Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding
Abstract
:1. Introduction
2. Results
2.1. Analysis of Mock Communities and Identification of the Best Primer Sets
2.2. Evaluation of the Taxonomic-Level Resolution
2.3. Application on Environmental Samples
3. Discussion
4. Materials and Methods
4.1. Identification of Primer Sets Targeting Walnut Pathogens
4.2. Fungal Mock Communities
4.3. Sampling and Total DNA Extraction
4.4. Illumina MiSeq Sequencing and Sequence Analyses
4.5. Comparative Evaluation of Primer Pairs
4.6. Evaluation of the Taxonomic-Level Resolution
4.7. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Fungal Species Included in Mock Communities | Mock1 | Mock2 | Mock3 | Mock4 | Mock5 | ||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ITS | ITS1 | GTAA | GTAAm | Kyo | ITS | ITS1 | GTAA | GTAAm | Kyo | ITS | ITS1 | GTAA | GTAAm | Kyo | ITS | ITS1 | GTAA | GTAAm | Kyo | ITS | ITS1 | GTAA | GTAAm | Kyo | |||||||||
Botryosphaeria dothidea | |||||||||||||||||||||||||||||||||
Diplodia seriata | |||||||||||||||||||||||||||||||||
Dothiorella omnivora | |||||||||||||||||||||||||||||||||
Lasiodiplodia theobromae | |||||||||||||||||||||||||||||||||
Neofusicoccum mediterraneum | |||||||||||||||||||||||||||||||||
N. parvum | |||||||||||||||||||||||||||||||||
Diaporthe amygdali | |||||||||||||||||||||||||||||||||
Dia. eres | |||||||||||||||||||||||||||||||||
Dia. foeniculina | |||||||||||||||||||||||||||||||||
Dia. novem | |||||||||||||||||||||||||||||||||
Dia. rudis | |||||||||||||||||||||||||||||||||
Epicoccum nigrum | |||||||||||||||||||||||||||||||||
Colletotrichum fioriniae | |||||||||||||||||||||||||||||||||
C. godetiae | |||||||||||||||||||||||||||||||||
Fusarium juglandicola | |||||||||||||||||||||||||||||||||
F. solani | |||||||||||||||||||||||||||||||||
Gibellulopsis nigrescens | |||||||||||||||||||||||||||||||||
Alternaria alternata |
Sample (Number of Expected ASVs) | Primer Set | Sensitivity | Precision | Similarity of Composition | Number of Obtained ASVs |
---|---|---|---|---|---|
Mock1 (6 ASVs) | ITS | 0.833 b | 0.045 c ± 0.001 | 0.739 c ± 0.016 | 113 ± 3.6 |
ITS1 | 0.833 b | 0.242 abc ± 0.007 | 0.845 a ± 0.003 | 21.7 ± 0.6 | |
GTAA | 1 a | 0.500 a | 0.846 ab ± 0.001 | 12 | |
GTAAm | 1 a | 0.250 ab | 0.820 abc ± 0.002 | 24 | |
Kyo | 0.833 b | 0.114 bc | 0.814 bc ± 0.004 | 45 | |
Mock2 (5 ASVs) | ITS | 1 a | 0.099 b ± 0.002 | 1 a | 50.3 ± 1.1 |
ITS1 | 1 a | 0.341 ac ± 0.014 | 1 a | 14.7 ± 0.6 | |
GTAA | 1 a | 0.500 a | 1 a | 10 | |
GTAAm | 1 a | 0.278 abc | 1 a | 18 | |
Kyo | 1 a | 0.135 bc | 1 a | 37 | |
Mock3 (17 ASVs) | ITS | 0.941 a | 0.178 ac ± 0.003 | 0.301 c ± 0.007 | 90.7 ± 1.5 |
ITS1 | 0.804 b ± 0.068 | 0.518 b ± 0.032 | 0.324 ac ± 0.005 | 27.3 ± 0.6 | |
GTAA | 0.882 ab | 0.454 ab | 0.710 ab ± 0.003 | 34 | |
GTAAm | 0.941 a | 0.253 abc ± 0.002 | 0.702 abc ± 0.010 | 64.3 ± 0.6 | |
Kyo | 0.882 ab | 0.115 c | 0.749 b ± 0.005 | 132 | |
Mock4 (18 ASVs) | ITS | 0.870 b ± 0.032 | 0.154 b ± 0.011 | 0.325 ac ± 0.003 | 103.7 ± 4.9 |
ITS1 | 0.685 b ± 0.085 | 0.457 a ± 0.056 | 0.292 c ± 0.001 | 28 | |
GTAA | 0.889 ab | 0.453 a ± 0.015 | 0.707 abc ± 0.006 | 36.3 ± 1.1 | |
