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Impact of Organic and Conventional

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 2321

Journal of Food Protection, Vol. 72, No. 11, 2009, Pages 23212325
Copyright G, International Association for Food Protection

Impact of Organic and Conventional Management on the

Phyllosphere Microbial Ecology of an Apple Crop
ANDREA R. OTTESEN,1* JAMES ROBERT WHITE,2 DEMETRA N. SKALTSAS,1 MICHAEL J. NEWELL,3
AND CHRISTOPHER S. WALSH1

1Department of Plant Science and Landscape Architecture, 2102 Plant Sciences, #036, and 2Center for Bioinformatics and Computational Biology,
Biomolecular Sciences, #296, University of Maryland, College Park, Maryland 20742, and 3Wye Research and Education Center,
P.O. Box 169, Queenstown, Maryland 21658-0169, USA

MS 09-165: Received 14 April 2009/Accepted 22 July 2009

ABSTRACT
Bacterial communities associated with the phyllosphere of apple trees (Malus domestica cv. Enterprise) grown under organic
and conventional management were assessed to determine if increased biological food safety risks might be linked with the
bacterial communities associated with either treatment. Libraries of 16S rRNA genes were generated from phyllosphere DNA
extracted from a wash made from the surfaces of leaves and apples from replicated organic and conventional treatments. 16S
rRNA gene libraries were analyzed with software designed to identify statistically significant differences between bacterial
communities as well as shared and unique phylotypes. The identified diversity spanned eight bacterial phyla and 14 classes in the
pooled organic and conventional libraries. Significant differences between organic and conventional communities were observed
at four of six time points (P , 0.05). Despite the identification of significantly diverse microfloras associated with organic and
conventional treatments, no detectable differences in the presence of potential enteric pathogens could be associated with either
organic or conventional management. Neither of the bacterial genera most commonly associated with produce-related illness
outbreaks (Salmonella and Escherichia) was observed in any of the libraries. The impressive bacterial diversity that was
documented in this study provides a valuable contribution to our developing understanding of the total microbial ecology
associated with the preharvest phyllospheres of food crops. The fact that organic and conventional phyllosphere bacterial
communities were significantly different at numerous time points suggests that crop management methods may influence the
bacterial consortia associated with the surfaces of fruits and vegetables.

