Research Collection. The identity and insect-mediated reproduction of systemic rust infections of Berberis vulgaris. Master Thesis.

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1 Research Collection Master Thesis The identity and insect-mediated reproduction of systemic rust infections of Berberis vulgaris Author(s): Naef, Andreas Publication Date: 2000 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library

2 THE IDENTITY AND INSECT-MEDIATED REPRODUCTION OF SYSTEMIC RUST INFECTIONS OF BERBERIS VULGARIS Diploma Thesis Andreas Naef September 2000 Referents: Prof. Dr. B. A. Roy & Dr. D. Siemens Geobotanical Institute, Swiss Federal Institute of Technology (ETH) Zürich, Switzerland

3 TABLE OF CONTENTS ABSTRACT...1 INTRODUCTION...2 MATERIALS AND METHODS...5 Hosts and pathogens...5 Fungal collection and identification...6 Molecular Methods...7 DNA Extraction...7 PCR amplification, cleaning, and sequencing...7 Sequence analysis...8 Field work...9 Infection incidence...9 Insect exclusion experiment...10 Insect observations...11 RESULTS...13 Identity of the systemic infection...13 The primary host...13 Phylogenetic analysis...14 Populations of infected Berberis vulgaris in Switzerland...18 Sequence variation of the pathogen...18 Infection incidence...19 Insect-mediated reproduction...22 Insect exclusion experiment...22 Insect observations...23 DISCUSSION...30 Identity of the systemic infection...30 The primary host...30 Phylogenetic analysis...31 Populations of infected Berberis vulgaris in Switzerland...31 Sequence variation...31 Infection incidence...32 Insect-mediated reproduction...33 Insect exclusion experiment...33

4 Insect observations...34 Conclusions...36 ACKNOWLEDGEMENTS...37 REFERENCES...38 APPENDIX A (Identification of rust fungi)...40 APPENDIX B (Sequence alignement)...41

5 ABSTRACT Witches brooms on Berberis vulgaris are relatively common in Switzerland. They are induced by a systemic infection of a rust fungus pathogen, called Puccinia arrhenatheri. Similar to stem rust of wheat, this rust fungus has a life cycle with host alternation to a species in the Gramineae. The grass Arrhenatherum elatius was reported as the only primary host in Switzerland but we found evidence, using molecular methods, that the fungus may also use Melica nutans as primary host. Sexual reproduction of rust fungi may depend on gamete transfer by insects. Witches brooms on B. vulgaris bear yellow discolored leaves upon which the fungus presents its gametes (spermatia) in a sugary nectar. During the reproductive phase these leaves spread a strong scent, most likely to attract insects. We used an insect-exclusion experiment to evaluate whether successful reproduction of P. arrhenatheri on B. vulgaris depends on gamete transfer by insects. Reproductive success was defined by the fungus ability to produce aeciospores. Aeciospores are dikaryotic spores that are distributed by wind to the Gramineae host. The production of aeciospores was significantly higher on witches brooms with insect visitation suggesting that the sexual reproduction of the pathogen requires out-crossing by insects. By excluding either crawling or flying insects, we could also show that crawling insects, such as ants, only play a minor role in the reproduction of the pathogen. In addition to the exclusion experiment, we conducted timed observations on witches brooms and simultaneously on leaves of similar sized uninfected branches of B. vulgaris. We observed a high diversity of visitors, mainly insects but also some spiders. The most frequently recorded taxa belonged to the Diptera and Hymenoptera, but depending on the locality, different genera and species dominated. Higher visitation rates and longer visits on witches brooms indicate that the leaves on witches brooms attract insects, and that the fungal nectar on them is an attractive food source. 1

6 INTRODUCTION Berberis vulgaris L. is the alternate host for at least four different grass rust fungus pathogens, including the stem rust of cereals (Gäumann, 1959). One rust species differs from the others in the pathogenic effect of its mycelium on B. vulgaris. The mycelium of this species grows systemically, which leads to the formation of witches brooms, whereas the mycelium of the other rusts is localized, and only forms spots on the leaves of B. vulgaris (Gäumann, 1959). Witches brooms on B. vulgaris are relatively common in Switzerland. The leaves on witches brooms appear earlier than those on the uninfected part of the bush. They are yellow in color, spread a sweet-smelling scent and have fungal sexual structures termed spermogonia on the upper surface. In the spermogonia a sugary nectar and fungal gametes (spermatia) are produced (Rathay, 1883). Spermatia, most likely need to be transferred from one self-incompatible mating type to another to fertilize the fungus (Craigie, 1927). Other rust fungi have been found to be outcrossed by insects (Craigie, 1927; Buller, 1950; Roy, 1993). It is possible that sexual reproduction of the witches broom-forming rust fungus is mediated by insects too. The rust fungus causing the witches brooms is macrocyclic, meaning that it proceeds through five possible spore stages on two different host plants. After successful sexual reproduction on B. vulgaris, the fungus forms aecia on the bottom side of infected leaves producing dikaryotic aeciospores. The aeciospores are distributed by wind to infect the primary host, a native grass. On the grass, dikaryotic urediniospores are produced that infect other grass plants. Near the end of the growing season another spore type is produced - the dikaryotic teliospores, which can overwinter. In spring, the nuclear pairs of teliospores fuse to form diploid nuclei followed immediately by meiosis. Then the teliospores germinate to form a hypha that produces monokaryiotic, haploid basidiospores. The basidiospores are distributed by wind, and can infect buds of the alternative host B. vulgaris. They give rise to haploid hyphae of one of the two mating types, which grows into the meristem of the branch causing abnormal growth of the branch (Gäumann, 1959). In the next year, the fungus grows into the leaves of the branch, generating the above mentioned yellowish discoloration and spermogonia, thereby completing the life cycle. Here we present the results of several studies designed to elucidate the identity of systemic infections of B. vulgaris and their natural history: First, we wanted to determine the identity and primary (grass) hosts of the systemic infection. For this we used molecular methods. We also wanted to analyze the phylogenetic relationships among the grass rusts that attack B. vulgaris. Peyritsch was the first who predicted host alternation with grasses for the systemic infection of B. vulgaris (in Magnus, 1894). He found a rust fungus on Arrhenatherum elatius L. underneath systemically 2

7 infected B. vulgaris and named it Puccinia magelhaenica Peyr. to match the name Aecidium magelhaenica Magn. used at this time for the aecia on witches brooms. Several authors reconfirmed host alternation to species of Arrhenatherum (reviewed in Urban and Marková, 1994). Based on these results, the fungus was renamed as Puccinia arrhenatheri (Kleb.) Erikss. This rust fungus was also found on B. vulgaris in arid sites in the Wallis, although no Arrhenatherum species could be found within a convenient distance. Gäumann (1934) could only infect A. elatius with aecia from these bushes. P. arrhenatheri also occurs on the primary host Arrhenatherum in regions where B. vulgaris is rare (Urban and Marková, 1994; Mayor, 1958). However, Mayor (1958) was able to infect B. vulgaris with teliospores from such a site in Neuchâtel (Switzerland). Although to date experiments suggest that Arrhenatherum is the primary host, it is nevertheless possible that this rust can infect other grass hosts. We collected samples of systemic and non-systemic infections on B. vulgaris from different localities in Switzerland and grasses with rust fungus infections underneath barberry bushes, and sequenced the internal transcribed spacer (ITS) region of the ribosomal DNA of the fungi. The ITS region has already been successfully used by other authors to analyze relationships among closely related fungal taxa and to examine genetic variation among different populations (Gardes and Bruns, 1993; Zambino and Szabo, 1993; Roy et al., 1998; Pfunder et al., in press). We compared the sequences of the ITS region of infections on B. vulgaris, and of rust fungi found on grasses to answer the following questions: 1) What is the identity of the pathogen causing systemic infection? 2) Can we detect the witches broom-forming rust fungus on grasses other than Arrhenatherum? 3) What is the phylogenetic relationship between the witches broom-forming rust fungus and other rust fungi found on native grasses? Second, we examined the genetic variation of the pathogen and the infection incidence in different populations in Switzerland. Populations of systemically infected B. vulgaris occur on the south slope of many alpine valleys and in warm areas in the midland of Switzerland. Large populations of infected B. vulgaris exist in the Wallis and in the lower Engadin valley. Since these populations are separated by about 200 km of high mountainous territory one might expect some genetic variation in the rust fungi, and also different amounts of infection in the different populations. It is also possible that different species of fungus cause the infections. We sequenced the ITS region of systemic infections, 3

