TOMATO

GENERAL INFORMATION

15_98/T Trial code

HOTI I BF000000_ACES_ 16 =======:--

Collaboration UNIVERSITY OF ALICANTE

Location Alicante, Spain

Start date October 2015

End date July 2016

Person in charge Elodie Brans and Abbas Caballero

OBJECTIVES

The objectives of this trial with use of the Stoller product BIOFORGE were:

• Evaluate the product's effect on the plant's physiologicalparameters.• Evaluate the most appropriate dose to alleviate thestress in this variety.

TRIAL DESIGN

T RIAL DESCRIPTION

Crop Tomato (Solanum lycopersicum)

Variety Mil.Seny

Trial design 5 Theses, 3 Stoller and 2 Control

Fertilization Hydroponic nutrient solution

Currently, stress caused by climatic factors is estimated to cause up to 50% of the annual global production losses. In a world

with ever-present climatic fluctuations, with hotter summers and warmer winters, questions regarding food security arise.

Plants are subjected to diverse types of stress throughout their growth cycle, such that their development is hindered and

thus their productive potential is affected. Therefore, it is essential to look for solutions to alleviate stress and thus avoid

any production losses. Stoller carne up with the innovative product BIOFORGE, a unique and patented solution to combat

stress in plants. In this trial, the effect of the product BIOFORGE is evaluated when applied at different doses on tomato

seedlings, grown in a hydroponic system and subjected to heat stress. In total 5 theses were established, a control in normal

conditions, a control in stress conditions and 3 treatments with BIOFORGE at the dose of 0.5, 1 and 2 L/Ha respectively.

The treatments were made directly into the hydroponic solution and the seedlings were grown in growing chambers with

different conditions. For each treatment, 9 repetitions were made and were distributed at random in the chambers.

Thesis:

C ____________ Control in normal conditions.

CT _________ Control in heat stress.

B 1 __________ BIOFORGE at the dose of 0.5 L/Ha.

B2 __________ BIOFORGE at the dose of 1.0 L/Ha.

Bl _________ BIOFORGE at the dose of 2.0 L/Ha.

PRO DUCTS AND TREATMENT DOSES

Nº TREATMENTS CONDITION DOSE

(Liters/Ha)

c CONTROL NORMAL

CT CONTROL STRESS

B I BIOFORGE 1 STRESS 0.5 L/Ha

B2 BIOFORGE 2 STRESS 1.0 L/Ha

B3 BIOFORGE 3 STRESS 2.0 L/Ha

METHOD

WATERING

WATERING

WATERING

lmage 1: Seedlings in the growing chambers

APPLICATION MOMENT

Every 7 days from the transplant, applied directly to the nutrient solution

Both theses treated and Control received the same nutritional treatments, with the only difference being the addition of the Stoller product in the treated thesis.

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The Stoller technology present in the product makes it unique in combating stress.

BIOFORGE is known to stimulate the complete recovery of the crops subjected to

different types of stress, inhibiting the synthesis of the stress hormone, Ethylene, thus

reducing the presence of ROS molecules. Furthermore, BIOFORGE has the ability to

stimulate root growth and the expression of photosynthetic genes, helping with crop

establishment after the transplant.

To carry out the trial, tomato seeds were germinated in a substrate consisting of silica sand and was watered with a nutrient

solution containing micro and macronutrients. One week after germination, the seedlings were transplanted into plastic

containers with a volume of 200 ml, where they were individually grown in a common growing chamber. lt is in this moment,

when the first application of BIOFORGE was made, being able to help with the initial root growth in the treated plants

(B 1, B2 and B3). One week after the transplant, the heat stress was applied to the plants by growing the stress theses in

another growing chamber.

lmage 2: Tomate seedlings transplanted into hydroponic containers

Both growing chambers had the same light and humidity levels, with the humidity levels being maintained at 70%. While the

light intensity was adjusted according to the simulated time of day. The temperature in normal conditions oscillated between

I SºC at night and 25ºC during noon. In the case of the chamber in stress conditions, the nocturnal temperature was I SºC

whereas the maximum diurna! temperature was of 42ºC during noon. The maximum temperature was maintained for 3 hours

daily, throughout the course of the trail.

TRIAL DEVELOPMENT

During the trial, different weight measurements were made on the seedlings so as to obtain a growth evolution throughout

the trial.After 20 days, the different analysis proposed for the trial were carried out.

