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# Hymenoscyphus fraxineus persistence in the ash litter

Simon Laubray, Marc Buée, Benoit Marçais

► To cite this version:

Simon Laubray, Marc Buée, Benoit Marçais. Hymenoscyphus fraxineus persistence in the ash litter. Plant Pathology, In press, 73 (7), pp.1937-1946. 10.1111/ppa.13948. hal-04581858

HAL Id: hal-04581858

https://hal.science/hal-04581858v1

Submitted on 21 May 2024

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# *Hymenoscyphus fraxineus* persistence in the ash litter

Simon Laubray, Marc Buée, Benoit Marçais

Université de Lorraine, UMR1136 Interactions Arbres/Microorganismes, F-54000 Nancy, France

Corresponding Author: Benoit.Marçais (benoit.marcais@inrae.fr)

## Abstract

*Hymenoscyphus fraxineus* causes ash dieback in Europe. It overwinters on ash leaf residues (rachis) within the forest litter. In late spring, the fungus produces apothecia on the rachises and releases ascospores to infect leaves. Previous studies reported that *H. fraxineus* was able to produce apothecia on rachis for 5 years after the leaf infection under artificial conditions. However, ash litter is known to decompose rapidly *in situ*. We therefore monitored the decomposition kinetics of ash leaves debris and the persistence of the pathogen, as well as its ability to produce apothecia in the forest litter. For this, leaves shed in autumn in stands affected by ash dieback were placed in mesh bags and left in the forest litter for 6, 18 and 30 months. At each sampling period, litter mass loss and level of colonization of the rachises by *H. fraxineus* were measured as was the pathogen's ability to produce apothecia on them. Despite high fragmentation, about 20% of the rachis dry weight remained in the mesh bags after 30 months, and the pathogen retained the ability to produce apothecia on these rachises. A simulation estimating the age composition of the colonized rachis present in the litter during the fruiting period was developed from these results. It shows that the persistence of *H. fraxineus* in old rachis of the litter represents a reservoir of inoculum that could compensate for the poor colonization of autumn leaves and revive local outbreaks after years of unfavourable weather for the development of the pathogen.

**Key words.** *Hymenoscyphus fraxineus*, pathogen persistence, inoculum reservoir, primary inoculum, ash dieback
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# 1. Introduction

Ash dieback is the most serious epidemic to hit European forests in recent decades. It began in the 1990s in Poland (Kowalski, 2006; Przybyl, 2002) and has invaded almost the entire range of common ash (*Fraxinus excelsior* L.) including the UK and Ireland, where it was first detected in 2012, and more recently in northern Spain (Stroheker et al., 2021). The impact of ash dieback on European forests has been devastating. Mortality was estimated to reach up to 60% (Coker et al., 2019), resulting in significant losses in biodiversity and ecosystem services. Ash trees are an important component of many European landscapes, whether in forests or hedgerows, and provide habitat for a wide range of wildlife species (Mitchell et al., 2014). Trees of all ages are affected (Madsen et al., 2021; Skovsgaard et al., 2010). Younger trees are more susceptible and die quickly: 10 years after the arrival of the disease, mortality observed in 10-year-old ash trees was about 65% (Coker et al., 2019). For older trees, the dieback is slower with 25% mortality observed after 10 years (Madsen et al., 2021).

The disease is caused by the fungus *Hymenoscyphus fraxineus* (Kowalski, 2006), which was probably accidentally introduced from northeast Asia (Sønstebø et al., 2017; Zhao et al., 2013). It causes leaf necrosis, branch mortality and crown dieback. *H. fraxineus* ascospores are released during the summer (Timmermann et al., 2011), germinate on leaf surfaces and penetrate the foliar tissues. In autumn, after leaf fall, *H. fraxineus* continues to develop in the remaining leaf tissue consisting of the midrib and petiole without leaflets (hereinafter referred to as the rachis). In winter, the pathogen creates a black pseudoclerotial plate in the rachis tissue. In late spring, the pathogen carries out sexual reproduction on the rachis with a pseudosclerotial plate (called pseudosclerotial rachises) and produces apothecia which release ascospores that infect new leaves. As apothecia only form on pseudosclerotial rachises (Gross et al., 2012), these are essential for the pathogen persistence and inoculum production.