8 and recorded the infection incidence in several populations of B. vulgaris to answer the following questions: 4) Is there sequence variation among systemic infections from different populations of B. vulgaris within Switzerland? 5) What is the infection incidence of different populations of B. vulgaris in Switzerland? The third objective was to test the role of insects for the fertilization of this fungus. Only a few studies have shown that insects are required as gamete vectors for rust fungi (Craigie, 1927; Roy, 1993; Pfunder and Roy, 2000; Schürch et al., 2000). Since the witches brooms on Berberis are caused by a systemic infection, all leaves on a witches broom are likely to be infected by the same mating type. Therefore spermatia may need to be transferred from another witches broom to fertilize the fungus. To our knowledge no fertilization experiment to test whether insects are required as gamete vectors has been done with the systemic infection on B. vulgaris. In previous years numerous ants were observed (Roy pers. obs.) on witches brooms. They seemed to be attracted by the sugary nectar on the leaves. Other authors have mentioned that ants use the sugary nectar of rust fungi as a food source (Buller, 1950; Schürch et al., 2000). We used an insect exclusion experiment and quantified observations to address the following questions: 6) Does the sexual reproduction of the fungus depend on insect visitation? 7) What role do flying insects and crawling insects, such as ants play in the reproduction of the fungus? 4

9 MATERIALS AND METHODS Hosts and pathogens The host of the systemic infection, Berberis vulgaris, is a southern european-west asian species that can grow into a 3m high bush. In Switzerland B. vulgaris grows in sunny and dry sites along forest edges and in scrub lands. It prefers Calcium rich soils (Hess et al., 1977). Robust fruiting individuals occur from elevations of 300 m to 2000 m, and nonfruiting individuals have been found in the upper Engadin valley even as high as 2660 m above sea level (Hegi, 1974). B. vulgaris is the alternate host at least four different grass rust fungus species. These rust fungi have been divided into two groups based on the different morphology of their telial stages on grasses (Gäumann, 1959): the Puccinia graminis Pers. complex in which the telia are exposed (contains the stem rusts of grasses and cereals) and the second group in which the telia are initially covered (contains Puccinia brachypodii Otth and its allies). The rust fungus that causes the systemic infection on Berberis was assigned to the second group, and is named Puccinia arrhenatheri (Kleb.) Erikss. according to its ability to infect Arrhenatherum species (Gäumann, 1959). In 1966 Cummins and Greene reduced the number of species within the second group because of morphological variability in the telial stage and named the same fungus Puccinia brachypodii f. sp. arrhenatheri Kleb. (see Table 1 for names and synonyms of these rust fungi). TABLE 1. Synonyms for grass rust species used in cited literature (names used in this study given in bold face) Name of grass rust species First and other authors Puccinia arrhenatheri Eriksson (1898), Gäumann (1959) Syn.: Puccinia magelhaenicum Peyritsch in Magnus (1894), Urban and Marková (1994) Puccinia brachypodii f. sp. arrhenatheri Cummins and Greene (1966) Puccinia brachypodii Otth (1870), Gäumann (1959) Syn.: Puccinia brachypodii f. sp. brachypodii Cummins and Greene (1966) Puccinia poae-nemoralis Otth (1870), Gäumann (1959) Syn: Puccinia brachypodii f. sp. poae-nemoralis Cummins and Greene (1966), Zambino and Szabo (1993) 5

10 Fungal collection and identification Table 2 lists rust fungus species, host species, and place of collection for all the sequenced samples. All collections of infected plants were made in the spring and summer of Infected leaves were dried at room temperature then stored at 4 C in plastic containers with Silica Gel (Merck). Urediniospores of two cereal stem rusts were supplied by the Swiss Federal Research Station for Agroecology and Agriculture (FAL). One was isolated from the summer-wheat cultivar Kolibri in Realta in 1982 and the other from the summer-oat cultivar Selma in Tenniken in Fig. 1 shows the geographic distribution of the localities on a map of Switzerland. TABLE 2. Sequenced samples of rust fungus species, host species and collection information. Rust species Host species Collection locality Number of samples Puccinia graminis Pers. Agropyron repens (L.) P. B. Ardez (A) 1 Puccinia graminis Pers. Agropyron repens (L.) P. B. Felsberg (F) 1 Puccinia arrhenatheri Erikss. Arrhenatherum elatius (L.) J. et C. Presl Felsberg (F) 1 Puccinia graminis Pers. Avena sativa L.* Tenniken (T) 1 undet. Brachypodium pinnatum (L) P. B. Felsberg (F) 1 Puccinia graminis Pers. Dactylis glomerata L. Felsberg (F) 1 undet. Melica nutans L. Felsberg (F) 1 Puccinia graminis Pers. Triticum aestivum L.* Realta (R) 1 Puccinia graminis Berberis vulgaris L. Ardez (A) 2 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Ardez (A) 4 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Piotta 1 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Felsberg (F) 3 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Kippel 1 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Hohtenn 1 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Merishausen (M) 1 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Quinten 1 Puccinia arrhenatheri Erikss. Berberis vulgaris L. Trimmis 3 * urediniospores supplied by the Federal Research Station for Agroecology and Agriculture (FAL) Identification of the pathogens was based on morphology and host association. Urediniospores or teliospores were measured with a Leica DMLB microscope. Based on these measurements (see Appendix A) and other attributes determined by using a binocular microscope (Wild, M5A, Heerbrugg) the species were identified according to Gäumann's (1959) Rostpilze Mitteleuropas. 6

11 Merishausen Tenniken Zürich Quinten Trimmis Felsberg Ardez Piotta Realta Kippel Hohtenn Fig. 1. Collection localities in Switzerland Molecular Methods DNA Extraction DNA was extracted from dried host leaves with visible fungal organs. We followed the 2xCTAB buffer protocol of (Gardes and Bruns, 1993), which we modified by shaking the samples together with a glass ball of 5 mm diameter in a 2 ml tube for 8 min, freezing and thawing three times, and shaking for an additional minute before adding the CTAB. PCR amplification, cleaning, and sequencing Double-stranded nuclear rdna was amplified directly from diluted extractions (1/10-1/1000) using polymerase chain reaction (PCR). From the extracts including plant DNA, we amplified the internal transcribed spacer region (ITS1 5.8S ITS2) of fungal DNA only, using primers that bind specifically to the conserved regions at the 3'-end of the 18S and the 5'-end of the 28S rdna genes of basidiomycetes. The primers used were ITS5 (White et al., 1990) and ITS4 (White et al., 1990). However, with some samples, amplification using the ITS4-u primer (Pfunder et al., in press) was more successful. 7

12 Each PCR reaction (10-50 µl) consisted of 1/2 volume diluted extracts and 1/2 volume of reaction mix, leading to the following final concentrations: 3 mm MgCl 2, O.25 units Taq DNA polymerase (Promega) per 10µl reaction, 1x polymerase buffer including 10 mm Tris-HCl and 50 mm KCl (Promega), 0.2 mm each of datp, dctp, dgtp and dttp (Promega) and 0.5 µm of each oligonucleotide primer. DNA thermal cyclers (Techne, models Progene or Cyclogene) were used for amplification, following the cycling program of (Gardes and Bruns, 1993). Double stranded PCR products were purified using the QIAquick PCR purification kit following the protocol of the manufacturers (Qiagen). The purified products were dissolved in elution buffer (Qiagen) and used directly for the amplification of single stranded DNA. Each PCR reaction consisted of ng DNA, diluted if necessary in dh2o, 0.16 µm primer, and 4 µl of dye terminator cycle sequencing ready reaction with AmpliTaq DNA Polymerase, FS (PE Applied Biosystems). The internal primers used were ITS2-u (Pfunder et al., in press) and ITS3r (Gardes and Bruns, 1993). The cycle program was used as described in Roy et al. (1998). The single stranded PCR products were purified with ethanol and dissolved in 12.5 µl template suppressant reagent (PE Applied Biosystems) before sequencing. Sequencing was performed on an ABI Prism 310 Genetic Analyser (PE Applied Biosystems). To combine the sequences from the different primers, the program ABPrism Sequence Navigator (PE Applied Biosystems) was used. Different samples that showed no differences in their nucleotide sequence are represented in the phylogenetic analysis by a single sequence. Sequence analysis We used the program Sequence Navigator v (PE Applied Biosystems) to align sequences through a combination of clustering algorithms (Clustal) and visual editing. Trees were generated with Paup version 4.0b4 (Swofford, 2000) using the following settings: heuristic search, random addition with 100 replicates, no limit to MaxTrees, and unweighted characters. Multi-bp gaps were assumed to represent single evolutionary events. Variable gaps longer than 2bp were coded in a separate matrix (0=no gap, 1=gap) which was analyzed simultaneously with the sequence data. The alignments used in this study can be seen in the Appendix B. Statistical support for clades was estimated by bootstrapping with the fast procedure of Paup on 100 replications (Swofford, 2000). To resolve the phylogenetic relationship among the different rust samples we also included known sequences of the ITS region of grass and cereal rusts by Zambino and Szabo (1993) 8

