Context-Dependent Fitness Trade-Offs in Penicillium expansum Isolates Resistant to Multiple Postharvest Fungicides
Abstract
1. Introduction
2. Materials and Methods
2.1. Selection of Penicillium expansum Isolates with Different Fungicide Sensitivity Phenotypes
2.2. Evaluation of Fitness In Vitro
2.3. Evaluation of Fitness on Apple Fruit
2.4. Expression Analysis of Fitness-Related Genes in P. expansum Isolates
2.5. Data Analysis
3. Results
3.1. Comparison of Fitness Capabilities of P. expansum Isolates In Vitro
3.2. Virulence, Sporulartion, and Resistance Stability in P. expansum Isolates on Detached Apples
3.3. Cumulative Phenotypic Fitness Changes in P. expansum-Resistant Isolates
3.4. Gene Expression of Fitness-Related Genes in P. expansum Isolates with Different Fungicide Sensitivity Phenotypes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
TBZ | Thiabendazole |
PYR | Pyrimethanil |
FDL | Fludioxonil |
TBZSPYRSFDLS | Wild-type isolates sensitive to all three fungicides |
TBZR | Single-resistant isolate to thiabendazole |
TBZRPYR | Dual-resistant isolate to thiabendazole and pyrimethanil |
PYRRFDLR | Dual-resistant isolate to pyrimethanil and fludioxonil |
TBZRPYRRFDLR | Triple-resistant isolates to all three fungicides |
Fc | Fitness change relative to wild-type isolates |
CFc | Cumulative fitness change |
FRAC | Fungicide Resistance Action Committee |
Gw or Gm | Generation at a given week or month following transfer of isolates |
OSS | Osmotic stress |
ROS | Reactive oxygen species |
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Sensitivity | Sensitivity | Year | Geographic Origin | Tested c | |||||
---|---|---|---|---|---|---|---|---|---|
Isolate ID | Phenotype a | Group | Isolated | Host | Cultivar | State b | County | In Vitro | In Vivo |
Pe-40 | TBZSPYRSFDLS | Sensitive to all | 2016 | Apple | Fuji | WA | Franklin | + | + |
Pe-1267 | 2017 | Apple | Fuji | WA | Grant | + | + | ||
Pe-2418 | 2017 | Apple | Gala | WA | Grant | + | − | ||
Pe-23 | TBZR | Single-Resistant | 2016 | Apple | Gala | WA | Okanogan | + | + |
Pe-184 | 2018 | Pear | Bosc | OR | Hood River | + | + | ||
Pe-219 | 2017 | Apple | Red Delicious | WA | Yakima | + | − | ||
