Annals of Agricultural Sciences 65 (2020) 107–115

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Annals of Agricultural Sciences

journal homepage: www.elsevier.com/locate/aoas

Impact of application of Trichoderma and biochar on growth, productivity

and nutritional quality of tomato under reduced N-P-K fertilization
Md. Nasir Hossain Sani a, Mahmudul Hasan b, Jasim Uddain a,c, Sreeramanan Subramaniam c,d,⁎
a
Department of Horticulture, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh
b
Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh
c
School of Biological Sciences, Universiti Sains Malaysia (USM), Minden Heights, 11800, Penang, Malaysia
d
Centre for Chemical Biology, Universiti Sains Malaysia, Bayan Lepas, Penang, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Extensive use of synthetic fertilizer to maximize the productivity often leads to depletion of essential soil nutri-
Received 21 March 2020 ents, environmental degradation and adversely affects soil rhizosphere microbiota. The present study investi-
Received in revised form 22 May 2020 gates the efficacy of Trichoderma and biochar co-application on tomato productivity, nutritional quality and
Accepted 15 June 2020
soil health improvements under reduced N-P-K fertilizer application. The study comprised of five treatments:
Available online 19 June 2020
(i) standard application of N-P-K, (ii) 50% dose of N-P-K (control), (iii) Trichoderma + 50% dose of N-P-K, (iv) bio-
Keywords:
char+50% dose of N-P-K and (v) biochar+Trichoderma + 50% dose of N-P-K. The growth, yield, and antioxidant
Trichoderma properties of tomatoes, as well as their mineral composition, were analysed. The results showed that the com-
Biochar bined application of Trichoderma and biochar increased the growth attributes positively and produced 101.45%
Synergistic effect and 11.33% higher yield compared to half dose and standard dose of N-P-K, respectively. The combined applica-
Synthetic fertilizer tion also elicited an increase in mineral contents, total soluble solids as well as bioactive molecules such as lyco-
Tomato pene and ascorbic acid, thereby increased the nutritional and functional quality of the tomato fruits. Collectively,
Trichoderma and biochar improved soil fertility, nutrient uptake and promoted the growth of rhizosphere fungal
and bacterial populations, which combined resulted in higher tomato yields, antioxidants, and minerals. There-
fore, the co-application of Trichoderma and biochar with a 50% dose of N-P-K can be considered an effective tech-
nique for the sustainable production of tomato with higher yield and superior quality.
© 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
The pressing challenge of food security in the face of climate change
poses a significant threat to agriculture in terms of sustainability, maxi-
mizing production per area per unit while minimizing environmental
consequences. In the past two decades, organically cultivated vegeta-
bles have attracted considerable scientific interest and consumer de-
mand as healthier products with safer characteristics. Also, sustainable
vegetable cultivation has often been identified as an eco-friendly pro-
duction system that can produce food with minimal risk to the environ-
ment (Dorais, 2007). Organic farming restricts the use of agrochemicals
and offers a way to reduce the adverse effects of chemical fertilization
(Aguilera et al., 2013; Aires et al., 2013). The most significant disadvan-
tage of organic crop production has been low yields compared to inten-
sive farming (Seufert et al., 2012). In an organic production system, the
slow release of mineral nutrients (N and P) from organic fertilizers often
leads to a mismatch between nutrient bioavailability and plant uptake,
⁎ Corresponding author at: School of Biological Sciences, Universiti Sains Malaysia
(USM), Minden Heights, 11800, Penang, Malaysia.
E-mail address: sreeramanan@usm.my (S. Subramaniam).
especially N during the peak growing period (Zhao et al., 2009; Lester
and Saftner, 2011). Therefore, farmers choose to use industrial synthetic
chemical fertilizers to grow vegetables. Large-scale use of inorganic fer-
tilizers can contribute to environmental pollution such as groundwater
contamination, eutrophication of waterways, soil acidification and in-
creased de-nitrification, resulting in higher emission of nitrous oxide,
which contributes to global warming (Molla et al., 2012). The continu-
ous use of chemical fertilizers in the soil is a growing concern, depleting
the soil from essential nutrients and leading to reduced production of
antioxidants and minerals in vegetables (Dumas et al., 2003). In addi-
tion, the extensive application of chemicals adversely affects soil bene-
ficial rhizosphere microbes (Meena et al., 2020). Agriculture must
consider priorities such as food quality, production costs, the environ-
mental impact of crops in addition to yield and productivity.
In this perspective, a promising sustainable approach could be the
use of microbial inoculants and organic amendments for vegetable pro-
duction. Numerous studies on the broad spectrum of horticultural crops
have shown that the use of beneficial microorganisms (e.g., the fungus
Trichoderma sp.) may promote primary or secondary plant metabolism
and boosts crop yield (Rouphael et al., 2017). The phyto-stimulatory ef-
fect of Trichoderma has several direct and indirect impacts on plants,

