Ampelomyces hyperparasites – occurrence and effect on the development of ascomata of Erysiphales species under conditions of anthropopressure

Species of the genus Ampelomyces Ces. ex Schltdl. are wide-spread hyperparasites found on more than 65 Erysiphales species [1]. Their hyphae penetrate the vegetative mycelium as well as conidiophores and conidia of the host fungus, thus forming inside them the spore-forming structures, i.e. pycnidia [2]. Hyperparasites may also colonize ascogonia and antheridia as well as young, immature chasmothecia of powdery mildews, transforming them into their own reproductive structures. Infested ascomata do not reach the stage of maturity, do not form appendages nor ascospores, which is linked with the reduction of the source of primary infections [3–7]. The capability of hyperparasites to infest different Erysiphales species, to suppress their sporulation as well as to inhibit the formation and development of chasmothecia has contributed to the advance of research addressing mainly the aspect of the application of Ampelomyces fungi in the biological control of powdery mildews [1,8–11]. From the economic point of view, Erysiphales species constitute a significant group of phytopathogens of multiple fruit plants and crops. Of high interest to researchers world-wide is also the problem of the genetic diversity of fungi of the genus Ampelomyces, which is still little known and requires in-depth examinations [12–16]. World and Polish literature provides a lot of information about presence Ampelomyces fungi on different Erysiphales species in natural condition ([11,17–24]. Relatively few investigations were focused on the ecology of these hyperparasites and their natural occurrence on various Erysiphales species under urban condition [25–27]. Therefore, the Department of Mycology of University of Warmia and Mazury in Olsztyn began studies on the occurrence of fungi from the genus Ampelomyces and their impact on the development Erysiphales in urban environment [28–30]. Data is also lacking on the effect of Ampelomyces fungi on the development of fruiting bodies of different species of powdery mildews infecting non-farming plants [29]. Works addressing this issue are devoted almost exclusively to a dangerous pathogen of grapevine – Erysiphe necator (Uncinula necator) – [3,5,6,31]. An analysis of the colonization of Erysiphales ascomata by Ampelomyces as well as the effect of mycoparasites on their development seems to be crucial in understanding the role of such antagonists in the natural dynamics of a powdery mildew population in its natural environment. The objective of the study was to evaluate the occurrence of fungi of the genus Ampelomyces and their effect on the development of ascomata of species of powdery mildews in the urban environment. Abstract


Introduction
Species of the genus Ampelomyces Ces.ex Schltdl.are wide-spread hyperparasites found on more than 65 Erysiphales species [1].Their hyphae penetrate the vegetative mycelium as well as conidiophores and conidia of the host fungus, thus forming inside them the spore-forming structures, i.e. pycnidia [2].Hyperparasites may also colonize ascogonia and antheridia as well as young, immature chasmothecia of powdery mildews, transforming them into their own reproductive structures.Infested ascomata do not reach the stage of maturity, do not form appendages nor ascospores, which is linked with the reduction of the source of primary infections [3][4][5][6][7].
The capability of hyperparasites to infest different Erysiphales species, to suppress their sporulation as well as to inhibit the formation and development of chasmothecia has contributed to the advance of research addressing mainly the aspect of the application of Ampelomyces fungi in the biological control of powdery mildews [1,[8][9][10][11].From the economic point of view, Erysiphales species constitute a significant group of phytopathogens of multiple fruit plants and crops.Of high interest to researchers world-wide is also the problem of the genetic diversity of fungi of the genus Ampelomyces, which is still little known and requires in-depth examinations [12][13][14][15][16].
World and Polish literature provides a lot of information about presence Ampelomyces fungi on different Erysiphales species in natural condition ( [11,[17][18][19][20][21][22][23][24].Relatively few investigations were focused on the ecology of these hyperparasites and their natural occurrence on various Erysiphales species under urban condition [25][26][27].Therefore, the Department of Mycology of University of Warmia and Mazury in Olsztyn began studies on the occurrence of fungi from the genus Ampelomyces and their impact on the development Erysiphales in urban environment [28][29][30].Data is also lacking on the effect of Ampelomyces fungi on the development of fruiting bodies of different species of powdery mildews infecting non-farming plants [29].Works addressing this issue are devoted almost exclusively to a dangerous pathogen of grapevine -Erysiphe necator (Uncinula necator) - [3,5,6,31].
An analysis of the colonization of Erysiphales ascomata by Ampelomyces as well as the effect of mycoparasites on their development seems to be crucial in understanding the role of such antagonists in the natural dynamics of a powdery mildew population in its natural environment.
The objective of the study was to evaluate the occurrence of fungi of the genus Ampelomyces and their effect on the development of ascomata of species of powdery mildews in the urban environment.