Pe-08 | TBZRPYRR | Dual-Resistant | 2016 | Apple | Gala | WA | Okanogan | + | + |
Pe-2311 | 2016 | Apple | Fuji | WA | Grant | + | + | ||
Pe-2483 | 2016 | Apple | Gala | WA | Grant | + | − | ||
Pe-153 | PYRRFDLR | 2016 | Apple | Gala | WA | Okanogan | + | + | |
Pe-2501 | 2016 | Apple | Gala | WA | Douglas | + | + | ||
Pe-2517 | 2017 | Apple | Gala | WA | Okanogan | + | − | ||
Pe-1020 | TBZRPYRRFDLR | Triple-Resistant | 2017 | Apple | Gala | WA | Okanogan | + | + |
Pe-2754 | 2017 | Apple | Gala | WA | Benton | + | + | ||
Pe-3045 | 2017 | Apple | Granny Smith | WA | Yakima | + | − |
Gene | Gene ID | Hypothesized Fitness Parameter | Primer Name | Sequence (5′-3′) | Reference |
---|---|---|---|---|---|
Ac1 | PEX2_003910 | Growth/Germination/Virulence | PeAc1-rt-F2 | AGTCACCCGTATCGAGGTATGC | This study |
PeAc1-rt-R2 | CTCGGTGATGGGTACAGCGT | ||||
Blistering1 | PEX2_008940 | Growth/Virulence/Patulin | R19_6445F | CACGGTCTTGCCACTTGCTCGT | [34] |
R19_6445R | TCGTTTCGAGTACGTCGCGCTG | ||||
BrlA | PEX2_076900 | Sporulation | dBrlA_F | CCTCGATGCCTCAATACA | [35] |
dBrlA_R | GTAAAGATTGGACGAGACAAG | ||||
LaeA | PEX2_005650 | Virulence/Sporulation/Patulin | PeLaeA-rt-F2 | TCCGATCGCAAGATACCCGA | This study |
PeLaeA-rt-R2 | GTCACACGGAAGCGGGTAGA | ||||
OS-2 | PEX2_061660 | Sensitivity to Osmotic Stress | PeOS-2-rt-F2 | CTGTAGCGAGAACACCCTCCG | This study |
PeOS-2-rt-R2 | CGAGCAAGTCAACCGCATCG | ||||
RacA | PEX2_019970 | Sensitivity to ROS/Patulin | PeRacA-rt-F1 | AACGTCAAAGCGAAGTGGTTC | This study |
PeRacA-rt-R1 | CACGGTCGTCCCTCAGATCG | ||||
VeA | PEX2_043190 | Growth/Virulence/Sporulation/Patulin | PeVeA-rt-F2 | CGGGAGCCAAGTCATCTGCT | This study |
PeVeA-rt-R2 | GCATCGTTAGCCGGATCGGA | ||||
28S | NG_069649 | Reference | 28SF | GGAACGGGACGTCATAGAGG | [32] |
28SR | AGAGCTGCATTCCCAAACAAC | ||||
CaM | DQ911134 | Reference | PeCMD-rt-F3 | CTCACCATGATGGCTCGTAAGA | [39] |
PeCMD-rt-R3 | GCGGAAATGAAACCGTTGTT |
Sensitivity | Spore Germination a | Mycelial Growth b | Sporulation c | Sensitivity to Exogenic Stress d | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Phenotype | Isolate | IM | WA | PDA | IM | PDA | IM | OSS | ROS | ||||||||
TBZSPYRSFDLS | Pe-40 | 99.9 ± 0.2 | a ef | 88.1 ± 5.1 | ab | 38.6 ± 4.2 | cde | 45.3 ± 2.1 | cde | 8.04 ± 0.2 | ab | 7.72 ± 0.2 | a | 5.4 ± 2.5 | de | 42.0 ± 6.2 | bcd |
