Chapter 5: DNA-DERIVED ASSESSMENTS OF FUNGAL COMMUNITY
5.3 RESULTS
5.3.3 Soil fungal community structure at Baynesfield Estate (site 1)
Fungal fingerprints from DGGE analysis of PCR amplicons (produced with FR1GC/FF390) of the various land uses at site 1, showed that the DNA banding profiles of the replicate subsamples of a single land use, were more similar to each other than they were to the subsample profiles of the other land uses (visual assessment) (Plate 5.3). This was confirmed by Quantity One analysis (Figure 5.2). In contrast, the banding patterns previously obtained when using primer pair FR1GC/NS1 were more similar across the gels regardless of land use (section 4.3.2).
In the current study, Quantity One analysis showed the number of bands in each lane varied from 4–18 (Figure 5.2), which was greater than the 5–10 bands previously produced by FR1GC/NS1. Nonetheless, this was fewer than the 15-44 bands in the bacterial community DGGE gels (section 3.3.2). Since each band was inferred to represent a single OTU, soil fungal richness at Baynesfield, as revealed by FR1GC/FF390, was greater than that shown by FR1GC/NS1, but lower than the bacterial richness at the same site. Some bands were again common to all lanes although the band intensities varied, indicating a difference in mass (Plate 5.3; Figure 5.2).
PLATE 5.3 DGGE gel (denaturing gradient 30–50%) of Baynesfield soil fungal amplicons from different land uses amplified with primer pair FR1GC/FF390.
Key: SC = sugarcane; M = maize; KIK = kikuyu pasture; NAT = native grassland; PF = pine plantation; W = wattle plantation. Arrow indicates bands excised for DNA elution.
FIGURE 5.2 Quantity One diagram of DGGE gel (Plate 5.3) showing bands not visible in the photograph.
Key: Lanes 1–3: sugarcane; lanes 4–6: maize; lanes 7–9: kikuyu pasture; lanes 10–12: native grassland; lanes 13–15: pine plantation; lanes 16–18: wattle plantation. Arrow indicates band 23, excised from all lanes for DNA elution and sequencing.
A DGGE gel (denaturing gradient, 35–45%) of Baynesfield samples, containing the bands produced by eluted fungal DNA from single excised bands, together with non- excised control DNA (to verify band position), is shown in Plate 5.4.
Analysis of the gel (Plate 5.4) with Quantity One (Figure 5.3) showed that multiple bands, varying from 2–7, (excluding control samples) were contained within each, apparently ‘single’, excised, dominant band. The distance migrated in the gel lanes by the native grassland control DNA (NAT C), and the pine plantation control DNA (PF3 C), was slightly shorter than that by the excised DNA (Plate 5.4). This was possibly because these control bands contained several sequences. In contrast, the kikuyu pasture control DNA (KIK C), contained a single band only, and therefore ran a similar distance in the lane, to that of the excised DNA. While the narrower denaturing gradient (35–45%) served to separate the multiple bands within each single excised band, the control DNA amplicons, produced fewer bands per lane than they had in the DGGE gels with a 30–50% denaturing gradient (Plate 5.3, Figure 5.2).
These results indicate that the narrower gradient would not have been suitable for the initial DGGE of mixed soil fungal communities in soil.
PLATE 5.4 DGGE gel (denaturing gradient 35–45%) to verify the position of some of the eluted fungal DNA samples, relative to non-excised controls from Baynesfield.
Key: SC1 = sugarcane, subsample 1; M2 = maize, subsample 2) KIK3 = kikuyu pasture, subsample 3; KIK C = control DNA from kikuyu subsample 3; lane 5, open; NAT2 = native grassland, subsample 2; NAT C = control DNA from native grassland subsample 2; lane 8, open; PF3 C = control DNA from pine plantation subsample 3; PF3 = pine plantation, subsample 3; W1 = wattle plantation, subsample 1; W1 C = control DNA from wattle plantation subsample 1. Arrow indicates bands excised for sequencing. Control DNA was not excised and sequenced.
FIGURE 5.3 Quantity One diagram of DGGE gel (Plate 5.4) showing bands not visible in the photograph.
Key: Lane 1, SC1: sugarcane; lane 2, M2: maize; lane 3, KIK3: kikuyu pasture; Lane 4, KIK C: control DNA, kikuyu pasture; lane 5: open; lane 6, NAT2 : native grassland) lane 7, NAT C: control DNA, native grassland; lane 8: open; lane 9, PF3 C: control DNA, pine plantation;
lane 10, PF3: pine plantation; lane 11, W1: wattle plantation; lane 12, W1 C: control DNA, wattle plantation. Arrow indicates excised bands (15).
The results obtained from sequencing the 18S rRNA gene fragments from the Baynesfield soil fungal communities under the different land uses (identified using the NCBI nucleotide database and the mega BLAST program), are presented in Table 5.2.