https://doi.org/10.1016/j.aoas.2020.06.003
0570-1783/© 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
108 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115
including the release of substances with auxin activity, small peptides,
organic acids, which appear to improve root system architecture and as-
similation of nutrients, thereby improving plant growth and productiv-
ity (Hermosa et al., 2012; Rouphael et al., 2017). Organic amendments
such as biochar can contribute to soil health through increased water re-
tention capacity, nutrient exchange capacity, and improved soil struc-
ture (Subhan et al., 2014). Biochar is a compound of black carbon
produced through pyrolysis at extremely high temperatures with no,
or limited, oxygen supply. The higher surface area of biochar facilitates
improved soil structure with higher water and nutrient holding
capacity.
Although research on Trichoderma and biochar has shown a substan-
tial potential to improve crop growth and productivity, most of the
studies to date were conducted mainly in the greenhouse or outdoor
pot experiments. Information on their potential benefits in field condi-
tions is minimal. At this time, it appears that neither microbial nor or-
ganic amendments can meet the requirements for a viable alternative
to synthetic mineral fertilizers. Recent research has shown that the
targeted combination of microbial inoculants with other non-
microbial substances may generate interactive synergistic effects that
cannot be achieved either alone (Adesemoye and Kloepper, 2009). Hu
et al. (2014) reported a 14.5% increase in Trichoderma spp. populations
due to the application of biochar. Muter et al. (2017) noted that biochar
applied with Trichoderma contributed to an increase in the germination
of maize (Zea mays L) and taller plants. Furthermore, Araujo et al.
(2019) found evidence for a synergistic effect of sewage sludge biochar
and Trichoderma harzianum on soybean (Glycine max L.) yield and re-
ported a 70% increase in fresh and dry mass in response to combined ap-
plication of these. More research is needed to develop an integrated
nutrient management approach that does not entirely remove chemical
fertilizers but reduces their use and associated environmental pollution.
Tomato (Lycopersicum esculentum L.) is a commonly grown and pop-
ular crop worldwide for flavour, colour and high nutritional value. Due
to its culinary value and high nutrient content, it occupies a vital posi-
tion among vegetables. Tomatoes are the primary dietary source of an-
tioxidants that are associated with many health benefits such as
decreased risk of cardiovascular disease and cancer (Perveen et al.,
2015). This study aims to obtain information useful for maximizing
growth, yield and quality of tomato in a sustainable and eco-friendly
way. The specific objectives are (i) to investigate the impact of
Trichoderma and biochar on growth, yield and nutritional quality of to-
mato on filed conditions, (ii) to quantify the efficacy of Trichoderma
and biochar co-application in minimizing N-P-K fertilizer application
and (iii) to investigate the effect of combined use of Trichoderma and
biochar on the soil health improvement in terms of nutrient availability
and microbial population.
2. Materials and methods
2.1. Description of the experimental site
The experiment was conducted between November 2017 and April
2018 in the horticulture research field of Sher-e-Bangla Agricultural
University, Bangladesh. The research field was at 24,090N latitude and
90,260E longitude at the height of 8.4 m above sea level, according to
the Global Positioning System. The field on the test site belongs to the
Shallow Red Brown Terrace soil series of Salna under Agro-ecological
Zone No. 28. The research field is situated in the subtropical climate
zone. The temperature ranged between 13 to 29 °C and the cumulative
precipitation during the growing season was 3.10 mm with an average
of 7.8 h of sunshine.
2.2. Raising of seedling, transplantation and management of crop
The seed of tomato (cv. BARI tomato 14) were obtained from the
Bangladesh Agricultural Research Centre, Gazipur, Bangladesh. Healthy
30-day old seedlings were used as planting materials. The seedlings of
tomatoes were grown in a 3 m × 1 m seedbed. The seedbed soil was
loosely mixed and dried to get fine tilth. The white polythene was
used to shade the young seedlings from intense sun or heavy rain. For
transplanting of seedling, the land in the main field was well prepared
and divided into 2.5 m × 2.0 m plots. Thirty-day-old seedlings of uni-
form height (20–25 cm) were selected and transplanted individually
into the plots; 16 plants per plot with a spacing of 50 cm × 60 cm. Stan-
dard management practices for growing tomatoes in the region were
practiced.
2.3. Properties of biochar and Trichoderma
The timber waste biochar was collected from Farm2Energy Pvt. Lim-
ited, Punjab, India. According to the manufacturer, the locally collected
Teak (Tectona grandis) timber waste (a mixture of woodchips, sawdust,
as well as leftover wood from sawmills) was used to produce biochar.
Initially, the timber waste was sun-dried and then pyrolyzed in a
specially-designed pyrolyzer at 400 °C for 80 min. Trichoderma
harzianum T22 powder was collected from the Ispahani Agro Limited
Laboratory, Dhaka, Bangladesh. The physiochemical properties of bio-
char and Trichoderma harzianum T22 are given in Table 1.
2.4. Experimental design and treatments
The field experiment was conducted with four replications in a Ran-
domized Complete Block Design (RCBD). The experiment comprised of
five treatments: To = standard doses of N-P-K, T1 = 50% doses of N-P-K
(control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50%
doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.
The standard dose of N-P-K corresponds to 135 kg N ha−1, 45 kg P
ha−1 and 75 kg K ha−1 respectively (BARC, 2012). At the final land prep-
aration of the plot, the entire dose of P and K was administered using
TSP (Triple Super Phosphate) and MOP (Muriate of Potash), respec-
tively. Three equal applications of nitrogen as urea were applied: the
final preparation of the plots, 25 and 50 days after the transplant.
Powdered biochar, at the rate of 300 g/plot and Trichoderma, at the
rate 250 g/plot were applied directly to the soil immediately before
transplanting of the seedlings, which were well watered after
transplanting.
2.5. Vegetative growth, chlorophyll content, dry matter production
and yield
Data from five randomly chosen plants were collected from each
plot in such a manner to minimize border effects. We measured plant
height, branch, leaf and flower number at 60 DAT (days after
transplanting). The leaf chlorophyll was determined using an automatic
Table 1
Physio-chemical composition of biochar and Trichoderma.
Component Value
Timber waste biochar
pH 7.07
EC 0.86 dS m−1
BET(surface area) 1045 m2g−1
Volatile matter 30. 10%
Ash content 9.80%
Fixed carbon 58.60%
CEC 16.2 mmol kg−1
Total N 0.30%
Total P 0.45%
Total K 1.32%
Trichoderma powder
Fungi spores (CFUg−1 powder) 1 × 108
