Bioresource Technology Reports 19 (2022) 101165

Contents lists available at ScienceDirect

Bioresource Technology Reports

journal homepage: www.sciencedirect.com/journal/bioresource-technology-reports

A stressing method for producing high-density Trichoderma spores in a

dual-layer by utilizing a starch-based medium in a reconditioning approach
Md. Abuhena a, 1, Md. Golam Kabir a, b, 1, Md. Faisal Azim a, 1, Jubair Al-Rashid a, b,
Noorain Munim Rasul a, b, Md. Amdadul Huq c, *
a
Department of Research & Development, Apex Biofertilizers & Biopesticides Limited, Gobindaganj 5740, Gaibandha, Bangladesh
b
Apex Biotechnology Laboratory, Apex Holdings Ltd., East Chandora, Shafipur, Kaliakoir, Gazipur 1751, Bangladesh
c
Department of Food and Nutrition, College of Biotechnology and Natural Resource, Chung-Ang University, Anseong, Gyeonggi-do 17546, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: A stressing method was adopted to produce Trichoderma harzianum TI21 spores in a dual-layer with an outer-
Trichoderma harzianum layer spore free of media-residue by reconditioning the starch-based rice agar medium. The essential parame­
Sporulation ters were explored and optimized using statistical methods. The single factorial test's findings demonstrated that
Fungal media
lower-pH and starch-based media encouraged sporulation while suppressing the growth of contaminants. The
Parameters optimization
Box-Behnken design identified the ideal levels as pH 4, 30 ◦ C, and 75 %. In the reconditioning approach, the first
Industrial production
Clogging harvest produced 4.91 (3.64 × 1011 CFU/g) of spores (g/L) after 5 days of cultivation, while the second and third
harvests produced 4.19 and 3.48 (respectively) after 4 days of cultivation, leaving 755 (g/L) (2.25 × 1010 CFU/g)
media-mixed spores. As compared to media-mixed spores, media residue-free spores had significantly longer
viability (18 months) with no clogging hazards. According to the findings, the method can be used industrially to
produce stable, high-density spores for widespread agricultural applications.

1. Introduction Recently, there has been a lot of interest in biocontrol products. Some
Trichoderma spp. have the potential to be biocontrol agents and can be
Trichoderma fungi, notably those in the clades of Viride and Harzia­ produced on a large volume. Adverse climatic situations, such as
num, are ubiquitous. They can be easily isolated from the soil, rotting extreme temperatures and drought, traditional farming practices, and
wood, plant roots and leaves, and other organic plant matter (Chaverri lack of technological expansion have considerably affected crop output.
et al., 2015; Hoyos-Carvajal and Bissett, 2011). Trichoderma improves The usage of agrochemical inputs has evolved as a result of the present
root growth and development, crop yield, tolerance to abiotic stressors, emphasis on environmentally friendly agriculture and the production of
and nutrient uptake and use (Sood et al., 2020). It can induce systemic high-value crops (Abuhena et al., 2022). In Bangladesh, Trichoderma-
resistance in plants throughout the growth period (Nawrocka and containing products suitable for use in crop production and disease
Małolepsza, 2013). Trichoderma strains have also been found to colonize management are not widely available. The use of biocontrol as an
plants' rhizosphere and enrich the soil microbiome diversity, and as a alternative to pesticides has arisen from the quest for cost-effective and
result, Fusarium spp. populations are significantly reduced (Sar­ ecosystem friendly strategies that improve plant growth and output.
avanakumar et al., 2017; Jahan et al., 2018). Lu et al. (2020) found Many commercial products containing Trichoderma spp. are marketed as
several Trichoderma strains that effectively controlled maize stalk rot, a biopesticides, biofertilizers, growth enhancers, and natural resistance
widespread disease in China. Wang et al. (2019) showed that synergistic stimulants. The number of Trichoderma-containing products in the in­
application of difenoconazole-propiconazole and T. harzianum SH2303 ternational market is increasing steadily. More than 250 are currently
reduced the Southern corn leaf blight (SCLB) disease in maize under available (Woo et al., 2014). Trichoderma harzianum is the most
both greenhouse and field conditions. commonly used species in commercial Trichoderma inoculums.

* Corresponding author.
E-mail addresses: abbl@apexbiofertilizer.com (Md. Abuhena), kabir@apexbiofertilizer.com (Md.G. Kabir), jubair@apexbiofertilizer.com (J. Al-Rashid),
amdadbge100@cau.ac.kr (Md.A. Huq).
1
Equal contributions.

https://doi.org/10.1016/j.biteb.2022.101165
Received 5 May 2022; Received in revised form 11 July 2022; Accepted 18 July 2022
Available online 27 July 2022
2589-014X/© 2022 Elsevier Ltd. All rights reserved.
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

