‘Shrooms in rooms: What fungi in the built environment can

tell us about fungi and buildings Rachel Adams

UC Berkeley

Leviticus, Chapter 14: Verses 33-57 Step Verse Instruction 1 36 Evacuation 2 37 Professional inspection 3 38 Quarantine for 1 week 4 39 Re-inspection 5 40 Mold removal 6 41 Scrape affected area 7 42 Repair with new materials 8 43 If mold persists, call for new inspection 9 44 Professional inspection 10 45 If expert confirms that mold persists, demolish the home and dispose of all

materials outside the city 11 46 Quarantine anyone who was in the house 12 47 Clean clothes of anyone who was in the house 13 48 If expert finds that mold is gone, declare the house habitable again 14 49-53 Perform ritual cleaning, effect atonement for the house, and assume it is

now clean

•  Damp and moldy buildings are consistently associated with negative health outcomes (Mendell et al. 2011)

•  The strongest association with health effects is the perception of mold odor by the occupants (Quansah et al. 2012, Jaakkola et al. 2013)

The actual dampness-related causal agents – biological or chemical – for these health

effects are unknown

•  Damp and moldy buildings are consistently associated with negative health outcomes (Mendell et al. 2011)

•  The strongest association with health effects is the perception of mold odor by the occupants (Quansah et al. 2012, Jaakkola et al. 2013)

Quicksilver77

The great indoors

Fungi in the indoor microbiome, and beyond

Fungi

Animals

Plants

Yeasts Filaments

Symbionts − +

Fungi in the indoor microbiome, and beyond

Fungi

Animals

Plants

Yeasts Filaments

Symbionts − +

Amend et al., PNAS, 2010

Indoor fungi are geographically patterned

UC Berkeley’s University Village

UC Berkeley’s University Village

Hypothesis: On a local scale, building characteristics, environmental conditions, and resident behaviors will

structure fungal communities.

UC Berkeley’s University Village

100m

5 hanging petri dishes •  Kitchen •  Living room •  Bedroom •  Bathroom •  Outdoor (balcony)

Collection ~ 1 month, summer 2011 (n=11) and winter 2012 sampling (n=8)

Ceiling-suspended (empty) Petri dish

T and Φ Sensor Survey information on house characteristics and resident behavior

UC Berkeley’s University Village – sampling scheme

Amplicon-based approach: Pyrosequencing of the ITS region of fungal DNA to define OTUs/species

UC Berkeley’s University Village – sampling scheme

Order Number of OTUs Description Primary source 1 Agaricomycetes 238 Mushrooms, polypores Outdoor 2 Dothideomycetes 208 Molds Mixed 3 Tremellomycetes 111 Yeasts Mixed 4 Leotiomycetes 54 Plant pathogens, saprobes Outdoor 5 Eurotiomycetes 48 Molds Mixed 6 Sordariomycetes 39 Plant pathogens, saprobes Mixed 7 Microbotryomycetes 35 Yeasts Mixed 8 Saccharomycetes 19 Yeasts Mixed 9 Chytridiomycetes 9 Aquatic saprobes & parasites Outdoor

10 Wallemiomycetes 8 Molds Mixed 11 Lecanoromycetes 7 Lichenized fungi Outdoor 12 Pezizomycetes 6 Mushrooms, molds Outdoor 13 Agaricostilbomycetes 5 Yeasts Outdoor 14 Glomeromycetes 3 Plant root biotrophs Outdoor 15 Taphrinomycetes 3 Plant pathogens Outdoor 16 Orbiliomycetes 2 Saprobes Outdoor 18 Cystobasidiomycetes 1 Yeasts Outdoor 19 Ambiguous 44 20 Incertae sedis 16 21 Unclassified 130 Total 986

Fungi indoors are diverse, coming from outdoors

Adams et al., 2013a

Order Number of OTUs Description Primary source 1 Agaricomycetes 238 Mushrooms, polypores Outdoor 2 Dothideomycetes 208 Molds Mixed 3 Tremellomycetes 111 Yeasts Mixed 4 Leotiomycetes 54 Plant pathogens, saprobes Outdoor 5 Eurotiomycetes 48 Molds Mixed 6 Sordariomycetes 39 Plant pathogens, saprobes Mixed 7 Microbotryomycetes 35 Yeasts Mixed 8 Saccharomycetes 19 Yeasts Mixed 9 Chytridiomycetes 9 Aquatic saprobes & parasites Outdoor