Pseudosclerotial rachises are functionally equivalent to sclerotia. In the fungal kingdom, sclerotia are protective structures that enable the fungus to survive harsh conditions such as frost, desiccation, enemy attack or microbial competition, and allow the organism to persist (Coley-Smith & Cooke, 1971; Townsend & Willetts, 1954; Willetts, 1971). This ability is found in many fungal phylogenetic lineages and functional guilds and is widespread among pathogens (Smith et al., 2014). *H. fraxineus* has been
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shown to persist and produce apothecia on pseudosclerotic spines for up to five years
(Gross & Holdenrieder, 2013; Kirisits, 2015). However, this persistence was observed in
artificial conditions (plastic box in a garden) and not studied in *in situ*, in the forest litter.
Moreover, the decomposition dynamics of pseudosclerotial rachises has not been
precisely quantified, and it is not known whether they differ from those of non
pseudosclerotial rachises. Ash leaves are known to decompose rapidly in forest litter
compared to leaves of other tree species such as oak or beech (Slade & Riutta, 2012),
due to a lower C/N and lignin/N ratio (Riutta et al., 2012; Steffens et al., 2012). Ash
leaves decompose 1,5 times faster than oak leaves (Riutta et al., 2012). Moreover, ash
leaf decomposition kinetic is affected by the stand composition. In mixed stands with
beech or oak, the decomposition rate of ash leaves is slower by a factor of 1.3
compared with ash-dominated stands (Dahlsjö et al., 2023). However, rachises which
account for up to 20% of ash leaf biomass decompose more slowly, probably due to
their different chemical composition and, in particular, their lower nitrogen concentration
(Niinemets, 1999)

Nevertheless, the ability of *H. fraxineus* to persist on old pseudosclerotial rachises
in litter should be an important feature of the pathogen's biology. The persistence of
seeds over several year is for example, known to be very important for many plant
species. This can lead to the existence of so-called seed banks and these have been
shown to play an important role for invasive plants, promoting population establishment
(Gioria et al., 2012, Gioria & Pyšek, 2016, Pyšek et al, 2015), enabling plants to survive
unfavorable conditions and limiting genetic erosion (Levin, 1990, Mandák et al., 2012).
Similarly, in the case of *H. fraxineus*, the persistence of pseudosclerotial rachises may
constitute a reservoir of inoculum and could be a key element in the epidemiology of
this invasive fungus.

The aim of this study was therefore to better document the dynamic of *H. fraxineus*
persistence in forest litter and its ability to produce apothecia over several years. We
tested the hypotheses: i. decomposition of ash rachises extends over several years *in situ* in forest litter. ii. the pseudosclerotial plate formed by *H. fraxineus* slows down the decay of colonized rachises compared with uncolonized rachises. iii. the capacity of pseudosclerotial rachises to produce apothecia remains constant over the years. To this end, we monitored the decomposition kinetic, the level of colonization by *H. fraxineus* and the fruiting potential of different cohorts of rachis placed in the forest litter over
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several years, and their relationship with stand and site parameters.

# 2 Material and method

## 2.1 Plot characterization

For this study, four locations with presence of *H. fraxineus* were selected in northeast France (Champenoux, Languimberg, Sélestat, Thionville). All ash trees present were *Fraxinus excelsior*. At each location, a plot was set-up in two types of ash stand, one with a high density of ash and the other with a low density (Table S1). In each of the eight plots, three subplots were set up near ash trees at a maximum distance of 20 m from the plot center (at least 20 m between sub-plots). Plots were characterized by their total basal area, tree diversity, ash density (basal area of ash), ash rachis density in litter, ash health status, soil chemistry and humus type. The values of the various parameters were averaged across the three subplots.

Basal area was estimated within three concentric circles. The diameter at breast height (DBH) of all tree stems over 7.5 cm was measured within a first circle of 7 m radius, the DBH of trees with stems over 22.5 cm within a second circle of 16 m radius, and DBH of trees with stem over 47.5 cm within a final circle of 21 m radius. Basal area in m².ha⁻¹ was the sum of the area of individual stems at breast height weighted by the area of the sampling circle. The species of trees measured was determined and the tree diversity was calculated from the basal area of each species present on the plot. Ash density corresponded to the basal area of the ash trees sampled. Ash health status was estimated by scoring the percentage of branch mortality: healthy, 0-10% (score 5), symptomatic, 10-50% (score 30), declining, 50-75% (score 62.5) and heavily declining, > 75% (score 82.5).

The density of rachis in the litter was measured in 2020 and 2022 within ten 0.1 m² areas distributed along two 10 m perpendicular transects (Laubray et al., 2023). The operation was repeated on each of the three sub-plots of each stand. The collected rachises were dried for 48h at 55°C and weighed. The rachis density is the average of the three subplots and is expressed in g.m⁻². The rachis density was divided by the ash density to compute the rachis mass per unit ash basal area.

The soil was sampled in the upper 20 cm using an auger (15 cm diameter) at the center of each of the three subplots in each plot. Samples were dried for 8 days at 35°C
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and then sieved through 2 mm mesh for further analyses. Sub-sample of 300 g for each
soil sample were sent to the LAS analysis platform (Arras, France) to measure the pH in
KCl, particle size distribution, amount of Carbon and of Nitrogen. Soil texture was
expressed as the sand / clay ratio determined by the particle size distribution. The
efficiency of organic matter degradation in litter was assessed by characterizing humus
in the field. This was done according to a humus determination key (Brethes et al.,
1995; Jabiol et al., 2007).