T. harzianum spores are very resilient. They can survive extreme climatic 2. Materials and method
conditions like desiccation, freezing, and UV radiation (Cai et al., 2020).
The production cost of Trichoderma products will be an important 2.1. Microorganism
aspect if they are to be sought in the least developed countries because of
the privation of raw materials and an optimum manufacturing process. 2.1.1. Isolation of Trichoderma harzianum strain TI21
It is necessary to define the culture parameters required for high-density For the isolation of Trichoderma harzianum strain TI21, environ­
spore production. The common and effective system of getting sub­ mental samples (decayed dry wood surface of palm tree) were collected
stantial amounts of spore is through low-cost by-products containing from Kaliakoir, Gazipur, Bangladesh (24◦ 05′ 50.0′′ N 90◦ 15′ 56.2′′ E).
nutrient sources. Carbon like sucrose and fructose, as well as grains like Following collection, samples were placed in sterile polythene zip-lock
rice, maize, and wheat, are common carbon sources (Abuhena et al., bags, labelled with their sources and locations, and kept in an aseptic
2022). A useful approach to cut production costs is to adjust cultivation condition. Upon collection, materials were taken to the Apex biotech­
time, use inexpensive media, reuse the media, and optimize the cultural nology laboratory (ABL) Gazipur, Bangladesh as soon as possible and
factors to obtain a greater spore density (Singh and Nautiyal, 2012). The securely maintained in a refrigerator at 4 ◦ C before being immediately
kinetics of the production process, on the other hand, are not well analysed. Strain TI21 was isolated using a dilution plate technique
known, and data on industrial production is limited. When the goal is to ranging from 10− 3 to 10− 5 fold dilutions. Diluted (1:10) samples of 0.1
produce Trichoderma spores, conditions that limit fungal enzyme pro­ mL were disseminated on Trichoderma specific medium (TSM) agar plate
duction and allow for prolific sporulation should be adopted. A suitable (in triplicates for each dilution) for Trichoderma isolation (Kumar et al.,
medium for industrial production not only supports the hyphal growth 2012). The plates were then incubated at 28 ± 2 ◦ C for 96 h. On Potato
of the fungi but also the successful production of spores. The applied Dextrose Agar (PDA) plates, morphologically distinct fungal bodies and
method requires generating spores swiftly and economically. colonies producing green conidia were selected and purified. Micro­
Different researchers have explored the production of Trichoderma scopic examination of morphological features such as conidia, conidio­
spp. at an industrial level following both liquid and solid-state fermen­ phore, and mycelia structure was performed using a phase-contrast
tation (Sala et al., 2021; Sachdev et al., 2018; Keswani et al., 2016). microscope (Axio Imager A1, Carl Zeiss, Germany) at 40×. The isolated
During liquid fermentation procedures, spore concentration and Trichoderma strain was maintain on PDA and stocked with 20 %
viability are frequently low, and contamination is common. In liquid glycerol.
culture, manufacturing is time-consuming, it is difficult to separate the
spores from the substrate, and spore viability is poor due to high mois­ 2.1.2. Molecular characterization of TI21
ture content. Spores produced in solid-state fermentation (SSF) are more Genomic DNA was isolated from a single colony of the TI21 isolate
robust and vigorous, have longer viability, and have higher resistance using the PrepMan® Ultra Sample Preparation Reagent Kit (Applied Bio-
(Naeimi et al., 2020). In this instance, semi-solid fermentation appears systems). PCR was performed on extracted genomic DNA from TI21 and
to be the most effective way for vigorous spore production. a positive control of Saccharomyces cerevisiae in a MyGene™ Series
Response surface methodology (RSM), which includes Box–Behnken Peltier Thermal Cycler. The Applied Biosystems' Fast MicroSeq® D2 LSU
design (BBD), is used to identify the crucial interactions among the rDNA Fungal PCR Kit was used as the PCR Master Mix. PCR products
defining components and determine their optimum levels. The BBD is were analysed in TAE buffer (1×) with 1.2 % Agarose gel. Purified PCR
employed to determine the growth parameters optimum for maximum product was observed in 1.2 % Agarose gel in 1 X TAE buffer after
production of the active ingredients. Li et al. (2016) used BBD to study ethanol precipitation. Cycle sequencing was performed with the purified
the interactions between, and determine the optimal levels of, several PCR product and the Forward Primer Reaction Mix of the MicroSeq® D2
factors in Trichoderma harzianum SH2303 chlamydospore production by LSU rDNA Fungal Sequencing Kit (Applied Biosystems). The purified
liquid fermentation. However, industrial production data on Tricho­ cycle sequencing product, obtained by ethanol precipitation, was dis­
derma harzianum using semi-solid fermentation is still inadequate. solved in Hi-Di™ Formamide (Applied Biosystems), denatured, and sent
It is crucial in the production process to ensure the safety of the for sequencing to NIB, Savar, Dhaka. It was analysed by a genetic ana­
active components (spores) against conditions with extreme pH, low lyser (ABI 3130XL 8-capillary sequencer) using big dye 3.1 sequencing
moisture content, toxins, and UV radiation. The formulation of a reliable chemistry. DNA similarity was analysed with the NCBI BLAST server
biocontrol product necessitates the addition of a suitable preservative (http://www.ncbi.nlm.nih.gov). The sequence was submitted to the
preparation to overcome environmental limits and give the antagonist a NCBI GenBank database and was given an accession number (Maitra
competitive benefit over disease-causing organisms and other micro­ et al., 2021).
flora. The formulations require including nutrients important for the
spore's stability, osmoregulation, and revival from initial biomass 2.2. Bioactivity of Trichoderma harzianum TI21
(Mulatu et al., 2021; Hewavitharana et al., 2018). Furthermore, the
biocontrol agents must endure steps of processing from harvesting to 2.2.1. Antagonistic activity against phytopathogens
delivery. In vitro tests of TI21 were performed to evaluate the antagonism
Hence, the current study was conducted to investigate the nutrients potential against phytopathogenic fungi following the Awad et al.
and cultural conditions for improving Trichoderma harzianum TI21 (2018) methodology with modifications. ABL provided four fungal
growth for large-scale spore production through semi-solid fermenta­ phytopathogens isolated from disease-infected samples and stored at
tion. Rice starch was used as the limiting nutrient to encourage TI21 ABL-culture collection (CCN-110-114): Sclerotium delphinii SCR5
sporulation. Response surface methodology, which includes Box- (MK478832), Fusarium equiseti PTR3 (MK478826), Curvularia spicifera
Behnken designs, was used to investigate the interaction of the critical BLR4 (MK478825) and Alternaria alternata ALT10 (MK478825). Two
elements and determine their optimum values. A reconditioning holes (5 mm) were made on a 90 mm sterile PDA plate containing 20 ml
approach was used to induce rapid initiation of sporulation in addition of media, maintaining a 60 mm distance between holes and 10 mm apart
to high spore yield. from the edge of the Petri plates. In the tests, the 5 mm diameter plugs
containing mycelium (five-day-old) from both the targeted phytopath­
ogenic fungi and Trichoderma harzianum TI21 were placed in the PDA
plate at a distance of 60 mm from each other. Dishes with only patho­
gens' discs were placed on one side as controls. All plates were incubated
at 28 ± 2 ◦ C for 7 days with three replications. These experiments were
repeated thrice, and the values were expressed as their mean. After 7

2
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

days of incubation, the growth inhibition of phytopathogens by TI21 2.3. Media

was measured by the following formula (Li et al., 2016) (Eq. (1)):
Potato dextrose agar (PDA; HIMedia) and rice agar (RA) media were
I = (C − T)/C × 100 (1)
utilized in this experiment. The PDA media contained potato infusion
where I denotes the percentage of phytopathogens' growth inhibition; C (200 g/L), dextrose (20 g/L) and agar (20 g/L), whereas the RA media
denotes phytopathogens' radial growth (cm) in the control plate; and T contained rice flour (20 g/L) and agar (20 g/L). The rice was collected
denotes phytopathogens' radial growth (cm) in the test Petri plates. from the BRRI (Bangladesh Rice Research Institute) regional centre in
Rangpur, Bangladesh. The rice was cleaned, dried, and stored in a dry
2.2.2. Quantitative estimation of Chitinase and β-1, 3-Glucanase activity place in the laboratory until utilization. Moreover, rice grains were
Chitinase and β-1, 3-Glucanase activities were determined using the milled using an automatic grain flour milling machine (Kingrunda,
modified Trichoderma liquid enzyme (TLE) medium following Gezgin model: 6FT-28), which yielded 0.08 mesh flour.
et al. (2020). The supernatant was separated from a 7-day-old culture of
TI21 (1.0 × 108) and kept at − 20 ◦ C until enzyme activity measurement. 2.4. Seed suspension preparation
Chitinase activity was assayed by using chitin (0.5 %) obtained from
shrimp shells (Sigma) as substrate in the modified TLE medium. Chiti­ To prepare a seed suspension, Trichoderma harzianum TI21 was
nase activity was measured in terms of the release of N-acetyl-D- revived from a glycerol (20 %) stock in a PDA plate. The retrieved cul­
glucosamine from chitin. β-1, 3-glucanase activity was measured using a ture was then subcultured twice. A single colony was seeded on PDA
25 % (w/v) suspension of laminarin dissolved in 50 mM acetate buffer plates (11 cm in diameter) from the second subculture and incubated at
(pH 5.0) at 40 ◦ C for 30 min. Reducing sugars liberated from the sub­ 28 ± 2 ◦ C with pH 5.8 ± 0.2. After 5 days of incubation, spores were
strate were estimated with dinitro salicylic acid (DNS) method. One unit scraped from the Petri plate's surface and placed in sterile DM (demin­
(U) of chitinase and β-1, 3-glucanase activity was defined as the amount eralized) water containing 0.9 % NaCl, then shaken in the incubator
of enzyme necessary to produce 1 μmol of N-acetylglucosamine (NAG) (Biobase B110) for 2 h. After shaking, the spore suspension was filtered
in 1 min and the amount of enzyme that released 1 μmol reducing sugar through cheesecloth to prevent mycelia from clogging the sprayer nozzle
in 1 min under the assay conditions, respectively. during inoculation. A seed suspension was prepared with a concentra­
tion of 1 ± 0.2 × 106 CFU/mL.
2.2.3. Quantitative estimation of available phosphate (P) solubilization
Trichoderma harzianum TI21 was cultured in NBRIP medium (Bononi 2.5. Tray design
et al., 2020) containing (g/L): glucose, 10.0; tricalcium phosphate
(TCP), 10.0; MgCl2⋅6H2O, 5.0; MgSO4⋅7H2O, 0.25; KCl, 0.2; (NH4)2SO4, During the tray design phase, two aspects were emphasized: the
0.1. Quantitative estimation of phosphate solubilization was carried out tray's overall surface area and its height. Surface area and medium
using Erlenmeyer flasks (100 mL) containing 50 ml of medium inocu­ height were bound to influence spore production since the amount of
lated in triplicate with three discs (5 mm in diameter) of actively media would vary with surface area and medium height. The tray was
growing cultures of TI21. For 5 days, the cells were incubated at 28 ± made of stainless steel (grade 304), and was designed to keep the media
1 ◦ C in a shaking incubator (Lab Tech, Korea) at 180 rpm. Estimation of from leaking out. To determine suitability, three potential tray sizes
phosphate solubilization was carried out using a 5-day old culture of were considered: S1 (20 × 20 × 0.5 in.3), S2 (22 × 30 × 0.5 in.3), and S3
TI21. An aliquot of 5 ml was withdrawn from the culture flask and (30 × 25 × 0.5 in.3). S2 was found suitable for handling during inocu­
centrifuged (Eppendorf, Germany) at 10,000 rpm for 10 min, and the lation, spore harvesting, and other maintenance (Supplementary
supernatant of culture was analysed for pH (pH meter, HANNA In­ Fig. F1).
struments) and phosphate concentration. Phosphate in the culture su­
pernatant was estimated using the Abuhena et al. (2022) and expressed
2.6. Rack design
as equivalent phosphate (μg ml− 1). The experiments were conducted in
triplicates and the values were expressed as their mean.
Height, width, length, and tray-to-tray distance were all considered
during the rack design stage. The height was set at 75 in. to allow 7 trays,
2.2.4. Quantitative estimation of Indole 3 Acetic Acid (IAA) production
and the width was set at 36 in. to accommodate 1 tray. To hold six trays,
Bader et al. (2020), with minor changes, was used to calculate IAA
the length was set at 135 in. As a result, each rack could hold 42 trays
output. Three 5 mm plugs were inoculated in potato dextrose broth
and was designed to keep the trays at the same (10-inch) distance from
(PDB) supplemented with 0.2 % filter-sterilized (Whatman) L-trypto­
one another. The rack is made of stainless steel (grade 304) and has a
phan solution from 5-day-old TI21 cultures and incubated at 28 ◦ C in a
groove for holding the tray. It was equipped with a wheel to allow it to
rotary shaker at 120 rpm for 7 days. The culture was centrifuged for 15
move about during inoculation and spore collection (Supplementary
min at 10,000 rpm after 7 days of incubation. Two milliliters of Sal­
Fig. F2).
kowski reagent were combined with 1 mL of supernatant (1 mL of 0.5 M
FeCl3 in 50 mL of 35 % HClO4). As a control, uninoculated medium has
been used. After one hour of incubation, a pink hue appeared, indicating 2.7. Room design
that IAA production had occurred. Preparing a set of fresh IAA solutions
(0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 65 g ml− 1 of pure IAA The space was divided into four sections in the room design plan:
in each solution) and measuring the absorbance with a double beam UV/ 482.5 ft2 for medium pouring and inoculation, 727.5 ft2 for two incu­
Visible spectrophotometer (BK-D590, Biobase, China) at 530 nm yielded bation rooms each, and 215 ft2 for spore harvesting (Supplementary
an IAA standard curve. The absorbance on the standard curve was Fig. F3). All of the rooms were the same height and length, measuring 9
plotted to determine the quantity of IAA produced by Trichoderma har­ and 25 ft, respectively. Each incubation room may hold up to 336 trays
zianum TI21. The quantity of IAA in the culture was measured in mi­ in eight racks in four rows. A total of 672 trays could be accommodated
crograms per mL. The trials were carried out in triplicate, and the results in the two incubation rooms. Incubation rooms were provided for
were expressed as the mean of the three results. temperature and humidity control. A humidifier was used to keep the
humidity in the rooms stable (Biobase; BKUH-03LB). The temperature
was controlled using a room heater (RFL; WRH-PTC001) and a chiller
(Biobase; KL-LSF30S). For disinfecting, the walls and floor were all
smooth surfaces.

