Research Article |
Corresponding author: Kamil Najberek ( najberek@iop.krakow.pl ) Academic editor: Tiffany Knight
© 2023 Kamil Najberek, Wojciech Solarz, Wojciech Wysoczański, Ewa Węgrzyn, Paweł Olejniczak.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Najberek K, Solarz W, Wysoczański W, Węgrzyn E, Olejniczak P (2023) Flowers of Impatiens glandulifera as hubs for both pollinators and pathogens. NeoBiota 87: 1-26. https://doi.org/10.3897/neobiota.87.102576
|
Flower infestation by pathogens may influence pollination effectiveness. At the same time, by sharing infested flowers, pollinators increase transmission of pathogens. In the presented study we identified fungi that colonised flowers of the invasive alien Himalayan balsam Impatiens glandulifera, one of the most nectar rewarding plants in Europe, as well as its pollinators. We determined factors (e.g., plant size, length of flower lower sepal and the width of its entry, air temperature and sun illuminance) that affect pathogen species presence and pollinators numbers. The study was conducted in three regions in Poland differing in time from the I. glandulifera invasion onset. It allowed embedding our results in the context of the evolution of increased competitive ability (EICA) hypothesis. With reference to this hypothesis we tested whether I. glandulifera from the two younger populations are more frequently pollinated than individuals from the old one, which may be a result of the higher infection prevalence in the flowers of individuals from the latter population. Harmful primary pathogens of I. glandulifera (e.g., Botrytis cinerea and Fusarium graminearum) were identified from its flowers. Although the knowledge of the impact of the recorded pathogen species on the pollinators that transmit them is still limited, these pathogens are known to cause devastating diseases of native plant species and to incur significant economic losses in crops. Therefore, the facilitation of their transmission by I. glandulifera in the invaded communities may pose a serious threat both to native biodiversity and nearby crop production. We did not find support for the EICA hypothesis that flower release from pathogens may increase the pollinator’s activity. Bombus hortorum was the most frequent visitor in the youngest surveyed population, while B. pascuorum was most frequent in the two others. So far the dominance of B. hortorum as a pollinator of I. glandulifera has not been recorded. A possible explanation is that flowers in the youngest population, with significantly wider entries than in the two older ones, were more accessible for this large bumblebee. We suggest that the shifts in flower dimensions may result from the evolutionary processes and/or phenotypic plasticity; however, this suggestion needs to be confirmed in further studies. At the same time, it can be expected that exceptionally frequent visits of B. hortorum in flowers of I. glandulifera in the youngest population may contribute to increasing transmission rate of pathogen species to the new native host plants that are particularly associated with this pollinator.
Bumblebee activity, EICA hypothesis, floral traits, Himalayan balsam, intraspecific and interspecific transmission, pathogen hotspot, post-introduction shifts, pathogen spillback
Himalayan balsam Impatiens glandulifera is native to the western Himalayas, while as an alien species it is known from Europe (35 countries), North America (Canada, United States, Mexico), Oceania (New Zealand) and Asia (Japan) (
I. glandulifera is characterized by traits that facilitate its invasion success. It was shown that it has a high photosynthetic capacity and growth rate (
Flowers are part of the phyllosphere which contains all aerial organs of plants. In general, in contrast to the rhizosphere and endo-rhizosphere, the phyllosphere microbiome was poorly studied to date (
In the presented study we selected three populations of I. glandulifera that significantly differed in age. The population age, determined on the basis of literature data documenting invasion of the species in Poland (
Moreover, these populations were tested for fungal pathogens in the flowers visited by pollinators in order to indicate the population with the highest infections prevalence. Basing on the EICA assumptions, we hypothesised that young and middle aged populations of the species should be less infected than the old one. Importantly, such dependence may drive higher pollination success in the two former populations. The flower pathogens aspect has never been tested using invasive alien plants from populations varying in age.
In the presented study, pollinators and flower pathogens of the one of the most nectar rewarding plants in Europe, invasive alien I. glandulifera (
The experiment was conducted in 2020 in southern Poland. Individuals of I. glandulifera were surveyed in three study regions – the Izerian Foothills, Kraków and Muszyna – differing in terms of the species introduction year (Table
Characteristics of localities of the invasive alien I. glandulifera differing in population age in three study regions (Izerian Foothills, Kraków and Muszyna) in southern Poland; the surveyed plants always occurred at riversides.
