechanisms that include improvement of plant nutrition, production and regulation of phytohormones, and suppression of disease causing organisms. While considerable research has demonstrated their potential utility, the successful application of plant growth promoting rhizobacteria (PGPR) in the field has been limited by a lack of knowledge of ecological factors that determine their survival and activity in the plant rhizosphere. To be effective, PGPR must maintain a critical population density of active cells. Inoculation with PGPR strains can temporarily enhance the population size, but inoculants often have poor survival and compete with indigenous bacteria for available growth substrates. PGPR often have more than one mechanism for enhancing plant growth and experimental evidence suggests that the plant growth stimulation is the net result of multiple mechanisms of action that may be activated simultaneously. The aim of this review is to describe PGPR modes of action and discuss practical considerations for PGPR use in agriculture.

Impact of Fumigants on Soil Microbial Communities

ABSTRACT Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity,H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, Hincreased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. andHeliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.

Bacterial functional redundancy along a soil reclamation gradient

ABSTRACT A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU). BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site. The number and diversity of bacterial groups responding to four carbon substrates (l-serine, l-threonine, sodium citrate, and α-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning.

Molecular basis of a bacterial consortium : Interspecies catabolism of atrazine

Pseudomonas sp. strain ADP contains the genes, atzA, -B, and -C, that encode three enzymes which metabolize atrazine to cyanuric acid. Atrazine-catabolizing pure cultures isolated from around the world contain genes homologous to atzA, -B, and -C. The present study was conducted to determine whether the same genes are present in an atrazine-catabolizing bacterial consortium and how the genes and metabolism are subdivided among member species. The consortium contained four or more bacterial species, but two members, Clavibacter michiganese ATZ1 and Pseudomonas sp. strain CN1, collectively mineralized atrazine. C. michiganese ATZ1 released chloride from atrazine, produced hydroxyatrazine, and contained a homolog to the atzA gene that encoded atrazine chlorohydrolase. C. michiganese ATZ1 stoichiometrically metabolized hydroxyatrazine to N-ethylammelide and contained genes homologous to atzB and atzC, suggesting that either a functional AtzB or -C catalyzed N-isopropylamine release from hydroxyatrazine. C. michiganese ATZ1 grew on isopropylamine as its sole carbon and nitrogen source, explaining the ability of the consortium to use atrazine as the sole carbon and nitrogen source. A second consortium member, Pseudomonas sp. strain CN1, metabolized the N-ethylammelide produced by C. michiganese ATZ1 to transiently form cyanuric acid, a reaction catalyzed by AtzC. A gene homologous to the atzC gene of Pseudomonas sp. strain ADP was present, as demonstrated by Southern hybridization and PCR. Pseudomonas sp. strain CN1, but not C. michiganese, metabolized cyanuric acid. The consortium metabolized atrazine faster than did C. michiganese individually. Additionally, the consortium metabolized a much broader set of triazine ring compounds than did previously described pure cultures in which the atzABC genes had been identified. These data begin to elucidate the genetic and metabolic bases of catabolism by multimember consortia.