Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere

Department of Plant Pathology, University of Wisconsin, Madison, WI, USA.
Journal of Applied Microbiology (Impact Factor: 2.48). 02/2011; 110(5):1284-96. DOI: 10.1111/j.1365-2672.2011.04975.x
Source: PubMed


Bacterial communities in the apple phyllosphere were examined quantitatively and qualitatively by applying culture-dependent and culture-independent methods.
Populations estimated by viewing cells stained with 4',6-diamidino-2-phenylindole generally were at least 100-1000 times greater than populations estimated by culturing on tryptic soy agar (TSA). Of the 44 operational taxonomic units (OTUs; cut-off threshold of 97%) detected in total, five bacterial orders containing 23 OTUs were identified by culturing on TSA, whereas nine orders containing 33 OTUs were identified by 16S rRNA gene cloning of DNA extracted from apple leaf surfaces. Twelve of the 44 OTUs were shared between cultured isolates and 16S rRNA gene clones and included the orders Burkholderiales, Pseudomonadales, Rhizobiales and Sphingomonadales. Three OTUs within the genus Sphingomonas accounted for 40% of isolates and 68% of clones. The Actinomycetales were found only among isolates, whereas the Bacteroidales, Enterobacteriales, Myxococales and Sphingobacteriales were represented in the 16S rRNA gene clone libraries but were absent among isolates.
Culture-independent methods revealed greater numbers and greater richness of bacteria on apple leaves than found by culturing.
This is the first study to directly compare culture-dependent and independent approaches for assessing bacterial communities in the phyllosphere. The biases introduced by different methods will have a significant impact on studies related to phyllosphere ecology, biological control of plant diseases, reservoirs of antibiotic resistance genes and food safety.

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Available from: Patricia Susan McManus, Sep 16, 2014
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    • "found no significant difference in species diversity between streptomycin-treated orchards and untreated (0.84 vs 0.74, respectively). While in an earlier study Yashiro et al. (2011) calculated values of D = 0.32 in two orchard systems. It is clear that the phyllosphere of apple is complex and dynamic with biotic and abiotic environmental factors, as well as management effects, influencing its composition. "
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    ABSTRACT: The phyllosphere of plant tissues is varied and dynamic. Pest management, time of sampling, proximity to immigration sources, tissue and tissue status such as leaf/fruit age and location within the canopy, and other environmental and biological factors interact to influence the composition and abundance of microbial species populating the phyllosphere of apple. The purpose of this study was to examine microbial variation in the apple phyllosphere that are influenced by production management while minimizing spatial variation from widely separated sites that could introduce environmental variation. Three production systems were evaluated in adjacent 1 ha production areas: (1) conventional using synthetic fungicides and insecticides (C), (2) organic using a kaolin-based insect repellent with sulfur and lime-sulfur for disease control (ORG), and (3) the C pest management program with the addition of kaolin (CK). Apple leaves and fruit were sampled 3 times (09/07, 08/08 and 09/08), washed with water to remove the microbial populations and DNA was extracted from the washed solution. DNA was probed with 16S bacterial primers and 18S fungal primers using 454 technology. Data from the top-hit BLASTn was then compiled and relative percentages of fungi at each taxonomic level were determined for each individual sample. Genus determination was based on >95% sequence identity. Simpson diversity index (D) and evenness (J) were calculated. A total of 356 bacteria and 373 fungal and yeast genera were detected in the 3 sampling studies of 3 pest management systems, however, not all genera were detected in all 3 sampling dates. Species diversity and evenness values were very small indicating very high diversity in all treatments and this is due to the detection of infrequent genera. The 3 pest management systems did not develop consistent and unique microbial populations. Nineteen bacteria and 24 fungal genera represented the core of the microbial phyllosphere diversity for the 3 sampling times. Eleven bacteria and 19 fungal genera were present in at least 50% of the plots on all sampling dates. Five bacteria (Geitlerinema, Massilia, Methylobacterium, Sphingomonas, Synechococcus) and 4 fungal (Alternaria, Aureobasidium, Catenulostroma, Phoma) genera were present in all plots on all sampling dates. In 5 samplings over 4 years, the use of kaolin in the ORG and CK treatments always increased recovery of DNA from the phyllosphere. Kaolin provided a habitat in which microbial populations were enhanced 2–5 times in both CK and ORG management systems and the recovered DNA from plant surfaces was best correlated with the growing season temperature 30–60 days before sampling. Kaolin may be a potential tool to aid in biological control efforts, by providing additional microbial habitats with reduced deleterious effects of UV on microbial populations.
    Scientia Horticulturae 02/2015; 183. DOI:10.1016/j.scienta.2014.12.009 · 1.37 Impact Factor
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    • "Therefore, the application of molecular biological techniques in microbial environmental studies for the assessment of uncultivable microorganisms has significantly improved our understanding of the composition of bacterial communities within plants (Whipps et al., 2006; Ranjard et al., 2000). The sizes of bacterial communities as determined using culture-independent methods might be 100-to 1,000-fold larger than communities uncovered via traditional isolation (Yashiro et al., 2011). "
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    • "Interesting among these are Bradyrhizobiaceae, Xanthomonadaceae, Enterobacteriaceae, Rhodobacterales, Pseudomonadales, Bacteriodetes and many groups of Actinobacteria (Fig. 5, Table S7). While it is tempting to attribute much of this unknown community to floral nectar or the phyllosphere (Jackson et al. 2006; Telias et al. 2011; Yashiro et al. 2011; Fridman et al. 2012; Alvarez-Pérez et al. 2012; Aizenberg-Gershtein et al. 2013; Aleklett et al. 2014), there are many unexplored microenvironments scattered throughout the hive, and within the mouth, pharynx, and "
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    ABSTRACT: Honey bee hives are filled with stored pollen, honey, tree resins, and wax, all antimicrobial to differing degrees. Stored pollen is the nutritionally rich currency used for colony growth, and consists of 40-50% simple sugars. Many studies speculate that prior to consumption by bees, stored pollen undergoes long-term nutrient conversion, becoming more nutritious “bee bread” as microbes pre-digest the pollen. We quantified both structural and functional aspects associated with this hypothesis using behavioral assays, bacterial plate counts, microscopy, and 454 amplicon sequencing of the 16S rRNA gene from both newly-collected and hive-stored pollen. We found that bees preferentially consume fresh pollen stored for less than three days. Newly-collected pollen contained few bacteria, values which decreased significantly as pollen was stored >96 hours. The estimated microbe to pollen grain surface area ratio was 1:1,000,000 indicating a negligible effect of microbial metabolism on hive-stored pollen. Consistent with these findings, hive-stored pollen grains did not appear compromised according to microscopy. Based on year round 454 amplicon sequencing, bacterial communities of newly-collected and hive-stored pollen did not differ, indicating the lack of an emergent microbial community co-evolved to digest stored pollen. In accord with previous culturing and 16S cloning, acid resistant and osmotolerant bacteria like Lactobacillus kunkeei were found in greatest abundance in stored pollen, consistent with the harsh character of this microenvironment. We conclude that stored pollen is not evolved for microbially mediated nutrient conversion, but is a preservative environment due primarily to added honey, nectar, bee secretions and properties of pollen itself.This article is protected by copyright. All rights reserved.
    Molecular Ecology 10/2014; 23(23). DOI:10.1111/mec.12966 · 6.49 Impact Factor
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