Material and methods
The mycological research was conducted in 2005-2009 in several cities of the northeastern Poland: Olsztyn, Frombork, Giżycko, Mrągowo and Kętrzyn.The experimental material were leaves of plants infested with Erysiphales fungi.The material were collected from different places of cities (parks, gardens, street and community lawns) always in the second half of the vegetative season (August-October), when mature fruiting bodies were visible on white mycelium.Ten leaves with symptoms of infestation by powdery mildews, randomly collected from each host plant, served as one sample.
Laboratory analyses were conducted to verify the presence of Ampelomyces fungi on the mycelium of the Erysiphales species examined.Stereomicroscope (Olympus SZX9) and light microscope (Olympus BX51) used to observe the mycelium, chasmothecia of Erysiphales species and hyperparasites from the genus Ampelomyces.Camera XC 50 and analytical software Analysis were used for graphic processing (documentation, measurement).

No.
Erysiphales species Host plants

Samples with
Erysiphales species
Each infested leaf in a sample was observed under a stereomicroscope.Once hyperparasites were detected (symptoms of the presence of Ampelomyces fungi: change of mycelium color into brown, the presence of pycnidia on the mycelium), microscopic analyses were conducted for 10 young (yellow and orange pigmentation, lack of appendages) and 10 mature ascomata (brown pigmentation, branched appendages) collected at random from the surface of mycelium non-infested and infested by the hyperparasites.These analyses involved determinations of: (i) morphological features of mature ascomata (diameter, wall peridium pigmentation, the number and length of appendages); (ii) the development of asci with ascospores of mature ascomata (the presence of asci with ascospores; asci without ascospores; a lack of asci and ascospores); (iii) the presence of spores of Ampelomyces fungi in young and mature ascomata.
The fungi were identified by means of Braun's key [32].The taxonomy and nomenclature were adopted after Braun and Takamatsu [33] and Braun et al. [34].The host plants were identified using keys by Rutkowski [35].The nomenclature was adopted after Mirek et al. [36].
In the case of all powdery mildews investigated, the analysis of the presence of Ampelomyces species in ascomata demonstrated the hyperparasites to infest young fruiting bodies without developed appendages.In 25 analyzed species of Erysiphales, fungi from the genus Ampelomyces infested only young ascomata.In turn, in two species: Erysiphe flexuosa and E. vanbruntiana var.sambuci-racemosae the hyperparasites were observed in young and mature chasmothecia with fully developed appendages (Fig. 1, Fig. 2).In the case of Erysiphe flexuosa, Ampelomyces fungi infested mature ascomata in 50% of the samples, whereas in the case of E. vanbruntiana var.sambuci-racemosae in 79%.
In both analyzed species some differences were noted in the morphological characteristics of mature ascomata collected from the surface of mycelium non-infested and infested by fungi from the genus Ampelomyces.The diameter of mature ascomata is smaller, wall peridium pigmentation is paler and the number of appendages is smaller in the infested ascomata than the un-infested ones (Tab.2).
On both surfaces, analyses demonstrated the prevalence of chasmothecia with fully-developed appendages (>80%).In contrast, differences were observed in the development of asci and ascospores in the ascomata.In the case of E. flexuosa, analyses showed 90% of ascomata with developed asci and ascospores on the surface of mycelium free of hyperparasites and 72% of these on the surface of mycelium colonized by Ampelomyces fungi.The latter mycelium was also characterized by 7% contribution of mature ascomata infested by the hyperparasites and 16% contribution of empty ascomata, without any asci nor ascospores (Tab.3).In the case of E. vanbruntiana var.sambuci-racemosae, the difference in the development of asci and ascospores turned out to be alike, i.e. 87% of the ascocarps containing asci and ascospores on the surface of mycelium without Ampelomyces fungi and as little as 60% of these on the surface of infested mycelium.In the latter, analyses showed additionally a very high percentage (38%) of Ampelomyces species in mature ascomata as well as 1% of empty chasmothecia (Tab.3), which results in nearly 40% reduction of ascomata.