Generally, the determinations were divided into two types, physical and physiological. The physical determinations were the

monitoring of the plant growth (weight) and root growth. On the other hand, the physiological determinations were subdivided

into different aspects.The general physiological state of the plant, by measuring photosynthetic pigments, membrane integrity

and transpiration intensity. The presence of oxidative stress was also analyzed by studying the indicators of this stress, reactive

oxygen species (ROS) as well as the activity of antioxidant enzymes. In brief, we have:

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PHYSICAL: Monitoring of the plant growth (weight) and root growth.

DETERMINATIONS C

ENERAL STATE:Transpiration, membrane integrity and chlorophylls. PHYSIOLOGICAL

OXIDATIVE ST RESS: Stress indicators, ROS and enzyme activity.

TOMATO

PHYSIOLOGICAL DETERMINATIONS

TRANSPIRATION INTENSITY

Regarding the general plant state, the transpiration intensity was studied for each thesis. That is, the intensity at which they

lose water through their stomata. Thus, 3 repetitions were chosen per thesis and the transpiration of their foliar tissues

were measured. The results of this measurement are shown below:

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Graph 1 :Transpiration intensity of the different thesis

According to the data in graph 1, in all the thesis treated with BIOFORGE (B 1, B2 and B3} statistically significant

differences were obtained against the heat stress Control (CT). In B3, similar results to those of the normal control

(C) were obtained, while in B I and B2, a reduction of the transpiration rates were detected in comparison to the normal

Control (C}, with the differences being statistically significant.Therefore, the addition of BIOFORGE produces a significant

decrease of the transpiration values compared to the heat stress Control (CT). In normal conditions, a regulated level of

transpiration exists, as is observed in the normal Control. In heat stress conditions, the plant opts for increasing water loss

as a cooling mechanism to avoid tissue damage owing to this type of stress. The seedlings treated with BIOFORGE had

a lower transpiration rate probably due to the fact that they did not suffer the consequences of heat stress, and

thus they developed a similar behavior to the Control (C} seedlings.

MEMBRANE INTEGRITY

To measure membrane integrity, roots from three seedlings were chosen at random. The electroconductivity of the fresh

samples and that of the samples after being subjected to a severe stress that ruptures their membranes. The difference

between both measurements indicated the initial membrane state of the cells. The results of the analysis are shown in the

following graph:

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Graph 2: Electrolyte leakage in different thesis

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The results in graph 2 show that there is a lower electrolyte leakage in the membranes of thesis B2 and B3,

achieving significant differences against the heat stress control (CT) while obtaining similar values to the normal

Control (C). B I treatment, attains the lower values to the heat stress Control without having statistically significant differences

between them.

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Diagram 1: Ion Exchange in the membrane in good state (left) and damaged membrane (right)

Therefore, in view of the results, there is a clear

reduction in the electrolyte leakage in the

treatments with BIOFORGE, with the most

notable effects being in the theses with higher

doses of the products (B2 and B3). In normal

conditions, a good ion exchange between the cell

and its medium exist, while in cases of stress, this

exchange can be affected due to damages in the

membrane, causing ion leakage. The membrane

in stress situations can have its fluidity affected,

giving way to a higher ion leakage from its interior

as can be seen in the following diagram.

PHOTOSYNTHETIC PIGMENTS

In this case, the chlorophyll content was analyzed in fresh leaves so as to determine the photosynthetic capacity of the plant.

Therefore, a spectrophotometric study was made in a solution containing chlorophyll extract, with the results being presented

in the following graph:

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Graph 3: Chlorophyll content from the different theses

lmage 3: Chlorophyll extract from each thesis

The data in graph 3 show that there is a considerable increase in chlorophyll content in the theses treated with

BIOFORGE, obtaining statistically significant differences in comparison to the heat stress Control {CT). Thesis

B I obtains higher values than the normal Control (C) with statistically significant differences. The results obtained, confirm

that BIOFORGE stimulates photosynthesis thanks to its influence on the expression of photosynthetic genes, thus increasing

the accumulation of photosynthetic pigments, specifically chlorophyll. The decrease in the chlorophyll content in the heat

stress Control can be due to the effect of oxidative stress owing to the accumulation of reactive oxygen species (ROS).

