mailto:herbycides@gmail.com



JUNE 2024


Study into the use of two novel bio stimulants in the Australian turf
industry to improve plant health and stimulate root growth.

Sunday 23 June 2024

Authors Jerry Spencer BSc Hons, and Cameron Smith CSTM.
jerry@gilbasolutions.com

Abstract: The use of biostimulants and turf pigments is gaining traction in the professional turf industry.
Combinations of salicylic acid, copper phthalocyanine, EO-PO block copolymers, 2,3- Butanediol and β-
cyclocitral were researched for their use in turfgrass and their effects on turf quality, colour or root growth.

A replicated block pot trial was run for 133 days on perennial ryegrass, looking at using these as stand-alone
applications and incorporating them with a combination of copper phthalocyanine pigment, salicylic acid
and a novel soil wetting agent formulation. Results were as follows:

1. Pigment applications gave significantly higher DGCI and Colour Quality readings than all other treatments.
2. 2,3 Butanediol gave a significantly lower Colour Quality result than the untreated control.
3. The pigment and pigment plus bio stimulants gave a significantly higher Percent Cover and Cover Quality
readings than all the other treatments.
4. At a depth of 0-2cm no significant differences in organic matter content were observed but all the
treatments gave higher levels than the untreated control.
5. At a depth of 2-4cm, although not significantly higher than the untreated control, the pigment plus bio
stimulant gave approximately 2x greater organic matter content than the untreated control.
6. Although not being significantly higher than the untreated control, the Wetting agent + bio stimulant gave
the highest organic matter contents at a depth of 4-6cm.
7. At a depth of 6-8cm, with the Wetting agent + bio stimulant and pigment plus bio stimulant treatments
gave the highest organic matter contents.

Keywords: biostimulant; turfgrass; organic matter; root growth; 2-3-butanediol; β-cyclocitral; salicylic acid

1. Introduction

Australian turf managers are constantly under pressure to produce the best possible turf surfaces within
budget, and while having minimal environmental impact. This is all whilst being subjected to increasing
levels of use and expectations from end users.

A key requirement to producing turf to the highest standard is a vigorous root system, with ideally this being
a result of minimal use of phosphorus. This is for two key reasons:

Firstly, heavy applications of phosphorus are thought to stimulate the growth of the undesirable weed grass
winter grass (Poa annua).


JUNE 2024



Secondly, excess phosphorus can run to waste and cause issues with algal blooms, and pollution of
waterways.

The use of bio stimulants to stimulate root growth is not new, and the use of seaweed-based products
containing high levels of cytokinins is a common approach adopted by many turf managers.

In recent years there have been major advances in bio stimulant technology. 2,3-Butanediol and β-
cyclocitral are two of these and are of particular interest as they have been shown to stimulate root growth
even when used at very low concentrations.

2,3-Butanediol

2,3-Butanediol, is a Bacterial Volatile Compound, and has been reported to enable plants to survive biotic
and abiotic stresses better.

Biotic Stress Management:
Synthetic Butanediol induced plant defence against R. solani for creeping bentgrass [1].

A two-year study in Canada [2] showed that Butanediol provided enhanced resistance to creeping bent
grass against the dollar spot pathogen in lab tests, with disease reduction in the range of 50% to almost
100%.

When combined with turf fungicides, 2,3-Butanediol increased efficacy at half rates [3].

Butanediol with half rates of fungicides significantly increased the disease control efficacy against dollar
spot and summer patch disease compared to the half concentration of azoxystrobin, and tebuconazole
alone.

Abiotic Stress Management:
Bent grass treated with 2,3-Butanediol shows faster recovery from high temperature stress than the
untreated control and shows increases in diameter/height/leaf index compared to untreated grass. [4]

In contrast under drought or cold temperature stress, an application of Butanediol in creeping bent grass
and tomato plants significantly reduced the negative impact of these abiotic stresses [5].

2,3-Butanediol treatment improves the survival rate of Arabidopsis, 3 times more than water treatment in
drought conditions [6].

Growth Response:
2,3-Butanediol increases the growth and size of red pepper [7].

Trials on Kentucky Bluegrass and Creeping Bent have shown a greening response when Butanediol is applied
every two weeks. The chlorophyll index was increased by 18% in Kentucky Blue grass and 8% in Creeping
Bent compared to the non-treated group.