 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115
SPAD (soil plant analysis development) meter (Minolta SPAD-502
meter). Three apical leaflets of a young fully extended leaf at the
flowering stage were taken for the SPAD reading. Root and shoot dry
matter weight (g) of the plant was determined at the final harvest (70
DAT). The harvested root and shoot were separated, washed, oven-
dried at 70 °C for 72 h and then weighed. Ripe tomatoes were harvested
at 3–4 days interval up to 70 DAT, counted, weighed, and yield was
determined.
2.6. Determination of mineral contents in tomato shoots
Two plants from each replicate were carefully harvested and washed
with clean water during final harvests to determine their mineral levels
(N, P, K, Fe and Zn). The collected plants were air-dried in the laboratory
at normal room temperature. Subsequently, the shoots and fruits were
oven-dried at 70 °C for 72 h until they reached a constant weight. The
dry matter was collected, ground and digested with concentrated
HNO3-H2O2 following the method reported by Yedidia et al. (2001).
The digested material was used to measure the mineral content using
atomic absorption spectrophotometer methods described in Piper
(1942).
2.7. Determination of TSS, protein and antioxidant content in ripe tomato
fruits
Immediately following harvest, total soluble solids (TSS) in ripe to-
mato fruits were measured with a hand refractometer that had been
calibrated with distilled water. The readings are reported in Brix (%)
levels. Crude protein was quantified as described by Lowry et al.
(1951). Immediately following harvesting of the ripe fruits, levels of
three antioxidants (ascorbic acid, lycopene, and β-carotene) were de-
termined. We measured ascorbic acid following the method described
by Ranganna (1986). A 10 g of blended fruit pulp from five randomly se-
lected red ripe tomato fruits served as a sample for estimation of ascor-
bic acid by using 2, 6-dichlorophenol indophenol titration. Four percent
oxalic acid was added to the sample and the volume was made up to
50 ml in a volumetric flask and filtered using Whatman No.4 paper.
Twenty-five milliliters of this filtrate was taken and titrated against 2,
6-Dichlorophenol-indophenol dye. The titration was continued until a
pink colour appeared. The dye was prepared using 50 mg of the sodium
salt of 2, 6-dichlorophenol-indophenol dye in approximately 200 ml of
double-distilled water containing 4.2 mg of sodium bicarbonate. It
was used for titration and standardizing ascorbic acid. Spectrometer
(Model 200-20, Hitachi, Japan) method as described by Nagata and
Yamashita (1992) was used to quantify lycopene and β-carotene. One
gram of blended ripe fruit sample was put into a mortar and pulp was
extracted repeatedly with acetone until the residue turned colourless.
The acetone extract was then transferred to separating funnel contain-
ing 10 to 15 ml of hexane layer by diluting the acetone with water.
The hexane-containing pigments were then transferred to a 25 ml vol-
umetric flask and carefully diluted to the mark with hexane. Then
1 ml of aliquot was further diluted to 4 ml with hexane and the absor-
bance was recorded in a spectrophotometer at 503 nm. For the determi-
nation of the mineral content, the fruits were sliced and then air-dried
aseptically for 72 h for determination of N, P, K Fe, and Zn for the ripe
fruit. The specimen was oven-dried at 70 °C, and then the digest was
used as an atomic absorption spectrophotometer to determine the min-
eral content as described by Piper (1942).
2.8. Soil chemical analysis
Initial samples of soil were taken from each plot using a soil auger to
a depth of 15 cm during plot preparation. Collected soil samples were
mixed thoroughly to prepare a composite sample from which approxi-
mately 250 g was taken as a working sample. The physiochemical prop-
erties of the initial experimental soil including initial N-P-K status were
109
evaluated by the Soil Research and Development Institute, Bangladesh
(Table 2).
After the final harvest of the tomatoes, soil samples were collected
again in the same way to the initial soil samples collection. The soil sam-
ples were air-dried at room temperature in the laboratory. The soil pH
was calculated using the glass electrode pH meter with a soil/water
ratio of 1:2.5 (Jackson, 1973). The Walkley-Black process was used
(Nelson and Sommers, 1982) to quantify soil organic matter. Kjeldahl
systems (Jackson, 1973) were used to calculate Total N (percent). P
was quantified using the Bray and Kurtz methods and Olsen methods
(Olsen and Sommers, 1982). The ammonium acetate extraction process
(Jackson, 1973) has been used to determine the exchangeable K.
2.9. Microbial populations in rhizosphere soil
At harvest, two plants were carefully uprooted with soil around the
roots from each replication. The soil still adhering to the roots was
regarded as the soil rhizosphere (Hervas et al., 1998). Each of the plants
was gently shaken to separate the soil from the roots. Ten grams of soil
was stored in an Erlenmeyer bottle after removal of all the debris, with a
sterilized liquid of up to 100 ml. The flasks were incubated and shaken
(180 r min−1), then serially diluted for 2 h at room temperature. Serial
dilutions from 10−1 to 10−6 were made for each soil sample following
the dilution plate count technique described by Hirte (1969). To quan-
tify fungi, 0.1 ml of each soil suspension was transferred to appropriate
Trichoderma selective media (TSM) reported by Elad et al. (1981). The
TSM consist of 0.2 g of MgSO4. 7H2O, 0.9 g of K2HPO4, 0.15 g of KCl,
1.0 g of NH4NO3, 3.0 g of glucose, 0.15 g of rose Bengal, 20 g of agar,
0.25 g of chloramphenicol, 9.0 ml of streptomycin, 1.2 ml of
propamocarb, 0.2 g of quintozene and 950 ml of distilled water. This
TSM favours the growth of Trichoderma strains over other rapidly grow-
ing fungi that may overlap it and hence ideal for easy identification
(Shah and Afiya, 2019). The Petri plates (11 cm) were incubated
under the fluorescent illumination at 28o C ± 2 °C and R.H. at 65% ±
5% for five to seven days. After 3 days, the number of colonies per
gram of the sample was counted up to 72 h. afterward and calculated
using the following formula-
CFU=mL ¼ ðno:of colonies  dilution factorÞ=volume of culture plate
For the quantification of bacteria, 10 folded serial dilutions were
made and 100 μl from each diluent were distribute on nutrient agar
(NA) medium (0.5% Peptone, 0.5% Beef extract, and 1.5% Agar, 0.5%
NaCl, pH adjusted to 7.4. At 28 °C for 48 h, the plates were incubated,
and then the CFU (colony-forming unit) was counted (Fischer et al.,
2007).
Table 2
Physiochemical properties of the experimental soil.
Textural class Silty clay loam to clay loam
Physical characteristics
Bulk density (g cm−3) 1.32
Particle density (g cm−3) 2.62
Porosity (%) 46.8
Chemical characteristics
pH 6.4
Organic carbon (%) 0.76
Organic matter (%) 1.14
Total N (%) 0. 091
Available P (μg/g) 18
Available K (meq/100 g) 0.33
110 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115

