The occurrence of Hymenoscyphus pseudoalbidus apothecia in the leaf litter of Fraxinus excelsior stands with ash dieback symptoms in southern Poland

The paper presents the results of a study performed in 28 ash stands located in Myśleniece and Dynow Forest Districts in southern Poland. The intensity of Fraxinus excelsior disease process was estimated based on the disease symptoms analysis of 1400 trees. The amount of infectious material of Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea ) was estimated by the leaf litter examination on 166 square (0.5 × 0.5 m) plots. The total number of ash leaf rachises and apothecia were counted. The analysis shows that the estimated number of H. pseudoalbidus apothecia may reach from 370 thousand to over 13 million per hectare at a time.


INTRODUCTION
For the past twenty years intense symptoms of European ash (Fraxinus excelsior L.) dieback could have been observed in Europe.While originally, this phenomenon was restricted to Poland and Lithuania (Sierota et al. 1993;Grzywacz 1995;Kowalski 2001;Stocki 2001;Przybył 2002;Vasiliauskas et al. 2006), the disease spread consequently to reach ashes in Scandinavia, western Europe, and some regions of southern Europe (Kirisits et al. 2009;Ioos et al. 2009;Timmermann et al. 2011;Hauptman et al. 2012).In 2012 the ash dieback symptoms were also reported from British Isles (Coghlan 2012).The fungus Chalara fraxinea T. Kowalski is believed to be the main causal agent of ongoing ash dieback process.This species was originally described based on cultures isolated from necrotic areas on ash shoots collected in Włoszczowa Forest District (Kowalski 2006(Kowalski , 2007)).The artificial inoculation of ash shoots proved its high pathogenicity (Bakys et al. 2009;Kowalski, Holdenrieder 2009a).The occurrence of Chalara fraxinea was confirmed in more than twenty European countries where the ash dieback symptoms were observed (Halmschlager, Kirisits 2008;Ioos et al. 2009;Timmermann et al. 2011).Further studies revealed that Chalara fraxinea is an anamorphic state of fungus from the genus Hymeno scyphus.It was initially believed that it was Hymenoscyphus albidus (Robergere ex Desm.)W. Phillips, known in Europe since 1851 (Kowalski, Holdenrieder 2009b).
This opinion was however changed on the basis of molecular analyses which showed that this species is comprised of two, genetically distinct, subpopulations.They are hardly distinguishable morphologically, but they differ significantly in their potential to generate disease symptoms.Eventually the highly virulent subpopulation of H. albidus was recognized to be the teleomorphic state of Chalara fraxinea and described as a new cryptic species Hymenoscyphus pseudoalbidus Queloz et al. (Queloz et al. 2011).It produce the apothecia on previous year ash leaf rachises in the stand floor.The molecular studies based on two-hundred and thirty apotheciaderived cultures originating from four regions of Poland, confirmed the presence of highly virulent H. pseudoalbidus in declining ash stands while H. albidus was not found (Kraj, Kowalski 2013).The apothecia of H. pseudoalbidus are produced mainly from early July to the end of September (Kowalski 2012).This is also the season when apothecia release the ascospores that infect ashes and initiate their disease process.The number of H. pseudoalbidus apothecia produced in the stand floor indicates the amount of infectious material and consequently the risk level for Fraxinus excelsior stands.
The primary goal of the study was the estimation of the overall number of H. pseudoalbidus apothecia that may be produced on ash rachises in the leaf litter in the selected stands in southern Poland.Additionally, in order to correlate their presence with the stand health, the intensity of the selected disease symptoms for this stands was estimated.

MATERIALS AND METHODS
The study was performed in August 2011 in 14 stands of Myślenice and 14 stands of Dynów Forest Districts.The selected stands represented mostly fresh upland broadleaved forest habitat, but they varied in age.The percentage of ash ranged from 40 to 90% for most stands, as only a few of them were characterized by lower proportion of ashes (Tabs 1, 2).The stands were selected randomly using information from forest management plans.Fifty centrally located neighboring trees of Fraxinus excel sior were examined for their health condition in each stand.Each examined tree was categorized as: i/ dead, ii/ with visible disease symptoms, or iii/ with no macroscopic disease symptoms.Furthermore, additional data were recorded for all trees classified as type ii: the presence of local trunk necroses, the presence of dead top, and the crown condition measured by the percentage of dead branches ranked as: below 25%, 26-50%, 51-75% and above 76% (Fig. 1).In total, the health condition evaluation was carried out for 1400 trees, 700 per each of two forest districts (Tabs 1, 2).

