CETvol87


 
 

 

                                                                    DOI: 10.3303/CET2187106 
 

 
 
 

 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Paper Received: 14 October 2020; Revised: 13 February 2021; Accepted: 3 April 2021 
Please cite this article as: Sandoval A.N., Sanchez L.J., Saavedra Y.M., Calla K.M., Alba R.A., Lloclla H., 2021, Reduction of Imidacloprid and 
Tebuconazole in Oryza Sativa Plantation Applying Strains of Trichoderma Spp., Chemical Engineering Transactions, 87, 631-636 
DOI:10.3303/CET2187106 

CHEMICAL ENGINEERING TRANSACTIONS 

VOL. 87, 2021 

A publication of 

The Italian Association 
of Chemical Engineering 
Online at www.cetjournal.it 

Guest Editors: Laura Piazza, Mauro Moresi, Francesco Donsì
Copyright © 2021, AIDIC Servizi S.r.l. 
ISBN 978-88-95608-85-3; ISSN 2283-9216 

Reduction of Imidacloprid and Tebuconazole in Oryza Sativa 
Plantation Applying Strains of Trichoderma spp. 

Ana N. Sandoval*a, Leidy J. Sánchezb, Yeravi M. Saavedrab, Kriss M. Callab, 
Rafael A. Albab, Herry Llocllab 
a Universidad Nacional de San Martín, Jr. Amorarca N° 334 Morales, San Martín, Perú 
b Universidad César Vallejo, Carr Marg Norte F.B.T KM 8.5 Sec. Maronilla, Cacatachi, San Martín, Perú. 
ansandoval@unsm.edu.pe 

The objective of this research was to assess the reduction of imidacloprid and tebuconazole in soil plantations 
of Oryza sativa applying strains of Trichoderma spp. Regarding the methodology, 3 treatments were used; 
strain 1 (TS1), 2 (T163+TS1) and 3 (T163), 19 pots of 15 x 15 cm with 3.5 kg soil were used. One pot was 
used for the control group (without treatment) and eighteen pots were used for experimental groups (6 pots for 
each strain). 5 seeds Oryza sativa of Esperanza variety (MQR78) were planted in each pot on 2 cm to the soil 
surface, irrigation was carried out using drench technique every 24 hours for 90 days. For Trichoderma spp 
inoculation it was added 40 ml of strain 1, strain 2 and strain 3 in 18 pots with Oryza sativa seeds, identifying 
that the root measured 2 centimeters (6 pots for each strain). Likewise, the fungus strains were inoculated 
after 45 days to nourish the soil and a soil sample was taken after 45 days to identify the absorption of 
tebuconazole (fungicide) and imidacloprid (insecticide), finally the height of the plant (cm) was measured in 
each experimental strain, the plant was measured from the ground level to the tip of the longest leaf. The 
results showed that strain 1 reduced 12.39 mg / kg of imidacloprid and 17.31 mg / kg of tebuconazole. 
Regarding the percentage of decrease efficiency, it was concluded that strain 1 is more efficient with 64.2% 
for imidacloprid and 64.5% for tebuconazole and finally, strain 3 showed greater height of the Oryza sativa 
plant with 79.8 cm. 

1. Introduction

Increase temperature caused by climate change and anthropogenic activities provide uncertainty to 
agriculture, likewise in absence of nutrients, the use of chemical products, floods, droughts, soil salinity, 
among others, affect food security. (Shobhit et al., 2017). Plants are surrounded by microorganisms, within 
them we find bacteria, fungi, oomycetes, nematodes, insects and viruses, these organisms can have a 
negative impact on the plant, destroying up to 30% of the crops through diseases and decomposition 
processes. (Simon et al., 2019). Others may have beneficial effects by improving the general fitness of the 
plant and eliminating diseases. (Vos et al., 2015). However, biological control provides a safe alternative for 
the monitoring of pathogens, due to its capacity as biocontrol which is helpful for the ecosystem, and they are 
considered potential agents in fungal diseases, because of they can interact directly with the roots, increasing 
plant growth, resistance to disease and tolerance to stress. (Silva et al., 2019). 
The growth of rice is given by the combination of the genotype and the environment, which allow to improve 
the soil fertility and its yield; however, the indiscriminate use of fertilizers and agrochemicals decrease 
production and deterioration of soil health; likewise, indiscriminate use of fertilizers and agrochemicals cause a 
decrease in production and a degradation of soil health; Therefore, new alternatives must be sought to 
improve fertility and stimulate its growth. It was researched that microbes are an essential element to maintain 
the quality of the soil, increasing its production and growth. (Doni et al., 2014, Darmawan et al., 2018, Sabki et 
al., 2019). 