## 2.2 Study design

Ash leaves were collected from late November to early December in 2019, 2020 and 2021 just after they had fallen. The rachises were selected randomly over the entire plot surface. An annual rachis sampling will hereafter be referred to as a cohort. The leaflets were removed, leaving only the rachis (petiole + midrib). Each year, a batch of 30 rachises per plot (10 per subplot) was immediately brought back to the laboratory to be dried at 55°C for 48h, weighed and ground. A sample of the ground rachises was then extracted for DNA in order to measure the quantity of *H. fraxineus* by qPCR (Laubray et al., 2023). For the 2019 cohort, nine batches of 20 rachises were placed in mesh bags and left *in situ* (3 at each of the subplot, mesh bags with 1 cm mesh aperture). The litter was removed, the mesh bag was placed directly on the soil and maintained in place with a stake then the litter was replaced on the mesh. One mesh bag per subplot was sampled in May after 6 months, 18 months and 30 months of incubation in forest litter. For the 2020 cohort, six batches of 20 rachises were incubated in litter *in situ* using the same method and sampled after six months and 18 months while for the 2021 cohort, only three batches were incubated in litter during 6 months. Once the mesh bags had been collected from the plots, the rachises from the three mesh bags of a plot x cohort x age combination were pooled and separated in two subsamples. A third of them (n=15) were placed in a wet chamber to estimate their potential to produce *H. fraxineus* apothecia. After completion of this fruiting assay, they were dried at 55°C for 48h and weighted. The remaining two thirds were dried at 55°C for 48h, weighed and analyzed by qPCR to determine their colonization by *H. fraxineus*. The total mass of rachis contained in each mesh bag was computed as the sum of the dry weight of rachises used for the fruiting assay and for *H. fraxineus* quantification by qPCR.
### 2.2.1 Fruiting potential and colonization rate

For each plot x cohort x age combination, fifteen rachises from mesh bags were placed in a wet chamber for 8 weeks at ambient temperature (for a total of 720). The bottom of the wet chamber was covered with a paper towel soaked with osmotic water and the lid was perforated to allow oxygenation. Each week, the number of apothecia was counted, the wet chamber was disinfected with 70% alcohol and the paper towel was changed.

After 8 weeks, the rachises were sorted according to their colonization status. Rachises that had developed pseudosclerotial plates (black melanized plectenchyma) were considered to be colonized by *H. fraxineus* while those without were considered as uncolonized. Sorted rachises were dried and weighed. The colonization rate corresponded to the dry mass of colonized rachis divided by the total mass. Fruiting potential was defined as the maximum number of apothecia counted per week divided by the dry weight of colonized rachis (Number of apothecia per g⁻¹).

### 2.2.2 *H. fraxineus* level in rachises

The level of colonization of rachises by *H. fraxineus* was quantified by qPCR. Dried rachises were cut into 5 mm-long pieces and ground using a pestle and porcelain mortar containing liquid nitrogen. From the powder obtained, 3 x 20 mg from each plot x cohort combination were placed in 2 ml microtube with two 3 mm tungsten beads. DNA extraction was performed using the DNeasy plant minikit supplied by Qiagen with some adjustments: 15 mg of PVPP were added for lysis phase and 200 µl of Guanidine thyocyanate were added for DNA precipitation phase to remove any polymerase inhibitors. The elution volume was of 200 µl. Before qPCR, samples were diluted to the tenth with H₂O. The level of colonization by *H. fraxineus* was determined with the qPCR method developed by loos et al. (2009). The 15 µl reaction mixture contained 1x Brillant II qPCR master mix and 0.03 µM ref dye supplied by Agilent Tachnologies. 0.01 U.µl⁻¹ of UDG (New England BioLabs) was added. 0.3 µM each Cfrax primers (Cfrax-F, 5'-ATTATATTGTTGCTTTAGCAGGTC-3' and Cfrax-R, 5'-TCCTCTAGCAGGCACAGTC-3'), 0.1 µM Cfrax-probes (Cfrax-P, 5'-FAM-CTCTGGGCGTCGGCCTCG-BHQ1-3') and 2 µl template DNA. The real time reaction was performed in a Quantstudio 6 thermocycler (Applied Biosystem). The qPCR reaction was initiated by first pre-cycling step at 37°C for 10 min for UDG activation and the initial denaturation step at 95°C for
15 min followed by 50 cycles of denaturation at 95°C for 15 sec and hybridization /elongation at 65°C for 55 sec. A plasmid DNA solution containing the CFRAX region of the *H. fraxineus* genome was cascade diluted by a factor of ten from 16.4 ng.µl⁻¹ of DNA to 1.64 x 10⁻⁷ ng.µl⁻¹ of DNA. This calibration serial dilution was then added to the qPCR tests to quantify *H. fraxineus* DNA.

