Selective screening: isolation of fungal strains from contaminated soils in Austria

Caroline Poyntner; Max Prem; Oliver Mann; Barbara Blasi; Katja Sterflinger

doi:10.1515/boku-2017-0014

Figures & Tables

Regression model of PAH ratio against fungal colonies and fungal colonies isolated from each sample. A: Ratio of high molecular weight (HMW) PAHs to total PAHs on the y-axis against the total number of fungal colonies (x-axis) was plotted. A linear regression was done (black line) using the samples with low HMW/total PAH ratio (blue dots) with a confidence interval of 95% (blue line). The excluded samples with high HMW/total PAH ratio (indicating high age) was colored in green and excluded from the linear regression. B: Number of fungal colonies isolated from each sample grown on toluene (red), PCB 126 (green) and hexadecane (blue).Abbildung 1. Regressionsmodell des PAK-Verhältnisses gegen die Anzahl der Pilzkolonien und der Gesamtanzahl der Pilzkolonien isoliert von den verschiedenen Proben. A: Verhältnis der hochmolekulargewichtigen (HMW) PAKs gegen die Gesamtanzahl der PAKs auf der y-Achse geplottet gegen die Gesamtanzahl der isolierten Pilzkolonien auf der x-Achse. Eine lineare Regressionsgerade wurde geplottet (schwarze Linie) mit Proben mit niedrigem HMW/total PAK Verhältnis (blau, runde Symbole) und ein Konfidenzintervall von 95 % berechnet (blaue Linie). Proben mit hohem HMW/total PAK-Verhältnisses wurden nicht in die lineare Regression genommen (grüne Punkte). B: Anzahl der Pilzkolonien isoliert von jeder Probe, welche in Anwesenheit von Toluol (rot), PCB 126 (grün) und Hexadekan (blau) gewachsen sind.

Fungal growth curves. Growth curves of the 11 selected fungal strains for identification grown in the presence of A hexadecane, B toluene and C PCB 126. Growth was measured through detected changes in the OD 700 using a Tecan reader.Abbildung 2. Wachstumskurven der Pilzstämme. Wachstumskurven der 11 identifizierten Pilzstämme, die in Gegenwart von A Hexadekan, B Toluol und C PCB 126 gewachsen sind. Das Wachstum wurde durch Änderung der OD 700 mit Hilfe eines Tecan Readers detektiert.

PAH concentrations and number of fungal colonies isolated from each sample. Top, left axis (green): Total sum of colonies isolated in the presence of toluene, PCB 126 or hexadecane, and wells containing glucose. Bottom, right axis: total concentration (pattern) and proportion of HMW PAHs (blue). X-axis presents the different samples.Abbildung 3. PAK-Konzentrationen und die Anzahl der von jeder Probe isolierten Pilzkolonien. Obere, linke Achse: grün: Gesamtanzahl der Kolonien isoliert in Anwesenheit von Toluol, PCB 126 oder Hexadecan als einzige Kohlenstoffquelle und Näpfchen mit Glukose. Untere, rechte Achse: Gesamtkonzentration (Muster) und Proportion an hochmolekulargewichtigen PAKs (blau). Die x-Achse zeigt die verschiedenen Proben.

GC Results: Measured US EPA PAH concentrations and standard deviations of the 12 soil samples (S1-12)_ Values are presented as mg/kg dry weight and standard deviations were calculated (mg/kg dryweight±standard deviation)Tabelle 2_ GC-Ergebnisse: Gemessene US EPA PAK-Konzentrationen und Standardabweichungen der 12 Bodenproben (S1-12) sind in der Tabelle dargestellt_ Die Werte sind also mg/kg Trockengeweicht dargestellt und die Standardabweichung wurde berechnet (mg/kg Trockengewicht±Standardabbweichung)