3
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

2.8. Media mixing, sterilization, and pouring facility parameters remained unchanged (Table 1).

It was crucial to ensure proper mixing of agar and to ensure no 2.9.1.4. Impacts of humidity. Experiments were conducted at humidity
coagulation occurred in the sterilized media. The medium was sterilized levels of 60 ± 2, 70 ± 2, 80 ± 2, and 90 ± 2 % to investigate the in­
using a mixing facility-equipped commercial media steriliser (800 L; fluence of humidity on sporulation, with other factors kept constant
Wenzhou Daysly Technology Co., Ltd.). The sterilization was carried out (Table 1).
at a temperature of 121 ± 2 ◦ C and a pressure of 15 ± 0.2 lbs. for 15 ± 2
min. To prevent the medium from solidifying, it was poured into the 2.9.2. Method optimization using Box–Behnken Design (BBD)
trays in a short time through an autoclavable container at 70–80 ◦ C. Response surface methodology (RSM) is a well-known method that
combines statistical and mathematical approaches to fit experimental
2.9. Stressing method for high-density media-free pure spore production data to a polynomial equation. BBD is an RSM method that can be uti­
lized for a variety of statistical optimizations. The optimal range of
The design of the tray, rack, and room was accomplished first, fol­ factors for the maximum yield of high density spore production in RA
lowed by installation. After that, the cultivation of seed cultures and the media was determined using BBD, a three-level model based on the
preparation of the suspension were accomplished five days ahead of the initial range of factors determined by a single-factor experiment. Tem­
inoculation of the tray media. In the stressing experiment, agar at a perature (X1), pH (X2), and humidity (X3) were the independent factors
concentration of 20 g/L was added to the media to harden it and allow selected, and each factor's experimental design and data analysis were
just the spores to be harvested by scraping without taking any media done using Design-Expert 13.0 software. Spore yield (g/L) was selected
residue. The medium was then properly mixed at 70 ◦ C to avoid coag­ as the response for the combination of the independent factors. The BBD
ulation prior to sterilization. After sterilizing, the media was thoroughly array and response values used to build the models are listed in Table 2.
mixed again at 300 rpm just before pouring. The trays were cleaned with Each condition was checked three times, with the mean values reported
0.1 % NaOCl and placed in the rack prior to media pouring. The media as observed responses. To reduce the impact of unspecified differences,
(3 L) was then poured into tray S2 (22 × 30 × 0.5 in.3), resulting in a all experiments were conducted in random order. Eq. (2) shows a non-
media height of 0.27 ± 0.01 in. and a media surface of 558.8 cm2/L. linear quadratic model generated by the design.
After 1 h of medium pouring, the media was inoculated with a 2 % seed Y =A₀ + A1 X1 + A2 X2 + A3 X3 + A4 X1 X2 + A5 X2 X3 + A6 X1 X3 + A7 X1 2
suspension using a sprayer machine with an initial spore concentration
+ A8 X2 2 + A9 X3 2
of 2.0 × 107 (CFU/mL) across the inoculation media surface of 558.8
cm2/L because the seed culture suspension had 1 ± 0.2 × 106 CFU/mL. (2)
The spore suspension was evenly sprayed on the media surfaces. In the In which Y denotes the calculated response of the dependent vari­
dark, the inoculation media was incubated to form a thick, deep green ables associated with each factor-level combination; A0 denotes the
spore mat. The day-by-day growth pattern was recorded, and the growth intercept; A1–A9 denote the regression coefficients; and X1, X2, and X3
of Trichoderma harzianum TI21 on the media's inner and outer surface denote the independent variables studied, as listed in Table 2. The
was examined. After 5 days of incubation, the spores were harvested fitness of the polynomial model equations was expressed by the coeffi­
from the media surface by scraping without getting any media residue. cient of determination R2 according to the analysis of variances. An F-
test was used to determine their statistical significance and the signifi­
2.9.1. Effects of intrinsic and extrinsic factors on sporulation of TI21 cance of the regression coefficients at a probability (P) of 0.001, 0.01, or
The fungus' sporulation response to intrinsic (pH and nutrients) and 0.05. Regression analysis and 3-D response surface plots were used to
extrinsic (temperature and humidity) factors was investigated using a determine the optimal spore production conditions (Iqbal et al., 2020).
single factorial test (Vrabl et al., 2019; Zhang and Yang, 2015). The
experiment was carried out in the dark for five days, with the evaluated 2.9.3. Verification
parameters having ranges of values and a constant level of control The validity of the statistical experimental procedures was then
variables (Table 1). confirmed in five additional verification trials at the determined levels,
utilizing T. harzianum TI21.
2.9.1.1. Effects of pH. In pH ranges of 3 to 6, the influence of pH on
sporulation was explored. The experiment was carried out at pH 3, 4, 5,
and 6, with other factors held constant (Table 1). The pH of the media
was adjusted with food-grade phosphoric acid. Table 2
BBD array and the response values.
2.9.1.2. Effects of nutrients. PDA and RA media were used to test the Run Factor 1 Factor 2 Factor 3 Response Y1: spore (g/
effect of nutrients on sporulation while other factors remained constant order X1: X2: pH X3: L)
(Table 1). In this experiment, rice flour in RA media contained 81.5 % temperature humidity