GTAAm | 0.944 a | 0.260 ab ± 0.002 | 0.751 ab ± 0.006 | 66.3 ± 0.6 | |
Kyo | 0.889 ab | 0.115 b | 0.769 b ± 0.007 | 140.7 ± 0.6 | |
Mock5 (17 ASVs) | ITS | 0.941 a | 0.178 ac ± 0.007 | 0.312 c ± 0.006 | 91 ± 3.5 |
ITS1 | 0.706 b ± 0.059 | 0.495 b ± 0.025 | 0.327 ac ± 0.020 | 25.3 ± 3.0 | |
GTAA | 0.882 ab | 0.454 ab | 0.731 ab ± 0.009 | 34 | |
GTAAm | 0.941 a | 0.255 abc ± 0.002 | 0.718 abc ± 0.005 | 63.7 ± 0.6 | |
Kyo | 0.882 ab | 0.117 c ± 0.004 | 0.764 b ± 0.009 | 129.7 ± 4.0 |
Fungal Species Included in Mock Communities | GTAAm | Kyo | ||||||
---|---|---|---|---|---|---|---|---|
w/ | wo/ | w/ | wo/ | |||||
Genus | Species | Genus | Species | Genus | Species | Genus | Species | |
Botryosphaeria dothidea | ||||||||
Diplodia seriata | ||||||||
Dothiorella omnivora | ||||||||
Lasiodiplodia theobromae | ||||||||
Neofusicoccum mediterraneum | ||||||||
N. parvum | ||||||||
Diaporthe amygdali | ||||||||
Dia. eres | ||||||||
Dia. foeniculina | ||||||||
Dia. novem | ||||||||
Dia. rudis | ||||||||
Epicoccum nigrum | ||||||||
Colletotrichum fioriniae | ||||||||
C. godetiae | ||||||||
Fusarium juglandicola | ||||||||
F. solani | ||||||||
Gibellulopsis nigrescens | ||||||||
Alternaria alternata |
Sample | Primer Set | Sensitivity | Precision | Similarity of Composition | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Genus | Species | Genus | Species | ||||||||
w/ | w/o | w/ | w/o | w/ | w/o | w/ | w/o | Genus | Species | ||
Mock1 | GTAAm | 0.5 a | 0.333 b | 0.167 a | 0 b | 0.143 a | 0.1 ab | 0.053 a | 0 b | 0.820 A ± 0.002 | 0.822 A ± 0.004 |
Kyo | 0.333 b | 0.5 a | 0.167 a | 0.167 a | 0.049 c | 0.071 bc | 0.025 ab | 0.025 ab | 0.814 B± 0.004 | 0.826 A ± 0.006 | |
Mock2 | GTAAm | 1 a | 1 a | 0.4 a | 0.4 a | 0.278 a | 0.278 a | 0.133 a | 0.133 a | 1 A | 0.790 A ± 0.004 |
Kyo | 1 a | 1 a | 0.5 a | 0.6 b | 0.135 b | 0.135 b | 0.059 b | 0.086 ab | 1 A | 0.817 B ± 0.004 | |
Mock3 | GTAAm | 0.823 a | 0.765 b | 0.235 ab | 0.176 a | 0.228 a ± 0.002 | 0.215 ab ± 0.002 | 0.078 a ± 0.001 | 0.060 bc ± 0.001 | 0.702 A ± 0.010 | 0.655 A ± 0.008 |
Kyo | 0.765 b | 0.823 a | 0.236 ab | 0.470 b | 0.101 c | 0.108 bc | 0.034 b | 0.065 ac | 0.749 B ± 0.005 | 0.745 B ± 0.001 | |
Mock4 | GTAAm | 0.833 a | 0.779 b | 0.278 ab | 0.222 a | 0.237 a ± 0.002 | 0.225 ab ± 0.002 | 0.094 a ± 0.001 | 0.076 ab ± 0.001 | 0.751 A ± 0.006 | 0.710 A± 0.003 |
Kyo | 0.778 b | 0.833 a | 0.245 ab | 0.5 b | 0.102 c | 0.109 bc | 0.039 c | 0.068 bc | 0.769 B ± 0.007 | 0.770 B ± 0.008 | |
Mock5 | GTAAm | 0.823 a | 0.765 b | 0.235 ab | 0.176 a | 0.229 a | 0.219 ab ± 0.002 | 0.078 a | 0.060 bc ± 0.001 | 0.718 A ± 0.005 | 0.672 A ± 0.004 |
Kyo | 0.765 b | 0.823 a | 0.235 ab | 0.470 b | 0.103 c ± 0.003 | 0.111 bc ± 0.003 | 0.034 b ± 0.001 | 0.066 ac ± 0.002 | 0.764 B ± 0.009 | 0.765 B ± 0.010 |
Classification | Species | Host (Species) | References | |||
---|---|---|---|---|---|---|
Walnut (Juglans regia) | Almond (Prunus dulcis) | Pistachio (Pistacia vera) | Olive (Olea europaea) | |||
Bionectriaceae | Geosmithia flava | X | [76] | |||
G. lavendula | X | [76] | ||||
G. morbida | X | [77,78] | ||||