13 and a crucifer rust by Roy et al. (1998). Our sequences included the ITS spacers 1 and 2, and the associated 5.8S and 28S genes. Unfortunately, the sequences by Zambino and Szabo (1993) only include 40bp of the ITS1 region and no 28S gene and are therefore about 230 bases shorter than ours. For this reason, we did two analyses, one with the whole length of our sequences and with missing data at the ends of the sequences from Zambino and Szabo (1993) and one with a subset of our sequences that corresponded to the length of their sequences. Field work Infection incidence The infection incidence (presence of witches brooms) was determined in 6 populations of B. vulgaris in Switzerland. To determine whether infection was size dependent, we measured the main trunk of the bush and counted the number of witches brooms of a randomly chosen sample of bushes in these populations. Since the populations were of different sizes and topology, the random sample of bushes was chosen with different methods. In the large populations of Ardez, Kippel, and Trimmis we measured all bushes along an imaginary transect following a point of the compass until we had a sample size of about 50 individuals. In the smaller populations of Piotta and Felsberg, we recorded all bushes found. In the steep territory of the south slope of the upper Wallis we chose an area of about m 2 close to Hohtenn in which we recorded all bushes. To test whether infection status is independent of size we conducted a χ 2 test with infection status and 10 categories of main trunk diameter. We also asked the opposite question, whether the infection status or site influences the main trunk diameter. This question was answered with a two-way analysis of variance (ANOVA) on mean trunk diameter with the infection status as a fixed factor and the site included as a random factor. The diameters of the main trunks were natural log transformed to comply with the normality requirements of ANOVA. In addition, we wanted to know whether the degree of infection was size dependent. To answer this question we analyzed the number of systemically infected branches using a two-way ANOVA with the site as a random factor and main trunk diameter as a continuous factor. We needed to log transform the number of systemically infected branches for the ANOVA. Type III mean squares were calculated with the program JMP, Version (SAS1994) and the F-values were calculated according to the tables in Zar (1996) for both ANOVAs. 9

14 Insect exclusion experiment To test whether insects fertilize the fungus, we conducted an insect exclusion experiment during the spring of We chose 25 bushes of Berberis vulgaris with at least four witches brooms in Ardez (Fig. 1) in the lower Engadin (co-ordinates / ). The bushes were spread over an area of about m 2 in dry calcareous grassland at 1410 m to 1460 m above sea level oriented an 20 angle and facing 190 south. Four witches brooms of each bush were assigned randomly to one of the following treatments: 1) no visitors the witches broom was caged with small-meshed florist gauze net (Kleen Test Products, division of Meridian Industries, Inc., Milwaukee, Wisconsin). The bags allow light, wind, and water to penetrate but exclude even the smallest insects. To prevent the cage from collapsing on to the infected leaves, we put a frame of chicken wire inside the cages. 2) crawling insects only we used the cages described above but instead of tying the florist bag around the stem of the witches broom, we left an opening of about 1 cm around the stem to allow insects to crawl in. 3) flying insects only to prevent crawling insects to visit we put a 5 cm wide tape around the stem of the witches broom and smeared it with an insect trapping adhesive (Tangle-trap from The Tanglefoot Company, Grand Rapids, Michigan) and tied other branches in a way they could not touch the witches broom. 4) all visitors uncaged control with natural insect visitation. All treatments were installed from 24 March 2000 to 10 April 2000 before buds on the witches brooms opened. During this period we replaced some treatments that were destroyed by a late snowfall. For the next month the field was visited once a week to record the stage of the leaves and to check whether the treatments excluded the proper insects. Treatments that allowed improper visitors were not included in the analysis. In addition, we lost some treated witches brooms because they were broken by grazing sheep or appeared to be dead, which could not be seen when the treatments were installed. Not all leaves on the witches brooms showed signs of infection. The first leaves appeared completely yellow and covered with spermogonia, the organs producing the fungal gametes (spermatia), whereas older leaves were yellowish and bore spermogonia only along the veins or were apparently healthy. This observation was also made by Gäumann (1959). Completely and partly infected leaves were harvested from the treated witches brooms on 17 and 18 June 2000 when they started to dry up. The reproductive success of the pathogen was defined by the fungus ability to produce aeciospores. Aeciospores are heterokaryotic spores that are distributed by wind to the grass host. The production of aeciospores is the result of successful fertilization and can therefore be compared to the production of seeds in plants. For each treated witches broom we counted the number of leaves with aecia, the fungal organs in which aeciospores are 10

15 produced, and the number of infected leaves without aecia. We based our analysis on the proportion of aecia-bearing leaves from the total of infected leaves. To determine the effect of our treatments, this proportion was analyzed with a factorial analysis of variance (ANOVA) with treatments and bushes as factors. Prior to analysis, the proportion was arcsine square root transformed to obtain normality and homogeneity of variance. The statistical analyses were performed with the program JMP, Version (SAS 1994). Insect observations We wanted to examine the attractiveness of the witches brooms for insects and the diversity of visiting insects during the stage when infected leaves spread a sweet-smelling scent and have spermogonia on the surface. Therefore, we recorded insect visitation for 20 min observation periods on a witches broom and simultaneously on leaves on a similar sized healthy branch of Berberis vulgaris. All observations were made by the same observer at four sites in alpine valleys in Switzerland. The four sites will be referred to as Trimmis, Piotta, Ardez, and Kippel (see Fig. 1) in this paper. The Trimmis site was in a grassland in the Rhine valley (co-ordinates / , 700 m above sea level). There we tallied the visits on 26 April 2000 during four observation periods of 20 min each made on a different bush. In Piotta, in the southern alpine valley Leventina (co-ordinates / , 1050 m above sea level), on 1 May 2000 we also counted visits during 20 min observation periods on four bushes. At the two other sites, in Ardez, at the same site as the exclusion experiment, and in Kippel in the western alpine valley Lötschental (co-ordinates / , 1400 m above sea level), we made six replicates of 20 min observation periods on three randomly chosen bushes per site. Each set of observations on the three bushes was made within a time block of about 70 min (20 min x 3 plus about 5 min time to change between bushes). To remove bias due to observing in chronological order, we systemically changed the order of the observed bushes after each time block. In Ardez, we observed for four time blocks on 3 May 2000, and two time blocks on 4 May 2000, and at the same time of day in Kippel for four time blocks on 9 May 2000, and two time blocks on 10 May In addition to the number of visits we measured the duration of each visit. All observations were made between and under clear to partly cloudy skies. The insect or spider visitors were identified as well as possible during the observations and identifications were verified after the experiment by catching representatives that were identified by B. Merz (Diptera), N. Neumeyer (ants) or A. Naef (other insects and spiders). 11

16 The insect observations in Ardez and Kippel were analyzed by mixed-model analyses of variance (ANOVA) on the number of visits per observation period, and the mean duration per visit. Observation site, time block (replicates), and bushes were included as random factors with bushes nested within site, and the kind of branch (systemically infected vs. uninfected) was included as a fixed factor. The number of visits and the duration per visit were natural log transformed to meet the normality and homogeneity assumptions of ANOVA. The three way interaction branch type * time block * bush was removed from both models because its inclusion was not supported by the analysis. The model for the number of visits per observation period was overfitted when this interaction was included. As for the duration per visit, the F-Value of the site-effect could not be approximated, which might have been caused by the unbalanced number of observations on infected and uninfected branches. Synthetic denominators, as calculated in JMP, Version (SAS1994) were used to calculate the F-ratios. 12

17 RESULTS Identity of the systemic infection The primary host We sequenced the ITS region of 24 samples: 15 of the systemic infection on Berberis vulgaris from 8 different localities in Switzerland, an additional two samples of non-systemic infections on B. vulgaris from Ardez, five samples of rust fungus infections on native grasses, and two samples of cereal rusts (see Table 2). We collected the samples of infected grasses underneath barberry bushes with witches brooms in Ardez and in Felsberg. The Swiss Federal Research Station for Agroecology and Agriculture (FAL) supplied urediniospores of the cereal rusts. We were able to analyze and align all amplified sequences except for the sample from Brachypodium pinnatum. For this sample the amplification with ITS5 and ITS4 resulted in electropherograms for the external primers that looked like two overlaid different sequences and therefore could not be analyzed. We tried to solve the problem with a method successfully used for the sample from Arrhenatherum elatius. We used the more specific ITS4-u primer and did a second PCR with the purified product of a first PCR in order to increase the amount of DNA product. For the A. elatius sample we could verify the resulting sequences with sequences from internal primers received by the normal protocol. However, for the B. pinnatum doing double PCR led to sequences from external primers that did not match the ones from internal primers. Interestingly, the sequences from the internal primers showed similarities with the sequence of the non-systemic infection on B. vulgaris whereas the sequences from external primers showed more similarities with the sequence of the systemic infection, suggesting that both Puccinia graminis and Puccinia brachypodii were present. P. brachypodii is assigned to the same group as the systemic infection (Gäumann, 1959). To our surprise, the only grass rust sequence matching sequences of systemic infections on B. vulgaris was the one found on Melica nutans in Felsberg. Unfortunately, we were not able to verify the identification of this fungus by microscopy. No telia and no uredia were found on the plant, probably because we did the microscopy after the molecular work, thus we might have scratched off all fungal material for DNA extraction. This happened also for the B. pinnatum sample where we did several extractions from the same plant to get better sequences. As for the M. nutans, within the timescedule of this undergraduate project we could not collect new material and repeat the result. However, we think that contamination is unlikely for the following reasons: first, with this extract we got the same sequence twice, second no sample of the systemic infection of B. vulgaris was 13