Enteric human pathogens such as Salmonella enterica
have been the cause of illness outbreaks linked to the
consumption of fresh produce dating back to the early
1900s. As early as 1912, R. H. Creel published Vegetables
as a Possible Factor in the Dissemination of Typhoid
Fever in the Public Health Reports, linking celery to an
outbreak of typhoid fever caused by Bacillus typhosus (S.
enterica serovar Typhi) (6). With produce-related outbreaks
on the rise in the United States over the past 10 years (2) and
the fact that enteric pathogens such as strains of Salmonella
and Escherichia coli can and do survive in the environment
(3, 9), epidemiological trace-back efforts have gone beyond
processing environments and back to agricultural settings.
The ecologically neglected milieu of the phyllosphere
(aerial surfaces of plants) (20) is at an intersection of public
health, microbial ecology, and food safety. This important
food safety niche is estimated to span 6.4 | 108 km2 and
contain as many as 1026 microorganisms (17).
Despite the fact that few studies have focused on the
total microbial ecology of preharvest environments, a
paradigm shift in research approaches is currently under
way. Recognition that human actions can drive the
* Author for correspondence. Tel: 240-277-4595 or 301-405-7543;
Fax: 301-314-9308; E-mail: andrea.ottesen@gmail.com.
evolution of pathogens and that even the smallest factors
may have unforeseen impacts on microbial communities
(14) suggests that a better understanding of the microbial
ecology associated with food crops, pre- and postharvest,
can help to improve the safe stewardship of agricultural
commodities. Understanding the natural dynamics of
microbial communities in agricultural settings may also
contribute to the identification of naturally occurring species
that may be capable of outcompeting potential pathogens in
field settings. Pathogen antagonists, already native members
of the ecological milieu, could potentially function as
biological controls for safe and effective pathogen suppres-
sion in the field.
Organic agriculture, a part of current efforts to
develop more healthful and sustainable models of agricul-
ture, has recently become a certified practice (1990 Organic
Food Production Act) (27) that prohibits the use of synthetic
pesticides, genetically modified organisms, sewage sludges,
irradiation, and other practices deemed to be detrimental to
society and the environment. With passage of the Organic
Food Production Act in the 1990 Farm Bill (27), the selling
of produce labeled organic means that a set of prescribed
practices have been followed. A completely different set of
pesticides and fertilizers are used to manage crops
organically, and it stands to reason that these materials
may differentially impact bacterial species and thus
2322 OTTESEN ET AL.
TABLE 1. Organic and conventional materials used to manage pest pressures in the experimental apple orchard at the Wye Research
and Education Center (2005 through 2007) in Queenstown, MD
Treatment Insecticides Fungicides
Organic Kaolin, pyrethrins, Copper, Kelp, fish emulsion, chicken
spinosad, azadirachtin sulfur manure, compost teas, 6-1-1
NPK, 5-3-4 NPK
Conventional Pyrethroid, carbamate, Carbamates Calcium nitrate, 15-0-0 NPK
organothiophosphates
biological food safety risks associated with surfaces of
organic and conventionally managed food crops.
Pest pressures and fertilization needs are different from
crop to crop and from region to region. We developed a
management schedule for a mid-Atlantic crop of apples by
substituting certified organic materials for the most
commonly used conventional pesticides and fertilizers at a
1:1 ratio. This rate was adapted throughout this 3-year study
(2005 through 2007) as our understanding of the efficacy of
each material was enhanced through trial and error. Table 1
shows a list of the materials that were used for organic and
conventional management.
One organic insecticide commonly used in organic
crops is made from kaolin clay. This insecticide does not
kill insects, as some of its conventional counterparts do with
cholinesterase inhibition and other forms of related
poisoning, but instead relies on the masking of visual and
olfactory cues associated with plant surfaces to which
insects normally respond. Studies of bacteria associated
with a specific brand of this type of material, preapplication
to the crop (unpublished data), have documented approx-
imately 11 different phyla, spanning both bacterial and
archaeal domains and 18 different taxonomic families. With
this type of bacterial and archaeal diversity associated with
