New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple
Abstract
1. Introduction
2. Materials and Methods
2.1. Fungal Isolates, Strains, Culture Media, and DNA Extraction
2.2. Primer and Probe Design, Specificity Testing, and RT-PCR Optimization
3. Results
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Taxon | Sample ID | Isolate | Host | Locality | |
---|---|---|---|---|---|
Colletotrichum acutatum species complex | |||||
C. acutatum s.s. | VT1108 | PJ51 | Lycopersicon esculentum tomato | Auckland, New Zealand [41] | |
C. fioriniae | VA-16 | VA-16 | Ginger Gold apple | Frederick Co. VA [22] | |
VA-44 | VA-44 | Honeycrisp apple | Madison Co. VA [22] | ||
VA-53 | VA-53 | Honeycrisp apple | Madison Co. VA [22] | ||
VA-1-6 | VA-1-6 | Wolf River apple | Berkeley Co. WV [22] | ||
VA-1-16 | VA-1-16 | pear | [22] | ||
VA-1-20 | VA-1-20 | Ginger Gold apple | Frederick Co. VA [22] | ||
VA-1-66 | VA-1-66 | Ambrosia apple | Rappahannock Co. VA [22] | ||
VA-1-99 | VA-1-99 | Honeycrisp apple | Rappahannock Co. VA [22] | ||
VA-2-9 | VA-2-9 | Gold Rush apple | Frederick Co. VA [22] | ||
VA-3-59 | VA-3-59 | Smokehouse or Rambo apple | Fauquier Co. VA [22] | ||
VA-3-75 | VA-3-75 | Yellow York apple | Bedford Co. VA [22] | ||
VA-3-96 | VA-3-96 | Golden Delicious apple | Bedford Co. VA [22] | ||
VA-4-32 | VA-4-32 | York apple | Bedford Co. VA [22] | ||
VA-4-99 | VA-4-99 | Golden Delicious apple | Rappahannock Co. VA [22] | ||
VT0787 | VT0787 | [22] | |||
C. godetiae | VT1111 | JA8 | Prunus dulcis almond | CA [40] | |
VT1112 | S1 | Rhododendron sp. | Helsingborg, Sweden [48] | ||
C. johnstonii | VT1114 | PJ49 | Citrus sp. | Clifton, New Zealand [41] | |
VT1115 | PJ50 | Citrus sp. | Clifton, New Zealand [41] | ||
C. lupini | VT1118 | PJ62 | Lupinus mutabilis | France [47] | |
VT1119 | PJ64 | Lupinus alba | Canada [45] | ||
C. nymphaeae | VA-1-22 | VA-1-22 | Ginger Gold apple | Frederick Co. VA [22] | |
VA-1-24 | VA-1-24 | Ginger Gold apple | Frederick Co. VA [22] | ||
VT1124 | FREC138 | Robinia pseudoacacia | Adams Co. PA [43] | ||
VT1125 | HC646 | Honeycrisp apple | Bourbon Co. KY [44] | ||
VT1126 | Rdl96 | Empire apple | Berks Co. PA [42] | ||
C. pyricola | VT1127 | PJ12 | New Zealand [46] | ||
C. salicis | VT1128 | FREC145 | Salix nigra black willow | Adams Co. PA [42] | |
VT1129 | FREC146 | Salix nigra black willow | Adams Co. PA [42] | ||
Colletotrichum gloeosporioides species complex | |||||
C. chrysophilum | VA-77 | VA-77 | Granny Smith apple | Madison Co. VA [22] | |
VA-1-83 | VA-1-83 | Idared apple | Frederick Co VA [22] | ||
VA-2-25 | VA-2-25 | Rappahannock Co. VA [22] | |||
VA-2-32 | VA-2-32 | Rappahannock Co. VA [22] | |||
VA-2-37 | VA-2-37 | Golden Delicious apple | Albemarle Co. VA [22] | ||
VA-2-67 | VA-2-67 | Law Rome apple | Albemarle Co. VA [22] | ||
VA-2-85 | VA-2-85 | Rappahannock Co. VA [22] | |||