starch as measured by Omar et al. (2016). 1 25 3 75 1.8 ± 0.03

2 30 3 90 3.79 ± 0.07
3 30 4 75 3.99 ± 0.12
2.9.1.3. Impacts of temperature. Temperature impacts on sporulation 4 30 3 60 4.27 ± 0.04
were assessed by incubating the inoculated media at temperatures of 20 5 30 4 75 5.39 ± 0.17
± 1 ◦ C, 25 ± 1 ◦ C, 30 ± 1 ◦ C, 35 ± 1 ◦ C, and 40 ± 1 ◦ C while all other 6 30 4 75 4.98 ± 0.09
7 35 5 75 1.63 ± 0.11
8 25 5 75 2.19 ± 0.05
Table 1 9 35 4 60 3.89 ± 0.03
Ranges of Trichoderma harzianum TI21 sporulation's intrinsic and extrinsic 10 35 3 75 4.78 ± 0.06
factors. 11 30 5 90 1.58 ± 0.04
12 25 4 90 2.43 ± 0.07
Factors Ranges value Constant value
13 30 4 75 4.9 ± 0.02
Intrinsic pH 3, 4, 5, 6 6 14 25 4 60 4.3 ± 0.04
Nutrients PDA, RA media RA 15 30 4 75 5.01 ± 0.10
Extrinsic Temperature (◦ C) 20, 25, 30, 35, 40 30 16 30 5 60 3.03 ± 0.07
Humidity (%) 60, 70, 80, 90 70 17 35 4 90 4.02 ± 0.04

4
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

2.9.4. Reconditioning approach gypsum, zinc sulfate, and boron were applied as a basal application at
The reconditioning approach was applied to the validated method to rates of 300, 250, 350, 100, 10, and 8 kg/ha, respectively. In mid-
recondition the media for the second and third harvests of spores from November 2020, tubers were sown (Abuhena et al., 2022). Each plot
the semi-solid media's outer layer. At the time of the first spore collec­ had a tuber-showing line-to-line distance of 60 cm and a plant-to-plant
tion, the pH of T. harzianum on starch (RA) media was 7.9 ± 0.3, up from distance of 25 cm. At harvest, yield and yield-contributing parameters
pH 4 at the start. Under certain conditions, extracellular alkalinisation were recorded to determine the field performance of media-free
has been reported in various fungal species. Carbon deficiency, which formulated spores.
happens in low-glucose conditions, is one of them (Vylkova et al., 2011).
According to Moreno-Mateos et al. (2007), pH affects a number of Tri­ 2.13. Analytical technique
choderma harzianum genes (chitinase, protease, glucose permease, and
the cell wall protein qid74) in a variety of ways. In T. harzianum, PacC The spore density was determined using the spread plate technique.
promotes chitinase and qid74 expression while lowering protease and Dilution factors from 10− 1 to 10− 7 were used to dilute cell suspensions.
glucose permease expression. PacC also prevents inhibitory metabolites CFU were counted after 48–72 h of incubation at 28 ± 2 ◦ C on PDA
from accumulating. So it was necessary to employ the reconditioning plates.
approach to adjust pH to the initial stage to start sporulation again. In
the reconditioning approach, the pH was adjusted to around 4 by 2.14. Statistical analysis
spraying diluted 3 M phosphoric acid on the upper side of the media
after the first harvest of spores from the outer layer. After the second For independent variable optimization, Design Expert software 13
harvest, the pH was adjusted using the same procedure, and the outer was utilized to create a BBD. The data from the SFT was analysed using
layer spores were collected. The exhausted medium was then taken and XLSTAT 2021. Using ANOVA in R 2021, a Tukey's HSD test was used to
blended uniformly. The density and quantity of spores collected were investigate significance variance in field data at the p < 0.05 level.
analysed.
3. Results
2.10. Viability evaluation of spores
3.1. Identification of Trichoderma harzianum TI21
Following harvesting, the media-free spore (1 %) and media-mixed
spore (1 %) were formulated separately, utilizing 57.5 ± 1 % talc This strain has deep green colonies (Supplementary Fig. F4) with a
powder, 40 ± 0.5 % dextrose, and 1.5 ± 0.1 % carboxymethylcellulose smooth edge and watery white floccose mycelial growth. Conidia were
(CMC), respectively. Throughout the formulation process, the moisture ellipsoidal and subglobose in shape and densely branched. Phialides that
content was maintained at 8 ± 2 % by drying in a vacuum dryer were flask-shaped and diverge into 2–4 whorls. The strain was identified
(Dongguan; YPO-027) at 30 ± 2 ◦ C. The formulation was then placed in as Trichoderma harzianum TI21 using D2 LSU rDNA sequences and given
the aluminium pack after proper mixing and placed under quality con­ the gene bank accession number MH084949.
trol to check the stability of the formulation. The viability of spores in a
formulation stored at 28 ± 2 ◦ C in dark conditions was studied using 3.2. Bioactivity of Trichoderma harzianum TI21
plate-count procedures at monthly intervals up to 18 months.
The quantitative analysis revealed that Trichoderma harzianum TI21
2.11. Clogging test of spores during foliar application exhibited chitinase and β-1,3-glucanase activities of 3.36 ± 0.14 (Units/
mL) and 2.34 ± 0.08 (Units/mL), respectively. TI21 showed P solubili­
Clogging of the spraying nozzle during foliar application is a com­ zation activity at a concentration of 321.36 ± 16.46 g/mL and generated
mon problem with fungal product application. Mycelia and media res­ IAA at a level of 44.17 ± 4.37 g/mL. On PDA media, Trichoderma har­
idue or substrate present in the formulation that appears to be zianum TI21 effectively inhibited the growth of phytopathogens. TI21
aggregated in the orifice will block the orifice in the spray nozzle. Spray had the most effective antagonistic activity against Sclerotium delphinii
patterns are interrupted, pressure rises, and flow is reduced as a result. (75.97 ± 8.06 %) and Fusarium equiseti (67.99 ± 4.80 %). The lowest
To clog tests on media-free and media-mixed spores, a hollow cone inhibition levels were found in Alternaria alternata (61.90 ± 4.58 %) and
nozzle with a 2 mm orifice size (Brand: MHM, Model: 3wf-3) VMD (200 Curvularia spicifera (61.66 ± 2.98 %).
μm) was used.
3.3. Stressing method for high-density media-free spore production
2.12. Field evaluation of media-free spores
In this method, the spores germinated after spraying the spore sus­
The experiment was carried out on potato (Solanum tuberosum) to pension on the media surface, and the hypha that emerged from the
demonstrate the performance of the formulated high-density spore of spore lengthened at the terminal. More branches sprouted along the
Trichoderma harzianum TI21 in field conditions. The experiment of po­ hypha. Being filamentous fungi, they proliferated in a branching way as
tato (SANTANA cultivar, Nederlands Potato Consultative Foundation: a result of continual iteration. The branches continued to branch,
GBR165) cultivation was carried out in the research field of ABBL (Apex forming mycelium, and a porous three-dimensional network of hyphae.
Bio-fertilizer & Bio-pesticides Ltd., Gobindaganj, Gaibandha, Fig. 1 shows a depiction of Trichoderma harzianum TI21 growth on the
Bangladesh: 25◦ 148′ N, 89◦ 89′ ). The experiment was set up in a Ran­ semi-solid media in a dual-layer. Initially, sparse mycelia were grown on
domized Complete Block Design (RCBD) with five replications. The the surface (layer 1), then randomly penetrated the media (layer 2)
following treatments were used: T1 (0% chemical fertilizers), T2 (0% because of its low water activity tolerance and ability to excrete bioac­
TSP + 100 % other chemical fertilizers), T3 (50% TSP + 100 % other tive compounds (Carlile, 1995). During semi-solid fermentation, Tri­
chemical fertilizers), T4 (100% all chemical fertilizers), T5 (T1 + TI21), choderma harzianum has a unique morphology that allows it to colonize
T6 (T2 + TI21), T7 (T3 + TI21), and T8 (T4 + TI21). The formulated and penetrate the semi-solid media surface in quest of nutrients. Mycelia
spore was applied in two ways; one with a compost (dose: 4 kg/ha) continued to grow, and as a result, the media surface was completely
applied in the soil before planting, and the other as a foliar spray (dose: covered by mycelia by day 2. On the third day, sporulation began at
5 g/L) at 15-day intervals throughout the crop's life cycle. Exception of layer 1, which quickly became dense after day 5. Because oxygen was
urea, all fertilizers were applied as a base dose during final land prep­ scarce in the semi-solid media, only thin mycelial and spore growth took
aration. Urea, Triple Super Phosphate (TSP), Muriate of Potash (MoP), place in layer 2. The growth pattern of Trichoderma harzianum TI21