### 2.2.3 Statistical analysis

The tree species composition of the stands was compared by Principal Component Analysis (PCA) with the basal area of each tree species. Humus type, soil pH, soil C/N, soil texture (clay/sand ratio), ash crown decline and rachis density per ash density were added to the PCA as supplementary variables.

Data from a plot x cohort x age combination was standardized by dividing them by the dry weight at leaf shedding of 60 rachises from the same cohort and plot. The value thus lies within the interval [0, 1] and is an estimate of the proportion of the initial rachis weight that remains at each sampling time. Weight loss was modelled by mixed glm with a logit link and a beta distribution. The fixed effect is the rachis age (incubation time in forest litter, in months) and selected plot characteristics (humus type, ash basal area) and the random factor are plot and cohort (year in which cohort was collected in autumn and plot of origin). The half-life time, corresponding to the number of months required to reach 50% of the initial dry weight was calculated. To assess whether pseudosclerotial rachises decayed at the same rate as non-pseudosclerotial rachises, we assumed that the initial colonization rate was the same for all samples in a cohort, whether left in the litter for 6, 18 or 30 months and we used the colonization rate after 6 months in the litter to characterize each cohort (3 cohorts x 8 plots). We then introduced this initial colonization rate as a fixed effect in the mixed model described above to assess the effect of the colonization rate on the dry weight dynamics.

Quantification of *H. fraxineus* DNA by qPCR and rachis colonization rate between cohorts, age and humus type were analysed by a mixed glm (gamma distribution with a log link and beta distribution with a logit link respectively) with plot of origin as a random effect. Fruiting rate as a function of rachis age and humus type was analyzed using a mixed glm (gamma distribution with a log link) with cohort and plot of origin as random effect.

We assessed the impact of *H. fraxineus* persistence on colonized rachis. To this
end, we developed a simple model to assess the annual cohort composition of the ash rachises colonized by *H. fraxineus* present in the litter in spring in a hypothetic stand located at Champenoux and producing the same quantity of ash rachises each autumn. We assumed that the colonization of the cohort of rachises shed in autumn would depend only on the previous summer's climate. It was shown by Marçais et al. (2023) that the proportion of rachises shed in autumn that are colonized by *H. fraxineus* in the next spring (p) increases with July-August rainfall (Rain78) according to the equation:

$$ \text{logit}(p) = 0.35 + 0.98 \times \text{Rain78} $$

This equation was used to estimate the level of colonization of fallen rachises each autumn, assuming, for simplicity, that the inoculum to which trees are exposed depends only on climate and not on the amount of ash rachis colonized in the litter of the stand and surrounding area. The proportion of rachises produced in a cohort each autumn then decayed at the rate modelled during this work. We assumed that both *H. fraxineus* colonized or uncolonized decay at the same rate, which is an acceptable assumption (see results). We used a boot strap procedure. For each prediction, a set of model parameters was generated both for the colonization rate of rachises shed in autumn and the decay rate in litter models. To do this, we assumed a multinormal distribution for the model parameters using the fitted parameter values and the variance-covariance matrix (R function mvrnorm). A prediction of the colonization rate of each cohort of rachises shed in autumn was done using rainfall data of the INRAE Champenoux weather station for the period 2013-22. The proportion of colonized rachis remaining after 6, 18, 30 and 42 months was computed for each annual cohort of rachises. Then the proportion of colonized rachises of 6, 18 and >=30 months-old in the litter was computed each year in May. This bootstrap procedure was repeated 10000 times.

# 3 Results

## 3.1. Relationship between plots characteristics and rachis density in the litter

The ash stands selected for this study had an ash basal area between 1.1 m².ha⁻¹ and 18.4 m².ha⁻¹ (average 8.1 m².ha⁻¹) and a total basal area between 10.4 m².ha⁻¹ and 36.7 m².ha⁻¹ (average 21.6 m².ha⁻¹). Other trees species in the stands were oak (*Quercus petraea* and *Q. robur*) and hornbeam (*Carpinus betulus*) for four plots, maple (*Acer pseudoplatanus*), lime tree (*Tilia cordata*) for two plots while the remaining two
plots were pure ash stands (Table S1). Soil parameters found in the different stands varied in terms of pH (range 4.7 - 7.1, median 6) and C/N ratio (range 10.6 - 14.2, median 12.5). Soil textures showed a dominance of clay over sand with values of clay/sand ratio below 1 (range 0.09 - 0.46, median 0.29). Humus types were related to

