A role for random, humidity-dependent epiphytic growth prior to invasion of wheat by Zymoseptoria tritici

- Zymoseptoria tritici is capable of over ten days of epiphytic growth on wheat leaves.
- Epiphytic growth and stomatal entry data are consistent with a random growth model.
- Z. tritici is easily dislodged from the leaf surface, by even gentle misting.
- Humidity promotes the hyphal growth form and facilitates rapid entry.
- Interpretation of “-omics” data must account for epiphytic growth, where appropriate.

Zymoseptoria tritici causes Septoria leaf blotch of wheat. The prevailing paradigm of the Z. tritici-wheat interaction assumes fungal ingress through stomata within 24-48 h, followed by days of symptomless infection. This is extrapolated from studies testing the mode of fungal ingress under optimal infection conditions. Here, we explicitly assess the timing of entry, using GFP-tagged Z. tritici. We show that early entry is comparatively rare, and extended epiphytic growth possible. We test the hypotheses that our data diverge from earlier studies due to: i. random ingress of Z. tritici into the leaf, with some early entry events; ii. previous reliance upon fungal stains, combined with poor attachment of Z. tritici to the leaf, leading to increased likelihood of observing internal versus external growth, compared to using GFP; iii. use of exceptionally high humidity to promote entry in previous studies. We combine computer simulation of leaf-surface growth with thousands of in planta observations to demonstrate that while spores germinate rapidly on the leaf, over 95% of fungi remain epiphytic, growing randomly over the leaf for ten days or more. We show that epiphytic fungi are easily detached from leaves by rinsing and that humidity promotes epiphytic growth, increasing infection rates. Together, these results explain why epiphytic growth has been dismissed and early ingress assumed. The prolonged epiphytic phase should inform studies of pathogenicity and virulence mutants, disease control strategies, and interpretation of the observed low in planta growth, metabolic quiescence and evasion of plant defences by Zymoseptoria during symptomless infection.