Table 3
Impact of Trichoderma and biochar on vegetative growth, chlorophyll content and dry matter production of tomato.

Treatments Plant height (cm) Branches (no. plant−1) Leaves (no. plant−1) SPAD (%) Dry matter weight (g plant−1)

Root Shoot

To 107.46 ± 1.76 b 11.50 ± 1.02 ab 66.96 ± 3.41 b 50.40 ± 1.06 a 5.17 ± 0.13 ab 73.91 ± 2.08 b
T1 92.67 ± 2.60 c 6.51 ± 0.09 c 55.75 ± 1.35 c 41.41 ± 1.14 c 3.99 ± 0.04 c 64.71 ± 1.23 c
T2 97.2 5 ± 1.65 c 8.65 ± 0.64 bc 62.29 ± 2.18 bc 47.13 ± 1.46 b 4.47 ± 0.28 bc 68.58 ± 1.64 b
T3 100.35 ± 1.42 bc 8.85 ± 1.03 bc 64.08 ± 0.92 bc 43.76 ± 0.74 bc 4.16 ± 0.11 c 71.66 ± 1.38 b
T4 112.75 ± 2.61 a 11.93 ± 0.23 a 71.38 ± 1.85 a 48.80 ± 1.22 a 5.56 ± 0.45 a 78.65 ± 0.90 a
LSD value 1.84 0.54 1.46 0.88 0.17 1.24
CV (%) 3.18 16.72 6.76 5.49 10.04 4.09

The data represent the mean values ± standard error. Different letters denote significant differences at p ≤ 0.05 by Duncan's multiple range tests. To = standard doses of N-P-K, T1 = 50%
doses of N-P-K (control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50% doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.

2.10. Statistical analysis
The data were analysed using the SPSS software package to assess
the difference in growth, yield characteristics, mineral content and the
nutritional quality of the tomato. Duncan's Multiple Range Test
(DMRT) was used to measure the value of the disparity between the
treatment combinations at p ≤ 0.05 level of significance. Chemical pa-
rameters of the soil and the chemical composition of the plants were
subjected to principal component analysis (PCA) to explore relation-
ships among variables and treatments using the software package R.
achieved with the combined application of Trichoderma and biochar
with a half dose of N-P-K (T4). However, the minimum number of
flower clusters per plant, the number of fruits per cluster, the number
of fruits per plant, the weight of individual fruit and yield per plant
were recorded in treatment T1. The single application of Trichoderma
and biochar also resulted in a significant increase in yield compared to
the half dose of N-P-K.
3.3. Nutritional quality and antioxidant properties of fruits