10 Wallemiomycetes 8 Molds Mixed 11 Lecanoromycetes 7 Lichenized fungi Outdoor 12 Pezizomycetes 6 Mushrooms, molds Outdoor 13 Agaricostilbomycetes 5 Yeasts Outdoor 14 Glomeromycetes 3 Plant root biotrophs Outdoor 15 Taphrinomycetes 3 Plant pathogens Outdoor 16 Orbiliomycetes 2 Saprobes Outdoor 18 Cystobasidiomycetes 1 Yeasts Outdoor 19 Ambiguous 44 20 Incertae sedis 16 21 Unclassified 130 Total 986

Fungi indoors are diverse, coming from outdoors

Adams et al., 2013a

Indoor surfaces are highly similar to indoor air

Adams et al., 2013b

But drains do show in situ growth

Adams et al., 2013b

Adams et al., 2013b

Exophiala spp.

Fusarium spp.

But drains do show in situ growth

Surfaces – including foreheads – are collectors of airborne fungi

Culture of my forehead: 4 morphospecies

10x that are hitchhiking on foreheads

Adams et al., 2013b

Surfaces – including foreheads – are collectors of airborne fungi

Fungi indoors are not influenced by residents

Categorical Unit Room type Unit age Floor number Number of bedrooms Number of bathrooms Number of residents Frequency of cleaning

Continuous Geographic distance Temperature Relative humidity Temperature variance Relative humidity variance

Adams et al., 2013a

Fungi indoors are not influenced by residents

Categorical Unit Room type Unit age Floor number Number of bedrooms Number of bathrooms Number of residents Frequency of cleaning

Continuous Geographic distance Temperature Relative humidity Temperature variance Relative humidity variance

Adams et al., 2013a

Fungi show dispersal limitation at a small scale

Adams et al., 2013a

Dispersal limitation and endemism in fungi

Release and dispersal of basidiospores from Amanita muscaria var. alba and their infiltration into a residence. Li (2005) Mycol. Res. 109 (11): 1235–1242

Dispersal limitation and endemism in fungi

30cm 5.2m ~10m

Release and dispersal of basidiospores from Amanita muscaria var. alba and their infiltration into a residence. Li (2005) Mycol. Res. 109 (11): 1235–1242

Dispersal limitation and endemism in fungi

30cm 5.2m ~10m

Release and dispersal of basidiospores from Amanita muscaria var. alba and their infiltration into a residence. Li (2005) Mycol. Res. 109 (11): 1235–1242

Day 3: 41,000 spores/m3 1,000 spores/m3 12 spores/m3

Dispersal and indoor fungi

•  In these non-water damaged buildings, airborne composite fungal samples were dominated by outdoor air

•  The source strength of deposition from the air to surfaces was much greater than the inverse

•  The outdoor pools varied in space (hundreds of meters) and seasonally

•  Surveys of the built environment support growing evidence for endemism in fungi

Dispersal and indoor fungi

•  In these non-water damaged buildings, airborne composite fungal samples were dominated by outdoor air

•  Do these long-term composite samples mask (interesting) short-term dynamics?

•  The source strength of deposition from the air to surfaces was much greater than the inverse

•  Does the same pattern hold on the short term?

Fungal composition on shorter time scales

Controlled Environment Chamber

Fungal composition on shorter time scales

Controlled Environment Chamber

Goal: Use quantity and composition of microbial communities to inform role of occupancy of airborne bioaerosols

Controlled Environment Chamber

Goal: Use quantity and composition of microbial communities to inform role of occupancy of airborne bioaerosols Treatment 0 people 1 person sitting 2 people sitting 2 people walking 8 people sitting

Flooring Carpet Sheeting

Location

Indoor Outdoor

Replicates

3 x x x

Controlled Environment Chamber

Filter cup

NIOSH BC 251

1.  Extraction negatives Lab contaminant (Boletus) and possible human

contaminant (Pyrenochaeta unguis-hominis). 2. Mock community

-- Extracted DNA of 18 taxa pooled and amplified along with rest of samples. -- Zero-ing all reads with an abundance less than 10 results in community of 20 or 21 taxa, recovery of all taxa