Wild-Type | Pe-1267 | 99.8 ± 0.4 | a | 92.7 ± 4.1 | a | 34.4 ± 4.8 | e | 44.8 ± 2.8 | de | 7.64 ± 0.5 | b | 7.73 ± 0.3 | a | 6.5 ± 0.6 | de | 27.9 ± 7.1 | ef |
Pe-2418 | 99.8 ± 0.4 | a | 92.8 ± 3.6 | a | 40.6 ± 2.8 | c | 46.5 ± 1.9 | abcde | 8.19 ± 0.1 | a | 7.58 ± 0.3 | a | 6.4 ± 0.5 | de | 43.6 ± 4.1 | bcd | |
Mean | 99.9 ± 0.3 | 91.2 ± 4.8 | 37.9 ± 4.8 | 45.5 ± 2.4 | 7.96 ± 0.4 | 7.68 ± 0.2 | 6.1 ± 1.6 | 37.8 ± 9.2 | |||||||||
TBZRPYRSFDLS | Pe-23 | 99.7 ± 0.6 | a | 68.8 ± 9.4 | fg | 39.9 ± 1.8 | cd | 47.4 ± 3.1 | abcde | 7.89 ± 0.3 | ab | 7.71 ± 0.1 | a | 7.6 ± 1.1 | bc | 45.0 ± 3.1 | b |
Single-Resistant | Pe-184 | 99.7 ± 1.0 | a | 74.7 ± 6.9 | def | 45.1 ± 3.1 | ab | 45.9 ± 3.8 | bcde | 7.90 ± 0.3 | ab | 7.64 ± 0.2 | a | 8.4 ± 1.1 | ab | 47.0 ± 4.0 | ab |
Pe-219 | 99.4 ± 0.8 | a | 80.1 ± 8.1 | cde | 47.1 ± 4.9 | a | 45.8 ± 3.5 | bcde | 8.01 ± 0.1 | ab | 7.60 ± 0.2 | a | 7.9 ± 0.5 | ab | 51.8 ± 3.8 | a | |
Mean | 99.6 ± 0.8 | 74.5 ± 9.3 | 44.1 ± 4.6 | 46.4 ± 3.5 | 7.96 ± 0.4 | 7.65 ± 0.2 | 8.0 ± 1.0 | 47.9 ± 4.6 | |||||||||
TBZRPYRRFDLS | Pe-08 | 99.9 ± 0.3 | a | 85.8 ± 6.6 | abc | 39.1 ± 4.5 | cd | 48.5 ± 2.2 | ab | 7.75 ± 0.2 | b | 7.53 ± 0.2 | a | 8.8 ± 0.7 | a | 37.2 ± 7.5 | cde |
Dual-Resistant | Pe-2311 | 99.9 ± 0.3 | a | 65.9 ± 5.7 | g | 40.7 ± 3.5 | c | 48.0 ± 1.8 | abc | 8.07 ± 0.1 | ab | 7.61 ± 0.2 | a | 7.4 ± 1.0 | bc | 41.8 ± 10.9 | bcd |
Pe-2483 | 99.7 ± 0.6 | a | 72.5 ± 10.6 | efg | 41.1 ± 3.3 | bc | 47.8 ± 2.3 | abcd | 8.00 ± 0.1 | ab | 7.61 ± 0.2 | a | 4.5 ± 3.5 | de | 44.3 ± 3.4 | bc | |
Mean | 99.8 ± 0.4 | 74.7 ± 11.4 | 40.3 ± 3.8 | 48.1 ± 2.1 | 7.96 ± 0.4 | 7.58 ± 0.2 | 6.9 ± 2.8 | 41.1 ± 8.3 | |||||||||
TBZSPYRRFDLR | Pe-153 | 99.8 ± 0.4 | a | 81.7 ± 4.8 | bcd | 38.0 ± 4.1 | cde | 44.6 ± 2.6 | e | 7.73 ± 0.2 | b | 7.75 ± 0.2 | a | 3.6 ± 3.8 | de | 42.8 ± 11.1 | bc |
Dual-Resistant | Pe-2501 | 99.8 ± 0.5 | a | 81.8 ± 7.1 | bcd | 40.1 ± 2.8 | cd | 45.4 ± 2.9 | cde | 7.67 ± 0.4 | b | 7.73 ± 0.1 | a | 4.3 ± 2.5 | e | 22.6 ± 23.3 | def |
Pe-2517 | 99.8 ± 0.4 | a | 72.3 ± 9.4 | efg | 39.1 ± 2.5 | cd | 45.0 ± 1.5 | cde | 7.90 ± 0.3 | ab | 7.64 ± 0.3 | a | 7.6 ± 0.5 | bc | 10.1 ± 19.7 | ef | |
