Tuesday, March 5, 2013

Unexpected Diversity during Community Succession in the Apple Flower Microbiome

I recently had the pleasure of reading about the succession of microbes in flowers as they emerge, open, and eventually fall from the tree. This interesting paper (published by the Handelsman group and found here:http://mbio.asm.org/content/4/2/e00602-12) used 454 pyrosequencing to identify bacterial community members in flowers across 5 different trees throughout time. I especially liked figure 4 (below):


They used the appearance of different groups at different parts of the season to identify "poineer", early, mid, late, climax and "generalist" microbes.  Although the list of sequences or microbes in each of these groups isn't provided, they go on to state: “Late” group members peaked when flowers had been open for 3 days and included a high abundance of Lactobacillusand Acetobacter taxa, whose occurrences aligned with previously reported conditions of flower decomposition by yeast"  I was surprised that beyond a casual mention in the paper's discussion, there was no attempt to probe whether or not the communities found on flowers resembled those found associated with pollinators.

As it turns out, the genera Lactobacillus and Acetobacter are also associated with sweat bees, honey bees and bumble bees (see http://aem.asm.org/content/early/2013/01/03/AEM.03681-12.abstract and http://www.biomedcentral.com/1471-2180/12/221).  I downloaded the entire apple flower dataset from MG-RAST and checked to see if any would affiliate with the bee specific sequences (using a 60% threshold).  Here is a quick table below, sorted alphabetically by "family" level. Out of a total of ~35,000 sequences, a subset (~5,600) have been previously found in bees. 


Family
Count
Acetobacetraceae
9
Acetobacteraceae
663
Actinomycetales
713
Actinomycetales_incertae_sedis
1271
alpha-1
53
alpha-2.2
11
alpha-2.2
47
Anaplasmataceae
1
beta
6
Bifidobacteriaceae
54
Brucellaceae
288
Burkholderiaceae
66
Burkholderiales_bacterium_YT0099
92
Comamonadaceae
38
Comamonadaceae_incertae_sedis
19
Comamonodaceae_incertae_sedis
194
Enterobacteriaceae
636
firm-4
3
gamma-1
3
Lactobacillaceae
293
Moraxellaceae
55
Neisseriaceae
19
Oxalobacteraceae
520
Staphylococcaceae
560

This picture changes when you classify the dataset using a more "complete" training set - the SILVA+Bees training set we developed (http://www.biomedcentral.com/1471-2180/12/221).  Sequences that found homes in the honey bee specific training set alone (within the honey bee specific clades), now find homes elsewhere and very few remain (highlighted below in red).  It's also worth mentioning that one major conclusion of the paper -- the "novel" sequences uncovered by the experiment -- was true in my hands as well. A very large fraction of the sequences  (~16,000) were "unclassified" at the family level, even at a 60% threshold using the NBC-RDPII and the SILVA + honey bee specific training set (see classifications below).  

Acetobacteraceae
821
Acholeplasmataceae
1
Acidimicrobiaceae
1
Acidothermaceae
5
Actinomycetaceae
16
Actinomycetales_incertae_sedis
1
Actinospicaceae
1
Actinosynnemataceae
1
Aerococcaceae
18
Aeromonadaceae
5
Alcaligenaceae
55
Anaplasmataceae
3
Aurantimonadaceae
15
Bacillaceae
344
Bacillales_incertae_sedis
7
Bacteriovoracaceae
29
Bdellovibrionaceae
19
Beijerinckiaceae
28
beta
4
Bifidobacteriaceae
1
Bradyrhizobiaceae
118
Brevibacteriaceae
6
Brucellaceae
8
Burkholderiaceae
89
Burkholderiales_bacterium_YT0099
3
Burkholderiales_incertae_sedis
91
Caldilineaceae
2
Campylobacteraceae
5
Carnobacteriaceae
13
Caryophanaceae
1
Caulobacteraceae
322
Cellulomonadaceae
90
Chitinophagaceae
1357
Clostridiaceae
113
Comamonadaceae
264
Conexibacteraceae
112
Coriobacteriaceae
10
Corynebacteriaceae
79
Cryomorphaceae
8
Cyclobacteriaceae
127
Cystobacteraceae
17
Cytophagaceae
1357
Deinococcaceae
696
Dermabacteraceae
3
Dermatophilaceae
32
Desulfovibrionaceae
1
Dietziaceae
21
Enterobacteriaceae
570
Enterococcaceae
24
Erysipelotrichaceae
69
Erythrobacteraceae
288
Eubacteriaceae
6
Flammeovirgaceae
12
Flavobacteriaceae
795
Fusobacteriaceae
2
gamma-1
1
Gemmatimonadaceae
236
Geobacteraceae
9
Geodermatophilaceae
75
Glycomycetaceae
1
Gracilibacteraceae
6
Haliangiaceae
1
Holophagaceae
4
Hydrogenophilaceae
3
Hyphomicrobiaceae
138
Hyphomonadaceae
13
Iamiaceae
161
Ilumatobacter
19
Incertae_Sedis_XI
33
Incertae_Sedis_XII
3
Incertae_Sedis_XIII
3
Incertae_Sedis_XIV
7
Incertae_Sedis_XVIII
2
Intrasporangiaceae
386
Kineosporiaceae
44
Lachnospiraceae
60
Lactobacillaceae
422
Leptotrichiaceae
5
Leuconostocaceae
21
Methylobacteriaceae
188
Methylocystaceae
3
Methylophilaceae
12
Microbacteriaceae
340
Micrococcaceae
178
Micrococcineae_incertae_sedis
5
Micromonosporaceae
23
Moraxellaceae
190
Mycobacteriaceae
31
Myxococcaceae
9
Nakamurellaceae
23
Nannocystaceae
18
Neisseriaceae
15
Nitriliruptoraceae
2
Nitrosomonadaceae
27
Nitrospiraceae
15
Nocardiaceae
45
Nocardioidaceae
406
Nocardiopsaceae
11
Oxalobacteraceae
396
Paenibacillaceae
90
Parachlamydiaceae
9
Pasteurellaceae
4
Peptococcaceae
11
Peptostreptococcaceae
117
Phyllobacteriaceae
110
Planococcaceae
162
Polyangiaceae
38
Promicromonosporaceae
1
Propionibacteriaceae
39
Pseudomonadaceae
21
Pseudonocardiaceae
92
Rhizobiaceae
42
Rhodobacteraceae
350
Rhodobiaceae
3
Rhodocyclaceae
38
Rhodospirillaceae
57
Rhodothermaceae
30
Rickettsiaceae
30
Rubrobacteraceae
4
Ruminococcaceae
35
Sanguibacteraceae
57
Saprospiraceae
17
Sinobacteraceae
86
Sneathiellaceae
1
Solirubrobacteraceae
44
Sphaerobacteraceae
10
Sphingobacteriaceae
401
Sphingomonadaceae
1673
Spirochaetaceae
2
Sporichthyaceae
4
Staphylococcaceae
19
Streptococcaceae
42
Streptomycetaceae
26
Streptophyta
28
Streptosporangiaceae
3
Syntrophobacteraceae
1
Syntrophomonadaceae
2
Thermoactinomycetaceae
77
Thermoanaerobacteraceae
7
Thermomonosporaceae
5
Trueperaceae
2107

Is this a surprising result? If pollinator-affiliated sequences were found in abundance on the flowers you could explain it by the simple fact that flowers are visited by these insects and therefore bacteria from bees might be shed onto the flower parts.  The relative paucity suggests instead that these bacteria, although inoculated into the environment by the bees, are specific to their hosts and perhaps do not thrive on the flower.