The presence, size and importance of bacterial communities on plant leaf surfaces are widely appreciated. However, information is scarce regarding their composition and how it changes along geographical and seasonal scales. We collected 106 samples of field-grown Romaine lettuce from commercial production regions in California and Arizona during the 2009-2010 crop cycle. Total bacterial populations averaged between 10(5) and 10(6) per gram of tissue, whereas counts of culturable bacteria were on average one (summer season) or two (winter season) orders of magnitude lower. Pyrosequencing of 16S rRNA gene amplicons from 88 samples revealed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the most abundantly represented phyla. At the genus level, Pseudomonas, Bacillus, Massilia, Arthrobacter and Pantoea were the most consistently found across samples, suggesting that they form the bacterial 'core' phyllosphere microbiota on lettuce. The foliar presence of Xanthomonas campestris pv. vitians, which is the causal agent of bacterial leaf spot of lettuce, correlated positively with the relative representation of bacteria from the genus Alkanindiges, but negatively with Bacillus, Erwinia and Pantoea. Summer samples showed an overrepresentation of Enterobacteriaceae sequences and culturable coliforms compared with winter samples. The distance between fields or the timing of a dust storm, but not Romaine cultivar, explained differences in bacterial community composition between several of the fields sampled. As one of the largest surveys of leaf surface microbiology, this study offers new insights into the extent and underlying causes of variability in bacterial community composition on plant leaves as a function of time, space and environment.

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pPfruB-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pP fruB-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and͞or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to Ϸ1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.

The phyllosphere is an ecologically and economically important ecosystem that hosts a large and diverse microbial community. Phyllosphere microbiota play a critical role in protecting plants from diseases as well as promoting their growth by various mechanisms. There are serious gaps in our understanding of how and why microbiota composition varies across spatial and temporal scales, the ecology of leaf surface colonizers and their interactions with their host, and the genetic adaptations that enable phyllosphere survival of microorganisms. These gaps are due in large part to past technical limitations, as earlier studies were restricted to the study of culturable bacteria only and used low-throughput molecular techniques to describe community structure and function. The availability of high-throughput and cost-effective molecular technologies is changing the field of phyllosphere microbiology, enabling researchers to begin to address the dynamics and composition of the phyllosphere microbiota across a large number of samples with high, in-depth coverage. Here, we discuss and connect the most recent studies that have used next-generation molecular techniques such as metagenomics, proteogenomics, genome sequencing, and transcriptomics to gain new insights into the structure and function of phyllosphere microbiota and highlight important challenges for future research.

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-␤-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by MichaelisMenten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.One intriguing type of interorganismal interaction is the manipulation by some microbes of their host's hormone system. A good example is given by bacteria of the genus Wolbachia, which convert male woodlice into females, supposedly by suppressing a gland that produces a masculinizing hormone (37). Another striking example is the microbial production of plant hormones such as auxin, cytokinin, and gibberellin, which allows certain bacteria and fungi to direct a plant's physiology toward their own advantage (5,7,14,20).Indole-3-acetic acid (IAA) is the main auxin in plants, controlling many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light and gravity (45). Bacterial IAA producers (BIPs) have the potential to interfere with any of these processes by input of IAA into the plant's auxin pool. The consequence for the plant is usually a function of (i) the amount of IAA that is produced and (ii) the sensitivity of the plant tissue to changes in IAA concentration. A root, for instance, is one of the plant's organs that is most sensitive to fluctuations in IAA, and its response to increasing amounts of exogenous IAA extends from elongation of the primary root, formation of lateral and adventitious roots, to growth cessation (8).The existence of BIPs was recognized very early in the research on auxin, and plant-associated BIPs have long been considered a source of contamination in measurements of IAA present in plant tissues (24, 51, 52). Later, BIPs were identified as the cause of symptoms associated with severe plant disease...

Aerial plant surfaces harbor large numbers of microbes, some of which are deleterious to plants whereas others are benign or beneficial. Commercial formulations of bacteria antagonistic to plant pathogenic microbes and ice nucleation active bacteria have been utilized as an environmentally safe method to manage plant disease and to prevent frost damage. Molecular genetic tools, microscopic examination and whole-cell bacterial biosensors have provided extensive information on these microbes, their complex associations and their habitat. The aerial habitat influenced by plants, termed the phyllosphere, is particularly amenable to studies of microbial ecology and the information gained should lead to more effective means of plant protection.

This review by a multidisciplinary team maps key components and emerging connections within the intellectual landscape of agroecology. We attempt to extend and preview agroecology as a discipline in which agriculture can be conceptualized within the context of global change and studied as a coupled system involving a wide range of social and natural processes. This intrinsic coupling, combined with powerful emerging drivers of change, presents challenges for the practice of agroecology and agriculture itself, as well as providing the framework for some of the most innovative research areas and the greatest potential for innovation for a sustainable future in agriculture. The objective of this review is to identify forward-looking scientific questions to enhance the relevance of agroecology for the key challenges of mitigating environmental impacts of agriculture while dramatically increasing global food production, improving livelihoods, and thereby reducing chronic hunger and malnutrition over the coming decades.