Mean | 99.8 ± 0.5 | 78.6 ± 8.5 | 39.1 ± 3.3 | 45.0 ± 2.4 | 7.77 ± 0.3 | 7.71 ± 0.2 | 5.1 ± 3.1 | 25.2 ± 22.9 | |||||||||
TBZRPYRRFDLR | Pe-1020 | 99.9 ± 0.2 | a | 65.1 ± 11.1 | fg | 40.8 ± 4.7 | c | 49.2 ± 2.3 | a | 7.91 ± 0.1 | ab | 7.62 ± 0.3 | a | 6.9 ± 0.4 | cd | 42.2 ± 3.5 | bcd |
Triple-Resistant | Pe-2754 | 99.9 ± 0.2 | a | 72.6 ± 5.7 | efg | 36.0 ± 2.8 | de | 47.1 ± 3.0 | abcde | 7.98 ± 0.1 | ab | 7.66 ± 0.2 | a | 6.2 ± 0.7 | de | 16.3 ± 16.9 | f |
Pe-3045 | 99.9 ± 0.3 | a | 74.2 ± 5.2 | efg | 41.5 ± 0.4 | bc | 47.3 ± 3.1 | abcde | 8.05 ± 0.1 | ab | 7.55 ± 0.0 | a | 4.7 ± 2.4 | e | 6.1 ± 17.7 | f | |
Mean | 99.9 ± 0.3 | 70.6 ± 8.7 | 39.4 ± 4.5 | 47.9 ± 2.9 | 7.98 ± 0.1 | 7.61 ± 0.2 | 5.9 ± 1.7 | 21.5 ± 21.0 |
Sensitivity | Spore Germination a | Mycelial Growth b | Sporulation c | Sensitivity to Exogenic Stress d | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Phenotype | Isolate | IM | WA | PDA | IM | PDA | IM | OSS | ROS | ||||||||
TBZSPYRSFDLS | Pe-40 | 22.4 ± 7.6 | bcdef ef | 41.8 ± 12.2 | ab | 29.7 ± 2.9 | bcde | 33.3 ± 1.2 | c | 8.41 ± 0.1 | ab | 7.76 ± 0.1 | a | 9.9 ± 2.8 | ab | 0.0 ± 0.0 | a |
Wild-Type | Pe-1267 | 25.8 ± 5.6 | bcde | 45.6 ± 14.3 | a | 23.0 ± 3.3 | gh | 34.3 ± 1.4 | abc | 8.20 ± 0.2 | b | 7.75 ± 0.1 | a | 5.0 ± 3.0 | de | 0.0 ± 0.0 | a |
Pe-2418 | 32.0 ± 7.7 | abc | 51.9 ± 13.0 | a | 33.8 ± 2.0 | ab | 35.8 ± 0.9 | abc | 8.37 ± 0.1 | ab | 7.74 ± 0.1 | a | 8.9 ± 2.3 | ab | 0.0 ± 0.0 | a | |
Mean | 26.7 ± 7.9 | 46.4 ± 13.6 | 28.8 ± 5.2 | 34.4 ± 5.1 | 8.33 ± 0.2 | 7.75 ± 0.1 | 8.0 ± 3.4 | 0.0 ± 0.0 | |||||||||
TBZRPYRSFDLS | Pe-23 | 20.0 ± 10.1 | cdefg | 31.3 ± 7.4 | abc | 23.5 ± 3.6 | gh | 32.8 ± 0.9 | c | 8.37 ± 0.1 | ab | 7.74 ± 0.2 | a | 7.5 ± 2.1 | bcd | 0.0 ± 0.0 | a |
Single-Resistant | Pe-184 | 5.6 ± 1.7 | gh | 14.6 ± 7.6 | ef | 24.8 ± 2.6 | fgh | 32.7 ± 1.4 | c | 8.36 ± 0.1 | ab | 7.84 ± 0.1 | a | 7.8 ± 1.4 | bc | 0.0 ± 0.0 | a |
Pe-219 | 17.4 ± 12.4 | defg | 12.3 ± 4.0 | f | 29.3 ± 1.9 | bcde | 34.8 ± 1.0 | abc | 8.34 ± 0.1 | ab | 7.81 ± 0.1 | a | 5.8 ± 1.4 | cde | 0.0 ± 0.0 | a | |
Mean | 14.3 ± 11.0 | 19.4 ± 10.7 | 25.9 ± 3.7 | 33.4 ± 4.8 | 8.36 ± 0.1 | 7.80 ± 0.1 | 7.0 ± 1.9 | 0.0 ± 0.0 | |||||||||