3. Taxonomy-based filtering

Remove all taxa that do not match to “fungus”

Quality control issues with amplicon sequencing

1.  Extraction negatives Lab contaminant (Boletus) and possible human

contaminant (Pyrenochaeta unguis-hominis). 2. Mock community

-- Extracted DNA of 18 taxa pooled and amplified along with rest of samples. -- Zero-ing all reads with an abundance less than 10 results in community of 20 or 21 taxa, recovery of all taxa

3. Taxonomy-based filtering

Remove all taxa that do not match to “fungus”

Quality control issues with amplicon sequencing

1.  Extraction negatives Lab contaminant (Boletus) and possible human

contaminant (Pyrenochaeta unguis-hominis). 2. Mock community

-- Extracted DNA of 18 taxa pooled and amplified along with rest of samples. -- Zero-ing all reads with an abundance less than 10 results in community of 20 or 21 taxa, recovery of all taxa

3. Taxonomy-based filtering

Remove all taxa that do not match to kingdom fungi

Quality control issues with amplicon sequencing

High occupancy – modest human signature – see a signal of the flooring in the indoor air

Low occupancy – similar to outdoor air

Hypotheses: Occupancy increases shedding & resuspension rates and structures airborne bioaerosols

Expectations for microbial composition

Fungal composition shows high indoor/outdoor overlap

Factor p

Variance explained

Date 0.01 0.39

Occupancy 0.05 0.14

Time 0.01 0.08 Floor -- --

Occupancy does not affect indoor-outdoor distance

Carpet Sheet

0.65

0.70

0.75

0.80

0.85

0.90

0.95

Com

mun

ity d

ista

nce

0 1 2 2w 8

0.65

0.70

0.75

0.80

0.85

0.90

0.95

p>0.05  

Factor p Variance explained

Date 0.01 0.39

Occupancy 0.05 0.14

Time 0.01 0.08

Floor -- --

Predictors of fungal composition

Indoor concentration generally tracks outdoor concentration

0 5000 10000 15000 20000 25000 30000

05000

10000

20000

Outdoor - GCN/m3

Indo

or -

GC

N/m

3

Outdoor  

Indoor concentration generally tracks outdoor concentration

0 5000 10000 15000 20000 25000 30000

05000

10000

20000

Outdoor - GCN/m3

Indo

or -

GC

N/m

3

Against this variable background, difficult for us to identify differences due to occupancy

Outdoor  

Carpet Indoor Outdoor Battarrea stevenii 0 160217 222 Trametes versicolor 140 47085 46720 UC1859899 voucher 34 31633 22716 Aureobasidium pullulans 6216 25089 53291 Cladosporium cladosporioides 5993 16128 42522 Cladosporium_sp_1_GP_2013 3145 13928 100781 Cladosporium cladosporioides 1797 11353 27356 Cladosporium sp. 1097 10107 44716 YNP777 voucher 0 9841 0 Rhodotorula mucilaginosa 1931 8689 4339 Pisolithus 73 7721 8406 Sistotremastrum 0 7685 7959 Cortinarius 0 7622 5000 Fungi 2428 6865 19330 Penicillium_sp_RM1_8 57 6852 11947 Cladosporium 1031 6528 30887 Cladosporium cladosporioides 1877 6300 22418 Cladosporium dominicanum 2113 6143 16402 Cryptococcus albidosimilis 510 5894 3753 Fungal_endophyte_sp_AP053 2371 5719 25075

Carpet Indoor Outdoor Battarrea stevenii 0 160217 222 Trametes versicolor 140 47085 46720 UC1859899 voucher 34 31633 22716 Aureobasidium pullulans 6216 25089 53291 Cladosporium cladosporioides 5993 16128 42522 Cladosporium_sp_1_GP_2013 3145 13928 100781 Cladosporium cladosporioides 1797 11353 27356 Cladosporium sp. 1097 10107 44716 YNP777 voucher (Yosemite) 0 9841 0 Rhodotorula mucilaginosa 1931 8689 4339 Pisolithus 73 7721 8406 Sistotremastrum 0 7685 7959 Cortinarius 0 7622 5000 Fungi 2428 6865 19330 Penicillium_sp_RM1_8 57 6852 11947 Cladosporium 1031 6528 30887 Cladosporium cladosporioides 1877 6300 22418 Cladosporium dominicanum 2113 6143 16402 Cryptococcus albidosimilis 510 5894 3753 Fungal_endophyte_sp_AP053 2371 5719 25075