Region | Year of introduction (references) | Locality with coordinates (latitude, longitude) | River name | Survey date |
---|---|---|---|---|
Izerian Foothills | 1890s ( |
Zgorzelec (51.103805, 14.982722) | Nysa Łużycka | 9/17/2020 |
Leśna (51.021625, 15.269387) | Bruśnik | 9/17/2020 | ||
Mirsk (50.973962, 15.365690) | Czarny Potok | 9/18/2020 | ||
Kraków and surrounding areas | 1980s ( |
Kraków (50.038255, 19.897358) | Wisła | 9/10/2020 |
Szczyglice (50.086645, 19.814899) | Rudawa | 9/10/2020 | ||
Zabierzów (50.097423, 19.806040) | Rudawa | 9/9/2020 | ||
Muszyna and surrounding areas | 2010s ( |
Andrzejówka (49.342193, 20.819197) | Poprad | 9/14/2020 |
Żegiestów (49.374850, 20.785117) | Poprad | 9/14/2020 | ||
Milik (49.347806, 20.851281) | Milik | 9/15/2020 |
At each locality 10 neighbouring individuals of I. glandulifera growing at the path margin were randomly selected and marked with a unique ID. Stem height and diameter of individuals were measured to include their size (Suppl. material
The surveys were conducted on warm and rainless days. During each survey data loggers (i-Button DS1921G) were used to measure air temperature at 10-minute intervals, while hand-held environmental metre (Extech 45170CM) measured sun illuminance (lux) and wind speed (m/s) at 20-minute intervals.
Over a 60-minute period during each survey the number of pollinators visiting flowers of the selected 10 plants was counted. The counting was always conducted in the morning (at 9:30 h) or in the afternoon (at 13:30 h). Pollinators were defined as all insects visiting flowers to collect pollen or nectar that may carry pollen from male (anther) to female (stigma) flower organs. Following the methodology adapted for I. glandulifera and I. parviflora in our previous study (
At the end of each survey 10 flowers (or less, depending on availability) of each plant individual were cut off and their profiles were photographed against a millimetre paper background (Canon EOS 60D, Canon EF 100 mm f/2.8 Macro USM lens and ring flashlight). The images were used to assess: the length of lower sepal and the width of flower entry. Subsequently, digital images were analysed with ImageJ software (ver. 1.51 k), and the area of one flower side was evaluated; this corresponds to 1/2 of the total flower area (
The cut off flowers were inserted in paper bags (one bag per one plant) and transported in a portable freezer. Molecular identification of fungal pathogen species was carried out in the laboratory of Department of Systematic and Environmental Botany of Adam Mickiewicz University in Poznań (Poland).
The flowers were surface-sterilized in 75% ethanol (30 s), 4.5% sodium hypochlorite (3 min 30 s), 75% ethanol (15 s), and then rinsed in sterile water. After sterilization, the flowers were cut into fragments and placed on Petri dishes with PDA medium and chloramphenicol (one flower per dish). To confirm the efficiency of the sterilisation process, 50 μl of rinse water was spread onto potato dextrose agar (PDA) and incubated at room temperature for 14 days. The addition of an antibiotic to the medium was used to exclude bacteria from the analysis, thus only pathogenic fungi were obtained from the plant tissues. In total, 585 flowers of 90 plant individuals were checked for the fungal presence. The dishes were placed in an incubator at 25 °C. They were observed every day, and emerging fungi were successively transplanted to new plates. For the identification of fungi, the fungal isolates were grouped into morphotypes based on macroscopic characteristics, such as the appearance and colour of the mycelium. Then, isolates representative of each morphotype were analysed using molecular methods.