3. Results Trichoderma and biochar also had significant effects on the

3.1. Plant vegetative growth, chlorophyll content and dry matter production
Application of Trichoderma and Biochar with half dose of N-P-K sig-
nificantly resulted in the greatest plant height, branches per plant, num-
ber of leaves, root and shoot dry matter weight (Table 3). The highest
plant height, the maximum branch number per plant, leaves per plant,
root dry matter weight and shoot dry matter weight were observed in
treatment (T4). However, the maximum SPAD was achieved by the
standard dose of N-P-K (T0) followed by the treatments T4 and T2,
which were statistically similar to each other. The lowest values of
plant height, branches per plant, number of leaves, SPAD, root and
shoot dry matter weight were recorded from the half dose of N-P-K (T1).
nutritional quality and antioxidant properties of tomato (Table 5).
Total soluble solids, protein (g), antioxidant compounds such as ascor-
bic acid, β-carotene, and lycopene were significantly affected by an in-
teraction between Trichoderma and biochar with half dose of N-P-K
(Table 5). The maximum total soluble solids were recorded in T4, but
statistically, similar reflection was also noted in T3 and T0. In case of pro-
tein content, however, both single and combined application of
Trichoderma and biochar showed a similar result to the standard dose
of N-P-K. A similar trend is observed in maximum protein, ascorbic
acid, β-carotene and lycopene in the T4 treatment. On the other hand,
the minimum total soluble solids, protein, ascorbic acid, β-carotene,
and lycopene were recorded from the half dose of N-P-K (T1).

3.2. Yield attributes and yield 3.4. Minerals in shoots

The yield-contributing variables differed significantly among the
treatments and were enhanced by the application of Trichoderma and/
or biochar (Table 4). However, the half dose of N-P-K with combined ap-
plication of Trichoderma and biochar showed better performance in en-
hancing the number of flower clusters per plant, number of fruits per
cluster, number of fruits per plant, the weight of individual fruit and
yield per plant (Table 4). Mean comparison showed that the highest
number of flower clusters per plant, number of fruit per cluster, number
of fruit per plant, the weight of individual fruit and yield per plant were
Mineral concentration in tomato shoots was determined at harvest
(Table 6). N, P, K, Fe & Zn content were found to be significantly higher
in shoots treated with Trichoderma, biochar and half dose of N-P-K (T4).
It is evident that the combined application of Trichoderma and biochar
with half dose of N-P-K showed better nutrient uptake than all other
treatments. It might be due to the synergistic effect of Trichoderma
and biochar, which might have facilitated higher N, P, K, Fe and Zn con-
tents in T4 compared to all other treatments. The lowest values of N, P, K,
Fe & Zn content were recorded from half dose of N-P-K (T1).

Table 4
Impact of Trichoderma and biochar on yield attributes and yields of tomato.

Treatments Number of flower cluster plant−1 Number of fruit cluster−1 Number of fruit plant−1 Weight of individual fruit (g) Yield plant−1 (Kg)

To 6.08 ± 0.22 b 5.83 ± 0.22 a 29.44 ± 0.54 a 80.04 ± 0.90 b 2.47 ± 0.14 a
T1 4.22 ± 0.27 c 4.38 ± 0.07 b 17.57 ± 0.96 c 70.74 ± 0.84 c 1.36 ± 0.07 c
T2 5.00 ± 0.42 bc 4.78 ± 0.30 b 21.87 ± 1.08 b 77.43 ± 0.93 b 1.78 ± 0.08 b
T3 5.34 ± 0.23 bc 4.55 ± 0.17 b 24.30 ± 0.76 b 79.33 ± 1.68 b 2.00 ± 0.10 b
T4 6.86 ± 0.31 a 5.94 ± 0.26 a 31.98 ± 1.44 a 85.21 ± 1.92 a 2.75 ± 0.14 a
LSD value 0.24 0.17 1.25 1.20 0.12
CV (%) 10.88 8.35 7.31 3.51 9.82

The data represent the mean values ± standard error. Different letters denote significant differences at p ≤ 0.05 by Duncan's multiple range tests. To = standard doses of N-P-K, T1 = 50%
doses of N-P-K (control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50% doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.
 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115 111

Table 5
Nutritional and antioxidant properties of tomato fruits (per 100 g of tomato) as influenced by sole or combined application of Trichoderma and biochar.

Treatments Total soluble solids Protein (g) Antioxidant compounds (mg)

Ascorbic acid β-Carotene Lycopene

To 6.00 ± 0.05 a 0.82 ± 0.02 a 15.55 ± 0.51 b 0.11 ± 0.001 a 0.10 ± 0.001 b
T1 5.04 ± 0.03 b 0.65 ± 0.07 b 11.12 ± 0.26 d 0.07 ± 0.003 c 0.07 ± 0.003 c
T2 5.08 ± 0.18 b 0.82 ± 0.01 a 13.40 ± 0.25 c 0.09 ± 0.001 b 0.09 ± 0.001 b
T3 6.02 ± 0.24 a 0.85 ± 0.01 a 14.47 ± 0.32 bc 0.11 ± 0.000 a 0.09 ± 0.005 b
T4 6.25 ± 0.17 a 0.90 ± 0.01 a 17.14 ± 0.47 a 0.12 ± 0.010 a 0.11 ± 0.003 a
LSD value 0.13 0.02 0.49 0.004 0.003
CV (%) 5.24 8.39 4.50 6.84 9.28

The data represent the mean values ± standard error. Different letter (s) corresponds to significant differences at p ≤ 0.05 by Duncan's multiple range tests. To = standard doses of N-P-K,
T1 = 50% doses of N-P-K (control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50% doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.

