Chapter 5: DNA-DERIVED ASSESSMENTS OF FUNGAL COMMUNITY
5.3 RESULTS
5.3.5 Soil fungal community structure at Mount Edgecombe (site 2)
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P K
Ca Mg
Acidity ECEC
pH
C
TF2 TF1 TF3 TFo1
TFo2 TFo3
BtoF1 BtoF2
BtoF3 BtoFo1
BtoFo2
BtoFo3 SOIL VARIABLES
SAMPLES
TF TFo BtoF BtoFo
PC1 (75.0%)
PC2 (16.0%)
FIGURE 5.7 Plot of samples and soil variables along the first two axes of a standardised and centred PCA of different land treatments at Mount Edgecombe.
The PC1 (horizontal) and PC2 (vertical) components accounted for 75.0% and 16.0%, respectively, and cumulatively for 91.0% of the total variance in the soil data.
Key: T = green cane harvested with trash retention; Bto = burnt with harvest residues removed; F = fertilized annually with N, P and K; Fo = unfertilized.
At Mount Edgecombe, MRPP tests of the soil PCA data showed that differences among the soil subsamples from the four land managements were highly significant (T = -4.976; p < 0.001).
lanes of the bacterial DGGE gels. This indicates that while primer pair FR1GC/FF390 revealed greater soil fungal community diversity than FR1GC/NS1, the communities were not as structurally diverse as those of the soil bacteria from the same site. Bands common to all replicates of the four treatments were observed (Plate 5.5). Band intensity varied, with some bands dominant and of higher intensity than others in the same lane (Figure 5.8).
PLATE 5.5 DGGE gel (30–50% denaturing gradient) of Mount Edgecombe fungal amplicons from soils under different management practices.
Key: T = green cane harvested with retention of a trash mulch; Bto = burnt cane harvested, either fertilized (F) or unfertilized (Fo); ML (marker lanes). Arrow indicates the bands excised for fungal DNA elution.
FIGURE 5.8 Quantity One diagram of gel (Plate 5.5) showing bands not visible in the photograph.
Key: Lanes 1 and 14: markers; lanes 2–4, TF: greencane harvested, fertilized; lanes 5–7, TFo:
greencane harvested, unfertilized; lanes 8–10, BtoF: burnt cane harvested, fertilized; lanes 11–13, BtoFo: burnt cane harvested, unfertilized. Arrow = excised bands.
A DGGE gel (denaturing gradient 35–45%) of Mount Edgecombe samples, containing the bands produced by eluted fungal DNA from single excised bands, together with non-excised control DNA (to verify band position), is presented in Plate 5.6, and a Quantity One diagram of this gel, in Figure 5.9.
PLATE 5.6 DGGE gel (35–45% denaturing gradient) to verify position of eluted DNA from excised bands relative to control DNA at Mount Edgecombe.
Key: TF (trashed and fertilized); TFo (trashed and unfertilized); BtoF (burnt cane harvested and fertilized); BtoFo (burnt cane harvested but unfertilized); lane 13 (control DNA). Arrow indicates bands excised for sequencing.
FIGURE 5.9 Quantity One diagram of DGGE gel (Plate 5.6) showing bands not visible in the photograph.
Key: Lanes 1–3, TF: greencane harvested, fertilized; lanes 4–6, TFo: greencane harvested, unfertilized; lanes 7–9, BtoF: burnt cane harvested, fertilized; lanes 10–12, BtoFo: burnt cane harvested, unfertilized; lane 13, control DNA. Arrow indicates bands excised for sequencing.
Analysis of the gel (Plate 5.6) by Quantity One (Figure 5.9) showed that 2–4 bands were contained within each apparently ‘single’ excised dominant band. As with the Baynesfield gels, the narrower 35–45% denaturing gradient separated the multiple bands within each excised band. The results of sequencing the 18S rRNA gene fragments from the excised bands of the soil fungal communities under the different treatments at Mount Edgecombe (identified using the NCBI nucleotide database and the mega BLAST program) are presented in Table 5.7. Of the 17 sequences excised, only five showed a 91–100% similarity to Genbank sequences. Of the remainder, three bands had a similarity of less than 90% and six showed no significant similarity, as their sequencing traces were either chimeric or mixed template traces. In addition, two sequences were identified as plant DNA, and one sequencing trace failed.
Consequently, these poor quality/chimeric sequences were eliminated from the analysis (Green et al., 2006).
TABLE 5.7 Identity of 18S rRNA gene sequences from excised DGGE bands of soil fungal communities from different land management practices at Mount Edgecombe
Sequence designation
Closest match from Genbank % sequence similarity (BLAST)
Genbank accession
no.
TF2 Uncultured soil fungus isolate DGGE gel band 12060835(SF01)FF390
93% EU647857.1
TF3* Cheilymenia coprinaria voucherHMAS69605 18S rRNA gene, partial sequence
83%* DQ787818.1
TFo1 Uncultured fungus clone Nikos_253 18S rRNA gene, partial sequence
99% HM104530.1
TFo2 Uncultured fungus partial 18S rRNA gene, isolate 9 91% FM202462.1 TFo3 Uncultured soil fungus isolate DGGE gel band
12060835(SF01)FF390
98% EU647857.1
BtoF1 Mortierella sp. 20006 18S rRNA gene 100% EU710842.1 BtoF2* Uncultured fungus clone DC_H09F 18S rRNA gene 88%* EU726151.1 BtoFo1* Knightiella splanchnirima small subunit rRNA
gene
86%* AF491856.1
* Sequence similarity values below 90% are not considered identical (Green et al., 2006)
Soil fungal community structure under the different sugarcane management practices at Mount Edgecombe is shown in an NMS plot (Figure 5.10), which clustered the replicate soil DNA subsamples from the four trash managements on the basis of the presence or absence of bands (OTUs). Soil subsamples from under TFo were the most
closely clustered of all the treatments, indicating the structural similarity of their intrinsic fungal communities. Although the three subsamples from under BtoF were separated by a similar distance to those of BtoFo (MRPP, av. distance ~0.5), the two treatments were associated with different fungal OTUs, and were clearly separated in the NMS biplot. This indicates that the intraspecies similarity of the three subsample communities within the same management (Bto) was greater than the interspecies similarity between the fungal communities from the fertilized (BtoF) and unfertilized (BtoFo) soils.