18 extracted in the same set, and finally none of five other grass samples extracted, amplified, and sequenced in the same set using the same stock solutions and equipment showed this sequence. They either showed sequences similar to the non-systemic infections of B. vulgaris, or did not amplify. The fungi found on Dactylis glomerata, Poa pratensis, and Agropyron repens were morphologically identified as P. graminis, whereas the fungi found on A. elatius was identified as P. arrhenatheri by its covered telia (see Appendix A) although its sequence matched with samples identified as P. graminis. Phylogenetic analysis To resolve the phylogenetic relationship among the different rust samples we also included published sequences of the ITS region of grass and cereal rusts, Puccinia poaenemoralis Otth (Syn.: P. brachypodii f. sp. poae-nemoralis Cumm. et H. C. Greene used by Zambino and Szabo), Puccinia striiformis Westend f. sp. tritici, Puccinia montanensis Ellis, Puccinia coronata Corda f. sp. tritici and several formae speciales of Puccinia graminis Pers. (all from Zambino and Szabo (1993)) and of the crucifer rust Puccinia monoica (Peck) Arth. on Arabis perennans S. Watson (Roy et al., 1998). Since the sequences from Zambino and Szabo are about 230 bases shorter than ours, we analyzed both the whole length of our sequences with missing data at the ends of their sequences, and we also shortened our sequences to match theirs so there was no missing data. The aligned full-length sequences of all 22 unique sequences contained a total length of 666 bp (see Appendix B). The gap matrix added additional 24 characters. The analysis was therefore based on 690 characters of which 99 were parsimony-informative. The unweighted parsimony analysis found 30 equally parsimonious trees of 225 steps with a consistency index (CI) of 0.84, a retention index (RI) of 0.91, and a rescaled consistency index (RC) of The phylogram of one randomly chosen tree for the full length of our sequences is shown in Fig. 2. The analysis of the shorter sequences was based on 431 characters divided into 418 bases of the sequence and 13 characters of a gap matrix. 51 characters were parsimonyinformative. This led to one single tree with a consistency index (CI) of 0.82, a retention index (RI) of 0.89, and a rescaled consistency index (RC) of The phylogram of this tree is shown in Fig

19 63 P. monoica on Arabis perennans ** Syst. Inf. on Berberis vulgaris (F) Syst. Inf. on Berberis vulgaris (A) Syst. Inf. on Berberis vulgaris (M) Inf. on Melica nutans (F) P. brachypodii f. sp. poae-nemoralis * P. striiformis f. sp. tritici * P. montanensis Non syst. Inf. on Berberis vulgaris (A) Inf. on Avena sativa (T) I II Inf. on Arrhenatherum elatius (F) Inf. on Dactylis glomerata (F) Inf. on Poa pratensis (F) III P. coronata f. sp. tritici * P. graminis f. sp. dactylis * 1 56 P. graminis f. sp. poae * P. graminis f. sp. avenae * Inf. on Triticum aestivum (R) Inf. on Agropyron repens (A) Inf. on Agropyron repens (F) P. graminis f. sp. tritici * P. graminis f. sp. secalis * IV 5 changes Fig. 2. Phylogram of one randomly chosen tree out of 30 equally-parsimonious trees based on the full length of our ribosomal DNA sequences (3 -end of 18S, ITS1, 5.8S, ITS2, and the 5 -end of 28S). Sequences marked with * are from Zambino and Szabo (1993) and have missing data at both ends. The sequence marked with ** is from Roy et al. (1998). The capital letters in brackets are for the collection localities as listed in Table 2. The number of changes is shown above the nodes, and bootstrap support (based on 100 repetitions) is shown below the nodes. 15

20 48 P. monoica on Arabis perennans ** Syst. Inf. on Berberis vulgaris (F) Syst. Inf. on Berberis vulgaris (A) Syst. Inf. on Berberis vulgaris (M) 4 90 Inf. on Melica nutans (F) 2 P. brachypodii f. sp. poae-nemoralis * 12 7 P. striiformis f. sp. tritici * P. montanensis * Non-syst. Inf. on Berberis vulgaris (A) I II Inf. on Avena sativa (T) Inf. on Arrhenatherum elatius (F) Inf. on Dactylis glomerata (F) Inf. on Poa pratensis (F) III P. graminis f. sp. avenae * P. graminis f. sp. dactylis* P. graminis f. sp. poae * 7 99 Inf. on Triticum aestivum (R) Inf. on Agropyron repens (A) 1 66 Inf. on Agropyron repens (F) P. graminis f. sp. tritici * IV 17 P. coronata f. sp. tritici * P. graminis f. sp. secalis * 5 changes Fig. 3. Phylogram of the most parsimonious tree based on a subset of our ribosomal DNA sequences chosen to match the lengths of sequences from Zambino and Szabo (1993)(part of ITS1, all of 5.8S, and all of ITS2). Sequences marked with * are from Zambino and Szabo (1993) and the sequence marked with ** is from Roy et al. (1998). The capital letters in brackets are for the collection localities as listed in Table 2. The number of changes is shown above the nodes, and bootstrap support (based on 100 repetitions) is shown below the nodes. The full length and the subset analyses both resulted in four main clusters. Group I is well supported by bootstrap values of 93 for the long sequences and 90 for the subset sequences. It contains the sequences of our systemic infections on B. vulgaris, the fungus found on M. nutans, and P. brachypodii f. sp. poae-nemoralis from Zambino and Szabo (1993). The other three clusters are similar to the ones that Zambino and Szabo (1993) found. In group II there is P. striiformis with no known aecial host and P. montanensis 16

21 found only in North America with aecia on Berberis fendleri Gray (Cummins and Greene, 1966). In both analyses the bootstrap values for this cluster are lower than 80. However, this relationship is not the objective of this study and will not be discussed further. Our rust samples from grasses, except for the one from M. nutans, were either in group III or IV. These two main clusters within the complex of P. graminis are well supported by bootstrap values of 92 or larger in both trees. Our sequences from native grasses were in the same cluster as the sequences of formae speciales from related cultivars published by Zambino and Szabo (1993). In group III our samples of P. graminis on Avena sativa, Dactylis glomerata and Poa pratensis cluster together with P. graminis f. sp. avenae, f. sp. dactylis and f. sp. poae and group IV contains our samples of P. graminis on Triticum aestivum and Agropyron repens as well as the formae speciales tritici and secalis. The non-systemic infection on B. vulgaris, and surprisingly, our sample of the P. arrhenatheri on Arrhenatherum elatius belong to group III. There are some differences within this group between the two phylograms. Table 3 shows that the separation of our samples from D. glomerata and P. pratensis in the full length analysis was based on deletions and base exchanges in the part of the sequence from which there is only missing data for the sequences from Zambino and Szabo (1993). Since this part was excluded in the analysis of the shorter sequences, this cluster does not show up there. The separation of the sequences from Zambino and Szabo (1993) in the full-length analysis is also due to the missing data. One would expect corresponding formae speciales and samples to cluster together, but with the short sequences for the formae speciales it is not possible to clarify the relationships. TABLE 3. Differences in the sequences within group III. Sequences marked with * are from Zambino and Szabo (1993). The capital letters in brackets are for the collection localities as listed in Table 2. Sample Base position in aligned sequences Non-syst. Inf. on Berberis vulgaris (A) T C A A A T Inf. on Avena sativa (T) T C A A A T Inf. on Arrhenatherum elatius (F) T C A A A T Inf. on Dactylis glomerata (F) T C gap gap T gap Inf. on Poa pratensis G T gap gap T gap Puccinia graminis f. sp. avenae * N N N N N N Puccinia graminis f. sp. dactylis * N N N N N N Puccinia graminis f. sp. poae * N N N N N N 17