3.5. Minerals in fruits
Total N content of fruits was significantly (p ≤ 0.05) higher in treat-
ment T4 than in the other treatments (Table 7). The highest P content
was recorded from the T4 which was statistically identical to all other
sole application of Trichoderma and biochar. Likewise, P, a similar kind
of reflection, was also noted in K content; however, maximum K content
was recorded from the single application of biochar (T3). The maximum
Zn content was also marked from the individual application of biochar
which was statistically similar to that T4. The combined application in-
creased not only the essential elements like N, P, K but also essential
trace element contents of fruits.
3.6. Nutrients abundance and microbial populations in the rhizosphere
Combined application of Trichoderma and biochar with half doses of
N-P-K had positive effects on soil properties (Table 8). Soil properties
such as organic matter, total N, available P and available K were signifi-
cantly affected by T4 treatment. Maximum organic matter, total N, avail-
able P and available K were achieved with the combined application of
Trichoderma, biochar and half dose of N-P-K in treatment T4. The highest
pH was recorded from the treatment T3, which was also statistically
similar to T4 treatment. On the other hand, minimum soil pH, organic
matter, total N, available P and available K was recorded from the half
dose of N-P-K (T1) treatment. The fungal and bacterial populations
were quantified at harvest, and fungal populations were significantly
(p ≤ 0.05) increased in the combined treatments compared to the sole
treatments. The maximum fungal population was recorded in T4,
which was closely followed by T0. Bacterial populations were also signif-
icantly higher in treatment T4. On the other hand, a substantially lower
number of microbial populations were noted in T1 treatment.
3.7. Principal component analysis (PCA)
Principal component analysis revealed that the variables were posi-
tively correlated within in two main components (PCs), which
accounted for 85.5% and 7.6%, of the overall variation. The distribution
of variation along PC1 was attributed to the application of Trichoderma
and biochar treatments. PC1 shows that all the quality and trace ele-
ments parameters were highly affected by the co-application of
Trichoderma and biochar (Fig. 1).
4. Discussion
The present study found a significant positive effect of field applica-
tion of Trichoderma and biochar on growth attributes of tomato. In con-
trast to single treatments and control, co-application of Trichoderma and
biochar (T4) showed improved performance. This might be due to syn-
ergistic effects of Trichoderma and biochar which exerted a considerable
growth promotion over the control. Previous studies have shown that
Trichoderma spp. may induce plant growth promotion by colonizing
roots, increasing the foliar area and secondary roots, as well as improv-
ing the root system architecture (Dorais, 2007; Chacón et al., 2007). The
promotion of plant growth and nutrition by Trichoderma and their
secondary metabolites released in the rhizosphere has also been
documented (Kotasthane et al., 2015; Zeilinger et al., 2016). The
newly emerging soil amendment biochar enhances soil fertility,
structure, and interaction with the soil microbiota (Hussain et al.,
2017). Due to having a higher surface area, biochar act as a medium
for enhanced growth and root colonization of beneficial fungi
through hyphal growth and elongation (Hussain et al., 2017). This
proliferation improves soil fertility and contributes to better uptake
of mineral nutrients for plants, thus boosting growth, as observed
in the current study.
Biochar has porous structure which facilitates better root prolifera-
tion and nutrient uptake, boosts root and shoot biomass. Application
of Trichoderma-based products resulted in a 35 and 43% increase of
root mass in well-watered and dry conditions, respectively (Rubin
et al., 2017). Therefore, the combined application of Trichoderma and
biochar might have interacted synergistically, facilitating higher root
and shoot biomass of tomato under the present investigation. Leaf
greenness is essential to the desired growth because it is directly related
to the absorption of nitrogen and the photosynthesis rate (Harman and
Shoresh, 2007). Our current research has shown the leaf greenness in

Table 6
Impact of Trichoderma and biochar on the concentration of minerals in shoots of tomato alone or in combinations.

Treatments N (%) P (%) K (%) Fe (mgkg−1) Zn (mgkg−1)

To 12.58 ± 0.42 a 0.31 ± 0.02 b 4.69 ± 0.02 c 230.00 ± 2.14 b 53.88 ± 1.56 b
T1 9.89 ± 0.03 b 0.23 ± 0.01 c 4.14 ± 0.03 e 207.46 ± 0.87 d 47.56 ± 0.83 c
T2 11.95 ± 0.44 a 0.34 ± 0.01 b 4.79 ± 0.02 b 235.10 ± 1.15 ab 56.84 ± 1.92 b
T3 12.17 ± 0.03 a 0.37 ± 0.01 ab 4.45 ± 0.01 d 220.16 ± 0.25 c 53.69 ± 0.37 b
T4 12.85 ± 0.43 a 0.42 ± 0.02 a 4.96 ± 0.01 a 237.94 ± 2.91 a 62.01 ± 1.50 a
LSD value 0.28 0.02 0.07 2.65 1.21
CV (%) 5.64 9.44 1.02 1.53 4.02

The data represent the mean values ± standard error. Different letters denote significant differences at p ≤ 0.05 by Duncan's multiple range tests. To = standard doses of N-P-K, T1 = 50%
doses of N-P-K (control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50% doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.
112 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115

Table 7
Mineral content of ripe tomato fruits as influenced by Trichoderma and biochar application alone or in combinations.