FIGURE 5.10 A NMS two-dimensional plot (rotated by PCA) of fungal communities (presence or absence of bands) at Mount Edgecombe. NMS stress = 0.07917.
Key: TF = green cane harvested with retention of crop residues, fertilized with N, P and K;
TFo = green cane harvested with retention of crop residues but unfertilized; BtoF = pre- harvest burnt sugarcane with tops raked off, fertilized with N, P and K; BtoFo = pre-harvest burnt sugarcane with tops raked off but unfertilized; B = band.
MRPP analysis of this data showed that the different land management practices had a significant effect on soil fungal community composition at this site (T = -1.853;
p = 0.04).
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TF1
TF2 TF3
TFo1
TFo2 TFo3
BtoF1 BtoF2
BtoF3
BtoFo1
BtoFo2
BtoFo3 B1
B2
B3 B4 B6 B5
B7
B8 B9
B10
B11 B12
B13
B14 B15
B17 B16
B18 B19
B20
B21 B22
B23
B24 B25
SAMPLES
TF TFo BtoF BtoFo
Axis 1
Axis 2
One-way ANOVA of soil fungal species richness (S) at Mount Edgecombe showed that the four different land management practices had an overall significant effect on the community species richness (p = 0.02) (Table 5.8). Comparisons of fungal species richness from soil treatments TF and TFo showed that they were not significantly different, nor did the richness of the soil fungi under these managements differ from that of the soil communities under BtoF or BtoFo. However, there was a significant difference in fungal species richness between the BtoF and BtoFo soils.
TABLE 5.8 ANOVA and land management means of soil fungal species richness (S) at Mount Edgecombe
Source of variation d.f s.s. m.s. f-ratio p-value
Land management 3 163.58 54.53 5.95 0.02
Residual 8 73.33 9.17
Total 11 236.92
Grand mean 8.08
Land management TFab TFoab BtoFb BtoFoa
Mean 6.67 9.62 13.00 3.00
Means with common superscript letters are not significantly different (LSD5% = 5.7).
Key: TF = green cane harvested, fertilized; TFo = green cane harvested, unfertilized; BtoF = burnt cane harvested, fertilized; BtoFo = burnt cane harvested, unfertilized.
No significant treatment effects were shown by one-way ANOVA on fungal community evenness (J) (F3,8 = 1.74, p = 0.25, grand mean = 0.85), nor did the Shannon Weaver diversity index (H′) (F3,8 = 3.82, p = 0.06, grand mean = 1.52) show any significant treatment effects on soil fungal community structural diversity.
The results of a CCA showing the relationship between selected (non-collinear) soil physicochemical variables and the soil fungal community (genetic) structure under the different sugarcane trash management practices at Mount Edgecombe are presented in Figure 5.11. CCA 1 accounted for 49.1% of variance due to environmental effects and 18.5% of total variance, and CCA 2 for 29.6% of variance due to environmental
effects and 11.2% of total variance. ECEC and pH were strongly correlated with CCA 1 and organic C with CCA 2. Trashed treatments, particularly TF but also TFo, were associated with a high soil organic C content, in contrast to the Bto treatments where the soil organic C content was lower. Both TF and BtoF treatments were correlated with a lower soil pH than the equivalent unfertilized treatments. Low ECEC was associated with the BtoF subsamples and high ECEC with the TFo subsamples.
Subsamples from the BtoF and BtoFo treatments were more closely clustered than those from the TF and TFo treatments.
FIGURE 5.11 Plot of samples (classified by land management) 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 Mount Edgecombe.
CCA 1 accounted for 49.1% of variance due to environmental effects and 18.5% of total variance. CCA 2 accounted for 29.6% of variance due to environmental effects and 11.2% of total variance.
Key: T = green cane harvested with trash retention; Bto = burnt with harvest residues removed; F = fertilized annually with N, P and K; Fo = unfertilized.
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TF1 TF2
TF3 TFo1
TFo2 TFo3
BtoF1 BtoF2
BtoF3
BtoFo1 BtoFo2
BtoFo3
ECEC pH
Organic C
SAMPLES
TF TFo BtoF BtoFo
ENV. VARIABLES
CCA 1 (49.1%)
CCA 2 (29.6%)
A plot showing the presence of species centroids (bands = fungal OTUs) associated with the different management practice soils in the CCA (Figure 5.11), is shown in Figure 5.12.
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B1
B2
B3 B4
B5 B6 B7
B8
B9
B10 B11
B12
B13
B14
B15
B16 B17
B18
B19
B20
B21 B22
B23 B24
B25 SPECIES
FIGURE 5.12 Plot of bands (centroids) in the CCA (Figure 5.11) showing the relationship of the different fungal OTUs to the land management soils at Mount Edgecombe.
Key: B = band.
Results of the Monte Carlo permutation test for significant relationships in the CCA data showed that along the first canonical axis p = 0.01 and along all canonical axes p = 0.008. This indicated that the selected soil variables had a significant effect on soil fungal community structure at this site (Appendix C, Table C2).