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The Trichoderma spp species are faulty filamentous fungus belonging to the Hypocreales order in the 
Ascomycete division, it is one of the most widely researched genera with several applications in agriculture, 
industry and in the environment, several species have the ability for decrease diseases, stimulate growth and 
productivity in plants. (Lee et al., 2016, Hyder et al., 2017).  
They release protein compounds which are similar to auxins thus stimulating the growth and development 
plants (Garnica et al., 2015; Contreras et al., 2016; Sachdev., Singh and Singh, 2018), as well as the quality of 
grape production (Pascale et al., 2017) wheat (Allen et al., 2017), soil improvement (Md et al., 2017, 
Shuangchen et al., 2020), and absorption of chemical agents from the soil (Zhenshuo et al., 2019). 
Some researches about the use of Trichoderma spp strains show it increases the speed of germination, 
allows the decrease of spots on seeds which produce incidences to control weeds, likewise the results allow 
to have a clearer idea about its importance and the effect of strains on growth rice plants, therefore it is 
considered applicable in agricultural production (Núñez and Pavone., 2014). Another study shows the need to 
identify the potential of the fungus for synthesizing metabolites, being a barrier strain for filtered agents which 
harm the plant. The results of the study concluded it improves each process from planting to growth and 
development, therefore it is being considered a sustainable procedure (Ortuño et al., 2013). Thus, they play 
an important role in ecology by participating in the decomposition of organic waste, as well as in 
biodegradation of artificial chemicals and the bioaccumulation of high amounts of several metals from 
wastewater and soil. (Nongmaithem and Bhattacharya, 2016); therefore, it is important to study this genus in 
order to provide alternative solutions for good rice production and absorption of agrochemicals. Finally, the 
objective was to evaluate the decrease of imidacloprid and tebuconazole in soil plantations of Oryza sativa 
applying strains of Trichoderma spp. 

2. Methods

2.1 Preparation of growing medium and growth of Trichoderma spp strains 

The strains were provided by the Microbiology Laboratory of the Peruvian Amazon Research Institute, they 
were stored in vials at a temperature below 12 ° C for 24 hours; strain 1 (TS1), strain 2 (TS1 + T163) and 
strain 3 (T163). CMA growing medium (Merck) was prepared by adding 2 g of penicillin to the medium to 
sterilize its content, 50 ml of medium was poured into 3 petri plates of 35 x 10 mm, and it was dried at 37 ° C 
for 30 minutes, then a fraction of each strain was extracted from the vials, placing them in the center of the 
petri plate, it was sealed with parafilm to prevent its contamination and growth for 5 days (3 without light and 2 
with light) at 35 ° C. The entire procedure was carried out in a laminar flow chamber (PB-48-115V) which 
meets the biosafety requirements. 

2.2 Preparation of enriched medium for Trichoderma spp strains 

The Peruvian Amazon Research Institute provided 1500 ml enriched medium of sugar cane molasses which 
was prepared by 45g of molasses and 7.5g of yeast. To sterilize the medium, we proceeded to selfclaved 
(BRAND) for 25 minutes, 121 ° C and 15atm, then it was allowed to cool for 3 hours, dividing the medium in 3 
glass containers with 500 ml each of them for adding a spore suspension of strain 1, strain 2 and strain 3 of 
Trichoderma spp with 3 ml (1x106 spores ml-1), to homogenize and allow their multiplication the digital stirrer 
(MIU-GS-30) was used for 4 days during 24 hours and 35 ° C, leaving it ready for being inoculated on the soil. 
(Sachdev et al., 2018) 

2.3 Soil and seed experimental conditions of Oryza sativa 

The experiments were performed in an unheated polyethylene greenhouse at the César Vallejo University in 
the Cacatachi district, San Martín Region, at 295 meters above sea level and 12 km to the north of Tarapoto 
(6°29'40 " south latitude and 76°27'57" west longitude), 19 pots of 15 x 15 cm with 3.5 kg of contaminated soil 
with 19.28mg / kg Imidacloprid and 26.81mg / kg Tebuconazole were used, one pot was used for control 
group (without treatment ) and 18 for experimental groups (6 pots for each strain), the soil was clay loam (74% 
clay, 18% silt and 8% sand), with 6.7 pH, electric conductivity 0.6 mS cm-1 and matter organic 1.35%. 
The seeds of Oryza sativa Esperanza variety (MQR78) were superficially sterilized with 75% ethanol, followed 
by 20% sodium hypochlorite, then they were washed with sterilized distilled water. (Doni et al., 2014, Lee et 
al., 2016). 5 seeds of Oryza sativa were planted in each pot on 2 cm to the soil surface being autoclaved in 
greenhouse conditions at a temperature of 31 ± 4 ° C, 318 ± 3 µmol of light intensity, 80 ± 3% humidity and 11 
h 15 m 17 s ± 9 s photoperiod, irrigation was carried out using drench technique with 50 ml of water every 24 
hours for 90 days. 

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0 5 10 15 20 25 30

Control group

Strain 1 (TS1)

Strain 2 (T163+TS1)

Strain 3 (T163)

19.28 

6.89 

9.67 

10.34 

26.81 

9.5 

13.78 

18.6 

T
ri

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Tebuconazole (mg/kg) Imidacloprid (mg/kg)

2.4 Trichoderma spp Inoculation and Oryza sativa growth 

40 ml of strain 1, strain 2 and strain 3 of Trichoderma spp were added in 18 pots with Oryza sativa seeds, 
identifying that root measured 2 cm (6 pots for each strain), likewise, the fungus strains were inoculated after 
45 days to nourish the soil. After 90 days a soil sample was taken to identify the absorption of tebuconazole 
(fungicide) and imidacloprid (insecticide) through the test analysis method of multi-residues of pesticides by 
QUECHERS in fruits and vegetables, juices, wines and food with high fat content using GC - MS and LC-MS / 
MS. (Stachniuk., 2019). Finally, the height of the plant (cm) was measured in each experimental strain, 
measuring from the ground level to the tip of the longest leaf. 

