The Abundance of Fungi, Bacteria and Denitrification Genes during Insect Outbreaks in Scots Pine Forests
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Site
2.2. Sampling
2.3. DNA Extraction
2.4. Standard Curves
2.5. Real-time PCR
2.6. Soil, Feces, Needle and Dead Larvaea Chemical Analyses
2.7. Statistical Analyses
3. Results
3.1. Fungal 18S and Bacterial 16S rRNA Gene Abundance in Soil
3.2. Denitrification Gene Abundance in Soil—nirK, nirS, nosZI, nosZII
3.3. Soil Chemical Properties
3.4. C and N in Dead Larvae, Feces and Needle Litter
3.5. Fungal 18S rRNA and Bacterial 16S rRNA Gene Abundance in Needles
3.6. The Abundance of Fungal 18S and Bacterial 16S rRNA Genes in Feces, Needle Litter, and Larvae
4. Discussion
4.1. The Soil Fungal and Bacterial Population Size Responds to Insect Derived Litter
4.2. Insect Outbreaks Lower the Soil C/N Ratio
4.3. The Varying Effects on Denitrification Genes
4.4. Litter, Feces and Larval Cadavers as Habitat for Microorganisms and N Source
4.5. Insect Outbreaks Increase Phyllosphere Bacteria Colonization
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Site | VL | Ol | Of | Oh | Ah |
---|---|---|---|---|---|
non-infested | 4.33 ± 0.10 | 3.91 ± 0.07 | 3.51 ± 0.07 | 3.33 ± 0.05 | 3.42 ± 0.06 |
nun moth infested | 4.57 ± 0.10 | 4.29 ± 0.07 | 3.94 ± 0.08 | 3.57 ± 0.06 | 3.57 ± 0.07 |
non-infested | 4.27 ± 0.10 | 3.97 ± 0.06 | 3.61 ± 0.05 | 3.34 ± 0.05 | 3.47 ± 0.06 |
pine-tree lappet infested | 4.21 ± 0.09 | 4.04 ± 0.04 | 3.74 ± 0.07 | 3.42 ± 0.04 | 3.58 ± 0.07 |
Target Gene | Final MgCl2 Concentration (mM) | Primer Concentration (µM) | Initial Denaturation | Denaturation | Annealing | Extension |
nirK | 2.5 | 0.5 | 95 °C, 120 s | 94 °C, 20 s | 63 °C–1 °C/cycle for 6 cycles, 58 °C for 35 cycles 30 s | 68 °C, 30 s |
nirS | 2.1 | 0.5 | 95 °C, 120 s | 94 °C, 20 s | 63 °C–1 °C/cycle for 6 cycles, 58 °C for 35 cycles 30 s | 68 °C, 30 s |
nosZI | 2.1 | 1 | 95 °C, 120 s | 94 °C, 20 s | 65 °C–1 °C/cycle for 6 cycles, 60 °C for 35 cycles 30 s | 68 °C, 25 s |
Target Gene | Final MgCl2 Concentration (mM) | Primer Concentration (µM) | 1 cycle | 35 cycles | ||
Initial Denaturation | Denaturation | Annealing | Extension | |||
BamoA | 2.0 | 0.3 | 95 °C, 120 s | 94 °C, 20 s | 62 °C, 30 s | 68 °C, 35 s |
AamoA | 30 | 0.3 | 95 °C, 120 s | 94 °C, 20 s | 61 °C, 30 s | 68 °C, 45 s |
nosZII | 2.5 | 2.7 | 95 °C, 120 s | 94 °C, 20 s | 60 °C, 30 s | 68 °C, 45 s |
18S | 2.6 | 0.3 | 95 °C, 120 s | 94 °C, 20 s | 59 °C, 30 s | 68 °C, 25 s |
16S | 2.5 | 0.3 | 95 °C, 120 s | 94 °C, 20 s | 63 °C, 30 s | 68 °C, 25 s |
Target Gene | Primer Name | Primer Sequence (5′–3′) | Amplicon Size (bp) | Reference |
---|---|---|---|---|