<figure><img src="image_2.png" /><figcaption>**Fig. 1:** Principal component analysis constructed with basal area of each tree species present in the studied stands with as quantitative supplemental data the soil pH, soil C/N ratio, the soil texture, the crown decline of ash and the ratio rachis density/ash density (Rachis / Ash.dens) represented by blue arrows and as qualitative supplemental data the humus type present in the stand (Eumull in red, mesomull in green and oligomull in blue).</figcaption></figure>

the tree species present. The humus type with the highest organic matter degradation efficiency (eumull) were found in stands with high ash, lime and maple densities, while the humus type with less efficient organic matter degradation was found in stands with a high oak and hornbeam densities (Fig. 1). Soil characteristics were related to humus
type. Eumull were found on soil with a higher pH, lower C/N and clay/sand ratio. In contrast, oligomull and mesomull were found on more acidic soil, with higher C/N and clay/sand ratios (Fig. 1). The ratio of rachis density per ash density depends on humus type. It was lower in stands with eumull than in those with mesomull (p-value < 0.001) or oligomull (p-value < 0.001), while no difference was observed between mesomull and oligomull (p-value= 0.7). Thus, fewer rachises were present in the litter at comparable ash densities in the faster-decomposing humus type. For 1 m².ha⁻¹ of ash basal area, rachis density was two times lower in eumull litter than in mesomull or oligomull. At the same period, crown decline increased significantly with ash basal area (coef 0.1 ± 0.04, p-value < 0.01) with no significant difference between humus types (p-value= 0.128). However, the eumull litter was found in stands with higher ash density (Fig. 1).

<figure><img src="image_3.png" /><figcaption>**Fig. 2:** Kinetics of rachis degradation in the litter. Proportion of dry weight of rachis remaining (persistence rate) as a function of time spent in the litter for the three annual cohorts of rachis (black:2019, red:2020 and green:2021, the 8 plots are sampled at each time x cohort combinations). The curve represents the modelled kinetic while the grey area represents the confidence interval (estimated half-life is highlighted by red dotted line).</figcaption></figure>

## 3.2. Dynamic of rachis decomposition

We observed a progressive degradation of the rachis in the forest litter (Fig. 2). After 6 months in the litter, the rachis still appeared intact (segments 7-14 cm long), whereas after 18 and 30 months, they were heavily fragmented (mainly segments 0.5-2 cm long).
Nevertheless, after 30 months, the rachis remaining in the mesh bags still represented 14 ± 5% of the dry weight of those initially present. Modelling of the degradation kinetics enabled us to estimate the half-life of the rachises in the litter at 6.9 months [4.3-12.4].

<figure><img src="image_4.png" /><figcaption>**Fig. 3:** Influence of stand characteristics on rachis degradation. a. Persistence rate of rachises in different ages as a function of ash density. b. Persistence rate of rachises in different ages as a function of humus type.</figcaption></figure>

Ash density had no effect on rachis degradation dynamics, as the residual rachis rate was comparable between low and high ash densities irrespective of rachis age (Fig. 3a). However, the type of humus present in the stand litter had an effect on the residual rachis rate. Indeed, after 30 months in the litter, the residual rachis rate was significantly lower (coef= 1.6, p-value= 9.8.10⁻³) in eumull (mean 0.10 ± 0.06) than in mesomull or oligomull (mean 0.18 ± 0.03) (Fig. 3b). The rate of rachises degradation was not
significantly related to the initial *H. fraxineus* colonization as assessed by the proportion of pseudoclerotial rachises at 6 months (p-values for the main effect of initial *H. fraxineus* colonization rate and its interaction with incubation time in the litter of 0.85 and 0.46).

<figure><img src="image_5.png" /><figcaption>**Fig. 4:** Level of *H. fraxineus* in rachises cohorts of the ash stands. a. Quantification of *H. fraxineus* DNA in rachis tissues just after fallen leaves during the three years of the study. b. Evolution of the rate of *H. fraxineus* colonization in the rachises of the three studied cohorts at different ages. (Red point: rachises from eumull litter, blue point: rachises from meso or oligomull litter, there is no data for 30 months for cohort 2020 and for 18-30 months for cohort 2021).</figcaption></figure>