5
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

Fig. 1. Day-wise growth progress of Trichoderma harzianum TI21 on starch-based media in the stressing method. A) Day 1; Media pouring, solidification inoculation.
B) Day 2; visual mycelial growth. C) Day 3 & 4; spore growth appears as a green layer. D) Day 5; a dense and thick spore mat formed. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

varied from day to day (Fig. 1). harzianum has the ability to produce a starch degrading enzyme
(α-amylase) that allows it to use starch (RA) (Mohamed et al., 2011). No
3.3.1. Effects of intrinsic and extrinsic factors on sporulation of TI21 other nutrition source that could encourage contaminant growth was
added to the RA media. Trichoderma can also obtain ATP by the meta­
3.3.1.1. Effects of pH. The spore yields at pH 3 and pH 4 were 4.476 g/L bolism of fibre material present in rice flour. Some Trichoderma sp.
and 4.622 g/L, respectively, with densities of 1.45 × 1011 CFU/g and produce highly efficient siderophores that chelate iron and stop the
1.95 × 1011 CFU/g, showing that the lower pH stress favoured sporu­ growth of other fungi (Benítez et al., 2004).
lation, as shown in Fig. 2 (A & E). During the cultivation period, the
sporulation process began on day 3, and by day 4, the entire surface was 3.3.1.3. Impacts of temperature. Temperatures of 30 ◦ C and 35 ◦ C pro­
covered in spore mat, with a thick spore mat by day 5. During the duced higher spore yields of 1.812 g/L and 1.626 g/L, with densities of
cultivation, no contaminants were found on the surface of the media, 2.24 × 1010 CFU/g and 1.81 × 1010 CFU/g, respectively, whereas
indicating that the lower pH had suppressed contaminant bacterial and temperatures of 20 ◦ C and 25 ◦ C produced relatively low spore yields of
fungal growth on the medium. At pH 5 and 6, spore yield and density 1.284 g/L and 1.394 g/L, with densities of 7.46 × 109 CFU/g and 8.59 ×
dropped to 2.082 g/L and 1.764 g/L, respectively, with densities of 7.75 109 CFU/g (Fig. 2C & G). The higher temperature of 40 ◦ C caused the
× 1010 CFU/g and 2.67 × 1010 CFU/g, respectively. At pH 5, there was media to dry out, resulting in no harvestable spore formation. Temper­
little contamination on the media surface, but at pH 6, there was a huge atures of 30 ◦ C resulted in increased spore production, but when the
amount, indicating that a higher pH encouraged contaminant growth temperature was elevated to 35 ◦ C, the spore yield decelerated
while limiting sporulation. The sporulation process began on day 3, and marginally, and temperature drops to 20 ◦ C and 25 ◦ C resulted in sig­
by day 4, the entire surface was covered in spore mat, with a thin spore nificant spore production declines. The stress of lower temperatures was
mat by day 5. not conducive to sporulation, whereas higher temperatures inhibited the
germination of inoculated spores on the media surface. Contaminant
3.3.1.2. Effects of nutrients. In the nutrients experiment, RA media had colonies were present on the surface of the inoculated media at all
a significantly greater spore yield of 1.854 g/L with a higher density of temperatures.
2.3 × 1010 CFU/g, whereas PDA had a significantly lower spore yield of
1.444 g/L with a density of 5.6 × 109 CFU/g (Fig. 2B & F). Both me­ 3.3.1.4. Impacts of humidity. Spore production was significantly higher
diums sporulated on day 3, and by day 4, the entire surface was covered at 70 % and 80 % humidity, with yields of 1.882 g/L and 2.01 g/L, and
in spore mat, but by day 5, it was only a thin spore mat. Because the PDA densities of 2.17 × 1010 CFU/g and 1.76 × 1010 CFU/g, respectively. At
medium included the readily available nutrient dextrose, it had a huge lower humidity levels of 60 % and higher humidity levels of 90 %, spore
amount of contaminants on the surface (Supplementary Fig. F5), production was significantly reduced, with yields of 1.338 g/L and
whereas the RA medium had a few contaminants. Trichoderma 1.558 g/L, and densities of 2.57 × 1010 CFU/g and 9.86 × 109 CFU/g,

6
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

Fig. 2. The influence of intrinsic and extrinsic factors on spore yield and spore density in SFT. Effects on spore yield (A. pH; B. Nutrients; C. Temperature; D.
Humidity) and spore density (E. pH; F. Nutrients; G. Temperature; H. Humidity).

7
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

respectively. Fig. 2D shows that spore yield increased as humidity derived from the regression equation.
climbed from 60 % to 80 %, but dropped as humidity reached 90 %. At
lower humidity levels, the spore mat was thin, but the spores were dry, 3.5. Verification of optimized method
resulting in a high density (Fig. 2H). The spore became wetter as the
humidity increased, the density decreased. At all humidity levels, The optimum values of the independent variables were predicted
contaminant colonies were detected on the surface of the inoculated using the design-expert software's point prediction function. Maximum
media. spore yield of 4.85 (g/L) predicted was at optimal values of temperature
30 ◦ C, pH 4 and humidity 75 %. Under the predicted optimal parameter
3.4. Method optimization using BBD values, the maximum experimental spore yield and density was 4.91 ±
0.12 (g/L) and 3.64 ± 0.08 × 1011(CFU/g).
The response factor was spore yield, and a multi-factors regression
analysis was performed. Table 3 displays the findings of the analysis. A 3.6. Reconditioning approach
quadratic regression polynomial was derived upon regression and
fitting. In contexts of code, the following is the response value to factors After the first harvest and pH reconditioning, hypha extension from
X1, X2, and X3 (Eq. (3)): the inner to outer surface occurred. The remaining spores germinated as
well, creating mycelia that spread across the surface of the medium. The
Y =4.85 + 0.4500X1 − 0.7762X2 − 0.4588X3 − 0.8850X1 X2 + 0.5000X1 X3
white mycelia covered the entire surface by the second day. On day
− 0.2425X2 X3 − 0.8808X1 2 − 1.37X2 2 − 0.3133X3 2
three, sporulation began, and on day four, a spore mat appeared. Fig. 4
(3) shows the sporulation of Trichoderma harzianum TI21 on the recon­
The computed mean response of the 17 runs, where Y was the spore ditioning media surface for the first, second, and third harvest. The
yield (dependent factor), was 4.85, and the assessed coefficients for second and third harvests yielded less spore than the first harvest, with
factors X1, X2, and X3 were 0.4500, − 0.7762, and − 0.4588, respectively. 4.19 ± 0.15 (g/L) and 3.48 ± 0.13 (g/L), respectively. However, there
The overall effects (X1 and X2) denoted the average of changing one was no significant reduction in spore density in the second and third
factor from a higher to a lower value at a time. The collaborative terms harvests when compared to the first harvest, with densities of 3.12 ±
(X1X2, X1X3, and X2X3) demonstrated how the response changes when 0.11 × 1011 CFU/g and 2.35 ± 0.16 × 1011 CFU/g, respectively. The
three factors are shifted at the same time. Nonlinearity was investigated exhausted media was reduced to 755 ± 21 g/L with a density of 2.25 ±
using polynomial terms (X21, X22, and X23). The quadratic regression 0.09 × 1010 CFU/g after the third harvest, containing spore and mycelia.
model's analysis of variance (Table 3) indicated that the model was
highly significant, as evidenced by the Fisher's F-test (Fmodel = 14.64) 3.7. Formulation evaluation
and a low probability value (Pmodel < 0.0001). The quadratic model also
suited the data well, as evidenced by the p value for “lack of fit” The variation in viable spore density of concentrated spores (media-
(0.8135). The model's R2 was found to be 0.9496, indicating that it was free) and media-mixed spores is shown in Fig. 5A. The concentrated
reliable. According to the modified R2, the three influence factors spore demonstrated extended viability with a density reduction of less
explained 88.47 % of the variance in the response factors. To better than one log from 4.4 × 109 to 9.3 × 108 (CFU/g) after 18 months, but
comprehend the interplay between independent factors, 3D response the media-mixed spore decreased two logs from 5.3 × 108 to 1.8 × 106
surfaces were created. Fig. 3 (A–C) depicts the response surfaces and (CFU/g) within six months. pH in concentrated media-free spores took
contour plots generated for the variation of spore yield as a function of 11 months to drop below 7.5, but pH in media-mixed spores took only 6
values of two factors, with the other factor fixed to their center values. months to drop below 7 (Fig. 5B).
The optimum values of the respective components are represented by
the coordinates of the center point within the highest contour levels in 3.8. Clogging test
each of the figures. Response surface curves and contour plots can be
used to determine the range of optimum conditions within the experi­ After 2–5 min of spraying with media-mixed spores, the inside of the
mental domain or to direct future experiments for better results. Fig. 3A nozzle had accumulated enough mycelia to restrict the orifice and
demonstrated that pH has a greater impact on spore yield than tem­ obstruct the flow. As a result, the nozzle developed a clog, which was
perature, with the optimal pH falling between 4 and 3. As shown in then pushed as aggregate. The nozzle clogged frequently during the
Fig. 3B and C, pH and humidity had a greater impact on spore yield than application of media-mixed spores. Since the medium mixed spores
temperature. These three figures have a maximum point. Temperature included mycelia, spores, and media substrate, the clogging formation
30 ◦ C, pH 4, and humidity 75 % are the optimization results of BBD was obvious. There was no clogging during the foliar application of the