Theses containing BIOFORGE, do not show this reduction in chlorophyll content, probably due to the absence of oxidative

stress in these seedlings and as such the chlorophyll content should not be affected.

OXIDATIVE STRESS

Oxidative stress is caused by an imbalance between the synthesis of reactive oxygen species (ROS) and the ability of the

biological system to detoxify the excess of these molecules. lf the organism is unable to detoxify the ROS, these molecules

can cause severe damages and alter the organism's metabolism. To study the presence of oxidative stress in the seedlings,

different parameters were studied, including the indicators of this type of stress, the concentration of hydrogen peroxide

(ROS) and the activity of antioxidant enzymes.

TOMATO

INDICATORS OF OXIDATIVE STRESS

In oxidative stress conditions, the accumulation of sorne substances able to alleviate the damage is produced.The osmolytes

as they are called, can be from sugars up to amino acids. In this trial, the concentrations of Malondialdehyde (MDA) a dialdehyde

produced during stress and an indicator of membrane damage is studied.

Proline, an amino acid that accumulates during oxidative stress due to its protective nature against damages produced from

this stress is also studied.

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Graph 4: MDA content in the different thesis

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Graph 5: Proline content in the different thesis

BIOFORGE 2 • BIOFORGE 3

As can be observed in graph 4, a significant accumulation of MDA is observed in the heat stress Control (CT) whereas

in all the theses treated with BIOFORGE, lower values of MDA are observed and are similar to those of the normal

Control (C), without presenting statistical differences between them.

Thesis B2 has the lowest content of this molecule. The data obtained in this study are in accordance with the results

obtained in the membrane integrity analysis. MDA is known to be an indicator of membrane damage, hence an increase

in its accumulation signifies more damage in the membrane. In both cases, the treated theses with BIOFORGE, present

a membrane in good state, a fact reinforced by the concentration of MDA present in the measured theses. In the heat

stress Control (CT) a higher accumulation of MDA is observed and thus representing more damage in the membrane as

can be seen in graph 2.

As observed in the case of MDA, the quantity of proline accumulate is statistically inferior in the treated theses with

BIOFORGE against the heat stress Control (CT) while equaling the results of the normal Control (C) (Graph 5). Once

more, the results provide proof that the treated seedlings were not in oxidative stress. Proline is a molecule that is

accumulated to alleviate the damage brought on by ROS, therefore, a lower accumulation of the amino acid, is an indicator

of the absence of oxidative stress.

To corroborate the lower incidence of oxidative stress in the treated seedlings, a ROS molecule is measured as is the case

of hydrogen peroxide (H2O2) y and the activity of enzymes responsible for the degradation of ROS such as Catalase and

Ascorbate Peroxidase.

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Hydrogen peroxide is considered as a reactive oxygen species (ROS), so in high concentrations, it forms part of the

phenomenon known as oxidative stress, thus, being toxic when in excess. In stress situations, cells suffer from the presence

of the high number of toxic compounds that are a direct result from the reduction of molecular oxygen, production

molecules such as hydrogen peroxide.

As can be observed in graph 6, there is an important reduction in the presence of hydrogen peroxide in the theses treated

with BIOFORGE.AII of the treated theses present statistical differences against the heat stress control, which has a high

concentration of this ROS molecule. Theses B2 and B3 achieve values similar to the normal Control, without

significant differences between them. Thesis B 1, presents the lowest concentration of the molecular with

statistical differences compared to the normal Control.

In summary, the lower presence of ROS molecules can be verified in the treated theses with BIOFORGE

and thus indicating that these seedlings are absent of oxidative stress. BIOFORGE, was indeed able to

inhibit the synthesis of the Stress hormone, Ethylene, so that the synthesis of ROS molecules is reduced

and thus, the stress damage is also reduced.

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Graph 6: H202 concentration in the different thesis

The results obtained in this measurement, are in accordance with the data observed in the chlorophyll content (graph 3),

as a lower content of the pigment is observed in the heat stress Control due to the increase in the presence of ROS molecules

(graph 6), while the opposite occurs in the normal Control and the BIOFORGE theses.