22 In group IV we have two sequences of P. graminis on A. repens, one from Ardez and one from Felsberg. They only differ for one base by substitution. The differences between the two phylograms in group IV are caused by the exclusion of the differences in position 167 and 177 in the analysis of the shorter sequence as shown in Table 4. As in group III, it is not possible to clarify the relationship between the sequences of the formae speciales from Zambino and Szabo (1993) and our sequences due to their short sequences. TABLE 4. Differences in the sequences within group IV. Sequences marked with * are from Zambino and Szabo (1993). The capital letters in brackets are for the collection localities as listed in Table 2. Sample Base position in aligned sequences Inf. on Triticum aestivum (R) T gap A A gap Inf. on Agropyron repens (A) A T C gap T Inf. on Agropyron repens (F) A C C gap T Puccinia graminis f. sp. tritici * N N A A T Puccinia graminis f. sp. secalis * N N A gap T Populations of infected Berberis vulgaris in Switzerland Sequence variation of the pathogen From the total of 15 samples of systemic infections on B. vulgaris from eight different populations, 13 samples showed the same ITS sequence, and two samples differed by only a single point mutation from a total of 636 bases. One of these unique samples was from Ardez where three other samples belonged to the large group with identical sequences. The other sample was from Merishausen in the north of Switzerland, from where we sequenced only this single sample. The two samples of non-systemic infections on B. vulgaris from Ardez were stem rust infections (see phylogenetic analysis). They had an identical ITS sequence that differed at 58 sites from the systemic infections of B. vulgaris. 18

23 Infection incidence We recorded a total of 266 bushes of Berberis vulgaris from six different populations in Switzerland. Table 5 shows for each population the number of recorded bushes, the infection incidence (proportion of infected individuals on the total of recorded bushes), and the mean and median diameter of the main trunk. TABLE 5. Recorded populations of Berberis vulgaris in Switzerland. Population Number of recorded Bushes Infection Diameter of main trunk (cm) Incidence Mean ± 1 SE Median Ardez (transect) ± Piotta (whole population) ± Felsberg (whole population) ± Hohtenn (selected area) ± Kippel (transect) ± Trimmis (transect) ± In Fig. 4 we present the distribution for our whole sample on 10 classes of main trunk diameter, and the proportion of infected bushes within these classes. It is quite obvious that infection incidence increases with the diameter of the main trunk and therefore with the age of the bush. The proportion of infected bushes was significantly different between the different classes of diameter range (χ 2 = 74.20, 9 df, P < ). 19

24 70 60 NUMBER OF BUSHES Infected Bushes Uninfected Bushes 10 0 RANGE OF MAIN TRUNK DIAMETER (cm) Fig. 4. Number of bushes of Berberis vulgaris recorded in six populations in Switzerland subdivided in 10 classes of main trunk diameter and the proportion of bushes with systemic infections within each class. We tested whether infection status or site is associated with the size of the bushes, expressed by the diameter of the main trunk (Table 6). The diameter of systemically infected bushes was significantly larger and the differences among the different populations were significant too. TABLE 6. ANOVA on the diameter of the main trunk of Berberis vulgaris (data log transformed) with site included as a random factor and infection status as a fixed factor. P values 0.05 are given in bold face. Source of variation DF MS F P Site < Infection status < Site * Infection status Error We also analyzed the relationship between the main trunk diameter and the number of systemically infected branches (witches brooms) per bush. An ANOVA (Table 7) showed that there is a significant effect of the main trunk diameter of the bush on the mean number of infections. In Fig. 5 the number of witches brooms of 111 infected bushes from different populations is linearly fitted against diameter. Looking at the figure, one might 20

25 think that the line is poorly fitted, but 45 points are crowded onto the one witches broom level. However, the correlation index of this fit is low, suggesting that the relationship is not truly linear. Separate linear regressions for the different site are not shown, since site and the interaction site * trunk diameter had no significant effect (Table 7), and correlation indices were lower than 0.4, decreasing with an increasing sample size. TABLE 7. ANOVA on the number of witches brooms on infected Berberis vulgaris (untransformed data) with site included as random factor and trunk diameter as a continous factor. P values 0.05 are given in bold face. Source of variation DF MS F P Site Main trunk diameter Site * main trunk diameter Error NUMBER OF WITCHES BROOMS f(x) = 0.37 * x R 2 = MAIN TRUNK DIAMETER (cm) Fig. 5. Relationship between the diameter of the main trunk and the number of systemically infected branches recorded on 111 infected bushes of Berberis vulgaris in 6 populations in Switzerland. The regression line and the equation are linear fitted for all points. 21

26 Insect-mediated reproduction Insect exclusion experiment From our total of 100 treated witches brooms on Berberis vulgaris, 80 were included in the analysis. 14 treatments were lost, either because grazing sheep broke them or because no leaves appeared on the witches broom. Two witches brooms, assigned to the treatment flying insects only were excluded, because they touched the grass, or another branch, and allowed crawling insects to visit. From the treatments with no visitors, four were not taken into account, because they did not exclude all insects. Flying insects were also found in some cages that should have excluded them. Since no holes were found in those bags, the flying insects must have crawled into the cages and thus were accepted as appropriate visitors. Fig. 6 shows the mean proportion of aecia-bearing leaves from the total of infected leaves for the remaining treatments. Even in the treatment with no insect visitation, some leaves bore aecia. Some of these leaves may have touched the florist bags, and the fungus might also have been fertilized through the bag by insects crawling around on the outside of the bag. We could not exclude these leaves from analysis because it was not possible to determine whether they had touched the bag during the spermatial phase after they started to dry up and shrink, or wether the aecia formed spontaneously. 1 PROPORTION OF LEAVES WITH AECIA a a n = 23 n = 21 b n = 19 c n = 17 All Visitors Crawling Insects Flying Insects No Visitors Only Only TREATMENT Fig. 6. Mean proportion of aecia-bearing leaves from the total of infected leaves (± SE) on differently treated witches brooms of Berberis vulgaris in an insect exclusion experiment. Treatments with the same letter do not differ significantly at P

27 The treatments excluding or including different groups of insects had a significant effect on the proportion of aecia-bearing leaves on the total of infected leaves (Table 8). A Dunnett s comparison test at P < 0.05 showed that the fungus had a significantly higher reproductive success in any treatment with insect visitation than in the treatment with no insect visitation. Using the same test with natural visitation as a control we found that the exclusion of flying insects significantly reduced the proportion of aecia-bearing leaves, whereas the exclusion of crawling insects did not have a significant effect. Individual bushes also had significantly different proportions of aecia (Table 8), but no spatial pattern was detected. TABLE 8. Factorial ANOVA on the proportion of aecia bearing leaves from the total of infected leaves on differently treated witches brooms of Berberis vulgaris in an insect exclusion experiment in Ardez (data arcsine-transformed). P values 0.05 are given in bold face. Source of variation DF MS F P Bush Treatment < Error Insect observations 27 visitors were observed in Piotta (Table 9) and in Trimmis 30 visitors (Table 10) were observed on four bushes per site during a total observation time of 80 min. For both sites more than 85 % of the visits were observed on witches brooms. The main visitors were insects of the classes Diptera and Hymenoptera. The ants counted in Trimmis were all tallied during one observation period of 20 min. They were densely patrolling on a witches broom, but no ants were present on the other observed witches brooms at this site. In addition to the insects at each site, one net spider was observed. 23

28 TABLE 9. Number of visitors observed in Piotta during 4 observation periods of 20 min on uninfected and systemically infected branches (witches brooms) of Berberis vulgaris. Class Order Family Species Uninfected branch Witches broom Arachnida Araneae Araneidae indet. 1 Insecta Coleoptera indet. indet. 1 Diptera Syrphidae Platycheirus albimanus Müller 1 1 Sepsidae Sepsis sp. 1 Drosophilidae Drosophila cameraria Halliday 3 Agromyzidae Agromyza sp. 11 Muscidae Eudasyphora zimi Hennig 2 Helina reversio Harris 1 2 Hymenoptera Formicidae Formica sp. 2 Ichneumonidae indet. 1 Total of visits 3 24 TABLE 10. Number of visitors observed in Trimmis during 4 observation periods of 20 min on uninfected and systemically infected branches (witches brooms) of Berberis vulgaris. Class Order Family Species Uninfected branch Witches broom Arachnida Araneae Araneidae indet. 1 Insecta Coleoptera indet. indet. 1 Diptera Sciaridae indet. 6 Lauxanidae indet. 1 Drosophilidae indet. 1 3 Muscidae Azelia sp. 2 indet. 2 Hymenoptera Formicidae Formica pratensis Retzius 13 Total of visits 3 27 At the Ardez and Kippel localities we made more extensive observations. In Ardez 148 visitors were counted during 360 min split into 18 observation periods of 20 min each. 81 % of the visits were counted on witches brooms. Table 11 shows the high diversity of visitors at this site. Diptera from eleven different families accounted for 81 % of all visits, Hymenoptera (mostly Formicidae) for 7 %, and spiders (mostly Thomisidae) for 10 %. The remaining 2 % of visitors belonged to the insect classes Coleoptera, Heteroptera and 24

