Are prohexadione calcium and Pseudomonas fluorescens a solution to limit the spread of annual bluegrass (Poa annua L.) on football pitches?

A greenhouse experiment was carried out at the Biotechnical Faculty in Ljubljana (46°04′ N, 14°31′ E, 299 m a.s.l.), Slovenia. Greenhouse temperatures at the time of the experiment were 17 °C during the day and 4 °C at night. Plants received artificial light (high-pressure sodium vapour light, Osram, luminosity of light: 550 nm, rated power: 400 W, lighting power: 178 W · m⁻², CO₂ concentration by light: 500 ppm) between 4:00 p.m. and 8:00 p.m. and were watered in 3 days interval with tap water. The pots were arranged in a split-plot design with three replications. The experiments consisted of 450 pots with a volume of 2 l, including untreated controls.

Turfgrass seeds were sown on 13^th January 2020 in pots filled with the special substrate ‘Biokompovit trava’ (supplier: Poljo Posavec Ltd., Croatia) for grass at a sowing rate of 0.3–0.7 g · pot⁻¹, which corresponds to the following sowing rates: 10 g · m⁻² for annual bluegrass and Kentucky bluegrass, and 35 g · m⁻² for perennial ryegrass (Turfgrass Seed, 2016). We seeded a mixture of perennial ryegrass. cv. ‘Fiesta IV’ and cv. ‘Rinovo’ (150 pots; supplier: Semillas Fitó Barcelona Ltd., Spain), Kentucky bluegrass cv. ‘Legend’ (150 pots; supplier: Poljo Posavec Ltd., Croatia; producer: ICL SF Ltd., France) and a population of annual bluegrass (150 pots; supplier: Naturescape British Wildflowers Ltd, Maple Farm, Coach Gap Lane, Langar, Nottinghamshire, NG13 9HP, England). Only a single grass species was sown in each pot. The substrate for sowing (‘Biokompovit trava’) was a mixture of quartz sand, white and black peat, and 100% organic materials without chemical ingredients that originated from Poljo Posavec Ltd. (Croatia).

Pots were fertilised with organomineral granular NPK (nitrogen-phosphorus-potassium) fertiliser DCM Vital green 14–4–8+3 MgO+Fe (at 0.9 g · pot⁻¹; 300 kg · ha⁻¹) producer: DCM Ltd.; Grobbendonkmesto, Belgium) and the soil improver DCM Antagon at a rate of 4 g · pot⁻¹ (20 kg · 100 m⁻²). Fertilisers were obtained from Vitalis Crop Care Ltd. (Samobor, Croatia).

Our study was divided into two different separated individual experiments. In Experiment 1, we were interested in the activity of PGRs on previously uncut grass, and in Experiment 2, we cut the grass with hand scissors (at 2.5 cm) before the first spraying. Respectively, each of mentioned experiments was performed in 225 pots; namely, each of three turfgrass species was grown in 75 pots (15 pots per specific treatment, including replications in time). Respectively, five pots were included in specific treatment. In both experiments, the grasses had developed 3–4 tillers at the time of the first spraying.

In both experiments, we have tested five treatments, i.e. three PGRs and positive and negative control, i.e. (1) P. fluorescens × Azospirillum brasilense (product Rhizoflo Premium; applied as 2.5% [45 ml · 1.8 l⁻¹ H₂O] in manuscript marked as Pf × Ab); (2) Pc 100 g · kg⁻¹ (product Regalis^® Plus; applied as 3.6 g Regalis^® Plus + 1.89 g Pehacid · 1.8 l⁻¹ H₂O, in manuscript marked as Pc); and (3) Eth 480 g · l⁻¹ (product Baia; applied as 7.5 ml · 1.8 l⁻¹ H₂O; in manuscript marked as Eth). Grasses in the positive control were sprayed with the herbicide Callisto^® 480 SC (a.i. mesotrione [Mes] 480 g · l⁻¹; applied as 1.4 ml · 1.8 l⁻¹ H₂O; in manuscript marked as Mes), while control treatment (in manuscript marked as Contr) was done only with sprayed with tap water. The biofertiliser Rhizoflo Premium is produced by CKC^® Argentina SA., the plant growth regulator Regalis^® Plus is produced by BASF SE Ltd. (Germany), the plant growth regulator Baia by ADAMA Agriculture B.V. Ltd. (Switzerland) and the herbicide Callisto^® 480 SC by Syngenta Crop Protection AG Ltd. (Switzerland). Mes, as the active substance in selected herbicide, was previously mentioned as successful against annual bluegrass encroachment by Park et al. (2019). To continue, Mes was initially labelled to use in corn production. More recently, it has been labelled to use in turfgrass production, and it is safe to use at cool-season turfgrass establishments (Venner et al., 2011).