bacterial ammonia monooxygenase α subunit (BamoA) | amoA-1F | GGGGTTTCTACTGGTGGT | 491 | Rotthauwe et al. (1997) [67] |
amoA-2R | CCCCTCKGSAAAGCCTTCTTC | |||
archaeal ammonia monooxygenase α subunit (AamoA) | CrenamoA23f | ATGGTCTGGCTWAGACG | 628 | Tourna et al. (2008) [68] |
CrenamoA616r | GCCATACABCKRTANGTCCA | |||
Cu-nitrite reductase catalytic subunit (nirK) | nirK876F | ATYGGCGGVAYGGCGA | 165 | Henry et al. (2004) [69] |
nirK1040R | GCCTCGATCAGRTTRTGGTT | |||
Cd1-nitrite reductase catalytic subunit (nirS) | cd3aF | GTSAACGTSAAGGARACSGG | 410 | Michotey et al. (2000) [70], Throbäck et al. (2004) [71] |
R3cd | GASTTCGGRTGSGTCTTGA | |||
nitrous oxide reductase catalytic subunit (nosZI) | NosZ2F | CGCRACGGCAASAAGGTSMSSGT | 267 | Henry et al. (2006) [72] |
NosZ2R | CAKRTGCAKSGCRTGGCAGAA | |||
nitrous oxide reductase catalytic subunit (nosZII) | 1153 nosZ 8F | CTIGGICCIYTKCAYAC | 698 | Jones et al. (2013) [73] |
1888 nosZ 29R | GCIGARCARAAITCBGTR | |||
fungal 18S rRNA (18S) | FR1 | AICCATTCAATCGGTAIT | 350 | Vainio & Hantula (2000) [74] |
FF390 | CGATAACGAACGAGACCT | |||
bacterial 16S rRNA (16S) | 341F | CCTACGGGAGGCAGCAG | 194 | López-Gutiérrez et al. (2004) [75] |
534R | ATTACCGCGGCTGCTGGCA |
Target Gene | Soil | Needle | Litter | Feces | Cadaver | |||||
---|---|---|---|---|---|---|---|---|---|---|
Nun Moth | Pine-Tree Lappet | Nun Moth | Pine-Tree Lappet | Nun Moth | Pine-Tree Lappet | Nun Moth | Pine-Tree Lappet | Nun Moth | Pine-Tree Lappet | |
18S | 87.8% | 76.7% | 79.2% | 22.9% | 100% | 100% | 100% | 100% | 100% | 100% |
16S | 65.6% | 65.0% | 93.8% | 89.6% | 100% | 100% | 100% | 100% | 33% | 100% |
nirK | 59.4% | 57.2% | NA | NA | NA | NA | NA | NA | NA | NA |
nirS | 13.9% | 19.4% | NA | NA | NA | NA | NA | NA | NA | NA |
nosZI | 32.8% | 35.6% | NA | NA | NA | NA | NA | NA | NA | NA |
nosZII | 72.2% | 63.9% | NA | NA | NA | NA | NA | NA | NA | NA |
Target Gene | VL | Ol | Of | Oh | Ah | ||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
spr | md | aut | spr | md | aut | spr | md | aut | spr | md | aut | spr | md | aut | |||||||||||||||||
inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | inf | non-inf | ||
Nun Moth Outbreak | 18S a | 5 | 5 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 5 | 5 | 6 | 6 | 6 | 6 | 5 | 4 | 6 | 6 | 4 | 3 | 5 | 6 | 5 | 5 | 2 | 3 |
16S b | 6 | 5 | 6 | 6 | 6 | 5 | 6 | 5 | 6 | 6 | 3 | 5 | 6 | 4 | 5 | 6 | 4 | 2 | 2 | 3 | 5 | 4 | 0 | 3 | 1 | 2 | 2 | 2 | 2 | 0 | |
nosZI | 2 | 4 | 2 | 3 | 0 | 1 | 1 | 3 | 3 | 1 | 2 | 2 | 2 | 1 | 2 | 2 | 2 | 2 | 3 | 3 | 1 | 2 | 0 | 1 | 2 | 2 | 2 | 3 | 2 | 3 | |
nosZII | 6 | 5 | 6 | 6 | 6 | 5 | 6 | 6 | 6 | 6 | 6 | 4 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 3 | 6 | 6 | 2 | 3 | 0 | 0 | 1 | 2 | 2 | 2 | |