ENZYME ACTIVITY (CATALASE A ND ASCORBATE PEROXIDASE)

The data presented regarding enzyme activity, show similar results for both the activity of Catalase and Ascorbate peroxidase

(PAX) In both cases, enzyme activity in the BIOFORGE theses does not show statistical differences from the

normal Control, indicating that the seedlings were not suffering from the Stress and thus are absent from

oxidative stress.

TOMATO

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Graphs 7 and 8: Enzyme activity in the samples of each theses

The heat stress Control seedlings show high levels of antioxidant enzyme activity with statistically significant

differences in comparison to the rest of the theses. This is due to the fact that the seedlings are in oxidative stress

(Graphs 4, 5 and 6) and therefore, require high activity of these enzymes to avoid any damages in their tissues. The Stolier

technology present in BIOFORGE, aids the seedlings in avoiding this situation as is observed in ali the oxidative stress

parameters measured. In ali the measured parameters, the results indicate that indeed the treated seedlings were absent

from oxidative stress.

PHYSICAL DETERMINATIONS

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Graph 9: Monitoring of seedling weights (g) of the different theses

Graph 9 shows the evolution of the seedling growth of each distinct thesis by measuring their weights (g) during the course

of the trial.As was expected, the normal Control, presents the highest weight, foliowed by theses B I and B2. B3 on the other

hand, shows a similar growth pattern to the heat stress Control. There were no important differences regarding the weight

of the seedlings in stress (B 1, B2, B3 y CT). Nevertheless, if the trial was carried out up to the fructification period, bigger

differences would be observed as the physiology of the seedlings would be different between the seedlings treated with

BIOFORGE and the heat stress Control as was observed in the physiological parameters studied.

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ROOT GROWTH

A quantitative study of the root growth was not performed as it was not the aim of this trial. However, due to the visually

important results observed, it was deemed relevant to present the differences.

In images 4 -8, differences in root development can be observed in the different seedlings. The seedling of the heat stress

Control (CT) show adventitious roots, that is, roots that grow on the stem, which is a clear symptom of stress. This is

not seed in any of the treated theses with BIOFORGE, but instead these seedlings show bigger root mass in comparison

to CT.

lmages 4 to 8: Root growth in the different thesis

CONCLUSIONS

With the application of BIOFORGE directly to the nutrient solution at different doses (0.5 L/Ha in B 1, 1 L/Ha in B2 and

2 L/Ha in B3), every 7 days from the transplant on tomato seedlings subjected to heat stress, we observe:

• CHLOROPHYLL: lncrease of the chlorophyll content, equaling the values of the normal Control.

• MEMBRANE INTEGRITY: Lower incidence of electrolyte leakage, indicating good membrane integrity.

• TRANSPIRATION: Adequate transpiration rates, without presenting alterations owing to stress.

• OXIDATIVE STRESS INDICATORS: Low concentrations of hydrogen peroxide and the indicators of oxidative

stress, indicating absence of oxidative stress.

• ENZYME ACTIVITY: Activity of antioxidant enzymes are inferior to the heat stress Control and equal to the

normal Control.

• ABSENCE OF OXIDATIVE STRESS.

• IMPROVED ROOT GROWTH.

• B2 WITH I L/Ha BEING THE MOST APPROPRIATE DOSE.

In this trial the Stolier technology in BIOFORGE was shown to be able to stimulate the physiological recovery of the

seedlings subjected to heat stress. In ali the physiological parameters studied, statistically significant differences were

observed between the treated theses and the heat stress Control, in such a way that the seedlings treated with BIOFORGE,

showed similar results to the seedlings of the normal Control, signifying that the treated seedlings did not suffer the stress.

Based on the global results, it can be concluded that B2 (BIOFORGE I L/Ha) is the most appropriate dose as it obtains

the most regular results in ali the analyzed parameters, from the absence of oxidative stress up to the increase in

photosynthetic pigments.

In conclusion, all the results from this trial verify the positive effect of BIOFORGE on the global physiology of the

seedlings, by inhibiting the synthesis of ethylene and reducing the presence of stress, thus restoring the seedlings to a

physiologically balanced state, despite being in an adverse medium, ali thanks to the unique and patented Stolier technology

in BIOFORGE.

TRIAL PREPARED BY:

DEPARTMENT OF BIOCHEMISTRY AND AGROCHEMISTRY, University of Alicante. ELODIE BRANS and ABBAS CABALLERO, Stoller Europe Technical Department.