Spraying dates, regarding selected products and Experiments 1 and 2, are presented in Table 1. In Experiment 1, mowing was performed on 3^rd and 27^th March, while in Experiment 2, mowing was performed on 24^th February and 20^th March. Mowing of turfgrass was performed to 2.5 cm (Manager's Guide to Natural Grass Football Pitches, 2021).

Measuring of plant height and counting the number of tillers in Experiment 1 were carried out on the following dates: 11^th February (only plant height); 18^th, 21^st, 25^th and 28^th February; 3^rd, 10^th, 13^th, 17^th, 20^th, 24^th, 27^th and 31^st March; and 3^rd, 7^th, 10^th, 14^th and 17^th April. In Experiment 2, both parameters were evaluated as follows: 28^th February; 3^rd, 10^th, 13^th, 17^th, 20^th, 24^th, 27^th, 31^st March; and 3^rd, 7^th, 10^th, 14^th, and 17^th April. The number of tillers per plant was assessed using a 9-point visual scale (BBCH MONOGRAPH, 2018), where a score of 1 represents the moment the first tiller penetrated the coleoptile, a score of 2 represents when the first tiller was unfolded, a score of 3 represents when 2 tillers were unfolded, a score of 4 represents when 3 tillers were unfolded, a score of 5 represents when 4 tillers were unfolded, and a score of 6 represents when 5 tillers were unfolded. A score of 7 marks the beginning of growth or when the first lateral tiller was detected, a score of 8 represents when two lateral tillers were detected, and a score of 9 represents when three lateral tillers were detected. The plant height was measured using a ruler in the middle of each of the pots. Counting of developed tillers was performed visually according to studied literature (Boyle et al., 2016; Tilley et al., 2019). The number of tillers was counted on five randomly selected plants in each of the pots. For purpose of calculation ratios (plant height, tiller ratio), we have divided Experiment 1 into three different periods (1^st period – 11^th February till 3^rd March, 2^nd period – 6^th March till 27^th March, 3^rd period – 31^st March till 17^th April) and Experiment 2 (1^st period – 24^th February till 20^th March, 2^nd period – 24^th March till 17^th April).

Analysis of variance (ANOVA) was conducted to determine the differences among different turfgrasses within treatments. Differences in plant height and number of tillers per plant were analysed with ANOVA. Before analysis, each variable was tested for homogeneity of variance, and nonhomogeneous data were log (Y) transformed before ANOVA. Significant differences (p ≤ 0.05) between the mean values were identified using the Student-Neuman Keuls honestly significant difference multiple range test. All statistical analyses were performed using Statgraphics Centurion XVI software (Statgraphics Centurion, 2009), and the results are presented as the untransformed mean ± the standard error (SE). Plant height ratio was calculated by dividing the average height of each turfgrass species on the last date of the evaluation period with the average height of plants on the first date of the evaluation period, while the tillers ratio was calculated by dividing the average number of tillers of each turfgrass species on the last date of the evaluation period with the average number of tillers on the first date of the evaluation period (Tilley et al., 2019).

We have detected a statistically significant impact of all main factors and their interactions on average plant height in Experiments 1 and 2. All values are presented in Table 2. Based on the general analysis in Experiment 1, plant height was significantly the highest in the control treatment (5.55 ± 0.13 cm) and Pf × Ab treatment (5.45 ± 0.12 cm). Significantly, the lowest average grass height was detected in the Pc treatment (3.83 ± 0.11 cm), which was approximately 2 cm lower than in the control treatment, and in treatment sprayed with Pf × Ab. Significantly, the lowest average plant height in Experiment 2 was recorded in the Pc treatment (3.75 ± 0.10 cm), while the highest average plant height was detected in the control treatment (5.13 ± 0.12 cm) and in the Pf × Ab treatment (5.09 ± 0.11 cm). On average, plants treated with Pc in Experiment 2 were 1.39–1.43 cm lower than plants in the control treatment and plants treated with Pf × Ab.

All main effects and their interactions, which have influenced plant height in Experiment 1, are presented in Table 3.

When annual bluegrass was exposed to Pc, an average height of 3.83 ± 0.18 cm was confirmed. Additionally, an average value of 5.91 ± 0.24 cm was recorded in the Pf × Ab treatment. The average plant height of perennial ryegrass under the Pc treatment reached 4.34 ± 0.25 cm, whereas a height of 5.84 ± 0.26 cm was found for the control treatment. The average grass height of Kentucky bluegrass ranged from 3.51 ± 0.16 cm in the Mes treatment to 5.19 ± 0.18 cm in the control treatment. For the Pc and Eth treatments, the average grass heights were 3.31 ± 0.09 cm and 3.56 ± 0.11 cm, respectively.