sample	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	S11	S12
Naphtalene	5.8±1.2	4.8±1.0	12.8±2.6	9.4±1.9	6.8±1.4	31.5±6.3	0.1±0.02	<0.3(0.0)	<0.3 (0.0)	0.1±0.01	<0.3(0.0)	<0.3(0.0)
Acenaphthylene	5.8±1.2	1.8±0.4	<2.7(0.00)	0.9±0.2	0.9±0.2	5.2±1.0	0.1±0.02	<0.3(0.0)	0.4±0.1	0.2±0.03	0.7±0.1	0.3±0.1
Acenatphene	6.9±1.4	4.3±0.9	11.2±2.2	8.5±1.7	6.8±1.4	24.9±5.0	0.6±0.1	0.4±0.1	0.4±0.1	0.1±0.02	<0.3 (0.0)	0.3±0.1
Flurene	10.1±2.0	6.3±1.3	14.8±3.0	10.3±2.1	8.3±1.7	34.3±6.9	0.5±0.1	0.4±0.1	0.4±0.1	0.1±0.02	0.5±0.1	0.6±0.1
Phenanthrene	55.20±11.0	28.3±5.7	70.2±14.0	48.3±9.7	36.5±7.3	154.0±31.0	5.9±1.2	4.4±0.9	4.1±0.8	1.0±0.2	6.4±1.3	5.7±1.2
Anthracene	22.5±4.5	11.4±2.3	22.2±4.4	18.1±3.6	14.4±2.9	67.7±13.5	1.4±0.3	1.2±0.2	1.6±20.3	0.4±0.01	2.1±0.4	1.4±0.3
Fluoranthene	78.0±15.6	29.1±5.8	48.2±9.6	39.0±7.8	30.8±6.2	141.0±28.0	7.5±1.5	12.6±2.5	10.0±2.0	2.9±0.6	17.5±3.5	9.8±2.0
Pyrene	77.8±15.6	30.4±6.1	47.9±9.6	41.6±8.3	32.2±6.4	108.0±22.0	7.5±1.5	24.4±4.9	10.0±2.0	2.3±0.5	14.5±2.9	7.7±2.0
Benz[a]anthracene	36.5±7.3	13.5±2.7	19.9±4.0	16.6±3.3	13.0±2.6	64.3±12.9	4.7±1.0	23.5±4.7	9.8±2.0	1.9±0.4	9.8±2.0	5.9±1.2
Chrysene	37.4±7.5	13.3±2.7	20.7±4.1	15.8±3.2	12.5±2.5	68.6±13.7	5.9±1.2	27.3±5.5	11.0±2.2	2.2±0.4	10.2±2.0	7.1±1.4
Benzo(b) fluoranthene	33.0±6.6	10.4±2.1	14.4±2.9	10.9±2.2	9.0±1.8	36.1±7.2	6.5±1.3	28.1±5.6	11.9±2.4	1.9±0.4	9.1±1.8	7.1±1.4
Benzo(k) fluoranthene	26.6±5.3	9.5±1.9	12.7±2.5	10.6±2.1	8.5±1.7	45.3±9.1	4.2±0.8	33.0±6.6	10.4±2.1	1.7±0.3	8.0±1.6	5.0±1.0
Benzo(a)pyrene	30.6±6.1	10.9±2.2	14.3±2.9	11.9±2.4	9.76±2.0	44.7±8.9	5.0±1.0	27.1±5.4	9.8±2.0	1.8±0.4	8.5±1.7	5.1±1.0
Indeno(1,2,3-cd)pyrene	21.3±4.3	7.0±1.4	8.5±1.7	8.3±1.7	6.0±1.2	20.1±4.0	3.1±0.6	19.6±3.9	8.2±1.6	1.7±0.3	5.2±1.0	2.8±0.6
Dibenzo(a,h) anthracene	5.9±1.2	1.7±0.4	<2.7(0.0)	1.6±0.3	1.3±0.3	4.8±1.0	1.1±0.2	5.5±1.1	2.0±0.4	0.5±0.1	1.2±0.2	0.7±0.1
Benzo(g,hi,i) perylene	19.6+3.9	6.5±1.3	7.8±1.6	6.4±1.3	5.2±1.1	17.2±3.4	3.4±0.7	22.7±4.5	7.2±1.5	1.8±0.4	4.2±0.8	2.7±0.5

sum	472.9±94.6	189.4±38.0	325.6±65.1	258.1±51.7	202.0±40.4	867.8±173.9	57.4±11.5	230.2±46.0	97.2±39.5	20.6±4.0	97.6±19.4	62.2±12.9

Growth pattern of the chosen fungal isolates for sequencing: + = good growth, ~ = very slow growth and - = no growth_ Hex = hexadecane, Tol = toluene, PCB = PCB 126Tabelle 4_ Wachstumsmuster der zum Sequenzieren ausgewählten Pilzisolate: + = gutes Wachstum, ~ = sehr langsames Wachstum und - = kein Wachstum_ Hex = Hexadekan Tol = Toluol, PCB = PCB 126

No	Hex	Tol	PCB
α 14	+	+	~
O	+	+	-
U	+	+	+
V	+	+	-
X 1	+	+	-
Y	+	+	-
Z	+	+	~
BL 3	+	+	+
BL 4	~	+	~
G	+	-	~
H	+	-	~