The amount of *H. fraxineus* DNA in rachises at the leaf shedding differed between the three cohorts analyzed. It was similar (p-value= 0.57) in the 2019 (8.6 10⁻⁶ ± 9.4.10⁻⁶ ng.mg⁻¹) and 2020 cohorts (4.5.10⁻⁶ ± 4.3.10⁻⁶ ng.mg⁻¹). On the other hand, in cohort 2021, amount of *H. fraxineus* DNA in rachises was higher with a mean value of 3.7.10⁻⁷
⁵ ± 2.2·10⁻⁵ ng.mg⁻¹ (p-value < 0.05, Fig 4a). The rate of rachis colonization was strongly correlated with the amount of *H. fraxineus* DNA detected by qPCR (coef= 0.23, p-value < 0.001). In addition, the quantification of DNA, in autumn in shed leaves, was a good predictor of the level of rachis colonization six months later in a given year (coef= 0.13 ± 0.04 p-value < 0.001, Figure S1). However, this highly depended on the year: Figure 4 shows that with a similar low level of *H. fraxineus* detected in autumn rachises, colonization in spring was very different for the 2019 and 2020 cohorts. After 6 months in the litter, the 2019 and 2021 rachis cohorts showed similar colonization rates (respectively 0.43 ± 0.15 and 0.39 ± 0.10, p-value= 0.5), while the 2020 cohort was significantly less colonized (0.20 ± 0.17 p-value < 0.001). The colonization rate decreases with rachis age for the 2019 cohort (mean of 0.30 ± 0.20 at 18 months and 0.18 ± 0.18 at 30 months, coef= -0.05 p-value < 0.001), while it remained stable for the 2020 cohort (mean 0.25 ± 0.65 at 18 months p-value= 0.5). Quantification of *H. fraxineus* DNA at leaf fall and colonization rate were not significantly related to humus type (respective p-values 0.96 and 0.77).

<figure><img src="image_6.png" /><figcaption>**Fig. 5:** Dynamic of *H. fraxineus* fruiting potential on colonized rachis incubated in the forest litter (expressed as the number of apothecia per dry weight of colonized rachis). Red point: rachises from eumull litter, blue point: rachises from meso or oligomull litter.</figcaption></figure>

The fruiting potential averaged 220.5 ± 107.6 apothecia.g⁻¹ for rachises 6 months old (Fig. 5). The fruiting potential of colonized rachises remained stable as the rachises aged, with a mean of 190.2 ± 103.2 apothecia.g⁻¹ for 18 months-old rachises (p-
value= 0.86) and 116.6 ± 84.6 apothecia.g⁻¹ for 30-months-old rachises (p-value= 0.54).

Humus type had no significant effect on fruiting potential (p-value= 0.53, Fig. 5).

<figure><img src="image_7.png" /><figcaption>**Fig. 6:** Simulation of cohort composition of rachises colonized by *H. fraxineus* present in the litter in spring of a hypothetical stand of Northeast France where the amount of shed ash rachis in autumn remains constant.</figcaption></figure>

**3.3. Modelling the impact of colonized rachis persistence**

The results of our model estimating the cohort composition of litter rachis in a
hypothetical stand in northeastern France supplying a similar quantity of ash rachis
each year are shown in Fig. 6. The relative importance of the most recent cohort (shed
in the previous autumn) depends strongly on the past climatic sequence, with values
ranging from 40% to 70%. In spring 2022, most of the colonized litter rachis came from
the previous autumn's cohort. The summer of 2021 was very rainy, and the estimate of
the percentage of rachis shed in autumn colonized by *H. fraxineus* was high, with a value
in the [90.6, 99.9] confidence interval. In contrast, the older cohorts of 2018-20 had been
poorly colonized after dry summers (colonization proportion in the [43.7, 69.4], [34.1,
54.9] and [18.1, 37.7] confidence interval respectively). Conversely, in spring 2021, the
different cohorts were equally represented in the litter: the 2020 cohort was poorly
colonized by *H. fraxineus* ([18.1, 37.7]), while the 2017 cohort had very well colonized
([77.5, 99.0]). Despite heavy weight loss after 4 years in the litter, the 2017 cohort was
still predicted to contribute significantly to the pool of colonized rachis in spring 2021.
The persistence of colonized rachises for several years should therefore strongly
stabilize the pool of colonized rachises in the litter, enabling sustained production of
*H. fraxineus* ascospore.

4 Discussion

The persistence of *H. fraxineus* on rachis over several years had already been
documented *ex situ* (Gross & Holdenrieder, 2013; Kirisits, 2015). The present study
provides new insights into the kinetics of degradation *in situ*, in forest litter, over 30
months and its potential modulation by several environmental factors. We show that
depending on the year, rachis cohorts older than two years can contribute as much to
the forest litter inoculum pool as the most recent rachis cohort, thus representing a
significant reservoir.

The density of rachis found in litter depended on humus type, with lower density in the eumulls for similar ash densities. This is consistent with the more rapid decay of rachises in this humus type, with a lower proportion of the initial rachis dry weight of rachis remaining after 30 months compared with other humus types (Ponge, 2003). However, the relationship between rachis density in litter and humus type is probably complex. Stands with eumull-type humus generally have a higher ash basal area, which, on average, leads to more severe ash dieback (Grosdidier et al., 2020; Laubray et al., 2023). While the amount of rachis produced should increase with ash basal area, increased ash dieback should decrease the amount of rachis produced. However, depending on past dieback dynamics and climate sequence, it is possible that colonized rachises from past cohorts may still be abundant in the litter.