Discussion
Fungi of the genus Ampelomyces are considered to be major antagonists of Erysiphales species [11].The range of their occurrence corresponds with the range of powdery mildews, which points to the ecological specialization of these hyperparasites.This has been confirmed in our study, where fungi of the genus Ampelomyces were noted on all investigated species, and with a high contribution in the samples (>50%).While infesting the host fungus, these hyperparasites colonize the mycelium, the conidial stages and young, yet immature fruiting bodies [3,5,17,25,32].Ample investigations [5,31,37] have shown that Ampelomyces species are capable of infecting chasmothecia only at the early stage of their development, before the formation of appendages and development of walls.Young fruiting bodies are, obviously, susceptible to infections, whereas the mature ones are seemingly resistant.However, results of these investigations are only in part consistent with our findings.In the powdery mildews analyzed, the hyperparasites were very often detected only on the perfect stage and infested mainly young ascomata in various developmental stages -from white to orange ones with undeveloped appendages or at the early stages of development.They were transforming them into their own reproductive structures, yielding abundant production of conidial spores.In contrast, in the case of two species, Erysiphe flexuosa on Aesculus spp.and E. vanbruntiana var.sambuci-racemosae occurring on Sambucus racemosa, the presence of Ampelomyces fungi was additionally detected in mature ascomata with fully developed appendages, with an especially high percentage reaching 38% in E. vanbruntiana var.sambuci-racemosae.This is the first documented case of this phenomenon.The worldwide literature does not provide any information on the colonization of fully mature fruiting bodies by the hyperparasites.The presence of Ampelomyces fungi on apparently mature fruiting bodies of Erysiphe necator (Uncinula necator) was mentioned by Füzi [11].This author described infested fruiting bodies as apparently mature due to the observed residual appendages and speculated on the terminated process of peridium formation before the infection with the hyperparasites.
Infested fruiting bodies may be recognized by their fawn, milk-brown color and smaller sizes [5,6].Results of our study confirm these changes of the colonized mature ascomata both in E. flexuosa as well as in E. vanbruntiana var.sambuci-racemosae.
However the infested ascomata possessed well developed appendages (>80%), whose development was very often synchronized with the development of asci and ascospores.In our study, apart from conidia of the hyperparasites, no other residues of those structures were observed in the ascomata.Therefore, it remains unclear when the chasmothecia are being infected.Being endoparasites, Ampelomyces fungi colonize structures of the host-fungi from the inside.The infection of fruiting bodies proceeds through thin hyaline hyphae of the mycelium that penetrate into the male sexual cell and ascogonium as well as into young chasmothecia, thus inhibiting their development [5].The presence of fully developed appendages probably indicates the infection of ascomata at the final stage of their development.
The colonization of mature fruiting bodies is, probably, a phenomenon pointing to the adaptation of Ampelomyces species to a better overwintering and dissemination.Falk et al. [4] report that the survivability of Ampelomyces fungi in young fruiting bodies reaches as little as 1-3%.Thus, it may, be speculated on a low effectiveness of these hyperparasites in their natural environments, which has been proved by annual epiphytoses induced by powdery mildews.The low survivability of Ampelomyces fungi is certainly due to the immature wall of ascomata which at this developmental stage are not capable of surviving low temperatures.This has been confirmed by Mmbaga [38], who reported that fruiting bodies with just partly developed walls are not capable of surviving the winter.Therefore, the stage of colonizing mature chasmothecia -that completed the process of peridium formation and possess well developed appendages -by the hyperparasites seems to be understandable.Probably it provides suitable conditions for the hyperparasites for a better overwintering and transmission in their environments.The improvement of the adaptation Tab. 3 Percentage participation of mature ascomata with different degree of development of asci and ascospores from surface of mycelium non-infested and infested by Ampelomyces fungi.
strategy of Ampelomyces fungi may lead to changes in the dynamics of an Erysiphales population.Thus, further researches of the occurrence of these hyperparasites of powdery mildews in their natural environment are advisable.They will enable recording the direction of those changes in a complicated system, viz.plant -parasite -hyperparasite.
The list of Erysiphales species and host plants.