The dataset on nodulation, biomass, relative growth rate (RGR), phosphatase activity (PME), and mycorrhizal colonization rate of Trifolium pratense fertilized by the gradient cow dung supply (sterilized and unsterilized). The results showed that the growth of T. pratense increased with increasing dung supply, while sterilization decreased growth by 15-20% (p=0.068). Root nodulation increased with increasing dung supply but was significantly lower with sterilized dung. Both root PME activity and mycorrhizal colonization decreased with increasing dung supply but were unaffected by sterilization.

Materials and methods

Cow dung, soil and Trifolium pratense plants

Dung of highland cow (Bos taurus L.) was collected in a coastal dune nature reserve, National Park Zuid-Kennemerland, in the Netherlands (52.3961°N, 4.5921°E) on 25 February 2020. Cow dung was collected from six different individuals. They were watched with binoculars until they excreted dung, after which it was directly collected in a plastic bag. The samples were kept in a cooled box during transportation to the Biology Department of the Vrije Universiteit Brussel (VUB) and then stored at 4°C until further processing. Next, subsamples of different dung samples (different individuals) were mixed, sieved, and divided into two parts. One part (about 100 g) was sterilized at 120 °C with an autoclave pressure steam sterilizer (Vapour Line 135-B, VER International, the USA). The second part was kept unsterilized. Small subsamples of sterilized and unsterilized dung (ca. 10 g) were dried in the oven at 70°C for 72 hours. 

Seeds of Trofolium pratense obtained from a local provider (EcoFlora) were germinated in potting soil 3 weeks before the experiment. Then seedlings were transplanted from the germination trays to the experimental pots. Their roots were washed in demi water before planting in the sand. The biomass of the seedlings at the start of the experiment was on average 4.0 ± 0.1 mg (dry weight determined of 5 seedlings from the germination tray). This average value was used for calculating the relative growth rate (RGR).

Experimental design and measurements

On 25 March 2022, T. pratense seedlings were transplanted to pots (0.4L) filled with a mixture of quartz sand with fresh cow dung, and 2 g of fresh soil. One seedling was planted per pot, and pots were placed on individual saucers. The quartz sand (obtained from Sibelco, type S32), had negligible N and P concentrations; levels below detection limits. The dung was supplied either in a sterilized or unsterilized treatment, and the dung supply level covered gradients of 1, 2, 4, 8, 12, 16, 24, 32, 48, and 64 mg N per pot. All treatments were duplicated. Thus, the total number of pots was: 10 dung supply levels × 2 dung sterilization treatments × 2 replicates = 40 pots. These pots were randomly distributed in a compartment of the greenhouse at the VUB Etterbeek campus in Brussels, and were watered with demi water supplied in the saucers addressing the demand of the plants, i.e. two or three times a week to prevent water limitation. After six weeks, leaf chlorophyll content represented by light absorption (SPAD values) was measured in three leaves of all T.pratense plants, using a Chlorophyll Meter 502-SPAD Plus (Konica Minolta, Munich, Germany).

After 7 weeks (12 May 2022) the plants were harvested by washing the sand from the roots, and separating plants into shoots and roots. The shoots were put in paper bags and dried at 70°C for 48 hours, after which dry mass was measured. The roots were carefully inspected for the presence of root nodules, and the number of nodules was recorded and used as a proxy for nodulation rate. After this step, the roots were dried with tissue paper and fresh root weight was measured. Subsequently, ca. 100 mg fresh roots (representative subsamples) were put in reaction tubes for the analysis of root PME activity. The root PME activity was determined using the para-nitrophenyl phosphate (pNPP) spectrophotometric method. Absorbance at 410 nm was measured with Genesys^TM 10 Series Spectrophotometer (Thermo Spectronic, Darmstadt, Germany), and the ‘μmol pNPP cleaved per g fresh root per hour’ was used to indicate root PME activity. A second subsample of the fresh roots was weighed (to enable dry mass calculation later) and put in a stored 50% glycerol solution. These subsamples were used to measure the root mycorrhizal colonization rate using the root segment colonization weighting method. Thereto, the roots were cleaned and stained, after which the mycorrhizal colonization was assessed for 25 root fragments (length 1 cm) for each root sample. Due to the limited quantity of root samples available, we lack data on the mycorrhizal colonization rates of two plants. Finally, a third subsample of the fresh roots was used to determine the moisture content of the fresh roots. Of these subsamples, both fresh weight and dry weight (after 48 hours at 70°C) were determined. The moisture contents of these subsamples were also used to calculate the dry weights of the subsamples used for root PME activity and mycorrhizal colonization. This enabled the calculation of total root biomass, total biomass, and root mass ratio (RMR = dry root mass / dry total plant mass). The RGR was calculated as (ln(dry plant mass at harvest) – ln(dry plant mass at the start) / (the number of days) (Hunt, 1990).

Statistics

Prior to conducting all statistical analyses, the dung supply was transformed by log(x+1), and the SPAD values representing leaf chlorophyll content by log(x) to meet assumptions of normality and homogeneity. We used the function ‘lmer’ in the lme4 package for linear mixed-effects modelling (LMMs) on RGR, root PME activity, mycorrhizal colonization rate, biomass, RMR and leaf chlorophyll (log(SPAD)), with the replicate as a random factor and obtained the ANOVA result by Type III Wald Chi-squared tests using the function ‘Anova’ in the car package (version 3). Additionally, we fitted a model of the number of root nodules by generalized linear mixed-effects modeling (GLMMs) with Poisson distribution using the function ‘glmer’ in the lme4 package. Moreover, we also assessed the relationship between RGR with the number of root nodules and with the mycorrhizal colonization rate under the two dung sterilization treatments and the gradient of dung supply by LMMs. Statistical analyses were conducted using R Statistical Computing version 4.3.2.