nirK | 6 | 5 | 6 | 5 | 6 | 5 | 6 | 4 | 6 | 6 | 3 | 5 | 6 | 4 | 5 | 6 | 4 | 2 | 2 | 2 | 4 | 3 | 0 | 2 | 1 | 0 | 2 | 1 | 0 | 0 | |
nirS | 6 | 1 | 5 | 3 | 2 | 0 | 4 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Pine-Tree Lappet Outbreak | 18S a | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 5 | 2 | 0 | 6 | 6 | 3 | 1 | 1 | 0 | 6 | 5 | 0 | 1 |
16S b | 6 | 6 | 6 | 6 | 6 | 6 | 5 | 0 | 6 | 6 | 5 | 6 | 3 | 4 | 5 | 4 | 3 | 4 | 0 | 3 | 5 | 6 | 1 | 2 | 1 | 2 | 5 | 4 | 0 | 1 | |
nosZI | 4 | 1 | 2 | 2 | 4 | 2 | 2 | 1 | 2 | 0 | 2 | 2 | 2 | 4 | 2 | 4 | 3 | 3 | 1 | 3 | 4 | 2 | 3 | 2 | 1 | 0 | 3 | 3 | 0 | 0 | |
nosZII | 4 | 4 | 6 | 5 | 3 | 5 | 4 | 5 | 5 | 5 | 6 | 6 | 6 | 4 | 6 | 6 | 4 | 4 | 3 | 2 | 5 | 6 | 2 | 1 | 0 | 2 | 1 | 4 | 0 | 1 | |
nirK | 5 | 6 | 6 | 6 | 6 | 5 | 4 | 0 | 6 | 5 | 5 | 6 | 3 | 4 | 5 | 4 | 3 | 2 | 0 | 2 | 5 | 6 | 1 | 2 | 1 | 0 | 1 | 3 | 0 | 1 | |
nirS | 0 | 0 | 5 | 4 | 0 | 0 | 2 | 4 | 0 | 4 | 0 | 0 | 0 | 0 | 6 | 5 | 0 | 0 | 0 | 0 | 2 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Target Gene | Nun Moth Outbreak | Pine-Tree Lappet Outbreak | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Spring | Main Defoliation | Autumn | Spring | Main Defoliation | Autumn | |||||||
Infested | Non-infested | Infested | Non-infested | Infested | Non-infested | Infested | Non-infested | Infested | Non-infested | Infested | Non-infested | |
fungal 18S rRNA | 6 | 4 | 8 | 5 | 7 | 8 | 0 | 1 | 2 | 0 | 6 | 2 |
bacterial 16S rRNA | 8 | 6 | 8 | 7 | 8 | 8 | 5 | 6 | 8 | 8 | 8 | 8 |
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Target Gene | Site | Nun Moth Infested | Pine-Tree Lappet Infested | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
VL | Ol | Of | Oh | Ah | VL | Ol | Of | Oh | Ah | ||
nirK | non-infested | 2.04 × 108 | 1.36 × 108 | 6.95 × 107 | 4.09 × 107 | 3.87 × 107 | 5.58 × 108 | 1.89 × 109 | 4.32 × 108 | 3.61 × 108 | 4.17 × 107 |
± 1.07 × 108 | ± 5.32 × 107 | ± 2.15 × 107 | ± 1.43 × 107 | - | ± 3.17 × 108 | ± 5.91 × 108 | ± 1.89 × 108 | ± 1.42 × 108 | ± 2.06 × 107 | ||
infested | 1.53 × 109 ** | 4.73 × 108 * | 1.64 × 108 | 2.84 × 108 | 1.40 × 108 | 8.52 × 108 | 1.39 × 109 * | 3.70 × 108 | 1.47 × 108 | 1.96 × 108 | |
± 2.16 × 108 | ± 2.20 × 108 | ± 5.95 × 107 | ± 1.90 × 108 | ± 2.51 × 107 | ± 1.81 × 108 | ± 8.25 × 108 | ± 1.19 × 108 | ± 1.01 × 108 | ± 1.28 × 108 | ||
nirS | non-infested | 9.93 × 107 | n.d. | 8.37 × 107 | n.d. | n.d. | 1.66 × 108 | 1.50 × 108 | 1.22 × 108 | 8.42 × 107 | n.d. |
± 3.66 × 107 | - | - | - | - | - | ± 4.36 × 105 | ± 1.29 × 107 | ± 7.29 × 106 | - | ||
infested | 1.06 × 108 | 7.47 × 107 | n.d. | n.d. | n.d. | 1.66 × 108 | 2.25 × 108 | 8.25 × 107 | 6.59 × 107 | 6.05 × 107 | |