VA-2-100 | VA-2-100 | Rappahannock Co. VA [22] | |||
VA-3-4 | VA-3-4 | Golden Delicious apple | Frederick Co. VA [22] | ||
VA-3-33 | VA-3-33 | Frederick Co. VA [22] | |||
VA-4-86 | VA-4-86 | Greening apple | Fauquier Co. VA [22] | ||
VA-5-13 | VA-5-13 | Granny Smith apple | Fauquier Co. VA [22] | ||
VA-6-19 | VA-6-19 | Winter Banana apple | Frederick Co. VA [22] | ||
C. fructicola | VA-1-32 | VA-1-32 | Red Delicious apple | Albemarle Co. VA [22] | |
VA-1-44 | VA-1-44 | Golden Delicious apple | Albemarle Co. VA [22] | ||
VA-1-49 | VA-1-49 | Granny Smith apple | Nelson Co. VA [22] | ||
VA-1-58 | VA-1-58 | Golden Delicious apple | Nelson Co. VA [22] | ||
VA-1-68 | VA-1-68 | Red Delicious apple | Albemarle Co. VA [22] | ||
VA-1-71 | VA-1-71 | Golden Delicious apple | Nelson Co. VA [22] | ||
VA-1-78 | VA-1-78 | Granny Smith apple | Nelson Co. VA [22] | ||
VA-1-79 | VA-1-79 | Golden Delicious apple | Albemarle Co. VA [22] | ||
VA-1-90 | VA-1-90 | Honeycrisp apple | Nelson Co. VA [22] | ||
VA-1-91 | VA-1-91 | Granny Smith apple | Nelson Co. VA [22] | ||
VA-2-21 | VA-2-21 | Golden Delicious apple | Albemarle Co. VA [22] | ||
VA-2-35 | VA-2-35 | Golden Delicious apple | Albemarle Co. VA [22] | ||
VA-2-54 | VA-2-54 | Rappahannock Co. VA [22] | |||
VA-3-39 | VA-3-39 | Harrison apple | Albemarle Co. VA [22] | ||
VA-3-44 | VA-3-44 | Gala Supreme apple | Frederick Co. VA [22] | ||
VA-3-52 | VA-3-52 | Yates apple | Albemarle Co. VA [22] | ||
VA-3-54 | VA-3-54 | Winter White Pearmain apple | Albemarle Co. VA [22] | ||
VA-3-73 | VA-3-73 | Gala Supreme apple | Frederick Co. VA [22] | ||
VA-3-87 | VA-3-87 | Bramtot apple | Albemarle Co. VA [22] | ||
VA-4-12 | VA-4-12 | Golden Delicious apple | Fauquier Co. VA [22] | ||
VA-4-41 | VA-4-41 | GoldRush apple | Bedford Co. VA [22] | ||
VA-4-53 | VA-4-53 | Winesap apple | Albemarle Co. VA [22] | ||
VA-5-86 | VA-5-86 | Red Delicious apple | Fauquier Co. VA [22] | ||
VA-5-88 | VA-5-88 | Royal Gala apple | Frederick Co. VA [22] | ||
VA-6-15 | VA-6-15 | Royal Gala apple | Frederick Co. VA [22] | ||
VA-6-16 | VA-6-16 | Buckeye Gala apple | Botetourt Co. VA [22] | ||
VA-6-28 | VA-6-28 | Pink Lady apple | Albemarle Co. VA [22] | ||
VA-6-56 | VA-6-56 | Buckeye Gala apple | Botetourt Co. VA [22] | ||
VA-6-59 | VA-6-59 | Buckeye Gala apple | Botetourt Co. VA [22] | ||
VT1109 | HC540 | Honeycrisp apple | Bourbon Co. KY [44] | ||
C. gloeosporioides s.s. | VT1104 | DLC8 | apple | Frederick Co. MD [43] | |
C. henanense | VT1105 | SHB6 | apple | Westmoreland Co. PA [45] | |
VT1113 | SHB5a | apple | Westmoreland Co. PA [43] | ||
C. kahawae clade | VT1116 | HC278 | Malus pumila | KY [3] | |
VT1117 | HC292 | Malus pumila | KY [3] | ||
C. noveboracense | VT1106 | AFKH109 | Idared apple | Columbia Co. NY [44] | |
VT1120 | PMBrms-1 | apple | Adams Co. PA [44] | ||
VT1121 | PMCMS-6751 | apple | Lehigh Co. PA [44] | ||