29 Homoptera. Table 11 also shows the mean time spent per visit for different species. Since the sample size for single species was small, the difference in duration per visit between infected and uninfected branches was significant only for the unidentified anthomyid flies (t-test, P < 0.05). Many visitors were observed licking the fungal nectar on the upper side of infected leaves and moving within the witches broom. Such movements within a branch, either infected or uninfected, were considered to be one visit. In contrast to this behavior, the sarcophagus Scatophagidae remained immobile up to the whole 20 min observation time until a prey approached. The spiders of the species Salticus scenicus were hunting insects and jumped often between the observed infected and uninfected branch, these changes between branches were counted as separate visits. TABLE 11. Visitors observed in Ardez during 18 observation periods of 20 min on uninfected and systemically infected branches (witches brooms) of Berberis vulgaris: Number of visits and mean time spent per visit in seconds (grand means ± SE). Class Order Family Species Uninfected branch Number Time per visit Numbe r Witches broom Time per visit Arachnida Araneida Araneidae indet Salticidae Salticus scenicus Clerck ± ± 106 Insecta Coleoptera Coccinellidae indet ± 0 Nitidulidae Epurea depressa Ill ± 443 indet. indet ± 147 Diptera Chironomidae indet. 2 4 ± ± 18 Bibionidae indet Syrphidae indet. 1 2 Sepsidae Sepsis sp ± 42 Lauxanidae Lauxania cylindricornis Fab Drosophilidae Drosophila nigrosparsa Strobl 3 4 ± ± 17 Acartophthalmida e indet ± 16 Scatophagidae Scatophaga stercoraria Linné ± 143 Anthomyiidae Anthomyia liturata Rob.-Desv. 3 4 ± ± 63 indet ± ± 31 Muscidae indet indet. indet Heteroptera indet. indet Homoptera indet. indet Hymenoptera Formicidae Formica rufa L ± ± 33 Ichneumonidae indet. 1 5 Total of visits ± ± 29 25

30 In Kippel, during the same amount of observation time as in Ardez, 195 visitors were observed. Similar to Ardez, 82 % of the visits were counted on witches brooms. The diversity of the visitors was slightly lower than in Ardez (Table 12). 98% of the visitors were Diptera from 9 different families. Hymenoptera (Argidae and Ichneumonidae) accounted for the remaining 2 %. In Kippel no ants were observed during the whole observation time, whereas in Ardez on one bush 10 individuals of Formica rufa were observed, 4 on the uninfected branch and 6 on the witches broom. For several insect species some differences were observed in the mean time spent per visit on infected and uninfected branches (Table 12). None of these differences were significant (t-test, P < 0.05), most likely because the sample size was too small. TABLE 12. Visitors observed in Kippel during 18 observation periods of 20 min on uninfected and systemically infected branches (witches brooms) of Berberis vulgaris: Number of visits and mean time spent per visit in seconds (grand means ± SE). Class Order Family Species Numbe r Uninfected branch Time per visit Numbe r Witches broom Time per visit Insecta Diptera Chironomidae indet ± 32 Bibionidae Bibio sp ± Sepsidae Sepsis sp ± 14 Lauxanidae Lauxania minor Martinek ± 48 Cnemacantha muscaria Fallén ± 246 Drosophilidae Drosophila nigrosparsa Strobl ± ± 86 Scatomyza flava Fallén ± 276 Acartophthalmidae Acartophthalmus nigrinus Zett Acartophthalmus pusio Frey ± ± 26 Scatophagidae Scatophaga stercoraria Linné ± ± 105 Anthomyiidae Hylemya variata Fallén ± 38 Tachinidae indet ± ± 122 indet. indet Hymenoptera Argidae Arge sp ± 63 Ichneumonidae indet. 1 3 Total of visits ± ± 26 26

31 MEAN NUMBER OF VISITS / 20 min A SITE Witches Broom Uninfected Branch B TIME BLOCK 0 Ardez Kippel C 450 D MEAN DURATION OF VISITS (s) Ardez Kippel Fig. 7. Observed visitors on uninfected branches and systemically infected branches (witches brooms) of Berberis vulgaris. (A) Mean number of visits during observation periods of 20 min (± 1 SE) in Ardez and in Kippel. (B) Mean number of visits during observation periods of 20 min (± 1 SE) for the different time blocks. (C)Mean time spent per visit (± 1 SE) in Ardez and in Kippel. (D) Mean time spent per visit (± 1 SE) for the different time blocks. The number of visits per observation period and the time spent on uninfected branches and witches brooms are shown in Fig. 7. The number of visits was logtransformed and analyzed with an ANOVA including the sites (Ardez or Kippel), the bushes nested within site, the time blocks, and the branch types (uninfected or witches broom) (Table 13). The untransformed data show higher visitation rates in Kippel than in Ardez (Fig. 7A) and some variation among time blocks (Fig. 7B), but only the type of observed branch had a significant effect on the number of visits per observation period. The 27

32 visitation rates were significantly higher on the witches brooms than on the uninfected branches. TABLE 13. ANOVA on the number of visits by insects and spiders during an observation period of 20 min on uninfected and infected branches of Berberis vulgaris (data log-transformed). Site, bush (nested within site), and time block are included as random factors and branchtype as a fixed factor.p values 0.05 are given in bold face. Source of variation DF MS F P Site Bush[site] Time block Branch type Site * time block Site * branch Type Bush[site] * time block Bush[site] * branch type Time block * branch type Site * time block * branch type Error We used the same model to analyze the log-transformed duration of visits (Table 14). Again, the branch type had a significant effect; the visitors spent significantly more time on witches brooms than on uninfected branches. As can be seen on untransformed data in Fig. 7C, the mean time spent per visit was longer in Kippel than in Ardez on both branch types, but this effect of the site was not significant and the tendency to spend more time on witches brooms was the same for both sites. The interaction between time blocks and branch types was also significant (Table 14). In Fig. 7D we show the untransformed data for this interaction. One can see, that the duration of visits on witches brooms is longer than on uninfected branches for all time blocks, except for the time block six. In this time block, during 6 observation periods of 20 min, only 8 visitors were counted on uninfected branches. One of them was a spider that stayed for the whole 20 min observation time, causing the high mean and the large standard error of the duration per visit in this time block. 28

33 TABLE 14. ANOVA on the time spent per visit on uninfected and infected branches of Berberis vulgaris (data log-transformed). Site, bush (nested within site), and time block are included as random factors and branchtype as a fixed factor. P values 0.05 are given in bold face. Source of variation DF MS F P Site Bush [site] Time block branch type Site * time block Site * branch type Bush [site] * time block Bush [site] * branch type Time block * branch type Site * time block * branch type Error

34 DISCUSSION Identity of the systemic infection The primary host Comparison of ITS sequences of fungi has been successfully used to analyze the relationship between closely related species (Gardes and Bruns, 1993; Zambino and Szabo, 1993; Roy et al., 1998; Pfunder et al., in press). We used this method to compare fungal infections on Berberis vulgaris with fungal infections found on grasses underneath infected bushes to identify the primary host. The result of this comparison on a molecular level differs completely from what is generally accepted based on infection experiments. J. Peyritsch was the first who presumed and tested the host alternation of the systemic infection to Arrhenatherum elatius (in Magnus, 1894). Additional experiments proving the connection of the witches brooms to Arrhenatherum were made by several authors in different countries (review by Urban and Marková, 1994). Among them was Gäumann (1934) who showed that aecia from B. vulgaris in arid sites in the Wallis (Switzerland), where no Arrhenatherum species could be found, caused infection only on A. elatius. Further Mayor (1958) was able to infect B. vulgaris with teliospores from a site in Neuchâtel (Switzerland) with no B. vulgaris in the vicinity. Our sequence data suggest that Puccinia arrhenatheri found on A. elatius is a morphological variation of Puccinia graminis that is closely related to the formae speciales found on Dactylis and Poa and that the grass host of the systemic infection is Melica nutans. As mentioned above, we could not verify the identification of the fungus on M. nutans due to the lack of spores post sequencing, and contamination of our samples with another fungus is unlikely. Gäumann (1934) made his inoculation experiment with species of Agropyron, Arrhenatherum, Bromus, Festuca and Poa but not with Melica nutans. To our knowledge no one has tested whether aecia from the witches brooms on B. vulgaris can infect M. nutans. As for the rust fungus on A. elatius, a simple explanation of the different identification based on morphological and molecular data would be a double infection with P. graminis and P. arrhenatheri on the same plant and a PCR amplification that favored P. graminis. We can not exclude this possibility since our first amplification using the ITS4 and ITS5 primer resulted in bad sequences for the external primers and the teliospores showed a large morphological variation (see Appendix A) which occurs for P. graminis as well as for P. arrhenatheri. On the other hand, we got good sequences for the internal primers that matched well the sequence of P. graminis and were later confirmed by using the ITS4-u and ITS5 primers. In addition, using the binocular microscope we found only covered telia on this sample. This is quite strong evidence that the fungus that was morphologically identified as P. arrhenatheri is genetically identical to P. graminis and is 30