Botryosphaeriaceae | Botryosphaeria dothidea | X | X | X | X | [11,12,79,80,81,82,83,84,85] |
Diplodia gallae | X | [70] | ||||
D. mutila | X | X | X | X | [80,83,84,86,87,88] | |
D. seriata | X | X | X | X | [7,80,81,82,83,86,87,89,90,91] | |
Dothiorella iberica | X | X | X | X | [80,83,87,91] | |
Dot. omnivora | X | [10,70] | ||||
Dot. plurivora | X | [70] | ||||
Dot. sarmentorum | X | X | X | [7,70,81,91] | ||
Dot. viticola | X | X | [70,87] | |||
Lasiodiplodia citricola | X | X | [70,80,91] | |||
L. mahajangana | X | X | [70,92] | |||
L. pseudotheobromae | X | X | [85,93] | |||
L. theobromae | X | X | X | X | [70,81,83,84,87] | |
Neofusicoccum hellenicum | X | [92] | ||||
N. luteum | X | [83,84] | ||||
N. mediterraneum | X | X | X | X | [7,80,81,83,85,87,88,91] | |
N. nonquaesitum | X | X | [80,81] | |||
N. parvum | X | X | X | X | [7,11,80,81,82,84,85,86,87] | |
N. vitifusiforme | X | X | X | X | [80,83,87,90,91] | |
Neoscytalidium dimidiatum | X | X | X | X | [80,87,94,95,96] | |
Diaporthaceae | Diaporthe amygdali | X | X | [7,82,97,98] | ||
Dia. australafricana | X | X | [86,87] | |||
Dia. biguttulata | X | [99] | ||||
Dia. capsici | X | [100] | ||||
Dia. cynaroidis | X | [86] | ||||
Dia. eres | X | X | [10,11,87,99] | |||
Dia. foeniculina | X | X | X | X | [7,11,80,85,90,98] | |
Dia. juglandicola | X | [101] | ||||
Dia. novem | X | X | [87], this study | |||
Dia. rudis | X | X | X | [83,91,102], this study | ||
Dia. shennongjiaensis | X | [103] | ||||
Didymellaceae | Epicoccum nigrum | X | X | [11,84] | ||
Glomerellaceae | Colletotrichum acutatum | X | X | X | X | [104,105,106,107,108] |
C. fioriniae | X | X | X | X | [5,11,105,107,109,110,111] | |
C. fructicola | X | [112] | ||||
C. gloeosporioides | X | X | X | [5,107,109,113,114] | ||
C. godetiae | X | X | X | [5,11,106,107,109,110] | ||
C. kahawae | X | X | [109,114] | |||
C. nymphaeae | X | X | X | [5,109,115,116,117] | ||
C. siamense | X | X | X | [108,113,114] | ||
C. viniferum | X | [104] | ||||
Gnomoniaceae | Ophiognomonia leptostyla | X | [118,119] | |||
Juglanconidaceae | Juglanconis appendiculata | X | [71] | |||
J. juglandina | X | [71,120] | ||||
Nectriaceae | Fusarium avenaceum | X | X | X | [121,122,123,124,125] | |
F. chlamydosporum | X | X | [125,126] | |||
F. culmorum | X | X | [121,123,125] | |||
F. equiseti | X | [125] | ||||
F. graminearum | X | [123] | ||||
F. incarnatum | X | X | [123,125,127] | |||
F. juglandicola | X | [128] | ||||
F. oxysporum | X | X | X | [121,123,125,126] | ||
F. proliferatum | X | X | [123,129] | |||
F. solani | X | X | X | X | [11,124,125,126,130,131] | |
Plectosphaerellaceae | Gibellulopsis nigrescens | X | [11] | |||
Pleosporaceae | Alternaria alternata | X | X | X | X | [79,84,121,132,133] |
A. arborescens | X | X | [132,133] | |||
A. tenuissima | X | X | X | X | [79,84,130,132,133] | |
Togniniaceae | Phaeoacremonium cinereum | X | X | [70] | ||
P. fraxinopennsylvanicum | X | [70] | ||||
P. italicum | X | X | X | [70,134] | ||
P. minimum | X | X | X | X | [70,85,90] | |
P. parasiticum | X | X | X | X | [70,90,134] | |
P. sicilianum | X | X | [10,134] | |||
P. tuscanum | X | [70] | ||||