± 1.73 × 107 | ± 1.24 × 107 | - | - | - | - | ± 3.68 × 107 | ± 6.75 × 106 | ± 2.84 × 107 | - | ||
nosZI | non-infested | 8.53 × 105 | 3.83 × 105 | 2.16 × 105 | 2.43 × 105 | 5.33 × 105 | 3.59 × 106 | 8.58 × 105 | 1.40 × 106 | 9.74 × 105 | 1.59 × 105 |
± 4.92 × 105 | ± 9.62 × 104 | ± 2.39 × 104 | ± 3.73 × 104 | ± 2.88 × 105 | ± 3.22 × 106 | ± 4.49 × 104 | ± 5.13 × 105 | ± 2.43 × 105 | ± 4.06 × 104 | ||
infested | 8.02 × 105 | 6.05 × 105 | 5.44 × 105 | 4.73 × 105 | 4.60 × 105 | 1.87 × 106 | 3.21 × 105 | 1.29 × 106 | 6.04 × 105 | 5.89 × 105 * | |
± 1.32 × 106 | ± 7.86 × 104 | ± 2.04 × 105 | ± 1.01 × 105 | ± 2.06 × 105 | ± 1.31 × 105 | ± 9.76 × 104 | ± 5.55 × 105 | ± 1.97 × 105 | ± 1.48 × 105 | ||
nosZII | non-infested | 2.53 × 107 | 2.38 × 107 | 9.37 × 106 | 4.17 × 106 | 2.62 × 106 | 1.96 × 108 | 4.75 × 107 | 3.62 × 107 | 1.51 × 106 | 4.45 × 108 |
± 1.68 × 107 | ± 1.04 × 107 | ± 3.79 × 106 | ± 2.69 × 106 | ± 1.70 × 106 | ± 8.85 × 107 | ± 1.97 × 107 | ± 2.47 × 107 | ± 6.85 × 105 | ± 5.77 × 108 | ||
infested | 3.26 × 107 | 1.93 × 107 | 1.13 × 107 | 6.58 × 106 | 1.32 × 106 | 2.32 × 108 | 3.75 × 108 | 7.97 × 106 | 2.94 × 106 | 2.04 × 105 | |
± 1.88 × 107 | ± 9.95 × 106 | ± 5.96 × 106 | ± 3.22 × 106 | ± 8.69 × 105 | ± 1.99 × 108 | ± 3.05 × 108 | ± 4.40 × 106 | ± 1.89 × 106 | - |
Type of Organic Input | Fungal 18S rRNA Gene Abundance | Bacterial 16S rRNA Gene Abundance | ||
---|---|---|---|---|
Nun Moth | Pine-Tree Lappet | Nun Moth | Pine-Tree Lappet | |
Litter | 4.15 × 109 * | 1.42 × 108 | 2.88 × 1011 * | 4.13 × 1010 |
± 1.56 × 109 | ± 7.95 × 106 | ± 1.22 × 1010 | ± 6.14 × 109 | |
Feces | 5.36 × 109 * | 1.58 × 109 | 3.04 × 1011 * | 1.57 × 1011 |
± 4.38 × 108 | ± 4.92 × 108 | ± 2.96 × 1010 | ± 3.46 × 1010 | |
Cadaver | 1.44 × 1010 * | 1.66 × 109 | 1.23 × 1012 ♦ | 7.34 × 1011 |
± 4.74 × 109 | ± 8.37 × 107 | - | ± 5.77 × 109 |
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Grüning, M.M.; Beule, L.; Meyer, S.; Karlovsky, P.; I.-M.-Arnold, A. The Abundance of Fungi, Bacteria and Denitrification Genes during Insect Outbreaks in Scots Pine Forests. Forests 2018, 9, 497. https://doi.org/10.3390/f9080497
Grüning MM, Beule L, Meyer S, Karlovsky P, I.-M.-Arnold A. The Abundance of Fungi, Bacteria and Denitrification Genes during Insect Outbreaks in Scots Pine Forests. Forests. 2018; 9(8):497. https://doi.org/10.3390/f9080497
Chicago/Turabian StyleGrüning, Maren Marine, Lukas Beule, Stephanie Meyer, Petr Karlovsky, and Anne I.-M.-Arnold. 2018. "The Abundance of Fungi, Bacteria and Denitrification Genes during Insect Outbreaks in Scots Pine Forests" Forests 9, no. 8: 497. https://doi.org/10.3390/f9080497