one material alone (preapplication to the crop), it seems
plausible that the introduction of such diverse consortia
could influence bacterial microflora associated with the crop
phyllosphere.
Additionally, organic fertilizers are often generated
from biological materials that almost certainly have a greater
microbial diversity than calcium nitrate, which is applied in
conventional settings. There are also inert ingredients
associated with insecticides and fertilizers. While they may
be inert in some regards, it is unlikely that they are
completely sterile. The better able we are to effectively
describe the microbial consortia associated with each
anthropogenic application to a food crop, the better able
we will be to manage and identify potential risk factors and
how they may be enhanced or diminished by human and
environmental pressures associated with the growing season
of a crop. Hence, the goal of this study was to assess the
impact of organic and conventional apple crop management
practices on the bacterial phyllosphere and presence of
foodborne pathogens on apple trees.
MATERIALS AND METHODS
Sample collection. At multiple time points throughout three
growing seasons (2005 through 2007), fruit and leaves of Malus
J. Food Prot., Vol. 72, No. 11
Fertilizers Bactericides Herbicides
Streptomycin Acetic acid, physical
barriers (plastics)
Streptomycin Glyphosate
domestica cv. Enterprise were aseptically collected from five
replicated blocks of organic and conventionally managed trees. A
total of 10 samples were collected at each time point: 5 organic
samples and 5 conventional samples. An independent replication
was composed of 20 leaves and two apples from one tree from an
individual block (each block contained nine trees). Samples were
not pooled. Collection time points were as follows: July and
August 2005, July 2006, August 2006, September 2006, July 2007,
and August 2007. Apples and leaves were placed in Ziploc bags by
using gloves, with ethanol disinfection of clippers between
samples. Leaves were systematically collected from all sides of
the tree, pooled, and then transported back to the laboratory in
sealed bags in a cooler at 4uC. Three hundred milliliters of
deionized sterile water was added to the bags, and samples were
sonicated for 5 min to dislodge phyllosphere microbial species.
The microfloral wash was transferred to centrifuge tubes and
centrifuged overnight at 30,000 | g for 12 h at 4uC. The long
centrifugation was necessary to effectively pelletize the phyllo-
sphere cells. The pellet was then transferred to a small
microcentrifuge tube and stored at 220uC until DNA extraction
was performed.
DNA extraction. Protocols preferential for the extraction of
gram-negative species (Promega Wizard DNA Extraction Kit)
were used according to the manufacturers specifications.
PCR for clone libraries. A 550-bp fragment of the V3 region
of 16S rRNA gene was amplified for cloning and sequencing
purposes. The forward primer was 59-CCTACGGGAGGCAG-
CAG-39 and the reverse primer was 907R; 59-CCCCGTCAA-
TTCCTTTGAGTTT-39 (18, 26). Reaction volumes of 50 ml were
prepared with 5 ml of 10| buffer (Takara), 2 ml of MgCl2, 1 ml of
deoxynucleoside triphosphates, forward primer, and reverse primer
at 25 pmol, 39.8 ml of water, and 0.2 ml of Taq (Takara). PCR
included a hot start of 95uC for 5 min and 30 cycles of denaturing
at 94uC for 1 min, annealing at 55uC for 1 min, and extension at
72uC for 1 min, with a final extension at 72uC for 5 min and
storage at 4uC.
Clone library construction. PCR products were ligated into
vector plasmids and then used to transform E. coli cells with the
Promega T-Easy Kit, according to the manufacturers specifica-
tions (Promega, Madison, WI). Plasmids were initially isolated
using the Wizard Plus Minipreps DNA Purification System
(Promega). In 2007 E. coli colonies were frozen in 200 ml of
Luria broth and 20% glycerol stock for direct sequencing (without
the miniprepping step).
Sequencing. Frozen 200-ml aliquots of the E. coli clones in
20% glycerol and Luria broth (Miller) solution, in 96-well plates,
were shipped on dry ice to Agencourt Genomic Services, Beverly,
MA, for sequencing. Alternatively, in 2005 and 2006, approxi-
mately 20 ml of miniprepped clones were shipped in 96-well plates
to Genewiz, South Plainfield, NJ.
J. Food Prot., Vol. 72, No. 11 CROP TREATMENT EFFECTS ON PHYLLOSPHERE MICROBIAL FLORA
FIGURE 1. Percentages of the eight most frequently encountered
bacterial classes in organic and conventional 16S rRNA gene
libraries from organic and conventional apple phyllosphere
samples.
Bioinformatic processing of 16S sequences. A total of