VT1122 | Coll940 | Juglans nigra | Cherokee Co. OK [44] | ||
VT1123 | PMEssl-10a | apple | Lycoming Co. PA [44] | ||
C. siamense | VA-6-10 | VA-6-10 | Granny Smith apple | Amherst Co. VA [22] | |
VT1130 | DLC6a | apple | Frederick Co. MD [43] | ||
VT1131 | KY146 | apple | Clinton Co. KY [44] | ||
VT1132 | KY8 | apple | Harlan Co. KY [44] | ||
C. theobromicola | VA-41 | VA-41 | Granny Smith apple | Nelson Co. VA [22] | |
Other fungi | |||||
Botryosphaeria dothidea | VT0745 | VT0745 | grape | Frederick Co. VA | |
Diaporthe sp. | VT0748 | VT0748 | grape | Frederick Co. VA | |
Diplocarpon coronariae | VT1136 | BMO8 | apple | Adams Co. PA [50] | |
VT1137 | BMO9 | apple | Adams Co. PA [50] | ||
VT1138 | Vtech4 | apple | Frederick Co. VA [50] | ||
VT1139 | Vtech5 | apple | Frederick Co. VA [50] | ||
Erysiphe necator | VT0688 | VT0688 | grape | Frederick Co. VA | |
Monilinia fructicola | VT1110 | Mfa1 | Jonamac apple | Lancaster Co. PA [51,52] | |
Neonectria ditissima | VT1133 | EUC1-T-1 | apple | Floyd Co. VA | |
Penicillium expansum | VT1135 | TDL12.1 | — | [49] | |
Pestalotiopsis maculans | VT0746 | VT0746 | grape | Frederick Co. VA | |
Phomopsis viticola | VT0005 | VT0005 | Vitis | Frederick Co. VA | |
Plasmopara viticola | VT0693 | VT0693 | Vitis | Shenandoah Co. VA | |
Plants | |||||
Malus domestica McIntosh | VT0695 | — | — | Frederick Co. VA | |
Vitis vinifera | GRAPE DNA | — | — | Frederick Co. VA |
Species | Gene Region | Primer and Probe | Sequence (5′-3′) | Final Concentration (nM) | Anneal T (°C) | Amplicon Size (bp) |
---|---|---|---|---|---|---|
C. chrysophilum | ladA | CHLADF2 | CAT CGT GGC TGT AAT TTT GGA TGT TTC | 300 | 72 | 164 |
CHLADR | CTT GCC GAA TCC TTC GCT GGT GGT CAC GGC CGA T | 300 | ||||
CHLADP | 6FAM-GAC ACC AGT CGC CTT GAC GTG G-MGBNFQ | 100 | ||||
C. fioriniae | calmodulin | FICALF | TTT ACG CAG CAA CCA CTG GCA ACC ATC | 600 | 69 | 182 |
FICALR | GTC TCT GAT TAG CAC TAT CTA CAT GC | 600 | ||||
FICALP | VIC-TTC AAG GTG AGA AGA TCT GGC GCA A-MGBNFQ | 200 | ||||
C. fructicola | ladA | FRLADF2 | TCT CAT GAC AGG AGC TTC CGA GAT TTC | 600 | 70 | 164 |
FRLADR | GCT GCC GAA CCC CTC ATT GGT GGT CAC GGC CGA C | 600 | ||||
FRLADP | VIC-AAC ACC AGT CGC CTT AAC GTG A-MGBNFQ | 200 | ||||
C. gloeosporioides s.s. | GAPDH | GLGF | CTC CAA GCT CGW CAT GAC TTC AC | 600 | 68 | 114 |
GLGR | GAT TTC AAT TGG CAT TAA TTC ATR ATG GCC | 600 | ||||
GLGP | 6FAM-GCC GCC CGC GTT TAG TAC AC-MGBNFQ | 200 | ||||
C. henanense | ApMat | HEAPF | TGA CTT GGT CAT CGA TTC GCT TCC CG | 300 | 65 | 141 |
HEAPR | GCG AGG ATG GTT CTC GAT TCG | 300 | ||||
HEAPP | VIC-CCT TGC GCC AGA AAC CAA CCC ACC T-MGBNFQ | 100 | ||||
C. noveboracense | ladA | NOLADF | GGG AAG TAT AGT CAG CGC ATT G | 300 | 68 | 357 |
NOLADR | TAA TCG CCG TCT CTC GTT CGT TCG AC | 300 | ||||
NOLADP | VIC-CGT CAT GAC TGG AAT TTG TGA TGT TCC-MGBNFQ | 100 | ||||
