Paul J. Hunter

Both Leaf Properties and Microbe-Microbe Interactions Influence Within-Species Variation in Bacterial Population Diversity and Structure in the Lettuce (Lactuca Species) Phyllosphere

ABSTRACT Morphological and chemical differences between plant genera influence phyllosphere microbial populations, but the factors driving within-species variation in phyllosphere populations are poorly understood. Twenty-six lettuce accessions were used to investigate factors controlling within-species variation in phyllosphere bacterial populations. Morphological and physiochemical characteristics of the plants were compared, and bacterial community structure and diversity were investigated using terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA gene clone libraries. Plant morphology and levels of soluble carbohydrates, calcium, and phenolic compounds (which have long been associated with plant responses to biotic stress) were found to significantly influence bacterial community structure. Clone libraries from three representative accessions were found to be significantly different in terms of both sequence differences and the bacterial genera represented. All three libraries were dominated by Pseudomonas species and the Enterobacteriaceae family. Significant differences in the relative proportions of genera in the Enterobacteriaceae were detected between lettuce accessions. Two such genera (Erwinia and Enterobacter) showed significant variation between the accessions and revealed microbe-microbe interactions. We conclude that both leaf surface properties and microbial interactions are important in determining the structure and diversity of the phyllosphere bacterial community.

Differences in microbial activity and microbial populations of peat associated with suppression of damping-off disease caused by Pythium sylvaticum.

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

Root traits and microbial community interactions in relation to phosphorus availability and acquisition, with particular reference to Brassica

Brassicas are among the most widely grown and important crops worldwide. Phosphorus (P) is a key mineral element in the growth of all plants and is largely supplied as inorganic rock-phosphate, a dwindling resource, which is likely to be an increasingly significant factor in global agriculture. In order to develop crops which can abstract P from the soil, utilize it more efficiently, require less of it or obtain more from other sources such as soil organic P reservoirs, a detailed understanding the factors that influence P metabolism and cycling in plants and associated soil is required. This review focuses on the current state of understanding of root traits, rhizodeposition and rhizosphere community interaction as it applies to P solubilization and acquisition, with particular reference to Brassica species. Physical root characteristics, exudation of organic acids (particularly malate and citrate) and phosphatase enzymes are considered and the potential mechanisms of control of these responses to P deficiency examined. The influence of rhizodeposits on the development of the rhizosphere microbial community is discussed and the specific features of this community in response to P deficiency are considered; specifically production of phosphatases, phytases and phosphonate hydrolases. Finally various potential approaches for improving overall P use efficiency in Brassica production are discussed.

A comparison of Olpidium isolates from a range of host plants using internal transcribed spacer sequence analysis and host range studies.

Olpidium brassicae is a ubiquitous obligate root-infecting fungal pathogen. It is an important vector of a wide range of plant viruses. Olpidium isolates that infected brassica plants did not infect lettuce plants and vice-versa. Host range tests, PCR amplification and sequencing of the internal transcribed spacer (ITS) and 5.8S regions of 25 Olpidium isolates from brassica, carrot, cucumber and lettuce originating from four continents revealed differences between isolates. Based on their ability to infect lettuce and brassicas and the differences between their ITS1, 5.8S and ITS2 regions they could be separated into a number of distinct groups. Comparisons with other published sequences revealed two distinct genetic groups of brassica-infecting isolates, two distinct groups of lettuce-infecting isolates, one of which contained a carrot-infecting isolate and a distinct group comprising a cucumber-infecting isolate and a melon-infecting isolate. The possibility of the isolates belonging to three distinct species is discussed.

Variation in Microbial Communities Colonizing Horticultural Slow Sand Filter Beds: Implications for Filter Function

The effect of microbial colonization on the function and rejuvenation of slow sand filters was investigated using culture-independent profiling. Colonization resulted in significant reduction in filter pore size, which may be important in order to fully remove pathogens, but was not associated with a specific microbial component. Communities were highly variable, and no common microbial groups were found in effective filters. Bacterial community composition was affected by sand particle size, although high levels of microbial turnover during filter maturation suggested that this was unlikely to have a major influence on community composition. The composition of microbial inoculum from a previous filter could not be maintained through a cycle of culture, storage and re-culture. Furthermore, no significant proportion of the inoculum persisted in filter maturity, and no advantages in terms of time to filter maturation or final filter efficiency were evident. These results may explain why filtration is such an effective and robust water treatment and emphasize the need for further research on the mechanisms involved in pathogen elimination.

Oxidative discolouration in whole-head and cut lettuce : biochemical and environmental influences on a complex phenotype and potential breeding strategies to improve shelf-life

Lettuce discolouration is a key post-harvest trait. The major enzyme controlling oxidative discolouration has long been considered to be polyphenol oxidase (PPO) however, levels of PPO and subsequent development of discolouration symptoms have not always correlated. The predominance of a latent state of the enzyme in plant tissues combined with substrate activation and contemporaneous suicide inactivation mechanisms are considered as potential explanations for this phenomenon. Leaf tissue physical properties have been associated with subsequent discolouration and these may be influenced by variation in nutrient availability, especially excess nitrogen and head maturity at harvest. Mild calcium and irrigation stress has also been associated with a reduction in subsequent discolouration, although excess irrigation has been linked to increased discolouration potentially through leaf physical properties. These environmental factors, including high temperature and UV light intensities, often have impacts on levels of phenolic compounds linking the environmental responses to the biochemistry of the PPO pathway. Breeding strategies targeting the PAL and PPO pathway biochemistry and environmental response genes are discussed as a more cost-effective method of mitigating oxidative discolouration then either modified atmosphere packaging or post-harvest treatments, although current understanding of the biochemistry means that such programs are likely to be limited in nature and it is likely that they will need to be deployed alongside other methods for the foreseeable future.

Older leaves of lettuce (Lactuca spp.) support higher levels of Salmonella enterica ser. Senftenberg attachment and show greater variation between plant accessions than do younger leaves

Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previous studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar. In this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the first and fifth to sixth true leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.