When comparing sequences from environmental samples with fully identified reference sequences, it is common to rely on threshold values (e.g. 97%) for determining sequence similarity (Ovaskainen et al., 2010). Of the 18 fungal sequences excised, 17 showed a similarity of 98–100% to Genbank sequences. Only band 2 from subsample W1 (W1/2) had a similarity of less than 90% and was, therefore, eliminated from the analysis (Green et al., 2006).
TABLE 5.2 Identity of 18S rRNA gene sequences from excised DGGE bands of soil fungal communities from different land uses at Baynesfield
Sequence designation
Closest match from Genbank % sequence similarity (BLAST)
Genbank accession no.
SC1 Uncultured fungus isolate 1 18S rRNA gene, partial sequence 99% AY769847.1
SC2 Uncultured fungus partial 18S rRNA gene, isolate 9 100% FM202462.1
SC3 Uncultured fungus isolate 1 18S rRNA gene, partial sequence 100% AY769847.1
M1 Uncultured fungus clone Nikos_35 18S rRNA gene, partial sequence 100% HM104558.1
M3 Uncultured fungus clone Nikos_35 18S rRNA gene, partial sequence 99% HM104558.1
KIK1 Uncultured fungus isolate 6 18S rRNA gene, partial sequence 99% AY769852.1
KIK2 Uncultured fungus clone Nikos_35 18S rRNA gene, partial sequence 100% HM104558.1
KIK3 Uncultured fungus clone Nikos_35 18S rRNA gene, partial sequence 99% HM104558.1
NAT1 Uncultured soil fungus isolate DGGE gel band 12060835(SF01)FF390 100% EU647857.1
NAT2 Uncultured fungus isolate 1 18S rRNA gene, partial sequence 100% AY769847.1
NAT3 Uncultured soil fungus isolate DGGE gel band 12060835(SF01)FF390 100% EU647857.1
PF1 Uncultured soil fungus isolate DGGE gel band 12060835(SF01)FF390 100% EU647857.1
PF2 Uncultured soil fungus isolate DGGE gel band 12060835(SF01)FF390 100% EU647857.1
PF3 Mortierella sp.20006 18S rRNA gene, partial sequence 100% EU710842.1
W1/1 Uncultured fungus clone Nikos_35 18S rRNA gene, partial sequence 98% HM104558.1
W1/2* Repetobasidium mirificum 18S rRNA gene, partial sequence 87%* AY293155.1
W2 Uncultured fungus isolate 1 18S rRNA gene, partial sequence 100% AY769847.1
W3 Uncultured soil fungus isolate DGGE gel band 12060835(SF01)FF390 99% EU647857.1
Sequence similarity values below 90% are not considered identical (Green et al., 2006)
Key: SC = sugarcane (burnt cane harvested); M = maize (conventional tillage); KIK = kikuyu pasture; NAT = native grassland; PF = pine plantation; W = wattle plantation.
Soil fungal community composition under the different land uses at this site, is shown in an NMS two-dimensional plot rotated by PCA (Figure 5.4). The various land use soil replicates were clustered on the basis of the presence or absence (Sörensen coefficient) of bands (sequence types). The replicate PF soil fungal communities were separated from those of the other land uses, as were the W fungal communities. Soil fungi under the arable crops, SC and M, clustered together indicating the presence of similar fungal OTUs in soils under these land uses. Similarly, KIK replicates were closely clustered with those of NAT. MRPP confirmed that subsamples from W (av.
distance 0.19, Sörensen) were the most closely correlated, indicating the structural similarity of their fungal communities. SC subsamples were also more closely clustered (0.27) than the NAT (0.33), PF (0.39), KIK (0.44) and M (0.66) subsamples, with those of KIK being more dissimilar than NAT and PF. However, the M subsamples were the most structurally dissimilar of all the land uses.
FIGURE 5.4 A NMS two-dimensional plot (rotated by PCA) of fungal communities (presence or absence of bands) at Baynesfield. NMS stress = 0.19955.
Key: SC = sugarcane; M = maize; KIK = kikuyu pasture; NAT = native grassland; PF = pine plantation; W = wattle plantation; B = band (OTU).
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
SC1 SC2
SC3
M1
M2 M3
KIK1
KIK2
KIK3 NAT1
NAT2 NAT3
PF1
PF2 PF3
W1
W2 W3
B1
B2
B3 B4
B5
B6
B7
B8 B9
B10
B11
B12
B13 B14
B15 B16
B17
B18 B20
B21
B22
B23 B24
B25 SAMPLES
SC M KIK NAT PF W
Axis 1
Axis 2
Analysis of species richness (S) (number of bands present = number of different groups of species) by one-way ANOVA of the soil fungal communities at this site, showed large differences between the various fungal communities (Table 5.3).
Pairwise comparisons made using Tukey’s test, indicated that soil fungal community richness under W differed from that of the soil fungi under M, SC and NAT, but not from that of the communities under PF or KIK (Table 5.3). Trends for richness followed the order: M < SC < NAT < PF < KIK < W.