C. nymphaeae | GAPDH | NYMGF | GAT AAC ACC AGC TTC GTC GAT ATC | 300 | 69 | 132 |
NYMGR | TCT GTC AGC AAG TTT TGT CTC GGC | 300 | ||||
NYMGP | 6FAM-GAT TGG GCT TGT TGT AAC GAC ACG-MGBNFQ | 100 | ||||
C. siamense | ApMat | SIAPF | ACT GAT ATC GGC GCT GCC AG | 300 | 70 | 168 |
SIAPR | GAA GGG AAT CGA TGG CCA GAT GTG | 300 | ||||
SIAPP | 6FAM-CGA CCT AAG GTT GTC TTT GTG TCC TAG-MGBNFQ | 100 | ||||
C. theobromicola | beta-tubulin | THTUBF | CTT TCA CCC GAG TTC CAT GTT CAC C | 600 | 65 | 181 |
THTUBR | GCG AGA GAT TAG CCC TTA GCC CTG C | 600 | ||||
THTUBP | 6FAM-CGT CAA TCC GAC CCC CTA CTG CG-MGBNFQ | 200 | ||||
Other primer or primer–probe sets tested but produced too much non-specific amplification: | ||||||
C. chrysophilum | APN2 | CHAPNF2 | GGC AAT CTA CAC CCG CAA CGC G | 300 | 72 | 131 |
CHAPNR | GGT ACC CGC CGA TAT GCT G | 300 | ||||
CHAPNP | VIC-CGT GGC GCG ACC TGC CCC CG-MGBNFQ | 100 | ||||
C. fioriniae | GAPDH | FIGF | TAC AAT AAC ACC AGC TTC ATC GGT AAC | 100 | 65 | 154 |
FIGR | TCT GTC AGC AAA TTT TGT TTG GGC | 100 | ||||
C. fructicola | APN2 | FRAPNF | GGC AAT CTA CAC CCG CAA CGC A | 100 | 65 | 131 |
FRAPNR | GGT ACC CGC CGA TGT GCT G | 100 | ||||
C. noveboracense | ApMat | NOAPF | GTG AGG ACC ATT GAT TTG CCC ACA TGT T | 100 | 65 | 116 |
NOAPR | GGA TCA GAC CTA GCT ATT CCC GTG ATG | 100 | ||||
C. nymphaeae | ACT | NYACTF | CGC AGA CCG CAA TCT TCT CCG TCA GG | 100 | 65 | 150 |
NYACTR | GCA GGA GAT GGC ATT GCC GCA GC | 100 |
Primer–Probe Set (Colletotrichum Species, Gene) | E | R2 | slope | y-Intercept | Cq at 1000 pg | LoD in pg (Cq) |
---|---|---|---|---|---|---|
CHLAD (C. chrysophilum, ladA) | 94.6% | 0.992 | −3.546 | 22.525 | 23 | 0.5 (36) |
FICAL (C. fioriniae, calmodulin) | 92.1% | 0.987 | −3.630 | 22.867 | 23 | 0.5 (36) |
FRLAD (C. fructicola, ladA) | 99.5% | 0.963 | −3.346 | 28.457 | 29 | 5 (36) |
GLG (C. gloeosporioides s.s., GAPDH) | 108.2% | 0.978 | −3.158 | 22.104 | 22 | 0.5 (33) |
HEAP (C. henanense, ApMat) | 92.1% | 0.991 | −3.529 | 22.851 | 23 | 0.5 (35) |
NOLAD (C. noveboracense, ladA) | 90.5% | 0.991 | −3.529 | 22.9851 | 24 | 1 (35) |
NYMG (C. nymphaeae, GAPDH) | 92.6% | 0.991 | −3.529 | 22.851 | 23 | 0.5 (35) |
SIAP (C. siamense, ApMat) | 93.2% | 0.983 | −3.713 | 22.752 | 22 | 0.5 (35) |
THTUB (C. theobromicola, beta-tubulin) | 101.3% | 0.921 | −3.226 | 27.215 | 27 | 5 (35) |
Taxon | CHLAD | FICAL | FRLAD | GLG | HEAP | NOLAD | NYMG | SIAP | THTUB | |
---|---|---|---|---|---|---|---|---|---|---|
Colletotrichum acutatum species complex | ||||||||||
C. acutatum s.s. (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
C. fioriniae (n = 15) | 3/14 | 13/15 | 0/14 | 0/15 | 0/15 | 0/14 | 2/15 | 0/15 | 0/15 | |
C. godetiae (n = 2) | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | |
C. johnstonii (n = 2) | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | |
