Abstract
Introduction:
Chlorophyll (Chl) degradation via the pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway produces linear tetrapyrrolic phyllobilins (PBs) during leaf senescence and infection by certain pathogens, such as phytoplasmas. Since leaf yellowing, or chlorosis, is a common symptom of fungal disease in apple (Malus × domestica Borkh.) and other plants, PB formation was analyzed during four fungal diseases to evaluate the general relevance of the PaO/PB pathway during infection.
Methods:
A comprehensive, targeted ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) method was used to profile a total of 79 known and putative PBs in healthy, naturally senescent, and symptomatic leaves infected with Diplocarpon coronariae, Alternaria alternata, Venturia inaequalis, or Podosphaera leucotricha, using five biological replicates per condition (n=5). Statistical analysis of Chl values was performed using one-way ANOVA followed by Tukey’s post-hoc test.
Results:
This method allowed us to detect 36 PBs, including eleven previously unknown PBs, one of which carries an unprecedented methoxy modification. Diplocarpon coronariae infection induced extensive chlorosis and a PB profile comparable to senescence, whereas A. alternata, V. inaequalis, and P. leucotricha did not substantially increase PB diversity or abundance, despite affecting photosynthetic tissues. These results are coherent with the Chl amount in leaves. In D. coronariae, an induced extensive chlorosis emerged in infection, with the total Chl reduced from 61.9 ± 5.3 µg cm⁻² in healthy leaves to 12.4 ± 5.3 µg cm⁻² in diseased leaves (p < 0.001, significant reduction), resulting in a PB profile comparable to natural senescence. In contrast, P. leucotricha (60.7 vs. 55.5 µg cm⁻²; p = 0.16, no significant difference) and A. alternata (41.4 vs. 49.0 µg cm⁻²; p = 0.15, no significant difference), showed no significant reduction in Chl and substantial increase in PB diversity.
Discussion:
Our findings indicate that engagement of the PaO/PB pathway in apple leaves is strongly associated with chlorosis, whether induced by natural senescence or specific pathogens, suggesting a general role of this catabolic route in chlorotic infections