P. viticola | X | X | X | X | [70,135] | |
Valsaceae | Cytospora atrocirrhata | X | [136,137] | |||
Cyt. californica | X | X | X | [87,88,136] | ||
Cyt. ceratosperma | X | [137] | ||||
Cyt. chrysosperma | X | [136,138] | ||||
Cyt. cincta | X | [138] | ||||
Cyt. gigalocus | X | [136,137] | ||||
Cyt. joaquinensis | X | X | [88,136] | |||
Cyt. nivea | X | [139] | ||||
Cyt. plurivora | X | X | X | X | [87,136] |
Barcode | Primer Name | Set Name | Direction | Sequence (5′-3′) | Amplicon Size (bp) | Primer Reference |
---|---|---|---|---|---|---|
ITS | ITS1F | ITS | Forward | CTTGGTCATTTAGAGGAAGTAA | 617 | [37] |
ITS4 | Reverse | TCCTCCGCTTATTGATATGC | [36] | |||
ITS1 | ITS1F | ITS1 | Forward | CTTGGTCATTTAGAGGAAGTAA | 295 | [37] |
ITS2 | Reverse | GCTGCGTTCTTCATCGATGC | [36] | |||
ITS2 | GTAA182f | GTAA | Forward | AAAACTTTCAACAACGGATC | 337 | [20] |
GTAA526r | Reverse | TYCCTACCTGATCCGAGGTC | [20] | |||
GTAA182fm | GTAAm | Forward | AAAACTTTYAACAACGGATC | 337 | This study | |
GTAA526r | Reverse | TYCCTACCTGATCCGAGGTC | [20] | |||
ITS3 | Kyo | Forward | GCATCGATGAAGAACGCAGC | 342 | [36] | |
ITS4_KYO1 | Reverse | TCCTCCGCTTWTTGWTWTGC | [38] |
Classification | Species | Strain No. | DNA Solutions Included in Mocks | ||||
---|---|---|---|---|---|---|---|
Mock1 e | Mock2 f | Mock3 g | Mock4 h | Mock5 i | |||
Plant | |||||||
Juglandaceae | Juglans regia | - | X | ||||
Fungi | |||||||
Botryosphaeriaceae | Botryosphaeria dothidea | P12N1I2_2020 a | X | X | X | X | |
Diplodia seriata | CBS112555 b | X | X | X | X | ||
Dothiorella omnivora | CBS140349 b | X | X | X | X | ||
Lasiodiplodia theobromae | CBS164.96 b | X | X | X | X | ||
Neofusicoccum mediterraneum | CBS121718 b | X | X | X | X | ||
N. parvum | P12N1I1_2020 a | X | X | X | X | ||
Diaporthaceae | Diaporthe amygdali | CBS126679 b | X | X | X | X | |
Dia. eres | P12N2I3_2020 a | X | X | X | X | ||
Dia. foeniculina | UBOCC-A-122019 c | X | X | X | X | ||
Dia. novem | UBOCC-A-122020 c | X | X | X | X | ||
Dia. rudis | P9N3I1_2020 a | X | X | X | X | ||
Didymellaceae | Epicoccum nigrum | P12N5I8_2020 a | X | X | X | ||
Glomerellaceae | Colletotrichum fioriniae | UBOCC-A-122017 c | X | X | X | ||
C. godetiae | UBOCC-A-122016 c | X | X | X | |||
Nectriaceae | Fusarium juglandicola | UBOCC-A-119001 d | X | X | X | ||
F. solani | UBOCC-A-122023 c | X | X | X | |||
Plectosphaerellaceae | Gibellulopsis nigrescens | UBOCC-A-122024 c | X | ||||
Pleosporaceae | Alternaria alternata | UBOCC-A-122015 c | X | X | X |
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Belair, M.; Pensec, F.; Jany, J.-L.; Le Floch, G.; Picot, A. Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding. Plants 2023, 12, 2383. https://doi.org/10.3390/plants12122383
Belair M, Pensec F, Jany J-L, Le Floch G, Picot A. Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding. Plants. 2023; 12(12):2383. https://doi.org/10.3390/plants12122383
Chicago/Turabian StyleBelair, Marie, Flora Pensec, Jean-Luc Jany, Gaétan Le Floch, and Adeline Picot. 2023. "Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding" Plants 12, no. 12: 2383. https://doi.org/10.3390/plants12122383