Table 3
ANOVA of the spore quantity (g/L) in a quadratic model.
Source Sum of squares Df Mean square F-value p-value Significance

Model 25.08 9 2.79 14.64 0.0009 Significant

A-temperature 1.62 1 1.62 8.51 0.0224
B-pH 4.82 1 4.82 25.33 0.0015
C-humidity 1.68 1 1.68 8.85 0.0207
AB 3.13 1 3.13 16.46 0.0048
AC 1 1 1 5.25 0.0556
BC 0.2352 1 0.2352 1.24 0.303
A2 3.27 1 3.27 17.16 0.0043
B2 7.94 1 7.94 41.72 0.0003
C2 0.4132 1 0.4132 2.17 0.1842
Residual 1.33 7 0.1903
Lack of fit 0.2562 3 0.0854 0.3175 0.8135 Not significant
Pure error 1.08 4 0.269
Cor total 26.41 16

Std. Dev. = 0.4363, Mean = 3.65, C.V. % = 11.97, R2 = 0.9496, Adjusted R2 = 0.8847, Predicted R2 = 0.7811, Adeq Precision = 10.3562.

8
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

Fig. 3. The 3D response surface and 2D contour plot display the interaction of all those components considered in the optimization. A) Interaction of temperature
and pH. B) Interaction of temperature and humidity. C) Interaction of humidity and pH.

media-free concentrated spore. Because the concentrated spores were 3.9. Field evaluation
free of mycelia and media substrate, no clog formed in the nozzle, which
then aggregated to hinder the flow. The media-free spores produced by the stressing starch media-based
method increased potato yield and yield-related measures (Fig. 6A–D),

9
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

Fig. 4. Sporulation of Trichoderma harzianum TI21 in a reconditioning approach on starch-based media in the stressing method.

Fig. 5. Changes in density and pH of media-mixed and media-free spores with time intervals. A) Variability in pH. B) The viability of spore density.

indicating that the spores' agronomic character remained unchanged. product on the market (Abuhena et al., 2022). The viability of an
Plant height was significantly improved in Dose0, Dose1, and Dose2, but active agent is also influenced by the active agent type, such as hyphae,
not in Dose3. Only Dose0, Dose1, and Dose3 showed a significant in­ mycelia, chlamydospore, and spore, as well as production methods,
crease in tuber numbers, but not Dose2. All doses resulted in significant formulation type, and so on (Hewavitharana et al., 2018). The method
increases in yield per plant and yield per meter square. As a result, we described here was designed to be applicable for the production of high-
can mix media-free spores with dose1, dose2, or dose3 to boost potato quality microbial products with diverse applications.
yields at the farmer level. The results of this method adoption revealed that pH and nutrients
appear to be the most important factors under a single factorial test.
4. Discussion Lower pH and nutritional stress induced sporulation, whereas sporula­
tion happened under normal growth conditions in terms of temperature
A series of challenges must be overcome in order to develop an and humidity. In terms of contamination-free spore production, lower
effective fungal product, including isolation steps, production method, pH and nutrient-deficient media (RA) were the principal factors rather
formulation type, and mode of application of the formulated product. To than temperature and humidity. The amount, density, purity, and
be a successful product, the active agent must be viable in the formu­ quality of spores are important considerations when choosing a medium
lation for a long period of time to be a cost-effective and high-quality for SSF. Simultaneously, RA was found to be a suitable medium for

10
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

Fig. 6. Two-way ANOVA illustrates the effect of media-free spores with different fertilizer doses. A) Plant height. B) Tubers per plant. C) Yields per plant. D) Yield
per m2. (ns p ≥ 0.05, * p < 0.05), data are means ± SD (n = 5). Bars without shared letters indicate significant differences.

maximum spore production under SSF, most possibly owing to the harvest and 4 days for subsequent harvests, compared to the other
presence of soluble sugar, nitrogen content, fibre, and starches that substrate-based SSF, which required 7–31 days (Mulatu et al., 2021;
Trichoderma utilizes (Mohamed et al., 2011). In the current study, the Naeimi et al., 2020; Sachdev et al., 2018; Zhang and Yang, 2015).
highest spore production was achieved on a single substrate of RA me­ By reconditioning and reusing the medium, the approach produced
dium, indicating a potential method for reusing the media to produce a two types of products: one of concentrated spore (3.64 ± 0.08 × 1011
greater quantity of high-density spore at a lower cost by using various CFU/g) with no media residual, and the other of exhausted media that
starch-based raw materials. contained the existing inner layer spores, hyphae, or mycelia. The spores
This method produced spores in two layers, with the inner layer produced on the outer layer were markedly better in terms of quantity,
containing less dense spores with hypha, mycelia, and exhausted media, density, and purity than those produced by traditional substrate-based
and the outer layer containing more dense pure spores with no media SSF (Mulatu et al., 2021; Naeimi et al., 2020; Sachdev et al., 2018;
residue. By lowering pH and nutrients, this method also handled the Zhang and Yang, 2015; Singh and Nautiyal, 2012).
problem of contamination in SSF that occurred with open-rack spore The quantity and density of the produced product are not the only
production. factors considered when determining whether a method is unique; the
According to the results of this method's statistical (BBD) optimiza­ shelf life of the product is also considered. Moisture is an important
tion, the effects of temperature, pH, and humidity on production were consideration in product formulation since it influences the product's
highly significant (p = 0.009). The optimal temperature and humidity shelf life. Moisture can increase the susceptibility of spores to stress by
were 30 ◦ C and 75 %, which were the same as those reported by Sachdev furthering unnecessary growth during storage. Maintaining a low
et al. (2018) and Zhang and Yang (2015) for substrate-based SSF. The moisture level can extend the life of a product. It is necessary to dry
majority of the substrate-based SSF for Trichoderma production did not products with a high initial moisture content to a low moisture level.
investigate the impact of pH on sporulation and contamination. How­ Rapid drying, on the other hand, will cause a certain percentage of cells
ever, Zhang and Yang (2015) observed the highest sporulation (8.64 × to die (Jin and Custis, 2011). The method used in this study produced a
109 CFU/g) at pH 6, but did not specify the contaminants' growth. In this unique product that did not involve drying to adjust the moisture con­
experiment, though, pH 4 demonstrated excellent sporulation (3.64 ± tent. By preventing contamination, this technique also reduced down­
0.08 × 1011 CFU/g), with no contaminants detected on the media sur­ stream spore viability decline and preserved the product's better quality.
face or in the harvested spores. In terms of incubation time, this method Past research has shown that aerial and dry spores are much more du­
demonstrated a significant reduction, requiring just 5 days for the first rable and have a longer shelf life than submerged cultures, which