approximately 1,500 sequences were obtained from all time points.
These were further culled using the following quality checks and
screens for contaminants such as eukaryotic rRNA, vector, and
chloroplasts. Quality scores of sequences were computed using
Phred (11). Sequences were subsequently trimmed for quality by
using LUCY (4), an open-source program developed by TIGR, and
then trimmed for any remaining cloning plasmid vector by using
NUCmer (7). Trimmed sequences were filtered for short lengths
and screened for vector, chloroplast, and 18S rRNA gene
contaminants by using BLASTN (1). Any sequences less than
400 bp were removed, as were those with BLASTN hits to
contaminants (chloroplasts and 18 S rRNA gene fragments from
Eukaryotes) with a bit score of greater than 300. Bellerophon was
used to identify potential chimeras (13).
Taxonomic assignment of 16S rRNA gene sequences. The
Ribosomal Database Projects (RDP II) unaligned release 9.57 (5,
28) of approximately 471,000 rRNA gene sequences was
downloaded to generate a database from RDP sequences
containing at least four taxonomic identification levels. Each 16S
sequence was assigned to its closest neighbor within that database
using the BLASTN (1) best bit score.
Alignment. Sequences were aligned using ARB (15).
Alignment was also done with MUSCLE (10) and NAST (8) to
ensure that observed differences were not due to artifacts of
alignment programs (29). Distance matrices were created using
ARB with Olsen distance correction.
Assignment to OTUs. DOTUR (Distance-Based OTU and
Richness) assigns sequences to operational taxonomic units
(OTUs) by the nearest-neighbor algorithm (22). Using the
frequency at which each OTU is observed DOTUR generates
rarefaction or collectors curves for designated measures of
richness and diversity and to determine the sampling depth needed
to accurately represent community members of any given
environment. The OTUs were clustered using the furthest-neighbor
algorithm for the recommended measurements of 3, 5, 10, and
20% difference. DOTUR also calculates the ACE and Chao1
2323
TABLE 2. P values associated with s-Libshuff analysis of
organic and conventional 16S rRNA gene libraries
P value
Conventional Organic
Date (as homologous) (as homologous)
July/August 2005
(pooled) 0.2357 0.0005a
July 2006 0.1434 0.4717
August 2006 0.0032a 0.2322
September 2006 0.2909 0.167
July 2007 ,0.0001a 0.0955
August 2007 0.0002a 0.4756
a
Significant difference between organic and conventional micro-
bial communities at a given time point.
nonparametric estimators for each specified evolutionary distance
along with the Shannon diversity index (22).
Shared OTUs. Distance matrices were also analyzed using
SONS, which implements nonparametric estimators for the fraction
and richness of OTUs shared by two communities (23).
Assigning a P value to observed differences. s-Libshuff
estimated the Cramer-von Mises statistic to test if two environ-
ments are drawn from the same underlying population by using a
Monte Carlo testing procedure. It evaluates differences between
each community (21).
RESULTS AND DISCUSSION
An impressive bacterial diversity was observed in the
phyllosphere of organically and conventionally managed
apples. Our total data set, after the removal of potential
chimeras, was composed of a total of 868 sequences.
Among those, we identified eight bacterial phyla and 14
classes as follows: Phylum Acidobacteria, Class Acidobac-
teria; Phylum Bacteriodetes, Classes Flavobacteria, Sphin-
gobacteria, and Bacteriodetes; Phylum Cyanobacteria,
Class Cyanobacteria; Phylum Deinococcus-Thermus, Class
Deinococci; Phylum Fusobacteria, Class Fusobacteria;
Phylum Proteobacteria, Classes Alpha-, Beta-, Gamma-,
and Deltaproteobacteria; Phylum Firmicutes, Classes
Clostridia and Bacilli; and Phylum Actinobacteria, Class
Actinobacteria. The percentages of the eight most prevalent
classes spanning four bacterial phyla are shown in Figure 1.
Classes Alpha-, Beta-, and Gammaproteobacteria of the
phylum Proteobacteria were by far the best-represented
taxonomic groups in our data set. Observed members of
phyla Acidobacteria, Cyanobacteria, Deinococcus-Ther-
mus, and Fusobacteria were represented by only a few
sequences and made up ,1% of the total library in most
cases (not shown in Fig. 1).
To describe potentially significant differences associat-
ed with treatment (organic and conventional management)
among 16S rRNA gene libraries, we generated distance
matrices and then used software (s-Libshuff) designed to
detect significant differences between communities based
on genetic distances. Four of six time points were
2324 OTTESEN ET AL. J. Food Prot., Vol. 72, No. 11