The DNA was isolated using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, USA) according to the manufacturer’s protocol and was stored at –20 °C. A pair of primers, ITS1F (
The data were analysed with generalised linear models (GLMs) using SPSS ver. 26.0 (
In the first model the number of all recorded pollinators, calculated per plant individual per survey (‘N pollinator records’), was a target variable (sample size = 216). In the base model (Suppl. material
The second GLM model was created only for bumblebee records as a target variable; as in the previous model, single records of B. lapidarius and P. vestalis were excluded from the analysis. This group of pollinators was most numerous. Data on bumblebees were also more precise than on other pollinators, thanks to the assessment of their size. Size assessment is particularly important in analyses of bumblebee effects, because meaningful size polymorphism occurs among workers (
In the last model the number of recorded pathogen species per each surveyed plant was used as a target variable. Moreover, the following variables were included in the model as fixed effects: the number of recorded pollinators (‘N pollinator records’; single records of B. lapidarius and P. vestalis were excluded from the analysis), ‘Region’ differing in population age, two weather variables (‘Temperature’, ‘Sun radiation’), ‘Flowering power’, three variables associated with flower dimensions (‘Flower area’, ‘Length of lower sepal’ and ‘Width of flower entry’) and a single variable representing size of the surveyed plants (‘Plant size’). In addition, the interaction between flowering power and study region differing in age was included in the model (‘Flowering power * Region’). 'Plant size' was obtained from principal component analysis (PCA), based on two variables: (1) stem height and (2) stem diameter. In PCA, the Kaiser-Meyer-Olkin measure of sampling adequacy was 0.50, with a p-value in Bartlett’s test < 0.001. The percentages of variance accounting for the two obtained components were 82% and 18%; the first component with an eigenvalue of 1.640 explained most of the variance.
Raw data used to perform statistical analyses are provided as a file with supporting information.
In total, we recorded 1828 pollinator visits (Table
The number of pollinators recorded at flowers of of I. glandulifera differing in population age in the three study regions (Izerian Foothills, Kraków and Muszyna) in southern Poland.
Pollinator species | N records | TOTAL: | ||
---|---|---|---|---|
Izerian Foothills | Kraków | Muszyna | ||
Bombus hortorum | 27 | 29 | 988 | 1044 |
Bombus pascuorum | 216 | 212 | 9 | 437 |
Apis mellifera | 134 | 33 | 11 | 178 |
Bombus terrestris | 20 | 82 | 0 | 102 |
Bombus lucorum complex | 0 | 31 | 0 | 31 |
Syrphidae | 3 | 5 | 6 | 14 |
Bombus sylvarum | 9 | 0 | 0 | 9 |
Vespula vulgaris | 0 | 6 | 0 | 6 |
Apoidea | 0 | 5 | 0 | 5 |
Bombus lapidarius | 1 | 0 | 0 | 1 |
Psithyrus vestalis | 0 | 1 | 0 | 1 |
TOTAL: | 410 | 404 | 1014 | 1828 |
In the statistical analysis conducted for all recorded pollinators, the model with eight fixed effects and one interaction was selected (Suppl. material
In the best-fit model, the three variables (pollinator species, flowering power and cover area) and the interaction between the regions differing in age and stem height played a significant role (Table
The estimated mean number of pollinators (± confidence intervals) recorded at flowers of I. glandulifera in the three study regions of different population age in southern Poland (Izerian Foothills, Kraków and Muszyna); groups with the same letter above the T-bars are not significantly different at p < 0.05; single records of Bombus lapidarius and Psithyrus vestalis were excluded from the analysis (see Methods).
The scatter plot with fit lines for the relationship between the number of visiting pollinators and stem height of I. glandulifera individuals in the three study regions of different population age in southern Poland (Izerian Foothills, Kraków and Muszyna). The coefficients of determination are R2 Linear: 0.011, Izerian Foothills: R2 Linear 0.007, Kraków: R2 Linear 0.002, Muszyna: R2 Linear 0.065.