11
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

validates and upholds the use of spores formed on the tray culture on RA the work reported in this paper.
by this method (Mulatu et al., 2021; Naeimi et al., 2020). The talc-based
formulation was chosen for this study because it is less susceptible to Acknowledgments
contamination and is widely recognized (Singh and Nautiyal, 2012). The
formulations incorporated carboxymethylcellulose (CMC) and dextrose. We appreciate the efforts of the entire team of Apex Biofertilizers &
CMC's anionic and amphiphilic backbone certainly aids in its stability Biopesticides Limited, Gobindaganj-5740, Gaibandha, Bangladesh and
(Krempel et al., 2019). Dextrose was employed to aid spore germination Apex Biotechnology laboratory, Apex Holdings Ltd., East Chandora,
after application and to protect the spores from desiccation during Shafipur, Kaliakoir, Gazipur 1751, Bangladesh for conducting labora­
storage. According to Morgan et al. (2006), sugar is used to protect tory experiments and implementing the field experiments.
microorganisms from changes in osmotic pressure and can aid with
protection and maintenance, especially after drying. Mishra and Sundari Appendix A. Supplementary data
(2016) also found that after 12 months of storage at 30 ◦ C, an intricate
formulation of B. bassiana based on skimmed milk powder, poly­ Supplementary data to this article can be found online at https://doi.
vinylpyrrolidone K-90, and glucose attained 100 % conidial germination org/10.1016/j.biteb.2022.101165.
and 78 % conidial viability. This method yielded longer viable spores
than the current substrate-based SSF method, reaching about 9.3 × 108 References
CFU/g after 18 months, up from 4.4 × 109 CFU/g at the start (Mulatu
et al., 2021; Hewavitharana et al., 2018; Kumar et al., 2014). Abuhena, M., Al-Rashid, J., Azim, M.F., Khan, M.N.M., Kabir, M.G., Barman, N.C.,
The clogging test revealed that the media residue-free spore did not Rasul, N.M., Akter, S., Huq, M.A., 2022. Optimization of industrial (3000 L)
production of Bacillus subtilis CW-S and its novel application for minituber and
cause blockage during foliar application, whereas the media-mixed industrial-grade potato cultivation. Sci. Rep. 12, 11153. https://doi.org/10.1038/
spore did. This demonstrated that the application restriction of s41598-022-15366-5.
substrate-based spores was overcome by this method. The field test re­ Awad, N.E., Kassem, H.A., Hamed, M.A., El-Feki, M.A., Elnaggar, M.A.A., Mahmoud, K.,
Ali, M.A., 2018. Isolation and characterization of the bioactive metabolites from the
sults confirmed the spore efficacy produced by this stressing method by
soil derived fungus Trichoderma viride. Mycolog. 9, 70–80. https://doi.org/
showing a significant yield increase in potato. 10.1080/21501203.2017.1423126.
Bader, A.N., Salerno, G.L., Covacevich, F., Consolo, V.F., 2020. Native Trichoderma
harzianum strains from Argentina produce indole-3 acetic acid and phosphorus
5. Conclusion
solubilization, promote growth and control wilt disease on tomato (Solanum
lycopersicum L.). J. K. S. Uni-Sci. 32 (1), 867–873. https://doi.org/10.1016/j.
Overall, the findings revealed that T. harzianum TI21 could be grown jksus.2019.04.002.
utilizing the nutrient deprivation stressing method at low pH with Benítez, T., Rincón, A.M., Limón, M.C., Codón, A.C., 2004. Biocontrol mechanism of
Trichoderma strains. Int. Microbiol. 7, 249–260.
considerable increases in spore yield and decreases in time. The recon­ Bononi, L., Chiaramonte, J.B., Pansa, C.C., Moitinho, M.A., Melo, I.S., 2020. Phosphorus-
ditioning approach was found to be a surprisingly good fit for maxi­ solubilizing Trichoderma spp. From Amazon soils improve soybean plant growth.
mizing the production of high-density spores through media reuse. The Sci. Rep. 10, 2858. https://doi.org/10.1038/s41598-020-59793-8.
Cai, F., Gao, R., Zhao, Z., Ding, M., Jiang, S., Yagtu, C., Zhu, H., Zhang, J., Ebner, T.,
RA media-based reconditioning approach could be the most effective Mayrhofer-Reinhartshuber, M., Kainz, P., Chenthamara, K., Akcapinar, G.B.,
method for the cost-effective mass production of pure Trichoderma spore Shen, Q., Druzhinina, I.S., 2020. Evolutionary compromises in fungal fitness:
for horticultural and agricultural uses. hydrophobins can hinder the adverse dispersal of conidiospores and challenge their
survival. ISME J. 14, 2610–2624. https://doi.org/10.1038/s41396-020-07090.
Carlile, M.J., 1995. The success of the Hypha and Mycelium. In: Gow, N.A.R., Gadd, G.M.
Funding (Eds.), The Growing Fungus. Springer, Dordrecht. https://doi.org/10.1007/978-0-
585-27576-5_1.
Chaverri, P., Branco, R., Fabiano, Aklitsch, W., Gazis, R., Degenkolb, T., Samuels, G.,
This study was supported by Apex Biofertilizers & Biopesticides 2015. Systematics of the Trichoderma harzianum species complex and the re-
Limited, Gobindaganj-5740, Gaibandha, Bangladesh and Apex identification of commercial biocontrol strains. Mycologia 107 (3), 558–590.
Biotechnology laboratory, Apex Holdings Ltd., East Chandora, Shafipur, https://doi.org/10.3852/14-147.
Gezgin, Y., Maral Gül, D., Sözer Şenşatar, S., Kara, C.U., Sargın, S., Sukan, F.V., Eltem, R.,
Kaliakoir, Gazipur 1751, Bangladesh.
2020. Evaluation of Trichoderma atroviride and Trichoderma citrinoviride growth
profiles and their potentials as biocontrol agent and biofertilizer. Turkish J.
CRediT authorship contribution statement Biochem. 45 (2), 163–175. https://doi.org/10.1515/tjb-2018-0378.
Hewavitharana, N., Kannangara, S., Senanayake, S.P., 2018. Isolation, identification and
mass production of five Trichoderma spp. on solid and liquid carrier media for
M.A.: Conceptualization, Fermentation plant operation, the experi­ commercialization. Int. J. Appl. Sci. Biotechnol. 6, 285–293. https://doi.org/
mental research work, Statistical analysis, Manuscript writing, revision, 10.3126/ijasbt.v6i4.22128.
and editing. Hoyos-Carvajal, L., Bissett, J., 2011. Biodiversity of Trichoderma in Neotropics. https://
doi.org/10.5772/23378.
M.G.K.: Conceptualization, Fermentation plant operation, the Iqbal, M.M.A., Abu Bakar, W.A.W., Toemen, S., Abdul Razak, F.I., Wan Azelee, N.I.,
experimental research work, Statistical analysis, Manuscript writing, 2020. Optimization study by Box-Behnken design (BBD) and mechanistic insight of
revision, and editing. CO2 methanation over Ru-Fe-Ce/γ-Al2O3 catalyst by in-situ FTIR technique.
Arabian J. of Chem. 13 (2), 4170–4179. https://doi.org/10.1016/j.
M.F.A: Conceptualization, Fermentation plant operation, the arabjc.2019.06.010.
experimental research work, Statistical analysis, Manuscript writing, Jahan, M., Abuhena, M., Azad, A.K., Karim, M.M., 2018. In vitro antibacterial and
revision, and editing. antibiofilm activity of selected medicinal plants and spices extracts against
multidrug resistant Pseudomonas aeruginosa. J. Pharmacogn. Phytochem. 7 (3),
J.A.-R.: Fermentation plant operation, the experimental research 2114–2121.
work, Manuscript editing. Jin, X., Custis, D., 2011. Microencapsulating aerial conidia of Trichoderma harzianum
N.M.R.: Manuscript reviewing, editing and Approval of the protocol. through spray drying at elevated temperatures. Biol. Cont. 56, 202–208.
Keswani, C., Bisen, K., Singh, V., Sarma, B.K., Singh, H.B., 2016. Formulation technology
M.A.H.: Manuscript reviewing, editing and submission of the
of biocontrol agents: present status and future prospects. In: Bioformul. Sustain.
manuscript. Agric. Springer, New Delhi, India, pp. 35–52. https://doi.org/10.1007/978-81-322-
All authors have read and agreed to the published version of the 2779-3_2.
manuscript. Krempel, M., Griffin, K., Khouryieh, H., 2019. 13 - Hydrocolloids as emulsifiers and
stabilizers in beverage preservation. In: Grumezescu, A.M., Holban, A.M. (Eds.),
Preservatives and Preservation Approaches in Beverages. Academic Press,
Declaration of competing interest pp. 427–465. https://doi.org/10.1016/B978-0-12-816685-7.00013-6.
Kumar, K., Amaresan, N., Bhagat, Madhuri, S.K., Srivastava, R.C., 2012. Isolation and
characterization of Trichoderma spp. for antagonistic activity against root rot and
The authors declare that they have no known competing financial foliar pathogens. Indian J. Microbiol. 52 (2), 137–144. https://doi.org/10.1007/
interests or personal relationships that could have appeared to influence s12088-011-0205-3.