Carpet Indoor Outdoor Battarrea stevenii 0 160217 222 Trametes versicolor 140 47085 46720 UC1859899 voucher 34 31633 22716 Aureobasidium pullulans 6216 25089 53291 Cladosporium cladosporioides 5993 16128 42522 Cladosporium_sp_1_GP_2013 3145 13928 100781 Cladosporium cladosporioides 1797 11353 27356 Cladosporium sp. 1097 10107 44716 YNP777 voucher (Yosemite) 0 9841 0 Rhodotorula mucilaginosa 1931 8689 4339 Pisolithus 73 7721 8406 Sistotremastrum 0 7685 7959 Cortinarius 0 7622 5000 Fungi 2428 6865 19330 Penicillium_sp_RM1_8 57 6852 11947 Cladosporium 1031 6528 30887 Cladosporium cladosporioides 1877 6300 22418 Cladosporium dominicanum 2113 6143 16402 Cryptococcus albidosimilis 510 5894 3753 Fungal_endophyte_sp_AP053 2371 5719 25075

Carpet Indoor Outdoor Battarrea stevenii 0 160217 222 Trametes versicolor 140 47085 46720 UC1859899 voucher 34 31633 22716 Aureobasidium pullulans 6216 25089 53291 Cladosporium cladosporioides 5993 16128 42522 Cladosporium_sp_1_GP_2013 3145 13928 100781 Cladosporium cladosporioides 1797 11353 27356 Cladosporium sp. 1097 10107 44716 YNP777 voucher 0 9841 0 Rhodotorula mucilaginosa 1931 8689 4339 Pisolithus 73 7721 8406 Sistotremastrum 0 7685 7959 Cortinarius 0 7622 5000 Fungi 2428 6865 19330 Penicillium_sp_RM1_8 57 6852 11947 Cladosporium 1031 6528 30887 Cladosporium cladosporioides 1877 6300 22418 Cladosporium dominicanum 2113 6143 16402 Cryptococcus albidosimilis 510 5894 3753 Fungal_endophyte_sp_AP053 2371 5719 25075

Air  samples  in  a  mycology  classroom:    a  unique  source  distorts  perceived  species  richness  

Adams et al., 2013c

Mycology  classroom  appears  to  be  less  rich  than  other  classrooms  but  also  have  higher  biomass  

0 2000 4000 6000 8000

0200

400

600

800

1000

B

AC

D

E

Individuals

Cha

o E

stim

ated

Ric

hnes

s

A B C D E

050

100

150

200

ClassroomP

enic

illiu

m s

pore

equ

ival

ents

Adams et al., 2013c

Composi?on  of  non-­‐mycology  classrooms  are  similar  

AB

CD

E

Proportion

Classroom

0 20 40 60 80 100

Adams et al., 2013c

Mycology  classroom  dominated  by  a  few  taxa  

AB

CD

E

Proportion

Classroom

0 20 40 60 80 100

Adams et al., 2013c

xx  PuEalls  dominate  mycology  classroom  

Pisolithus,  aka  dog  turd  fungus   Ba+arrea,  tall  s?ltball  

Lycoperdon,  common  puEall  

Implications for amplicon-based studies

Culturing greatly undersamples the diversity that is there, but the efficiency of current culture-independent techniques can make it difficult to identify true residents Differences in biomass across samples can skew community assessments and community comparisons

Implications for studying fungi in the built environment

The far majority of what we encounter in indoor air does not originate indoors

Different in other climates? May still have impacts on human health

Interpretation of indoor air samples are greatly aided when there is a comparable outdoor sample Short-term air sampling can be idiosyncratic Effect of human occupancy/activity is largely swamped by outdoor dynamics

BIMERC: Berkeley Indoor Ecology Research Consortium

Rachel Adams John Taylor Tom Bruns

Gary Anderson Steve Lindow Ed Arens

Allen Goldstein Bill Nazaroff Seema Bhangar

Any questions?