Invasive plants apply new selection pressures on neighbor plant species by different means including allelopathy. Recent evidence shows allelopathy functions as remarkably influential mediator for invaders to be successful in their invaded range. However, few studies have determined whether native and non-native species co-occurring with invaders have evolved tolerance to allelopathy. In this study, we conducted germination and growth experiments to evaluate whether co-occurring native Juncus pallidus and non-native Lolium rigidum species may evolve tolerance to the allelochemicals induced by Cyanara cardunculus in Australian agricultural fields. The test species were germinated and grown in pots filled with collected invaded and uninvaded rhizosphere soil of C. cardunculus with and without activated carbon (AC). Additionally, a separate experiment was done to differentiate the direct effects of AC on the test species. The soil properties showed invaded rhizosphere soils had higher total phenolic and lower pH compared with un-invaded soils. We found significant reduction of germination percentage and seedling growth in terms of above-and below ground biomass, maximum plant height and root length of native in the invaded rhizosphere soil of C. cardunculus, but little effect on non-native grass species. Even soil manipulated with AC showed no significant differences in the measured parameters of non-native except aboveground biomass. Taken together, the results indicate allelochemicals induced by C. cardunculus exert more suppressive effects on native than non-native linking the co-evolved tolerance of those.

MATERIALS AND METHODS

Our experiments were set to test the allelopathic effects of C. cardunculus on germination and growth of grass species that co-occurs in agriculture field in Victoria, Australia. We used field-collected rhizosphere soils to imitate natural conditions through exposing of target species that minimize uncertainties in plant extract concentrations or exclusion of other possible edaphic effects (Gómez-Aparicio & Canham, 2008). As the allelopathic effects are species-specific (Callaway & Ridenour, 2004; Gomez-Aparicio & Canham, 2008), we also devised the experiments to determine the evolved tolerance of associated native versus non-native grass species to the allelopathic potential of C. cardunculus in its invaded community. In addition, chemical properties (total phenolic content and pH) of C. cardunculus rhizosphere were assessed to test the differences among the populations. Overall, this study aimed to evaluate allelopathy and co-evolutionary effects of rhizosphere soil induced by C. cardunculus on co-occurring native and non-native grass species in its invaded range.

Measurement of field soil chemical properties

To test whether invasive plant species C. cardunculus changes chemical soil properties, we collected rhizosphere soil at the end of November, 2018 during its highest biomass production from three invaded and three nearby non-invaded populations in agricultural land at 26.7 m above sea level in Werribee South, Victoria, , Australia (37°54'44.5"S 144°42'00.8"E). We designed our sampling protocol to minimize the effects through selection of homogenous soil profile, and plant density and diversity in uninvaded and invaded sites of each population, which were also close to each other. The location of each population including soil from both invaded and nearby non-invaded was separated from the others by a distance of at least 500 m. At each location, we collected five samples from five individuals of C. cardunculus as invaded soil samples and five from nearby non-invaded population where other species such as Juncus pallidus, Maireana decalvans, Themeda triandra etc. except C. cardunculus were present. After that, the soil samples were transported into the laboratory, sorted and homogenized for composite sample. Then, we took five sub-samples from each population for measurement of soil properties and measured total phenolic content (TPC) and pH in soil as it is assumed those properties might play a significant role in testing allelopathy, though rhizosphere has also numerous aspects (Scavo, Abbate, et al., 2019). Soil pH was determined with a pH meter (Pocket digital pH meter, 99559, Dick Smith Electronics, Australia) in a 1: 2.5 w/v (soil: distilled water) ratio (Paz‐Ferreiro, Trasar‐Cepeda, Leirós, Seoane, & Gil‐Sotres, 2007). Soil phenolics was determined by sampling 100 mg of air dried soil following the Folin-Ciocalteu method (Blainski, Lopes, & De Mello, 2013).