TBZRPYRRFDLS | Pe-08 | 25.9 ± 13.6 | bcde | 23.8 ± 3.5 | cd | 23.9 ± 2.8 | fgh | 33.9 ± 0.6 | bc | 8.41 ± 0.1 | ab | 7.82 ± 0.1 | a | 10.8 ± 1.9 | a | 0.0 ± 0.0 | a |
Dual-Resistant | Pe-2311 | 6.6 ± 2.6 | fgh | 20.8 ± 7.7 | de | 26.5 ± 2.7 | efgh | 33.5 ± 0.5 | c | 8.37 ± 0.1 | ab | 7.89 ± 0.1 | a | 8.2 ± 2.3 | abc | 0.0 ± 0.0 | a |
Pe-2483 | 1.2 ± 1.9 | h | 15.7 ± 6.4 | ef | 28.4 ± 1.5 | cdef | 33.8 ± 0.9 | abc | 8.38 ± 0.0 | ab | 7.77 ± 0.2 | a | 7.2 ± 1.8 | bcd | 0.0 ± 0.0 | a | |
Mean | 10.8 ± 13.2 | 20.1 ± 6.9 | 26.3 ± 3.0 | 33.8 ± 2.9 | 8.39 ± 0.1 | 7.83 ± 0.1 | 8.8 ± 2.5 | 0.0 ± 0.0 | |||||||||
TBZSPYRRFDLR | Pe-153 | 51.8 ± 10.1 | a | 20.6 ± 7.4 | de | 31.2 ± 6.8 | abcd | 35.6 ± 0.9 | abc | 8.44 ± 0.1 | a | 7.77 ± 0.1 | a | 4.4 ± 1.7 | ef | 0.0 ± 0.0 | a |
Dual-Resistant | Pe-2501 | 30.8 ± 9.1 | abcd | 29.0 ± 10.9 | bcd | 27.0 ± 5.8 | defg | 35.2 ± 2.9 | abc | 8.42 ± 0.1 | a | 7.74 ± 0.2 | a | 3.3 ± 3.4 | efg | 0.0 ± 0.0 | a |
Pe-2517 | 35.7 ± 7.3 | ab | 10.7 ± 3.7 | f | 31.1 ± 5.0 | abcd | 33.9 ± 0.8 | bc | 8.46 ± 0.0 | a | 7.60 ± 0.2 | a | 9.8 ± 3.0 | ab | 0.0 ± 0.0 | a | |
Mean | 39.4 ± 12.5 | 20.1 ± 10.8 | 29.8 ± 6.1 | 34.9 ± 3.5 | 8.44 ± 0.1 | 7.70 ± 0.2 | 5.8 ± 4.0 | 0.0 ± 0.0 | |||||||||
TBZRPYRRFDLR | Pe-1020 | 31.2 ± 9.7 | abcd | 21.6 ± 10.3 | de | 22.0 ± 5.8 | h | 35.3 ± 1.0 | abc | 8.36 ± 0.1 | ab | 7.83 ± 0.1 | a | 2.1 ± 1.2 | fg | 0.0 ± 0.0 | a |
Triple-Resistant | Pe-2754 | 26.0 ± 5.4 | bcde | 21.6 ± 8.9 | de | 31.6 ± 4.2 | abc | 39.0 ± 1.2 | a | 8.44 ± 0.0 | a | 7.75 ± 0.1 | a | 1.6 ± 0.8 | g | 0.0 ± 0.0 | a |
Pe-3045 | 14.3 ± 2.4 | efgh | 21.4 ± 6.4 | de | 34.5 ± 4.3 | a | 37.5 ± 0.8 | ab | 8.45 ± 0.1 | a | 7.77 ± 0.2 | a | 1.8 ± 1.2 | fg | 0.0 ± 0.0 | a | |
Mean | 23.9 ± 9.6 | 21.5 ± 8.4 | 29.4 ± 7.2 | 37.3 ± 4.4 | 8.41 ± 0.1 | 7.78 ± 0.1 | 1.8 ± 1.1 | 0.0 ± 0.0 |
Cumulative Relative Fitness Change (CFC) in the Resistant Isolates to Sensitive Isolates | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Resistance | WA b | IM | PDA | On Detached Apples | |||||||||||
Resistance Group | Isolate | Phenotype a | 1.5 °C | 20 °C | 1.5 °C | 20 °C | 1.5 °C | 20 °C | Control | TBZ | PYR | FDL | Fitness Gain | Fitness Loss | |
Single-Resistant | Pe-23 | TBZR | −39 | −28 | −34 | 4 | −13 | 42 | 4 | 393 | 0 | −200 | 0 | 0 | |