35 therefore not the fungus that causes the systemic infection on B. vulgaris. However, we will try to verify our results with new samples from another locality. Phylogenetic analysis Zambino and Szabo (1993) resolved the phylogenetic relationship of different grass and cereal rust fungi based on the sequence of the ITS region of rdna. Their analysis included rusts found on cultivars, including formae speciales of Puccinia graminis and Puccinia poae-nemoralis with aecia on Berberis vulgaris. Our phylogeny of rusts on native grasses supports their main clusters. The genotypes of P. graminis found on different telial host plants in Switzerland are in the same group as their formae speciales. The published sequence of P. poae-nemoralis (Zambino and Szabo, 1993) is closely related to the systemic infections of B. vulgaris found in Switzerland. This supports the accepted systematics that names the systemic infection P. arrhenatheri, which is assigned to the group of grass rusts with aecial host B. vulgaris and long-time covered telia including Puccinia brachypodii, P. poae-nemoralis and Puccinia pygmaea Eriks. (Cummins and Greene, 1966; Urban and Marková, 1994). Populations of infected Berberis vulgaris in Switzerland Sequence variation We found three slightly different sequence types of systemic infections on Berberis vulgaris in Switzerland and only in Ardez was there variation within a population. A few other studies have examined intraspecific sequence variation in intragenic spacers on rust fungi (Gardes and Bruns, 1993; Zambino and Szabo, 1993; Roy et al., 1998; Pfunder et al., in press) and all found variation within species. Relatively large variation was found within species complexes such as Puccinia graminis (Zambino and Szabo, 1993) and Puccinia monoica (Roy et al., 1998). Some of this variation could be associated with the different telial hosts. Since our samples were all extracted from the same aecial host and so far only Arrhenatherum elatius, and possibly Melica nutans are known as telial hosts (we discussed this above) it is not too surprising that we found much smaller variation. The fungi causing systemic infection on the mediterranean Berberis species aetnensis C. B. Presl., cretica L. and hispanica Boiss. et Reuter, are also considered to be Puccinia arrhenatheri. Whether these fungi occurring on different aecial hosts show greater sequence variation would be an interesting future question. 31

36 Infection incidence Systemic infection is common in most of the larger populations of Berberis vulgaris in Switzerland. The only population of B. vulgaris without systemic infections was found on the Lägern, a foothill of the Swiss Jura. We recorded the infection incidence and the number of witches brooms on B. vulgaris in six populations in Switzerland, where systemic infection was abundant. The higher infection incidence within classes of larger bushes (Fig. 4) and the significant association between infection incidence and main trunk diameter (Table 7), indicate an increasing susceptibility with aging of the bush. However, the mean of the main trunk diameters differ significantly among populations, and a closer look on the different populations shows an inconsistent picture. In Trimmis, where we recorded more small plants than in other populations (lower median of main trunk diameter), the infection incidence was only 0.13, compared to very similar infection incidences in Ardez, Felsberg, Hohtenn and Kippel in a range of 0.44 to This supports the proposed relationship between age and susceptibility, but the populations with similar infection indices had different age structures (different mean and median diameters of main trunks), suggesting that infection incidence is independent of age. The infection incidence in Piotta was higher (0.78), but was calculated based only on a sample of nine bushes. In this small population no bushes with a trunk diameter smaller than 0.8 cm were found, possibly because the population was in an agronomically used grassland that is cut frequently. The lack of very small plants with low infection incidence (Fig. 4) could again explain the higher infection incidence within this population. In addition, the significant effect of the diameter on the number of witches brooms indicates a higher degree of infection on older bushes. Our results show that the infection incidence differs between populations and give some evidence that larger bushes are more often and stronger infected than younger ones. However, our examined populations were not chosen by random, because we focused on populations with herbarium evidence for rust infections. Therefore, the relationship between age of bush and infection status might be valid only for some populations of B. vulgaris in Switzerland and not for all populations of B. vulgaris. In addition, the diameter of the main trunk might be an inaccurate attribute for age of bush among different sites. To establish a correlation between age and infection status of B. vulgaris, if there is any, further research is needed on the growth rates of B. vulgaris in different sites and on the quantification of the infection status. 32

37 Insect-mediated reproduction Insect exclusion experiment The question, whether insects were required for sexual reproduction of the rust fungus, was answered with an exclusion experiment. The mean proportion of aecia-bearing leaves in treatments allowing insect visitation was significantly higher (39% for crawling insects only, 79 % for flying insects only, and 85 % for natural visitation) than on witches brooms without insect visitation (11 %) (Fig. 6). If sexual reproduction depends on insect visitation, how can we explain a mean proportion of aecia-bearing leaves of 11 % in treatments with no insect visitation? Out of 17 witches brooms within this treatment, 16 had aecia bearing leaves only at the edge, where contact with the bags and fertilization through the bags was likely. We did not exclude these leaves in the analysis, because many infected leaves were dried up and shrunken, and did not touch the bags any more, when the bags were opened. Cages of a size preventing any contact of the leaves with the bag, could not have been installed because the branches Berberis vulgaris grow too densely, and the weight of the chicken wire frame would have caused stability problems. Choosing impermeable plastic bags instead of gauze net would have caused other problems. Such bags would have created a different microclimate in these treatments and holes made by thorns of B. vulgaris, would have tended to become larger on plastic bags. Gauze net in contrary, allows water and wind to penetrate, but remains impermeable for insects even when thorns punch through. However, one of 17 witches brooms with no insect visitation had a proportion of aecia bearing leaves of 63 %, which is inexplicable by the design of the experiment. Other studies have also found a certain degree of fungal reproduction on heterothallic rust fungi when visitors were excluded (3-19%) (Craigie, 1927; Pfunder and Roy, 2000; Schürch et al., 2000). A low level of self-compatibility, infection by two fungi of different mating type, or errors in the experimental work, such as undiscovered visitors, can explain these results. In consideration of these findings, we conclude that the rust fungus causing the witches brooms on B. vulgaris is a nearly obligate out-crosser and successful reproduction of the pathogen normally depends on gamete transfer by insects. Numerous ants were seen on witches brooms in previous years. The treatment crawling insects only was designed mainly to test whether ants play an important role for the reproductive success of the fungus. In these treatments, the reproductive success of the fungus was significantly higher than in treatments without insect visitation. However, in the spring of 2000 only a few ants were seen on witches brooms of B. vulgaris, although many ants of the species Formica rufa were present in the vicinity. The treatments crawling insects only were designed in a way that inhibits flying insects to land on infected leaves, but flying insects, once landed, also crawl around, and some of them found the way into the 33

38 cages. Therefore, the reproductive success of the fungus in these treatments could be the result of fertilization by winged insects instead of fertilization by unwinged crawling insects such as ants. Compared to natural insect visitation, the exclusion of flying insects significantly reduced the proportion of aecia bearing leaves, whereas exclusion of crawling insects did not have a significant effect. These results suggest that unwinged insects, such as ants only play a minor role in the fertilization of the fungus on B. vulgaris. Flying insects are more mobile and therefore more likely to have visited a witches broom of the other mating type before. Schürch et al. (2000) conducted an exclusion experiment with the rust fungus Uromyces pisi Pers. on Euphorbia cyparissias L. Unlike us, they observed many ants, but no winged visitors in the treatments crawling insects only. Flying insects are probably more likely to find an opening around a branch of B. vulgaris than a 1 cm gap on the ground, which was left open on the cages they installed on the perennial plant E. cyparissias. Since the reproductive success of U. pisi was even lower with ant-visitation than with no insect visitation, they concluded that ants are bad out-crossers. Hickman (1974) proposed ten traits that adapt plants for pollination by ants. Among others, he suggested that the population must be dense, and plants must be short or prostrate, or if erect, flowers must be sessile to allow readily interplant access to non-flying insects. Assuming that conditions are similar for out-crossing of rust fungi, witches brooms on B. vulgaris clearly do not favor a gamete transfer by ants. In addition the metapleural gland secretions of ants may negatively affect fungal spores (Beattie et al., 1986). Other observed unwinged visitors of witches brooms in Ardez were thomisid and salticid spiders. Outcrossing by spiders presumably is of minor importance, as it is for plant pollination, where only one probable case of pollination by a thomisid spider is reported (Vroege et al., 1987). Insect observations We recorded insect visitation at four different sites in Switzerland to examine the diversity of visitors, and to test whether witches brooms on Berberis vulgaris attract insects during the spermatial phase of the fungus. At all four observation sites the witches brooms had higher mean visitation rates ( visits per hour) than similar sized uninfected branches ( visits per hour). This result suggests that insects are attracted by the leaves on witches brooms. In spring, the leaves on witches brooms appear earlier than leaves on uninfected branches, and they are yellow to orange in color. The dense growth structure of witches brooms increases the spatial concentration of infected leaves, thus forming a conspicuous yellow patch. This yellow patch, caused by the fungus may work as a visual cue for insect, comparable to pseudoflowers induced by some crucifer rusts (Roy, 1993). Roy and Raguso (1997) showed that odor may also be an important attractant 34