GLM best-fit model for the number of recorded pollinators. The following variables were included in the model: pollinator species (Table
Effect | F | df | p |
---|---|---|---|
Pollinator species | 7.31 | 205 | < 0.001 |
Region | 0.45 | 199 | 0.6 |
Flowering power | 8.30 | 198 | 0.004 |
Flower area | 0.74 | 198 | 0.4 |
Stem height | 0.29 | 198 | 0.6 |
Temperature | 1.60 | 198 | 0.2 |
Sun radiation | 2.83 | 198 | 0.094 |
Cover area | 3.44 | 198 | 0.065 |
Region * Stem height | 3.18 | 199 | 0.044 |
In the analysis carried out solely for bumblebees, the model with all seven fixed effects was selected (see base model in Suppl. material
GLM best-fit model for the number of recorded bumblebees. The bumblebee species (Suppl. material
Effect | F | df | p |
---|---|---|---|
Bumblebee species | 8.82 | 233 | < 0.001 |
Pollinator size | 58.79 | 231 | <0.001 |
Flowering power | 18.45 | 230 | <0.001 |
Flower area | 1.60 | 230 | 0.2 |
Temperature | 12.90 | 230 | <0.001 |
Sun radiation | 64.38 | 230 | <0.001 |
Wind speed | 7.38 | 230 | 0.007 |
There were 16 species of fungal pathogens (Table
The number of fungal species records revealed in three study regions differing in population age (Izerian Foothills, Kraków and Muszyna).
Species | N of records | TOTAL: | ||
---|---|---|---|---|
Izerian Foothills | Kraków | Muszyna | ||
Alternaria alternata | 1 | 6 | 8 | 15 |
Epicoccum nigrum | 1 | 7 | 5 | 13 |
Fusarium boothii | 0 | 8 | 0 | 8 |
Fusarium sporotrichioides | 3 | 5 | 0 | 8 |
Alternaria tenuissima | 0 | 1 | 3 | 4 |
Boeremia exigua | 0 | 0 | 4 | 4 |
Epicoccum tritici | 1 | 2 | 1 | 4 |
Fusarium acuminatum | 1 | 2 | 1 | 4 |
Botrytis cinerea | 0 | 0 | 3 | 3 |
Cladosporium cladosporioides | 0 | 1 | 2 | 3 |
Fusarium avenaceum | 1 | 2 | 0 | 3 |
Fusarium equiseti | 1 | 2 | 0 | 3 |
Mucor hiemalis | 1 | 0 | 2 | 3 |
Nigrospora oryzae | 0 | 1 | 1 | 2 |
Fusarium graminearum | 0 | 1 | 0 | 1 |
Stagonosporopsis ligulicola | 0 | 0 | 1 | 1 |
TOTAL: | 10 | 38 | 31 | 79 |
In the analysis of pathogen species records, the model with two fixed effects – study region and flowering power – and interaction between these two variables was selected (Table
GLM best-fit model for the number of recorded pathogen species. The pathogen species (Table
Effect | F | df | p |
---|---|---|---|
Region | 3.03 | 86 | 0.054 |
Flowering power | 6.80 | 85 | 0.01 |
Flowering power * Region | 4.92 | 86 | 0.01 |
The number of pathogen species in flowers of I. glandulifera from the Izerian Foothills (the old population) was lower than that in flowers from the two younger populations from Kraków or Muszyna (contrasts respectively: Estimate = -0.81, SE = 0.187, t = -4.358, p = 3.692E-5; Estimate = -0.84, SE = 0.20, t = -4.157, p = 7.737E-5; Fig.
The estimated mean number of pathogen species (± confidence intervals) recorded from flowers of I. glandulifera in the three study regions of different population age in southern Poland (Izerian Foothills, Kraków and Muszyna); groups with the same letter above the T-bars are not significantly different at p < 0.05.
The relationship between the number of pathogen species and flowering power for different regions. Flowering power was calculated by PCA (combined N of flowers and N of seed capsules); the measurements were carried out in the three study regions of different population age in southern Poland (Izerian Foothills, Kraków and Muszyna).