On the first evaluation date (11^th February), the average plant height in annual bluegrass treatment was ranging from 5.44 ± 0.22 cm in Eth treatment to 4.92 ± 0.30 in Contr treatment. On 6^th March, the highest average plant height was recorded in Pf × Ab treatment (4.1 ± 0.11 cm). On the last evaluation date (17^th April), the average plant height was significantly the lowest in Pc treatment (2.60 ± 0.10 cm). All values are presented in Figure 1.

Figure 1

At the start of the evaluation period (11^th February), there was no significant difference detected between the five treatments on perennial ryegrass. On the first evaluation date after the first mowing, the average plant height was the lowest among all treatments. Suppression of plant growth by Mes was detected after 27^th March. All values are presented in Figure 2.

Figure 2

When Kentucky bluegrass was exposed to five different treatments during the growing period, the average plant height was the highest 3 days after first mowing, ranging from 3.90 ± 0.17 cm at Contr treatment to 2.50 ± 0.10 cm at Mes treatment. When Kentucky bluegrass was sprayed with Pc, growing stopped after second mowing. All values are presented in Figure 3.

Figure 3

All main effects and their interactions, which have influenced plant height in Experiment 2, are presented in Table 3. We detected an average plant height of 3.86 ± 0.27 cm for Kentucky bluegrass, 3.86 ± 0.17 cm for perennial ryegrass, and 3.81 ± 0.15 cm for annual bluegrass when plants were exposed to Pc treatment.

The average plant height of annual bluegrass was the highest on Contr treatment during first and second mowing, ranging from 5.10 ± 0.15 cm on 28^th February to 8.10 ± 0.15 cm on 20^th March. After second mowing, the average plant height of annual bluegrass did not reach >2.5 cm. Detailed values are presented in Figure 4.

Figure 4

After first mowing till second mowing, the average plant height of perennial ryegrass reached the highest values when sprayed with Pf × Ab. After the second mowing, the highest average plant height was recorded on 3^rd April (5.00 ± 0.11 cm) at Eth treatment and did not grow additionally till the end of the experiment. All detailed values are presented in Figure 5.

Figure 5

We have detected that average plant height at Kentucky bluegrass stands out through the entire growing season, where the grass was sprayed with Pf × Ab and at Contr treatment. When sprayed with Pf × Ab, the average plant height was ranging from 7.30 ± 0.15 cm on 6^th March to 8.00 ± 0.15 cm on 20^th March. After mowing, the average plant height was ranging from 2.50 ± 0.15 cm after days after mowing to 4.60 ± 0.10 cm on 10^th April. Detailed values are presented in Figure 6.

Figure 6

We have detected a statistically significant impact of all main factors and their interactions on the average number of tillers per plant in Experiments 1 and 2. All values are presented in Table 4 based on the general analysis in Experiment 1, the average number of tillers per plant was significantly the highest in the Pc treatment (4.40 ± 0.09). Significantly, the lowest average number of tillers was detected in the Mes treatment (3.00 ± 0.05), which is >1 tiller per plant lower than on turfgrass treated with Pc. Significantly, the lowest average number of tillers per plant in Experiment 2 was recorded in the Mes treatment (3.31 ± 0.07), while the highest average number of tillers per plant was detected in the Eth treatment (4.32 ± 0.10) and in the Pc treatment (4.20 ± 0.08). Therefore, the average tiller number on plants in positive control reached >1 tiller per plant less, than plants exposed to Eth and Pc.

All main effects and their interactions, which have influenced the average number of tillers in Experiment 1, are presented in Table 5. In general, the highest average number of tillers was detected on turfgrass, which was treated with Pc. Significantly the lowest number of tillers per plant was detected at Mes treatment (3.06 ± 0.05). In general, the highest number of tillers per grass was recorded, when grass was sprayed with Pc (4.40 ± 0.09). When annual bluegrass was sprayed with the product Eth, 3.45 ± 0.12 tillers per plant were detected, while 4.06 ± 0.12 tillers per plant were detected when annual bluegrass was sprayed with Pc. On the other hand, when perennial ryegrass was treated with Eth, 4.41 ± 0.18 tillers per plant were detected, while 4.47 ± 0.14 tillers per plant were detected when the Pc was used. Eth and Pc, as active substances, reached the same effect.