In addition, the tree species present in the stand affect litter composition. Maple,
lime and ash leaves provide fast-decomposing litter compared to oak, beech or
hornbeam (Mitchell et al., 2016). Although ash rachis decomposes much more slowly
than leaflets, their rate of decomposition still depends on the overall composition of the
litter. In mull-type humus, especially eumull, litter fragmentation is mainly carried out by
macroinvertebrates (Jabiol et al., 2007; Ponge, 2003). Indeed, we observed that
rachises became strongly fragmented between months 6 and 18. Dahlsjö et al. (2023)
have shown that ash leaves are highly consumed by macroinvertebrates (woodlouse,
earthworm and millipedes), while rachis fragmentation is reduced mixed with less
suitable leaves from oak or beech. These two species have leaves with higher lignin
composition and C/N ratio than ash (Steffens et al., 2012), making them less palatable
to macroinvertebrates. Conversely, lime leaves have a lignin composition similar to that
of ash, which improves the palatability of the litter (Steffens et al., 2012). Under
controlled conditions, ash leaves decompose more rapidly when mixed with lime leaves
while they decompose more slowly when mixed with hornbeam leaves (Bartha et al.,
2017). In addition, there may be a "Gadgil effect" in stands with numerous
ectomycorrhizal fungi (ECM). The Gadgil effect is a reduction in litter decomposition
associated with presence ECM fungi (Gadgil and Gadgil, 1975). While saprotrophic
fungi mobilize carbon from litter and soil organic matter, ECM fungi are supplemented
with carbon by their host. This symbiosis makes them more competitive than
saprotrophic fungi to extract nitrogen from litter. The presence of ECM fungi tends to
increase the C/N ratio of the litter and may release chemical inhibitors limiting the
development of saprotrophic fungi (Fernandez & Kennedy, 2016; Gadgil & Gadgil,
1971). While ash and maple are primarily associated with endomycorrhizal fungi, oak,
hornbeam and lime are ECM species (Brundrett & Tedersoo, 2020). Interannual
variability in decomposition kinetics could be explained meteorological conditions,
particularly winter precipitations during the first months of decomposition. For leaf litter,
low precipitation reduces the rate of mass loss (Austin & Vitousek, 2000; O'Neill et al.,
2003; Staley et al., 2007) and leaching losses are reduced when precipitation is low
(Salamanca et al., 2003).

The production of a pseudosclerotic plaque on rachises colonized by *H. fraxineus*
was expected to reduce the rate of decomposition. The pseudosclerotial plate is
probably due to the synthesis of a melanin pigment by the fungus (Butler & Day, 1998),
which plays a protective role in preventing desiccation and UV damage to the fungal
hyphae (Butler & Day, 1998; Campbell, 1934; Campbell, 1933; Pearce, 1991). It also
protects the pseudosclerotial rachis from the development of antagonistic fungi (Adams
& Roth, 1969). However, no conclusive effect of the proportion of pseudosclerotial
rachises on the rate of decomposition was found. The persistence of *H. fraxineus* on the
rachises should necessitate the consumption of organic matter and contribute to their
decomposition. On the other hand, rachises not colonized by *H. fraxineus* should harbor
more efficient saprotrophic fungi decompose more rapidly. It should be noted that
between 6 and 18 months, we observed a high fragmentation of the rachises,
presumably due to macroinvertebrates in the litter. The pseudosclerotial plate may be
much less effective in protecting the rachis from macroinvertebrate activity. The effect of *H. fraxineus* colonization on rachis degradation may depend on various factors, such as soil pH. Soil moisture favors rachis colonization by *H. fraxineus* (Grosdidier et al., 2020) and impact melanization (Tudor et al., 2013), but also litter decomposition (Krishna & Mohan, 2017) and rachis palatability for macroinvertebrates (earthworms, millipedes and woodlice) (Dahlsjö et al., 2023).

We found that the quantity of *H. fraxineus* DNA in autumn was poorly related to the colonization rate of the pathogen in spring, 6 months later. Low level of *H. fraxineus* DNA in fallen rachises in autumn may result in very different colonization rate in the next spring depending on the year cohort. It would appear that the final colonization of rachises by *H. fraxineus* depends on climatic parameters not only during the season, but also after shedding in late autumn and winter. The colonization of the rachises during the following spring is highly dependent on rainfall in July and August, a favorable condition for increasing ascospore production and, consequently, leaf infection (Marçais et al., 2023). Conversely, leaf colonisation is limited by summer temperatures above 35°C (Grosdidier et al., 2018; Hauptman et al., 2013). Winter conditions could also be decisive for the colonization of rachises by *H. fraxineus* in the litter. Other parameters may affect the colonization of the rachis by *H. fraxineus* such as microbial community present on the leaves before and after the shedding.