Infected plants may be less attractive to pollinators because pollinators can usually recognise contaminated flowers and thus they are able to avoid getting infected (
The interspecific transmission of pathogens in I. glandulifera may determine the lower pollination rate of co-occurring native plants. The transmission probably occurs because native species have lower nectar content than I. glandulifera and it is probable that they are not able to mask the presence of pathogens so efficiently as this alien species. Its domination in terms of nectar production may also be manifested in neighbouring crops, whose yield success usually depends on the availability of pollinators (
In Muszyna, the cosmopolitan pathogen Botrytis cinerea was also recorded from the sampled flowers of I. glandulifera. This pathogen causes grey mould disease in more than 200 crop species and could be transmitted by insects (
Another fungal species detected during the study was a secondary pathogen Mucor hiemalis (recorded in the Izerian Foothils and Muszyna). It is a harmless saprophytic species producing secondary metabolites that are toxic to Thielaviopsis paradoxa – a pathogen that causes inflorescence brown rot disease; however, the same metabolites could be harmful to insects that may transmit them (
In the presented study the aspect on floral traits that may influence transmission of pathogens between plant flowers was also taken into consideration. Because flower shape could play a significant role in pathogens transmission (
In our experiment, bumblebees were the most frequently noted pollinator group. Two species dominated – Bombus pascuorum (in Kraków and Izerian Foothills) and B. hortorum (in Muszyna). The former species was recorded as the dominant pollinator of I. glandulifera also in other European studies (
Brain size of bumblebees increases with their body size and it was revealed that individuals with larger brains have superior cognitive abilities to smaller ones (
In the presented study the associations between the EICA hypothesis (
Pollinators facilitate the spread of primary pathogens with invariably negative impact on plants, as well as less harmful secondary plant pathogens (their presence may even benefit a plant) and saprotrophs. In the presented study we demonstrated that the invasive alien Impatiens glandulifera may play a significant role in this process. The nectar-rich flowers of this species are very attractive for common pollinators and should be considered as a hot spot in intraspecific and interspecific pathogens transmission in the invaded communities. We identified each of three types of pathogens from the species flowers: primary pathogens, secondary pathogens and also saprotrophs. The most dangerous plant pathogens were Botrytis cinerea and Fusarium graminearum. These two fungal pathogens cause devastating diseases of native plant species and hundreds of crop species worldwide and are included in the list of top 10 fungal pathogens in molecular plant pathology (
The surveyed I. glandulifera flowers were pollinated mainly by two bumblebee species, Bombus pascuorum and B. hortorum. The former one is known from its association with this plant species, while the latter has never been recorded as its dominant pollinator. Its domination was recorded in the youngest surveyed population of I. glandulifera, in which the flowers had wider flower entries than in two other populations. B. hortorum is a large bumblebee and narrower flower entry may be a barrier for this species. We suppose that the revealed shifts in flower shape of I. glandulifera may have occurred after its introduction in Europe. Nevertheless, this supposition needs to be confirmed in further experiments testing a post-introduction shift of floral traits of I. glandulifera. It should also be noted that exceptionally frequent visits of B. hortorum to I. glandulifera in the population where plants have probably undergone a post-introduction shift may contribute to a significant increase in the transmission rate from I. glandulifera flowers to new native host plants that it also frequently pollinates.
It is noteworthy that the results of our study could not be explained based on the assumptions of the EICA hypothesis. We found that population age of plants surveyed in different regions was not associated with the numbers of recorded pollinators; moreover, the results of flowers’ infestation by primary pathogens were even contrary to these assumptions. In addition, in the presented study the evidence of negative impact of recorded pathogen species on host plants (including crops) is provided, whereas still little is known on the impact of those pathogens on the pollinators that transmit them. Single studies demonstrated that the interactions between plant pathogens and pollinators could be beneficial (e.g., B. cinerea may have nutritional value for Apis mellifera;
We thank Dr Zygmunt Dajdok for his help in I. glandulifera detection in the Izerian Foothills. We also thank Dr Andrzej Kosior for sharing his data on the host plants of B. pascuorum and B. hortorum in Southern Poland. This work was supported by the Institute of Nature Conservation, Polish Academy of Sciences (Cracow, Poland), through statutory funding.
Raw data used to perform statistical analyses
Data type: Tables
Explanation note: Raw data contains datasets on recorded pollinators and floral pathogens used in statistical analyses.
Data on I. glandulifera individuals, bumblebees pollinating them, and additional information on statistical analyses
Data type: Tables, plots
Explanation note: Supporting tables contain data on the size of the surveyed I. glandulifera individuals as well as on the statistical models selection. Supporting figures demonstrate data on bumblebee species recorded from Impatiens glandulifera as well as the influence of weather conditions on the obtained results.