When sprayed with Pc, the average number of tillers at annual bluegrass was ranging from 3.20 ± 0.16 on 28^th February, 5.6 ± 0.10 on 27^th February to 4.80 ± 0.15 on 3^rd April. At Mes treatment after first mowing, the average number of tillers reached 3.6, meanwhile 3 days after last mowing 3 tillers were on average recorded at annual bluegrass. Additional values are presented in Figure 7.

Figure 7

The average number of tillers per perennial ryegrass was the highest in treatments, where we have used Eth and Pc. What's more, on several dates of evaluation (27^th March, 3^rd April, 7^th April, 17^th April) we have detected on average 6 tillers per plant. At Mes treatment, the stagnation in tiller formation was detected 14 days before the second mowing. Detailed values are presented in Figure 8.

Figure 8

Where Kentucky bluegrass was sprayed with Pc, the highest average number of tillers was measured in almost all evaluation dates. Almost 6 tillers per plant were measured on the day of the second mowing. All additional values are presented in Figure 9.

Figure 9

All main effects and their interactions, which have influenced the average number of tillers per plant on specific turfgrass, are presented in Table 5. In general, the average number of tillers ranged from 3.48 ± 0.04 for Mes, 4.32 ± 0.09 for Eth, 3.65 ± 0.05 for the Control, 4.20 ± 0.08 for Pc, and 3.55 ± 0.05 tillers per plant for Pf × Ab. When annual bluegrass was treated with Eth, 3.27 ± 0.07 tillers per plant were detected; in contrast, 5.20 ± 0.18 tillers per plant were detected for perennial ryegrass. When Kentucky bluegrass was sprayed with Pc, 3.88 ± 0.09 tillers per plant were detected. Additional data are presented in Figures 10–12.

When annual bluegrass was sprayed in Experiment 2, spraying with Pc proved the highest average number of tillers per grass, reaching almost 5 tillers per plant on 3^rd and 7^th June. Additional values are described in Figure 10.

Figure 10

When perennial ryegrass was sprayed either with Pc or Mes, the highest tiller number was detected in almost all dates of evaluation throughout the entire evaluation period. When perennial ryegrass was sprayed with Mes, the average tiller number per plant was the lowest in almost all dates. All detailed numbers are presented in Figure 11.

Figure 11

After second mowing, when Kentucky bluegrass was sprayed either with Eth or Mes, the average number of tillers per plant was either the highest or the lowest. Detailed data are presented in Figure 12.

Figure 12

Table 6 presents the plant height ratio of three turfgrass species across five treatments over three growth periods. Regarding the first two periods of growth, Pc most efficiently restrained the growth of annual bluegrass, as the average plant height increased by only 28% in the first period, while in the second period, the annual bluegrass did not grow at all after mowing. Pc also showed a restraining effect on the growth of perennial ryegrass and Kentucky bluegrass in the first period, in which the plants were 6% shorter before the first mowing or only 9% taller than at the first measurement, respectively. Pc also showed a restraining effect on the latter two turfgrass species in the second period, and 3 weeks after mowing, the plants grew to only 20% of their original height, but Eth also showed a significant inhibitory effect on both species.

However, interestingly, Eth did not show an effect on annual bluegrass. The herbicide Mes did not have any effect on annual bluegrass in the first two periods, but it affected the complete cessation of the growth of perennial ryegrass in the first period. The perennial ryegrass sprayed twice with this herbicide collapsed in the third growth period, while similarly treated Kentucky bluegrass collapsed in the second period.

When we compared the number of tillers per plant of annual bluegrass in the first evaluation period, the ratio was the highest for the control treatment and the lowest for the Pf × Ab treatment. In the second evaluation period, the tiller number ratio was the lowest for plants sprayed with Eth. The most uniform decline in the tiller number ratio overall assessment periods was observed for Pf × Ab and Pc, with the largest decrease in the number of tillers found for Pf × Ab. All detailed values are presented in Table 7.

In Experiment 2, in the first and second assessment periods, the lowest growth ratio for annual bluegrass was found for the Pc treatment. This product also showed an inhibitory effect on the growth of the other two turfgrass species. The Mes treatment (herbicide) showed better performance, since, in the second period of growth, it caused the collapse of both turfgrass species. The remaining values are presented in Table 8.

Tillers ratio measured on 20^th March for annual bluegrass plants sprayed with Pc × Ab was 7% lower than the tillers ratio on 28^th February. Additionally, in the second period, the tillers ratio under this treatment increased only slightly (6%). In both periods, comparable indices were found for the Pc × Ab and Mes treatments. After comparing the two evaluation dates in the second evaluation period for annual bluegrass sprayed with Eth, the tillers ratio increased by 63%. Pc × Ab also showed satisfactory performance on the development of a small number of tillers for annual bluegrass in the first period. The remaining details are presented in Table 9.