C. lupini (n = 2) | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 2/2 | 0/2 | 0/2 | |
C. nymphaeae (n = 5) | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 | 2/5 | 5/5 | 0/5 | 0/5 | |
C. pyricola (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
C. salicis (n = 2) | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | |
Colletotrichum gloeosporioides species complex | ||||||||||
C. chrysophilum (n = 13) | 13/13 | 0/13 | 4/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | 0/13 | |
C. fructicola (n = 30) | 8/30 | 0/30 | 30/30 | 0/30 | 0/30 | 0/30 | 0/30 | 0/30 | 0/30 | |
C. gloeosporioides s.s. (n = 1) | 0/1 | 0/1 | 0/1 | 1/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
C. henanense (n = 2) | 0/2 | 0/2 | 0/2 | 0/2 | 2/2 | 0/2 | 0/2 | 0/2 | 0/2 | |
C. kahawae clade (n = 2) | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | |
C. noveboracense (n = 5) | 0/5 | 0/5 | 0/5 | 0/5 | 0/5 | 5/5 | 0/5 | 0/5 | 0/5 | |
C. siamense (n = 4) | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | 1/4 | 4/4 | 0/4 | |
C. theobromicola (n = 1) | 1/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 1/1 | |
Other fungi | ||||||||||
Botryosphaeria dothidea (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Diaporthe sp. (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Diplocarpon coronariae (n = 4) | 0/4 | 0/4 | 0/4 | 0/3 | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 | |
Erysiphe necator (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Monilinia fructicola (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Neonectria ditissima (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Penicillium expansum (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Pestalotiopsis maculans (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Phomopsis viticola (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Plasmopara viticola (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Plants | ||||||||||
Malus domestica (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | |
Vitis vinifera (n = 1) | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 | 0/1 |
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McHenry, D.J.; Aćimović, S.G. New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple. Microorganisms 2024, 12, 878. https://doi.org/10.3390/microorganisms12050878
McHenry DJ, Aćimović SG. New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple. Microorganisms. 2024; 12(5):878. https://doi.org/10.3390/microorganisms12050878
Chicago/Turabian StyleMcHenry, Diana J., and Srđan G. Aćimović. 2024. "New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple" Microorganisms 12, no. 5: 878. https://doi.org/10.3390/microorganisms12050878
APA StyleMcHenry, D. J., & Aćimović, S. G. (2024). New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple. Microorganisms, 12(5), 878. https://doi.org/10.3390/microorganisms12050878