The persistence of *H. fraxineus* on pseudoclerotial rachises is even more important for the pathogen as such rachises retain ability to produce apothecia and thus inoculum for at least 30 months. Indeed, this study shows that one gram of pseudosclerotial rachis has the same potential for spore production whether it is 6, 18 or 30 months old. It had been demonstrated that ash pseudosclerotial rachises kept in bulk in plastic containers could produce apothecia up to the age of 5 years (Gross & Holdenrieder, 2013; Kirisits, 2015) but not pseudosclerotial rachises left in the forest litter and subject to optimal conditions for decay. The accumulation, year after year, of *H. fraxineus* pseudosclerotial rachises that retain their ability to produce inoculum constitutes a reservoir that favor disease development. Populations of *H. fraxineus* are subject to dependence on the density of colonized rachises in the litter for apothecia production (Laubray et al., 2023). A low density of colonized rachis in the litter reduces apothecia production (Allee effect). The long persistence of pseudosclerotial rachises in the litter should help *H. fraxineus* to reach the Allee threshold above which production of
apothecia no longer depends on colonized rachises density. Our model is very simple and does not take into account all important mechanisms. In particular, if less rachises colonized in the litter are present after dry years, we may expect a lower number of fruiting bodies and thus less spores to which the trees are exposed even if climate is favorable. This is probably the larger shortcoming of the model and should led to underestimation of the lag effects of dry years. Despite that, the model clearly shows that a long persistence of colonized rachises in the litter may have the effect of stabilising the inoculum pool. The level of colonization of leaves shed in autumn strongly depends on summer rainfall. NE France, experienced very dry and hot summers in the 2018-20 period, which resulted in poor colonization by *H. fraxineus* of the rachis cohort produced these years. The presence of an inoculum reservoir of pseudosclerotial rachises in the litter probably allowed the epidemic to be rekindled after such unfavorable year for the pathogen's foliar development. This was observed in NE France 2021 after the very wet summer.

We thus confirm that *H. fraxineus* survives in rachises incubated *in situ* in the forest litter and keep the ability to produce apothecia for several years. However, the mass loss of rachis is large, in particular associated with a high fragmentation after the first year. We showed that this has important consequences for the biology of the pathogen, stabilizing the inoculum level after unfavorable years (dry summers).

# 5 Acknowledgements

We wish to thank Olivier Caël, Anaïs Gillet, Arielle Beltran and Nelly Perrachia for their technical assistance.

This work was funded by the project HOMED from the European Union's Horizon 2020 Program for Research & Innovation under grant agreement No 771271. The UMR1136 research unit is supported by a grant managed by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (ANR-11-LABX-0002-01, Laboratory of Excellence ARBRE).

The authors have no conflict of interest to declare.

Data availability statement: The data that support the findings of this study are available on request from the corresponding author.
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## Supplemental information

<table><caption>**Table S1.** Plot characteristics</caption><thead><tr><th>Site</th><th>Ash presence</th><th>Coordinates (EPSG 4326)</th><th>Ash basal area (m².ha⁻¹)</th><th>Humus</th><th>Soil pH</th><th>Tree species present</th></tr></thead><tbody><tr><td>Champenoux</td><td>low</td><td>6.31986, 48.75379</td><td>1.1</td><td>Oligomull</td><td>5.8</td><td>C. betulus<br>Quercus spp.</td></tr><tr><td>Champenoux</td><td>high</td><td>6.31881, 48.74247</td><td>18.3</td><td>Eumull</td><td>7.1</td><td>C. avellana</td></tr><tr><td>Languimberg</td><td>low</td><td>6.88423, 48.72461</td><td>1.6</td><td>Oligomull</td><td>4.7</td><td>C. betulus<br>Quercus spp.</td></tr><tr><td>Languimberg</td><td>high</td><td>6.88955, 48.72244</td><td>7.5</td><td>Mesomull</td><td>5.5</td><td>C. betulus<br>Quercus spp.</td></tr><tr><td>Sélestat</td><td>low</td><td>7.47778, 48.22509</td><td>1.5</td><td>Eumull</td><td>6.3</td><td>A. pseudoplatanus<br>T. cordata</td></tr><tr><td>Sélestat</td><td>high</td><td>7.46157, 48.20983</td><td>11.6</td><td>Eumull</td><td>6.7</td><td>A. pseudoplatanus</td></tr><tr><td>Thionville</td><td>low</td><td>6.09203, 49.38827</td><td>5.6</td><td>Mesomull</td><td>5.8</td><td>C. betulus<br>Quercus spp.</td></tr><tr><td>Thionville</td><td>high</td><td>6.09643, 49.38599</td><td>17.6</td><td>Eumull</td><td>6.7</td><td>-</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>**Fig. S1:** Relation between the level of *H. fraxineus* in rachises cohorts at 6 months according to quantification of *H. fraxineus* DNA in rachis tissues just after leaf shedding for the 3 cohort with their regression curve and dotted confidence interval (red:2019, green:2020 and blue:2021)</figcaption></figure>