2.5 Determination adsorption efficiency of the experimental group 

The percentage difference formula was used to determine the percentage of efficiency, considering the control 
group and the experimental group as it is detailed below: 

(1) 

Where: 
CGC: Control group concentration 
CGE: Experimental group concentration 

3. Results and discussion

3.1 Analysis of results in Trichoderma spp strain in soil 

Figure 1a shows the data obtained from the control group by the analysis performed on the soil of the Oryza 
sativa plantations with 19.28 mg / kg of imidacloprid and 26.81mg / kg of tebuconazole before the treatment; in 
the experimental group the Trichoderma spp strains were applied, finding in strain 1 (TS1) 6.89 mg / kg of 
imidacloprid and 9.5 mg / kg of tebuconazole, in strain 2 (T163 + TSI) 9.67 mg / kg of imidacloprid and 13.78 
mg / kg of tebuconazole and finally in strain 3 (T163) 10.34 mg / kg of imidacloprid and 18.6 mg / kg of 
tebuconazole; it is important to mention that Trichoderma spp strains play a very important role to achieve the 
production efficiency, therefore people must know their function in decomposing organic matter, capacity of 
the circulation of nutrients, decrease of chemical load by agro-fertilizers in the soil, therefore this organism 
would be beneficial for making the agriculture more sustainable. (Colla et al., 2017). 

Figure 1.a: Tricoderma spp strains versus Imidacloprid and Tebuconazole control group (mg/kg)  

Adsorption efficiency (%)= �𝐶𝐶𝐶𝐶𝐶𝐶−𝐶𝐶𝐶𝐶𝐶𝐶
𝐶𝐶𝐶𝐶𝐶𝐶

� x 100 

633



0

10

20

30

40

50

60

70

80

Control group Strain 1 (TS1) Strain 2
(T163+TS1)

Strain 3
(T163)

51.3 

61.8 
69.5 

79.8 

O
ri

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a

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a

ti
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h
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ig
th

 (
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m
) 

Trichoderma spp strains 

0 20 40 60 80

Strain 1 (TS1)

Strain 2 (T163+TS1)

Strain 3 (T163)

64.2 

49.8 

46.3 

64.5 

48.6 

30.6 

%Decrease efficiency of Imidacloprid and Tebuconazole 

T
ri

c
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a
 s

p
p

 S
tr

a
in

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Tebuconazole Imidacloprid

Figure 1b shows the percentage of decrease imidacloprid and tebuconazole in Oryza sativa plantations, 
finding in strain 1 (TS1) 64.2% of imidacloprid and 64.5% in tebuconazole, strain 2 (T163 + TS1) 48.6% of 
tebuconazole and 64.5% in imidacloprid and finally in strain 3 (T163) 30.6% of tebuconazole and 46.3% of 
imidacloprid. It is important to improve the sowing processes by applying new biological control techniques 
that show their growth and better harvest, considering a sustainable procedure will not only help in production, 
but also in the recovery of contaminated soils. (Fiorentio et al., 2018). 

Figure 1.b: Percentage of decrease efficiency of Imidacloprid and Tebuconazole with Trichoderma spp. 
Strains. 

3.2 Analysis of results in Oryza sativa plant 

Figure 2 shows the control group (without treatment) where the height of Oryza sativa plant was 51.3 cm, in 
strain 1 (TS1) 61.8 cm, strain 2 (T162 + TS1) 69.5 cm and in the strain 3 (T163) 79.8 cm. It is necessary to 
look for alternatives which improve the growth of the plant, the microbes are resistant to the pathogens 
therefore they are an ecological way to maintain a clean ecosystem without pollutants; likewise, it is shown 
that fungi are growth promoters due to their ability to produce siderophores and phytohormones. (Doni et al., 
2014) 

Figure 2: Oryza sativa plant height before and after applying Trichoderma sp strains 

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a) Increase in strains of Trichoderma spp. b) Nursery construction and implementation. c) Contamination with
agrochemicals to the substrate. d) Soil sample for corresponding analysis. e) Cane molasses culture medium 
with inoculated fungi. f) Incorporating microorganisms into the pots. g) Presence of treatment. h) Evenly 
watering the pots with rice. 

 Figure 3: Photographic summary 

4. Conclusions

It is evidenced that strain 1 (TS1) had a higher percentage of decrease efficiency of imidacloprid and 
tebuconazole in Oryza sativa plantations, strain 3 (T163) provides plant growth compared to the control group, 
reaching an approximately height of 79.8 cm. Therefore, Trichoderma spp strains are an alternative to be used 
as a bioremediation technique of pesticide-laden soils, thus the productivity of crops would be improved in a 
sustainable way and the contamination decrease in some way.  

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