JUNE 2024
β-cyclocitral.
β-cyclocitral is an effective root growth promoter in monocots and potentially a valuable tool to enhance
growth under environmental stress.
In turfgrass systems strong root growth and branching are highly desirable, as they enhance the plants
ability to take up nutrients and water. β-cyclocitral enhances root growth and branching [8].
Salicylic acid.
Plants rich in SA and its derivatives, have been used in medicine for 1000’s of years. For example. aspirin is
based on a derivative of Salicylic acid.
Salicylic acid (SA) is found in plants, and plays a role in plant growth, and helps fight against both biotic and
abiotic stress such as heat stress recovery [9].
Salicylic acid is a plant growth regulator [10], that modulates the antioxidant defence system and decreases
oxidative stress [11].
Plant-water relations in abiotic stress affected plants were regulated by SA [12], and it was found that SA
regulates plant water relations and increases heat tolerance [13].
Salicylic acid also reduced Alternaria solani in tomatoes and enhanced plant growth [14].
Turf specific research.
SA has also been shown to enhance heat tolerance of Kentucky Bluegrass, which regrew faster following
heat stress. An SA concentration of 0.25 mmol [15] was most effective in enhancing heat tolerance.
In perennial ryegrass [16] research looked at how SA affected this under drought stress. It was found that
foliar SA increased chlorophyll a, b content, and reduced electrolyte leakage, proline accumulation and
antioxidant enzyme activity. All these factors suggested that SA can be used to reduce the negative impacts
of drought stress.
2.Materials and Methods
A tube trial was established on October 1st 2023, using perennial ryegrass var Grand Slam GLD sown at the
equivalent of 35g/m2 into a clean USGA specification sand. This was a replicated block trial with 6
treatments including an untreated control and 5 blocks (30 tubes in total). The treatments were 1) 2,3-
butanediol 1L/Ha, 2) β-cyclocitral 300ml/Ha, 3) wetting agent plus β-cyclocitral and 2,3-butanediol at
12.5L/Ha, 4) Vertmax Duo plus β-cyclocitral and 2,3-butanediol at 1L/Ha, 5) Vertmax Duo at 1L/Ha and (6)
an untreated control.
The 2,3-butanediol was supplied by GS Caltex Corporation, the β-cyclocitral by JH Chemical HK Co., Ltd
Hangzhou and the Vertmax Duo from Gilba Solutions, Australia.


JUNE 2024


Following seeding, the pots were allowed to establish until the first treatment was made on the 29th
October 2023. Subsequent applications were made on the 7th of November 2023, 17th November 2023,
2nd February 2024 and 1st March 2024.
For the duration of the trial irrigation was applied daily via an onsite misting system and no fertilizer was
applied.
Assessments were as follows:
1. Turf DGCI.
2. Turf Colour Quality.
3. Density Quality.
4. Percent Cover.
5. Cover Quality.
6. Organic Matter by Loss on Ignition.

Digital Image Analysis.

Turf Quality Analysis was carried out using a light box. Images were taken with a Panasonic DMC-TZ80, and
then analysed using Turf analyzer software to determine the DGCI, Colour Quality, Density Quality, Percent
Cover and Cover Quality.

Loss on Ignition (LOI).
The individual rootzones were taken and carefully removed from their respective tubes. The top 1cm was
then cut off and removed. This was to ensure that any organic matter results were due to root development
rather than surface thatch.

These rootzones were then split into sections 0-2 cm, 2-4 cm, 4-6 cm and 6-8 cm. The samples were then
dried in a muffle furnace for 48 hours at 60°C, and then ashed at 440°C for 4 hours. Weights were taken
after drying and ashing, with the loss of ignition then being calculated. This corresponds to the organic
matter content at the respective depth. The ash content is expressed as a percentage of the mass of the
oven-dried sample.

% Ash Content = (C X 100)/B
where: C = ash weight in g, and B = oven-dried test specimen, weight in g.
The calculation to determine the amount of organic matter by difference, is as follows:
% Organic matter = 100.0 - D
where: D = ash content, %.
If these products were increasing root growth, organic matter contents would increase over the control. The
assumption was that as this was initially a clean USGA spec sand, any increases in organic matter would be
due to increases in root growth.

Tubes were monitored regularly using digital image analysis in combination with Image J. Statistical analysis
was carried out using Studio R, agricolae package. All data were subjected to two-way ANOVA (analysis of


JUNE 2024


variance) to determine the Treatment effects. Treatment means were separated using the Duncan’s
multiple range test at the P = 0.05 level of probability.

3. Results

Image 1. (A) Image taken on 29th October 2023 before treatments applied. (B) Image taken on the 7th November 2023.

A

B











JUNE 2024


Digital Image Analysis.