TABLE 3. Phyla and genera observed in organic and conven- abundance OTUs, comprising 5.3 and 9.3% of the
tional phyllosphere 16S rRNA gene libraries with BLASTN identity respective libraries. Underrepresented organisms in meta-
scores above 95% genomic data sets are sometimes referred to as the rare
biosphere of a biome (25) and are thought to serve
Proteobacteria Fusobacteria unrecognized but important functions in community dy-
Acidovorax Leptotrichia namics. They could be products of ecological shifts that
Acinetobacter Actinobacteria maintain the potential to become dominant in response to
Afipia Actinomyces shifts in environmental conditions (25), or they could be
Aquabacterium Bifidobacterium
Aurantimonas
keystone species in unidentified phyllosphere consortia
Corynebacterium (19). The 48 shared OTUs represented 85.4% of all
Bartonella Curtobacterium
Burkholderia sequences. At D ~ 0.03, coverage of gram-negative species
Flavobacterium
Anaeromyxobacter in this environment was estimated at 91% for the
Frigoribacterium
Devosia Kineococcus conventional library and 92% for the organic library.
Enterobacter Leifsonia To describe and manage risks while simultaneously
Erwinia Mycobacterium streamlining sustainable agricultural objectives, it will be
Haemophilus Quadrisphaera essential to have a comprehensive inventory of the
Hyphomicrobium Rothia microbial players that constitute agriculturally impacted
Janthinobacterium Bacteriodetes biomes associated with food crops. The present study
Labrys
Alistipes identified a list of organisms so complex that its
Legionella
Massilia Cardinium implications are still uncertain. Our sequence libraries,
Methylobacterium Chryseobacterium though the largest currently assembled for a phyllosphere
Moraxella Dyadobacter environment (and the only work to date addressing the
Dysgonomonas impact of organic and conventional practices on this
Novosphingobium
Pantoea Hymenobacter environment), are still less than 1/100 to 1/1,000 the size
Pelomonas Firmicutes of metagenomic data sets of the day (12, 13, 16, 25, 30).
Pseudomonas Anaeroglobus One of the reasons that an apple crop was selected,
Ralstonia Bacillus given that there are certainly higher-risk crops such as
Rhizobium Clostridium
lettuce, spinach, green onions, cantaloupes, tomatoes, and
Rhodobacter Coprobacillus
Hespellia
peppers, was due in part to the fact that agricultural grades
Rhodopila
Lactobacillus of antibiotics (streptomycin and occasionally oxytetracy-
Roseomonas
Schineria Sporobacter cline) are still applied to apple crops to manage Erwinia
Serratia Staphylococcus amylovora, a gram-negative bacterial pathogen responsible
Sphingomonas Deinococcus-Thermus for fireblight. Erwinia is in the same family (Enterobacte-
Zymobacter Deinococcus riaceae) as E. coli and Salmonella. Documentation of the
bacterial members, especially those in close genetic
proximity to known pathogens, provides valuable informa-
significantly different from each other with regard to tion as we assess the sustainability of using pesticides that
organic and conventional treatments (P , 0.05) (24) may exert selective pressures for antibiotic resistance in
(Table 2). A Bonferroni correction was used to account agroecological settings.
for multiple comparisons (0.05/12) so our alpha was set at The full list of taxonomic identities and the phyloge-
0.004. s-Libshuff uses the Monte Carlo testing procedure, netic delineations of families documented in this data set is
which optimizes the information available in small sequence extremely interesting from an ecological perspective. The
libraries. use of 550 bases of a highly conserved gene (16S rRNA
No detectable differences in the presence of potential gene), however, provides only a low-resolution (with regard
enteric pathogens were associated with either treatment: no to species) examination of the microbial taxonomy of this
Salmonella or Escherichia was found among any of the 868 phyllosphere environment. To provide an understanding of
sequences. The class with the most commonly encountered the diversity present in this environment, we have reported
pathogens implicated in produce-related health outbreaks is all genera that were observed with a BLASTN (1) similarity
Gammaproteobacteria. Enterobacteriaceae, home to Esch- score of 95% or greater (Table 3).
erichia and Salmonella, is a member of this class. There It is important to note that our 16S rRNA gene libraries
were no significant compositional differences between include genera that contain established and emerging
treatments in members of Gammaproteobacteria or Entero- pathogenic species within their ranks. A brief list of genera
bacteriaceae except at one time point in 2005. that contain pathogenic species that were found in both
A total of 136 OTUs (D ~ 0.03 or 97% similarity) organic and conventional treatments includes Haemophilus,
were found across both libraries; 48 of those OTUs were Pseudomonas, Pantoea, Staphylococcus, Acinetobacter, and
shared between organic and conventional, 51 were unique to Burkholderia. Genera that were seen only among sequences
conventional, and 37 were unique to organic. Despite the from conventional treatments include Enterobacter, Schi-
high number of unique OTUs associated with organic and neria, Bartonella, Xanthomonas, Alistipes, and Clostridium.
conventional treatments, these were actually very low Genera that were uniquely associated with organic treat-
 J. Food Prot., Vol. 72, No. 11 CROP TREATMENT EFFECTS ON PHYLLOSPHERE MICROBIAL FLORA
ments included Legionella, Serratia, Ralstonia, Moraxella,
Enterococcus, Mycobacterium, and Chryseobacterium.
The use of additional diagnostic sequencing methods in
future studies will help determine whether or not this
environment may serve as an agricultural reservoir for such
pathogens as Cronobacter sakazakii, Legionella pneumo-
philia, Haemophilus influenzae, or Chryseobacterium
meningosepticum or whether the observed genera simply
represent nonpathogenic environmental members.
Our results demonstrate how complex an epiphytic
biome can be and suggest that agricultural inputs associated
with organic or conventional management have the potential
to influence the associated microbial consortia. It is likely that
our findings are only the proverbial tip of the iceberg with
important ramifications for future assessment of preharvest
food safety risks and development of best agricultural
practices to meet the worlds urgent challenge of creating
sustainable agricultural practices while being ever mindful of
their impact on public and environmental health.
ACKNOWLEDGMENTS
We thank the Harry R. Hughs Center for AgroEcology, the Joint Institute
for Food Safety and Applied Nutrition (JIFSAN), and the Sustainable Agriculture,
Research and Education (SARE) program of the USDA for funding assistance.
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