Treatments N (%) P (%) K (%) Fe (mgkg−1) Zn (mgkg−1)

To 1.53 ± 0.06 c 0.09 ± 0.013 b 4.21 ± 0.09 b 185.66 ± 3.77 b 76.10 ± 4.06 b
T1 1.63 ± 0.03 d 0.07 ± 0.005 b 4.12 ± 0.01 b 146.60 ± 4.95 c 55.09 ± 0.59 c
T2 1.84 ± 0.01 c 0.11 ± 0.005 a 4.63 ± 0.01 a 201.28 ± 8.56 b 82.59 ± 0.98 b
T3 2.31 ± 0.09 b 0.12 ± 0.004 a 4.76 ± 0.01 a 203.49 ± 3.19 b 104.29 ± 0.78 a
T4 2.62 ± 0.01 a 0.14 ± 0.004 a 4.75 ± 0.01 a 241.18 ± 1.12 a 101.24 ± 1.33 a
LSD value 0.09 0.006 0.06 7.28 4.19
CV (%) 5.12 13.24 1.67 4.82 5.01

The data represent the mean values ± standard error. Different letters denote significant differences at p ≤ 0.05 by Duncan's multiple range tests. To = standard doses of N-P-K, T1 = 50%
doses of N-P-K (control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50% doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.

Trichoderma + biochar treated plants to be significantly higher com-
pared to the half dose of N-P-K. Danish and Zafar-ul-Hye (2019) re-
ported an increase of 114% of total chlorophyll content of wheat
through the combined application of Bacillus amyloliquefaciens + bio-
char as compared to the untreated control. Abid et al. (2017) reported
that the application of biochar improved the production of photosyn-
thetic and accessory pigments of tomato plants under various irrigation
waters.
Crop productivity was shown to have increased 101.45% over the
control and even 11.33% over the standard doses of N-P-K in response
to the combined application of Trichoderma and biochar with half dose
of N-P-K. Biochar directly adds nutrients in the soil as well as increases
bioavailability, whereas Trichoderma has been reported to increased
nutrient availability, uptake, and assimilation in plants. Therefore, the
co-application of biochar and Trichoderma might have synergistically fa-
cilitated improved growth which might have the reason for the higher
number of flowers cluster in tomato plants under the current research.
In addition, various proteins associated with photosynthesis were
shown to be up-regulated in plants during Trichoderma-plant interac-
tion, which could increase the photosynthesis ability of the plants
resulting in higher crop yield (Harman, 2000). Molla et al. (2012) re-
ported 50% increase in tomato yield due to the application of
Trichoderma along with standards doses of N-P-K. The highest perfor-
mance in T4 could, therefore, be due to Trichoderma's synergistic interac-
tion with biochar.
Combined application of Trichoderma and biochar increased the nu-
tritional and antioxidant properties of the tomato fruits. The co-
application of Trichoderma and biochar (T4) showed the highest value
of TSS, but statistically similar results were also noted in T3, which is
the sole treatment of biochar. Therefore, it may be assumed that biochar
has a positive impact on increasing the TSS value of tomato fruits.
Caliman et al. (2010) reported the higher value of titratable acidity
and total soluble solid in tomato cultivated on substrates amended
with various biochar. Almaroai and Eissa (2020) reported a 29% increase
in the TSS content of tomato due to the application of biochar. Biochar
may increase the fruit quality by increasing photosynthesis and translo-
cation of sucrose and organic acid to the fruits that may contribute to a
higher concentration gradient of sucrose in fruits (Wang et al., 2014).
Nitrogen fertilizer has been demonstrated to improve protein content
(Rembiałkowska, 2007). Consistent with this, our study showed maxi-
mum protein content in the standard dose of N-P-K. Brandt and
Mølgaard (2001) observed that plants cultivated with a lower ready-
to-use level of nitrogen in field conditions appear to contain a lower
level of nitrate and protein content of the majority of foodstuffs. Khan
et al. (2017) reported increased antioxidant compounds such as ascor-
bic acid, lycopene and β-carotene in ripe tomato fruits due to the appli-
cation of 100% Trichoderma bio-compost in field conditions, and our
findings are entirely consistent with this. However, a contradictory re-
sult has been reported by Petruccelli et al. (2015), with lower lycopene
and β-carotene content in tomato when amended with only biochar
compared to non-amended control. However, they pointed out that fur-
ther research is required in field conditions to confirm their greenhouse
findings. The content of β-carotene and lycopene of tomatoes was
stated to be significantly affected by the environmental and agricultural
factors (Dumas et al., 2003; Violeta et al., 2013). Therefore, tomato anti-
oxidant content in field conditions remains a worthy area for further
study.
The combined use of Trichoderma and biochar appears to enhance
nutrient uptake more than either alone. This could be due to a combined
effect from Trichoderma and biochar, which could have led to higher N,
P, K contents in T4, with an increased nutrient solubility, nutrient absor-
bance, mobilization, and assimilation. The findings of Samolski et al.
(2012) and Zhao et al. (2009) reported that Trichoderma spp. improves
nutrients absorption through root architecture modulation or through
exudation of nutrients availability-enhancing substances such as
siderophores and organic acids and that are fully consistent with our
findings. Moreover, the capacity of biochar to adsorb nutrients also
may minimize losses of N and thus improve uptake by plants (Younis
et al., 2014). The high surface areas of biochar may be a key reason for
improving the exchange of cations in soils which leads to better nutrient
bioavailability (Chan et al., 2008). It has been reported that biochar im-
proves soil fertility by retaining nutrients for a longer period, thus min-
imizing nutrient leaching to the surrounding environment (Cao et al.,
2018).

Table 8
Nutrients and microbial populations in rhizosphere soils as influenced by Trichoderma and biochar alone or in combinations.