TABLE 5.3 ANOVA of species richness (number of bands produced using primer pair FR1GC/FF390) and land use means in the fungal communities at Baynesfield
Source of variation d.f s.s. m.s. f-ratio p-value
Land use 5 96.44 19.29 6.09 0.005
Residual 12 38.00 3.17
Total 17 134.44
Grand mean 7.56
Land use SCa Ma KIKab NATa PFab Wb
Mean 5.33 5.00 8.33 7.00 7.67 12.00
Means with common superscript letters are not significantly different. (LSD5% = 3.166).
Key: SC = sugarcane; M = maize; KIK = kikuyu pasture; NAT = native grassland;
PF = pine plantation; W = wattle plantation.
ANOVA of soil fungal community evenness (J) under the different land uses at Baynesfield, showed a marked overall difference in the evenness of the fungal species present (Table 5.4). Wattle soil fungal evenness differed from that of the communities under SC, M and KIK, but not from that of NAT and PF soil fungal communities.
Trends for evenness were: M < KIK ≤ SC < NAT ≤ PF < W.
TABLE 5.4 ANOVA and land use means of soil fungal species evenness (J) at Baynesfield Estate
Source of variation d.f s.s. m.s. f-ratio p-value
Land use 5 0.11 0.02 13.14 < 001
Residual 12 0.02 0.00
Total 17 0.13
Grand mean 0.83
Land use SCabc Ma KIKab NATbcd PFcd Wd
Mean 0.79 0.72 0.78 0.88 0.89 0.94
Means with common superscript letters are not significantly different. (LSD5% = 0.072).
Key: SC = sugarcane; M = maize; KIK = kikuyu pasture; NAT = native grassland; PF = pine plantation; W = wattle plantation.
Analysis of soil fungal diversity by the Shannon Weaver diversity index (H′) showed an overall difference in community structural diversity, with communities under W being separated from those of SC and M, but not from those of KIK, NAT or PF.
Communities under M and SC could not be separated from each other nor from those under KIK, NAT and PF (Table 5.5). Trends for fungal diversity followed the order:
M < SC < KIK < NAT < PF < W.
TABLE 5.5 ANOVA and land use means of soil fungal species diversity (H′) at Baynesfield Estate
Source of variation d.f s.s. m.s. f-ratio p-value
Land use 5 2.64 0.53 7.14 0.003
Residual 12 0.89 0.07
Total 17 3.53
Grand mean 1.65
Land use SCa Ma KIKab NATab PFab Wb
Mean 1.29 1.15 1.64 1.70 1.77 2.34
Means with common superscript letters are not significantly different. (LSD5% = 0.484).
Key: SC = sugarcane; M = maize; KIK = kikuyu pasture; NAT = native grassland; PF = pine plantation; W = wattle plantation.
The results of a CCA of the relationship between the effects of selected (non- collinear) soil physicochemical variables, and the soil fungal community (genetic) structure under the different land uses at Baynesfield Estate are presented in Figure 5.5. CCA 1 accounted for 43.8% of the variance due to environmental effects, and 17.5% of the total variance. CCA 2 accounted for 30.4% of the variance due to environmental effects, and 12.1% of the total variance. CCA 1 was correlated with pH, whereas organic C, P and ECEC were correlated with CCA 2. Subsamples of M and SC were clustered together on the basis of a strong correlation with high soil P values as a result of fertilizer applications. PF replicate subsamples were correlated with a low soil pH. Soils with a higher pH and level of ECEC were associated with W, and organic C and ECEC with NAT and KIK soils. A plot showing the presence of species centroids (bands) in the CCA (Figure 5.5) is shown in Figure 5.6.
FIGURE 5.5 Plot of samples (classified by land use) and soil variables along the first two axes of a CCA of the effects of selected soil variables on fungal community composition (band presence) at Baynesfield Estate.
CCA1 accounted for 43.8% and CCA2 for 30.4% respectively, of the variance due to environmental effects.
Key: SC = sugarcane; M = maize; KIK = kikuyu pasture; NAT = native grassland; PF = pine plantation; W = wattle plantation.
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5
-2.0 -1.0 0.0 1.0 2.0 3.0
SC1 SC2
SC3 M1 M2 M3
KIK1 KIK2
KIK3 NAT1
NAT2 NAT3
PF1
PF2
PF3
W1 W2
W3
P
pH ECEC Organic C
SAMPLES
SC M KIK NAT PF W
ENV. VARIABLES
CCA 1 (43.8%)
CCA 2 (30.4%)
FIGURE 5.6 Plot of bands (centroids) in the CCA (Figure 5.5) showing the relationship of the different fungal OTUs to the land use soils at Baynesfield.
Key: B = band.
The Monte Carlo Permutation test for significant relationships within the CCA data showed that soil variables had affected fungal community structural diversity at this site. (Appendix C, Table C1).