Over the duration of the trial, Digital Image Analysis showed significant P<0.05) differences between the
treatments. DGCI readings were highly significant for the Pigment (5) and Pigment plus 2,3-butanediol and
cyclocitral (4) treatments compared to the control and other treatments.

Figure 1. Dark Green Colour Index. Values with different letters are significantly different (P < 0.05).

With Colour Quality, the Pigment (5) and Pigment plus 2,3-butanediol and cyclocitral (4) treatments showed
significant P<0.05) results compared to the control and other treatments.
Figure 2. Colour Quality. Values with different letters are significantly different (P < 0.05).



JUNE 2024



With the Cover Quality both the Pigment (5) and Pigment plus 2,3-butanediol and cyclocitral (4) treatments
continued to give significant P<0.05) results compared to the control and other treatments.

Figure 3. Cover Quality. Values with different letters are significantly different (P < 0.05).


Figure 4. Percent Cover. Values with different letters are significantly different (P < 0.05).





JUNE 2024



Loss on Ignition and Organic Matter %.

This shows that Wetting agent + Butanediol + β-cyclocitral (3), Vertmax Duo + Butanediol + β-cyclocitral (4),
and Vertmax Duo (5) treatments gave the highest organic matter readings at the 0-2cm depth. However,
none of these were significantly different from the Control.


Figure 5. Organic Matter % at 0-2 cm and 2-4 cm. Values with different letters are significantly different (P < 0.05).



There were no significant differences between the treatments at the 2-4cm depth. The 2,3-Butanediol (1),
β-cyclocitral (2) and Vertmax Duo + Butanediol + β-cyclocitral (4) Treatments had the highest organic matter
levels at the 2-4cm depth.


Figure 5. Organic Matter % at 4-6 cm and 6-8 cm. Values with different letters are significantly different (P < 0.05).




JUNE 2024


There were no significant differences between treatments at the 6-8cm depth at the 0.05% level. However,
the wetting agent + Butanediol + β-cyclocitral (4) and Vertmax Duo + Butanediol + β-cyclocitral (4) showed
the highest organic matter contents.
4. Discussion.
2,3-butanediol and β-cyclocitral do appear to stimulate root growth and development, even when used at
these low rates. However, the economics of their use must also be taken into consideration.
Currently 2,3-Butanediol is a significantly lower-cost option in comparison to β-cyclocitral.
Bearing in mind Vertmax Duo has already been shown to stimulate root growth and lateral branching of
turfgrass the incorporation of 2,3-butanediol has merit for increasing stress tolerance of turfgrass when
incorporated into this product.
Most pigment applications are made in the winter months to mask warm season turfgrass dormancy or
throughout the year to golf greens in conjunction with fungicides. In the latter case the pigment serves two
purposes.
Firstly, it acts as a tracking colourant so you can see where spray is applied, and you can avoid chemical
overspray.
Secondly, the biostimulants in the pigment has additional benefit such as stimulating root growth or
improving overall plant health.
The fact that 2,3-butanediol has also been shown to reduce fungicide applications by up to 50% is worth
serious consideration to its future use in spray applications.
In combination with the Vertmax Duo pigment plus salicylic acid, statistically the results are not different
from the Vertmax Duo treatment as a standalone treatment. However, two issues require further testing.
The first is people’s perception of colour. What statistically is no different, in the field may result in
completely different feedback from the customer.
The second is drying time. A unique property of Vertmax Duo is the fact that once dried it doesn’t go back
into solution. This trait needs to be tested to ensure that when Vertmax Duo is combined with 2,3-
Butanediol or cyclocitral that it is not negatively affected.

5. Conclusions

The results showed that there is significant potential for the use of newer generation biostimulants in the
turf industry. Opportunities exist for these as stand-alone applications and to incorporate them with
commonly applied products.
Further work needs to be looked at regarding standalone applications at increased rates before moving onto
field trials.
The other point worth considering is that is there a similar effect on other grasses such as couch or
bentgrass?

mailto:herbycides@gmail.com


jerry@gilbasolutions.com
JUNE 2024

1

Acknowledgments: We would also like to thank Craig Geeves, Course Superintendent at Duntry Leagues Country Club and
formerly 2IC at Bonnie Doon Golf Club with his help.
Funding: “There are no sources of funding to declare.”
Conflict of interest: "The author(s) declare no conflict of interest."
Data availability statement: "Data supporting these findings are available upon request."





















https://doi.org/10.1186/1471-2229-10-34



JUNE 2024


References:

[1] Shi Y, Niu K, Huang B, Liu W, Ma H. Transcriptional responses of creeping bentgrass to 2, 3-butanediol, a
bacterial volatile compound (BVC) analogue. Molecules 2017, 22, 1318.