Treatments pH Organic matter Total N Available P Available K Microbial populations (CFU)

(g kg−1) (kg−1) (mg kg−1) (c-mol kg−1) Fungi(×105) Bacteria(×109)

To 6.30 ± 0.021 b 9.85 ± 0.10 b 1.09 ± 0.01 b 5.46 ± 0.02 b 0.31 ± 0.01 a 13.28 ± 0.22 b 14.89 ± 2.73 b
T1 6.26 ± 0.004 b 9.83 ± 0.76 b 1.08 ± 0.02 b 5.32 ± 0.30 c 0.26 ± 0.01 b 10.33 ± 0.51 c 13.58 ± 1.00 c
T2 6.27 ± 0.006 b 13.24 ± 0.01 a 1.75 ± 0.05 a 6.95 ± 0.02 b 0.31 ± 0.01 a 15.02 ± 0.26 b 24.21 ± 0.06 ab
T3 6.35 ± 0.005 a 13.92 ± 0.31 a 1.82 ± 0.01 a 7.30 ± 0.14 ab 0.31 ± 0.01 a 14.53 ± 0.02 b 24.67 ± 0.04 ab
T4 6.34 ± 0.006 a 14.67 ± 0.11 a 1.85 ± 0.01 a 7.67 ± 0.01 a 0.32 ± 0.01 a 17.22 ± 0.75 a 25.76 ± 0.04 a
Pre-soil 6.21 9.60 0.90 5.40 0.27 4.5 9.74
CV (%) 0.35 6.08 2.95 4.60 7.29 6.27 12.11

The data represent the mean values ± standard error. Different letters denote significant differences at p ≤ 0.05 by Duncan's multiple range tests. To = standard doses of N-P-K, T1 = 50%
doses of N-P-K (control), T2 = Trichoderma + 50% doses of N-P-K, T3 = biochar+50% doses of N-P-K and T4 = biochar+Trichoderma + 50% doses of N-P-K.
 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115 113

Fig. 1. Principal component analysis (PCA) loading plot (A) and scores of principal component analysis (B) of chemical parameters of the soil and the chemical composition of tomato fruits
as a function of Trichoderma, biochar and N-P-K treatments. TSS, total soluble solids; BC, β-carotene; Lyco, lycopene; Ptn, protein; Btria, Bacteria; Fgi, fungi, OM, organic matter; Avp,
available phosphorous; Avk, available potassium; TN, total nitrogen; Ns, Ps, Ks, Fes, Zns, nitrogen, phosphorous, potassium, iron and zinc content of shoots and Nf, Pf, Kf, Fef, Znf,
nitrogen, phosphorous, potassium, iron and zinc content of fruits respectively.

Colla et al. (2015) reported that Trichoderma spp. can enhance iron
solubility and hence uptake and translocation by the plant. Our results
are similar, with higher Fe content in shoots of tomato with a single ap-
plication of Trichoderma, which was statistically similar to the combined
application. An increased P availability in rhizosphere grounds was re-
ported due to the application of the Trichoderma-enriched biofertilizer
(Cai et al., 2015). Addition of biochar to the soil increase availability of
basic cations as well as phosphorus and total nitrogen concentrations
(Glaser et al., 2002; Lehmann et al., 2003). A positive correlation was re-
ported between higher mineral content in crop plants with a higher
amount of rhizosphere soil microorganisms (Worthington, 2001). Our
PCA analysis demonstrated a similar pattern.
Microbes, including beneficial fungi and bacteria of the soil rhizo-
sphere, play a vital role in the provision of nutrients by solubilizing or-
ganic nutrients. These rhizosphere microorganisms also increase the
availability of nutrients for plants and thereby promote plant growth
and productivity by enhancing soil fertility (Kaewchai, 2009). Improved
water retention following the application of biochar in soil is well
established and this may positively affect the rhizosphere microbes in
the soil. Biochar offers an adequate habitat for a broad and diverse
group of microorganisms, although the interaction between biochar
and microorganisms in the soil is complex (Busscher et al., 2010) and
not fully understood. Our study shows that the co-application of
Trichoderma and biochar resulted in an increased abundance of fungal
114 M.N.H. Sani et al. / Annals of Agricultural Sciences 65 (2020) 107–115
and bacterial populations compared to the control (half dose of N-P-
K). This abundance may explain the positive effect on the production
of tomato fruits, minerals, and antioxidant content.
5. Conclusion
Co-application of Trichoderma and biochar with half dose of N-P-K
increased not only the plant growth and yield but also permitted a
50% reduction in the requirement for synthetic N-P-K fertilizer, without
compromising yield. The combined application also enhanced the anti-
oxidant contents (e.g., ascorbic acid, β-carotene, and lycopene) as well
as minerals in shoots and fruits. It also increased soil nutrient availability
and fertility by increasing fungal and bacterial growth in the rhizo-
sphere. Co-application of Trichoderma and biochar along with 50%
doses of N-P-K can be considered as a sustainable strategy for the pro-
duction of tomato in the field with higher yield and superior quality.
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgments
This project was funded by the Ministry of Science and Technology,
Bangladesh. The research facilities were provided by Sher-e-Bangla Ag-
ricultural University, Dhaka, Bangladesh and Universiti Sains Malaysia.
We thank Professor Jacob Weiner (Department of Plant and Environ-
mental Sciences, University of Copenhagen) for valuable comments on
the manuscript and English language editing.
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