Results of the second microtiter plate screening: Number of colonies of having the same growth pattern of all 93 fungal isolates on toluene, hexadecane and PCB 126_ + = good growth, ~ = very slow growth and - = no growth_ Hex = hexadecane, Tol = toluene, PCB = PCB 126Tabelle 3_ Ergebnisse des zweiten Mikrotiterplatten-Screenings: Anzahl der Kolonien mit demselben Wachstumsmuster innerhalb der 93 isolierten Kolonien in Gegenwart von Toluol, Hexadekan und PCB 126_ + = gutes Wachstum, ~ = sehr langsames Wachstum und - = kein Wachstum_ Hex = Hexadekan Tol = Toluol, PCB = PCB 126

colony numbers	Hex	Tol	PCB
21	+	+	-
14	+	+	~
9	+	+	+
8	+	-	-
6	+	~	~
5	+	-	~
4	-	-	-
3	~	+	-
3	+	~	-
2	-	+	-
2	~	-	-
2	~	-	~
2	~	~	~
2	~	+	~
2	+	~	+
2	~	~	+
1	-	~	-
1	-	-	~
1	-	~	~
1	~	~	-
1	-	~	+
1	~	~	~

Phylogenetic classification of the ITS/18S rDNA coding sequences of the fungal isolatesTabelle 1_ Phylogenetische Klassifikation der ITS/18S rDNA kodierenden Sequenzen der Pilzisolate

No	Primer Pair	Closest identified phylogenetic relatives [EMBL accession numbers]	Query cover	Ident	ACBR strain No	accession No
α 14	NL1/NL4	Doratomyces purpureofuscus (Cephalotrichum purpureofuscum) genomic DNA sequence contains 28S rRNA gene, strain UAMH 9209 [LN851018.1]	99%	99%	MA6020	KY454753
O	NS5/NS8	Roussoella intermedia strain CBS 170.96 18S ribosomal RNA gene, partial sequence [KF443390.1]	99%	100%	MA6025	KY454758
Y	NL1/NL4	Purpureocillium sp. JCM 28545 gene for 28S ribosomal RNA, partial sequence [LC134235.1]	100%	99%	MA6015	KY454754
BL 3	NL1/NL4	Ochroconis longiphorum strain CBS 435.76 28S ribosomal RNA gene, partial sequence [KF156135.1]	96%	99%	MA6021	KY454755
BL 4	NL1/NL4	Ochroconis longiphorum strain CBS 435.76 28S ribosomal RNA gene, partial sequence [KF156135.1]	99%	99%	MA6017	KY454756
V	ITS1/ITS4	Penicillium janthinellum [EF634422.1]	100%	99%	MA6016	KY454760
Z	ITS1/ITS4	Pyrenochaeta inflorescentiae [EU552153.1]	97%	98%	MA6019	KY454761
H	ITS1/ITS4	Penicillium canescens [AY373901.1]	100%	99%	MA6018	KY454762
	NL1/NL4	Pyrenochaeta inflorescentiae culture-collection CBS:119222 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence	99%	99%		KY454757
U		[EU552153.1]			MA6024
	NS5/NS8	Pyrenochaeta sp. GMG_PPb7 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence [FJ439593.2]	99%	99%		KY454759
X1	ITS1/ITS4	Purpureocillium lilacinum	99%	100%	MA6022	KY454763
G	ITS1/ITS4	Penicillium canescens [AF033493.1]	99%	99%	MA6023	KY454764

Selective screening: isolation of fungal strains from contaminated soils in Austria

Figures & Tables

Figure 1

Figure 2

Figure 3

Phylogenetic classification of the ITS/18S rDNA coding sequences of the fungal isolatesTabelle 1_ Phylogenetische Klassifikation der ITS/18S rDNA kodierenden Sequenzen der Pilzisolate

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colony numbers	Hex	Tol	PCB
21	+	+	-
14	+	+	~
9	+	+	+
8	+	-	-
6	+	~	~
5	+	-	~
4	-	-	-
3	~	+	-
3	+	~	-
2	-	+	-
2	~	-	-
2	~	-	~
2	~	~	~
2	~	+	~
2	+	~	+
2	~	~	+
1	-	~	-
1	-	-	~
1	-	~	~
1	~	~	-
1	-	~	+
1	~	~	~

colony numbers	Hex	Tol	PCB
21	+	+	-
14	+	+	~
9	+	+	+
8	+	-	-
6	+	~	~
5	+	-	~
4	-	-	-
3	~	+	-
3	+	~	-
2	-	+	-
2	~	-	-
2	~	-	~
2	~	~	~
2	~	+	~
2	+	~	+
2	~	~	+
1	-	~	-
1	-	-	~
1	-	~	~
1	~	~	-
1	-	~	+
1	~	~	~

colony numbers	Hex	Tol	PCB
21	+	+	-
14	+	+	~
9	+	+	+
8	+	-	-
6	+	~	~
5	+	-	~
4	-	-	-
3	~	+	-
3	+	~	-
2	-	+	-
2	~	-	-
2	~	-	~
2	~	~	~
2	~	+	~
2	+	~	+
2	~	~	+
1	-	~	-
1	-	-	~
1	-	~	~
1	~	~	-
1	-	~	+
1	~	~	~