Germination experiment

However, the multiple populations provide the possibility to check the variation among populations through increasing the reliability of the results, and better representing the condition of each treatment, but we avoided using multiple populations in this occasion due to complexity in research design and the possibility of confusion. The collected homogenized rhizosphere soil from only one population was used in the experiment as no attempt was made to check variation among populations. Half of the soil (invaded and non-invaded) was thoroughly mixed with activated carbon (AC) and the mixture was placed into the pots as a treatment. The remaining soil was considered as a treatment without AC. The AC has special characteristics with high absorptivity of organic complexes such as allelochemicals, and weak affinity for inorganic molecules, such as those present in nutrient solution. Many allelopathic studies related to C. stoebe including C. maculosa, C. rhenana, C. muretii, C. vallesiaca and C. stoebe subsp. have demonstrated through reducing the suppressive effects of root exudates (Callaway, Ridenour, Laboski, Weir, & Vivanco, 2005; Lyytinen & Lindstrom, 2019; Ridenour & Callaway, 2001). The 100 g soils were placed in punnets (9.75 cm by 6.75 cm) with five replicates. The punnets were moistened and kept at room temperature for 24 hours to activate the soil microbes. The sterilized seeds (collected from fields invaded by C. cardunculus L.) of native Juncus pallidus and non-native Lolium rigidum grass species were then seeded at a rate of 20/pot. The tested seeds were selected due to the following reasons: i) they are co-occurring plant species of C. cardunculus L.; ii) allelopathic interactions between invasive alien and coexisting plant species (native and non-native) seem to be one of the underlying mechanisms for the invasion success of some invaders; and iii) testing the evolved tolerance of the co-existing species. We followed a complete randomized block design in this study. The pots were kept in shade for 3 days, and then transferred to the natural lit greenhouse at 23 ± 3° C day and 12 ± 2° C night temperatures. The pots were watered with automated irrigation system twice in a day. Pots were randomly shuffled every week to minimize the spatial effects. Then, after four weeks, the germinant was counted for each treatment.

Growth and establishment experiment

The soils were prepared as per the abovementioned germination experiment and the 100g soils were placed in punnets (9.75 cm by 6.75 cm) for growth experiment. However, the size of the punnets may seem tighten for growth of whole period of the experiment, but the plants can adopt with it, and it may not interfere the findings as it was applicable for all replicates of both tested species. About 300 (three hundred) sterilized seeds of each grass species (native and non-native) were germinated in a tray (30 cm by 28 cm) filled with sterilized sand. One seedling of both species was then transplanted into the prepared punnet by twenty-one replicates and pots were grouped by target species following a randomization across treatments and kept as per the abovementioned condition. Dead seedlings were replaced after a period of two weeks, to allow the experiment to continue. The pots were watered using an automated irrigation system twice in a day and fertilized fortnightly with liquid fertilizer (Hyponex, N-P-K, 6-10-5, Hyponex Inc., Japan) at a concentration of 2ml/1 of water. Pots were randomly shuffled every week to minimize the spatial effects, and after 3.5 months, the plants were harvested for biometric parameters including maximum plant height and root length, aboveground biomass (AGB), belowground biomass (BGB), total biomass (AGB plus BGB), and AGB-BGB ratio

We conducted a separate experiment along with the main experiments to assess the direct effect of AC on seedlings growth of native and non-native used grass species with sterilized sand and AC treatments. We used five replicates per treatment (with and without AC) for both of grass species. The direct effect of AC on plant growth has been assessed due to its complication on allelopathy (Lau et al., 2008). This experiment was carried out adopting the same procedure as the aforementioned experiment and measured biometric parameters (Appendix S1).

Data analyses

An independent sample t-test was performed to check the differences of soil phenolics and pH level among un-invaded and invaded sites for each population of C. cardunculus L. Two-way ANOVAs were also conducted to test the differences of those variables as functions of populations and invasion status (un-invaded and invaded). We again conducted two-way ANOVAs to test the effect of soil allelopathy as functions of activated carbon (with and without AC) and invasion status (un-invaded and invaded) on AGB, BGB, AGB-BGB ratio, total biomass, maximum plant height and root length of native and non-native grass species. Target species (native and non-native) were not considered as third fixed factor in the univariate general linear model due to avoid complexity. For example, the test does not provide which specific species is significantly different from each other. As a result, separate analysis (species-specific) was done to get direct understanding of the allelopathic effects on each of them. Moreover, an independent sample t-test was performed to compare the significant differences between AC treatments (with vs. without carbon) and species (native vs. non-native) in both of germination and growth experiments including separate growth experiment. To maintain homogeneity of variances and normality of the used data for these analyses, we used data transformations techniques:: a square root (soil phenolics, AGB, BGB, total biomass for native; AGB fornon-native) and an arcsine-square root function (germination percentage for native) We also applied Levene’s test for homogeneity of variance and Komolgorov-Smirnov (K-S) test for normality of the used data. All data were analyzed using SPSS version 20.0 (IBM Corporation, NY, USA).