DISCUSSION
The characteristic symptoms of ash dieback were observed in all analyzed stands of Myślenice and Dynów Forest Districts.The nature of these symptoms was the same as the ones observed in the other parts of Poland (Kowalski, Czekaj 2010), or in other European countries (Ioos et al. 2009;Kirisits et al. 2009;Hauptman et al. 2012).However, the intensity of disease process, measured by the number of trees with high proportion of dead branches, trees with dead tops, or entirely dead trees varied significantly between two forest districts or even between stands of particular forest districts.These differences result from the age of trees, habitat properties, and the ash susceptibility (Gil et al. 2006;Jaworski 2011;McKinney et al. 2011;Kowalski et al. 2012;Stener 2013).Local conditions are also an important factor that affects the formation of the infectious material reservoirs of H. pseudoalbidus (anamorph: Chalara fraxinea), the ash dieback causal agent (Kirisits et al. 2009;Holdenrieder 2012;Kowalski et al. 2012).
According to previous research, the Chalara fraxinea conidia cannot germinate and the F. excelsior trees are infected exclusively by wind-borne ascospores, that are produced in apothecia on ash leaf residues in the litter (Kirisits et al. 2009;Kowalski 2012;Gross, Holdenrieder 2013).Only occasionally apothecia may develop on dead ash shoots (Kowalski et al. 2010;Holdenrieder 2012).Beside shoots, also leaves are infected by ascospores.The H. pseudoalbidus infected rachises of fallen leaves darken due to formation of melanized, stroma-like plectenchyma that functions as protection against competing microorganisms, and against unsuitable environmental conditions (Gross, Holdenrieder 2013).All H. pseudoalbidus apothecia-carrying rachises, as well as many apothecia-free ones, were covered by plectenchyma.The total numbers of ash rachises on experimental plots of particular analyzed stands were varied.This resulted mainly from the various proportion of ash in the species composition of the stand, but also from the health condition of trees, especially from the level of foliage reduction.On the other hand, the relationship between the total number of rachises on plots and the number of apothecia-carrying rachises may be affected by many factors.
The development of apothecia may be delayed or even prevented by excessively dry conditions (Holdenrieder 2012;Kowalski et al. 2012).Furthermore, H. pseudo albidus is a heterothalic fungal species, thus the apothecia formation requires the infected rachis to contain both mating types (Gross et al. 2012).Finally, the proportion of apothecia-carrying rachises depends on the on-tree leaf infection frequency by this fungus.If the leaves do not fall due to disease process but naturally, or the premature leaf falling is the result of the trunk-or the basal-branch-part necrosis, the proportion of H. pseudoalbidus infected rachises is lower.The above factors were most probably the reasons why the total number of apothecia counted on 82 plots in Myślenice Forest Districts exceeded threefold their number on 84 plots in Dynów Forest District.Those factors probably affected also the number of apothecia per 1 m 2 estimated for various stands, as they varied significantly in both forest districts.These differences are evidenced by data showed in tables 1 and 2. The data, when extrapolated to the area of 1 hectare, show even more explicitly that the amount of infectious material in stands with the presence of F. excelsior is huge.In fact, the number of the apothecia produced in a given stand is even greater, as the apothecia formation on the particular rachis is not a one-off exercise but a continuous process.While some apothecia are fully developed and release ascospores, some others are in initial, hardly visible, stage that can be seen on Figure 6.In Poland, the H. pseudolabidus apothecia are produced for about 3 months usually from July to September.

Fig. 6 .
Fig.6.Fragment of ash leaf rachis with one mature apothecium and several apothecia in the initial stage of development.

Table 1
The occurrence of disease symptoms on Fraxinus excelsior and the occurrence of the H. pseudoalbidus apothecia on ash leaf rachises in the litter in Myślenice Forest District