39 for insects on pseudoflower-forming rust fungi. Similar to pseudoflowers, infected leaves of B. vulgaris spread a sweet-smelling scent. The fragrances are distinct from those of uninfected leaves (Roy, unpubl. data) and therefore must be induced by the fungus, most likely to attract insects. The total observation time in Ardez and in Kippel was 360 min, whereas in Piotta and Trimmis we made observations only for 80 min. Thus, we can not compare the diversity of the visitors among all four sites. However, at all sites the diversity of observed insects was high. The most frequently recorded taxa belonged to the Diptera and Hymenoptera, but depending on the locality, different genera and species have dominated. In Kippel we made an extraordinary observation - the most tallied species was Acartophthalamus pusio, a small fly that has not been collected in Switzerland before (pers. communication with B. Merz). Diptera of the family Drosophilidae (mainly Drosophila nigrosparsa) were recorded at all four sites, whereas anthomyid and scatophagid flies were common visitors only in Ardez and in Kippel. Species of these two families were also found to be visitors of pseudoflowers induced by rust fungi on crucifers (Roy, 1994; Roy, 1996). Halictid bees, also frequent visitors of pathogen-induced pseudoflowers on crucifers, were never observed on witches brooms of B. vulgaris although they are present in Switzerland. The mostly observed Hymenoptera were ants. Different species of Formica ants were observed in Ardez, Piotta and Trimmis but not in Kippel. The ants were usually present in similar quantities on uninfected branches as on witches brooms. Only on one bush in Trimmis, ants were crowded on a witches broom, as observed in previous years in Ardez. We also looked for ant occurrence on witches brooms in four other localities, where we collected fungal material for sequencing, but again, only a few ants were seen. More attractive food sources may have co-occurred during the spermatial stage of the fungus in spring However, we do not know what caused the higher attractiveness of witches brooms in previous years in Ardez, but the weather might have played a role. In Ardez and Kippel we measured also the time spent per visit. The visitors stayed significantly longer on witches brooms than on uninfected branches, indicating that the sugary nectar, produced by the pathogen, was an attractive food source. It has been shown that the attraction of pathogen-induced pseudoflowers on insects can affect the insect visitation on co-occurring flowers of the host and other plants (Roy, 1994; Pfunder and Roy, 2000). We did not quantify such effects in this study, but the witches brooms may also have an effect on the visitation of co-occurring flowers. The visitation rate on flowers of the host may not be affected in this case, since the flowers of B. vulgaris appear after the spermatial stage of the rust fungus. However, witches brooms mostly dry up before they bloom, and do not contribute to the reproductive success of the 35

40 plant. It is difficult to quantify the reduction of the fitness of B. vulgaris by the systemic infection, since the proportion of infected branches changes for each bush and additional non-systemic infection by stem rust is fairly common. Conclusions We showed that witches brooms in different populations of B. vulgaris in Switzerland are common and caused by the same rust fungus, called Puccinia arrhenatheri. We found only small intraspecific variation in the sequence of the ITS region of ribosomal DNA in different populations. On an indivdual of Melica nutans, growing underneath an infected barberry bush, we found a pathogen that had an identical ITS sequence as most systemic infections on B. vulgaris. Although we could not verify this result, this is evidence that M. nutans may also be used as primary host, in addition to the experimentally proven host alternation to Arrhenatherum elatius. Successful reproduction of the fungus depends on gamete transfer by insects between witches brooms of different mating type. The fungus attracts insects during the spermatial phase, most likely by the yellow discoloration of infected leaves and the sweet-smelling scent. Flying insects, mainly Diptera, are the most important gamete vectors, and crawling insects, such as ants, only are of minor importance. An extraordinary attraction of ants by witches brooms, as observed in previous years in Ardez, was not observed in any locality in We do not know what caused the difference, many factors can affect the attractiveness of a food source, but we will pay further attention to this phenomenon. 36

41 ACKNOWLEDGEMENTS I would like to thank B. Roy for enabling me to do this many-sided project and for her support and advice during the whole work, E. Rohacek for introducing me to the molecular methods, D. Siemens for his comments on the manuscript, B. Merz and R. Neumeyer for insect identifications, S. Züllig for helping with the leaf harvest on thorny barberry bushes, and all other people who contributed in any way to this study. Special thanks also to F. von Planta and the community of Ardez for permission to work on their properties. 37

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43 PFUNDER, M., S. SCHÜRCH AND B. A. ROY. in press. Sequence variation and distribution of pseudoflower-forming rust fungi (Uromyces pisi s.l.) and related taxa in Switzerland. Mycological Research RATHAY, E Untersuchungen über die Spermogonien der Rostpilze. Denkschrift der Kaiserlichen Akademie der Wissenschaften XLVI: ROY, B. A Floral mimicry by a plant pathogen. Nature 362: ROY, B. A The effects of pathogen-induced pseudoflowers and buttercups on each other's insect visitation. Ecology 75: ROY, B. A A plant pathogen influences pollinator behavior and may influence reproduction of non hosts. Ecology 77: ROY, B. A. AND R. RAGUSO Olfactory versus visual cues in a floral mimicry system. Oecologia 109: ROY, B. A., D. VOGLER, T. BRUNS AND T. SZARO Cryptic species in the Puccinia monoica complex. Mycologia 90: SCHÜRCH, S., M. PFUNDER AND B. A. ROY Effects of ants on the reproductive success of Euphorbia cyparissias and associated pathogenic rust fungi. Oikos 88: SWOFFORD, D. L PAUP: Phylogenetic analysis using parsimony. URBAN, Z. AND J. MARKOVA The rust fungi of grasses in Europe.2. Puccinia brachypodii Otth and its allies. Acta Universitatis Carolinae Biologica 38: VROEGE, P. W., A. D. J. MEEUSE AND R. VINKENOOG A probable case of arachnophily involving Euphorbia. Journal of Plant Science Research 3: WHITE, T. J., T. BRUNS, S. LEE AND J. TAYLOR Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp In M. Innis, D. Gelfand, J. Sninsky and T. White (eds.), PCR protocols: A guide to methods and applications. Academic Press, New York. ZAMBINO, P. J. AND L. J. SZABO Phylogenetic relationships of selected cereal and grass rusts based on rdna sequence analysis. Mycologia 85: ZAR, J. H Biostatistical Analysis. Prentice Hall, Upper Saddle River, New Jersey. 39

44 APPENDIX A (Identification of rust fungi) Table A: Identification of the rust fungi based on morphology and host association according to Gäumann's (1959) Rostpilze Mitteleuropas. Morhology of telia, if there were any, and size of teliospores or urediniospores measured with a Leica DMLB microscope. Sizes are given in µm as means ± 1 SE of 20 measurements. Host plant species Arrhenatherum elatius Poa pratensis Dactylis glomerata Agropyron repens Rust fungus species Puccinia arrhenatheri Puccinia graminis Puccinia graminis Puccinia graminis Picture Telia covered none none uncovered Teliospores: Length 46.4 ± ± 0.5 Width 17.5 ± ± 0.3 Stem length 29.8 ± ± 1.0 Wall thickness (side) 2.2 ± ± 0.1 Wall thickness (top) 8.7 ± ± 0.3 Urediniospores: Length 30.0 ± ± ± 0.5 Width 14.0 ± ± ± 0.2 Wall thickness 1.9 ± ± ± 0.1 Pictures of rust fungus spores were taken with a Nikon CoolPix 990 digital photo camera on a Leica DMLB microscope. Magnification is about 500x, but may vary depending on the focus of the camera. Pict. 1: Teliospores of Puccinia arrhenatheri on Arrhenatherum elatius Pict. 3: Urediniospores of Puccinia graminis on Agropyron repens Pict. 4: Urediniospores of Puccinia graminis on D. glomerata Pict. 5: Teliospores and urediniospores of Puccinia graminis on Poa pratensis 40