Pe-184 | TBZR | −104 | −20 | −135 | 0 | 9 | 70 | 11 | 393 | 200 | 267 | 5 | −5 | ||
Pe-219 | TBZR | −116 | −13 | −40 | −1 | −94 | 80 | - | - | - | - | 25 | −25 | ||
50 | −50 | ||||||||||||||
Mean | −86 | −20 | −70 | 1 | −33 | 64 | 7 | 393 | 100 | 33 | 100 | −100 | |||
200 | −200 | ||||||||||||||
Dual-Resistant | Pe-08 | TBZRPYRR | −64 | −6 | −4 | 4 | −44 | 34 | −3 | 393 | 400 | −200 | |||
Pe-2311 | TBZRPYRR | −76 | −32 | −122 | 4 | −65 | 38 | −7 | 393 | 400 | −200 | ||||
Pe-2483 | TBZRPYRR | −99 | −23 | −184 | 4 | −89 | −6 | - | - | - | - | ||||
Mean | −80 | −20 | −103 | 4 | −66 | 22 | −5 | 393 | 400 | −200 | |||||
Pe-153 | PYRRFDLR | −77 | −11 | 67 | −1 | 36 | −42 | −23 | 60 | 0 | 391 | ||||
Pe-2501 | PYRRFDLR | −46 | −11 | 16 | 0 | −67 | −86 | −4 | 55 | 200 | 0 | ||||
Pe-2517 | PYRRFDLR | −125 | −23 | 25 | −2 | 17 | −92 | - | - | - | - | ||||
Mean | −83 | −15 | 36 | −1 | −5 | −73 | −14 | 57 | 100 | 196 | |||||
Triple-Resistant | Pe-1020 | TBZRPYRRFDLR | −73 | −33 | 19 | 7 | −161 | 30 | −5 | 393 | 400 | 379 | |||
Pe-2754 | TBZRPYRRFDLR | −73 | −23 | 10 | 3 | −118 | −85 | −10 | 393 | 400 | 394 | ||||
Pe-3045 | TBZRPYRRFDLR | −71 | −21 | −51 | 2 | −135 | −160 | - | - | - | - | ||||
Mean | −72 | −26 | −8 | 4 | −138 | −71 | −7 | 393 | 400 | 386 |
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Puglisi, J.T.; Amiri, A. Context-Dependent Fitness Trade-Offs in Penicillium expansum Isolates Resistant to Multiple Postharvest Fungicides. Microorganisms 2025, 13, 1846. https://doi.org/10.3390/microorganisms13081846
Puglisi JT, Amiri A. Context-Dependent Fitness Trade-Offs in Penicillium expansum Isolates Resistant to Multiple Postharvest Fungicides. Microorganisms. 2025; 13(8):1846. https://doi.org/10.3390/microorganisms13081846
Chicago/Turabian StylePuglisi, Jonathan T., and Achour Amiri. 2025. "Context-Dependent Fitness Trade-Offs in Penicillium expansum Isolates Resistant to Multiple Postharvest Fungicides" Microorganisms 13, no. 8: 1846. https://doi.org/10.3390/microorganisms13081846
APA StylePuglisi, J. T., & Amiri, A. (2025). Context-Dependent Fitness Trade-Offs in Penicillium expansum Isolates Resistant to Multiple Postharvest Fungicides. Microorganisms, 13(8), 1846. https://doi.org/10.3390/microorganisms13081846