[2] Goodwin PH and Hsiang T. (2011). Stimulating host defences for control of turfgrass diseases. Ontario
Turfgrass Research Foundation Final Report, June 2011.

[3] Duraisamy K, Ha A, Kim J, Park AR, Kim B, Song CW, Song H and Kim JC. (2022). Enhancement of Disease
Control Efficacy of Chemical Fungicides Combined with Plant Resistance Inducer 2,3-Butanediol against
Turfgrass Fungal Diseases The Plant Pathology Journal · June 2022 DOI: 10.5423/PPJ.OA.02.2022.0022.

[4] Shi Y, Niu K, Huang B, Liu W, Ma H. Transcriptional responses of creeping bentgrass to 2, 3-
butanediol, a bacterial volatile compound (BVC) analogue. Molecules 2017, 22, 1318.

[5] Park AE, Kim J, Ha A, Son JY, Song CW, Song H, and Kim JC. Exogenous Bio-Based 2,3-Butanediols
Enhanced Abiotic Stress Tolerance of Tomato and Turfgrass under Drought or Chilling Stress.
Microbiol. Biotechnol. 2022. 32(5): 582–593.

[6] Cho, Song & Kang, Beom & Han, Song & Anderson, Anne & Park, Ju-Young & Lee, Yong-Hwan & Cho, Baik
& Yang, Kwang-Yeol & Ryu, Choong-Min & Kim, Young. (2008). 2R,3R-Butanediol, a Bacterial Volatile Produced
by Pseudomonas chlororaphis O6, Is Involved in Induction of Systemic Tolerance to Drought in Arabidopsis
thaliana. Molecular plant-microbe interactions: MPMI. 21. 1067-75. 10.1094/MPMI-21-8-1067.

[7] Hyun GK, Teak S, Kim TH, and Ryu CM. (2018). Stereoisomers of the Bacterial Volatile Compound 2,3-
Butanediol Differently Elicit Systemic Defense Responses of Pepper against Multiple Viruses in the Field.
Front Plant Sci. 2018; 9: 90.
[8] Dickinson AJ, Lehnera K, Mid J, Jiad KP, Mijara M, Dinnenyc J, Al-Babilid S and Benfey PN. (2019) β-
cyclocitral is a conserved root growth regulator. PNAS, May 21, 2019, vol. 116, no. 21, 10563–10567
[9] Wang, LJ., Fan, L., Loescher, W. , Duan W, Liu GJ, Cheng JS, Luo HB and Li SH. Salicylic acid alleviates
decreases in photosynthesis under heat stress and accelerates recovery in grapevine leaves. BMC Plant
Biol 10, 34 (2010). https://doi.org/10.1186/1471-2229-10-34

[10] Raskin I. (1992). Role of salicylic acid in plants. Annu. Rev. Plant Biol. 43 439–463. 10.1146/
annurev.pp.43.060192.002255
[11] Shirasu K, Nakajima H, Rajasekhar VK, Dixon RA, Lamb C. (1997). Salicylic acid potentiates an agonist-
dependent gain control that amplifies pathogen signals in the activation of defence mechanisms. Plant Cell 9
261–270. 10.1105/tpc.9.2.261
[12] Miura K, Tada Y. (2014). Regulation of water, salinity, and cold stress responses by salicylic acid. Front.
Plant Sci. 5:4 10.3389/fpls.2014.00004
[13] Larkindale J, Knight MR. (2002). Protection against heat stress induced oxidative damage in Arabidopsis
involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol. 128 682–695. 10.1104/pp.010320
[14] Aslam M, Habib A, Sahi ST and Khan RR. (2019). Comparative Study of Bion and Salicylic Acid Applied
through foliar and seedling root dipping in Tomato against Alternaria Solani. January 2019. Applied Ecology
and Environmental Research 17(1):561-574


JUNE 2024


[15] He, Yali & Liu, Youliang & Cao, Weixing & Huai, Mingfang & Xu, Baogang & Huang, Bingru. (2005).
Effects of Salicylic Acid on Heat Tolerance Associated with Antioxidant Metabolism in Kentucky Bluegrass.
Crop Science - CROP SCI. 45. 10.2135/cropsci2003.0678.

[16] Hosseini, S.M., M. Kafi and M. Arghavani. 2015. The effect of salicylic acid on physiological
characteristics of Lolium grass (Loliumperennecv. "Numan") under drought stress. Int. J. Agron. & Agric.
Res., 7(1): 7-14.