12
Md. Abuhena et al. Bioresource Technology Reports 19 (2022) 101165

Kumar, S., Thakur, M., Rani, A., 2014. Trichoderma: Mass production, formulation, Omar, K., Salih, B., Abdulla, N., Hussin, B., Rassul, S., 2016. Evaluation of starch and
quality control, delivery and its scope in commercialization in India for the sugar content of different rice samples and study their physical properties. Indian J.
management of plant diseases. Afr. J. Agril. Res. 9 (53), 3838–3852. https://doi.org/ Nat. Sci. 6, 11084–11093.
10.5897/AJAR2014. 9061. Sachdev, S., Singh, A., Singh, R.P., 2018. Optimization of culture conditions for mass
Li, Y., Sun, R., Yu, J., Saravanakumar, K., Chen, J., 2016. Antagonistic and Biocontrol production and bio-formulation of Trichoderma using response surface
potential of Trichoderma asperellum ZJSX5003 against the Maize Stalk Rot methodology. Biotech 8, 360. https://doi.org/10.1007/s13205-018-1360-6.
Pathogen Fusarium graminearum. Indian J. Microbiol. 56 (3), 318–327. https://doi. Sala, A., Barrena, R., Sánchez, A., Artola, A., 2021. Fungal biopesticide production:
org/10.1007/s12088-016-0581-9. process scale-up and sequential batch mode operation with Trichoderma harzianum
Lu, Z.-x., Tu, G.-p., Zhang, T., Li, Y.-q., Wang, X.-h., Zhang, Q.-g., Song, W., Chen, J., using agro-industrial solid wastes of different biodegradability. Chem. Eng. J. 425,
2020. Screening of antagonistic Trichoderma strains and their application for 131620 https://doi.org/10.1016/j.cej.2021.131620.
controlling stalk rot in maize. J. Integr. Agric. 19 (1), 145–152. https://doi.org/ Saravanakumar, K., Li, Y., Yu, C., Wang, Q.-q., Wang, M., Sun, J., Gao, J.-x., Chen, J.,
10.1016/S2095-3119(19)62734-6. 2017. Effect of Trichoderma harzianum on maize rhizosphere microbiome and
Maitra, P., Al-Rashid, J., Barman, N.C., Khan, M.N.M., Mandal, D., Rasul, N.M., biocontrol of Fusarium stalk rot. Sci. Rep. 7, 1771.
Chowdhury, A., El-Sappah, A.H., Jia, L., 2021. Sand particle size and phosphorus Singh, P.C., Nautiyal, C.S., 2012. A novel method to prepare concentrated conidial
amount affect Rhizophagus irregularis spore production using in vitro propagated biomass formulation of Trichoderma harzianum for seed application. J. Appl.
spore as a starter inoculum in rhizosphere of maize (Zea mays) plantlets. J. Fungi 7 Microbiol. 113 (6), 1442–1450. https://doi.org/10.1111/j.1365-2672.2012.05426.
(10), 846. https://doi.org/10.3390/jof7100846. x.
Mishra, N., Sundari, S.K., 2016. Designing low cost SSF strategy for mass production of Sood, M., Kapoor, D., Kumar, V., Sheteiwy, M.S., Ramakrishnan, M., Landi, M.,
bioinoculant Trichoderma harzianum KSNM with longer shelf life. Asian J. Araniti, F., Sharma, A., 2020. Trichoderma: the "Secrets" of a Multitalented
Microbiol. Biotechnol. Environ. Sci. 18, 447–458. Biocontrol Agent. Plants. 9 (6), 762. https://doi.org/10.3390/plants9060762.
Mohamed, S., Azhar, E., Ba-Akdah, M., Tashkandy, N., Kumosani, T., 2011. Production, Vrabl, P., Schinagl, C., Artmann, D., Heiss, B., Burgstaller, W., 2019. Fungal growth in
purification and characterization of α-amylase from Trichoderma harzianum grown batch culture – what we could benefit if we start looking closer. Front. Microbiol. 10,
on mandarin peel. Afr. J. Microbiol. Res. 5, 1018–1028. https://doi.org/10.3390/ 2391. https://doi.org/10.3389/fmicb.2019.02391.
2Fmolecules19068027. Vylkova, S., Carman, A.J., Danhof, H.A., Collette, J.R., Zhou, H., Lorenz, M.C., 2011. The
Moreno-Mateos, M.A., Delgado-Jarana, J., Codón, A.C., Benitez, T., 2007. pH and Pac1 fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising
control development and antifungal activity in Trichoderma harzianum. Fungal extracellular pH. MBio 2 (3), e00055-11. https://doi.org/10.1128/mBio.00055-11.
Genet. Biol. 44 (12), 1355–1367. https://doi.org/10.1016/j.fgb.2007.07.012. Wang, S.-q., Ma, J., Wang, M., Wang, X.-h., Li, Y.-q., Chen, J., 2019. Combined
Morgan, C.A., Herman, N., White, P.A., Vesey, G., 2006. Preservation of micro-organisms application of Trichoderma harzianum SH2303 and difenoconazole-propiconazolein
by drying: a review. J. Microbiol. Methods 66 (2), 183–193. https://doi.org/ controlling Southern corn leaf blight disease caused by Cochliobolus heterostrophus
10.1016/j.mimet.2006.02.017. in maize. J. Integr. Agric. 18 (9), 2063–2071. https://doi.org/10.1016/S2095-3119
Mulatu, A., Alemu, T., Megersa, N., Vetukuri, R.R., 2021. Optimization of Culture (19)62603-1.
Conditions and production of Bio-Fungicides from Trichoderma Species under Solid- Woo, S., Ruocco, M., Vinale, F., Nigro, M., Marra, R., Lombardi, N., Pascale, A.,
State Fermentation using Mathematical Modeling. Microorganisms. 9, 1675. https:// Lanzuise, S., Manganiello, G., Lorito, M., 2014. Trichoderma-based products and
doi.org/10.3390/microorganisms9081675. their widespread use in agriculture. Open Microbiol. J. 8, 71–126. https://doi.org/
Naeimi, S., Khosravi, V., Varga, A., Vágvölgyi, C., Kredics, L., 2020. Screening of Organic 10.2174/1874437001408010071.
Substrates for Solid-State Fermentation, Viability and Bioefficacy of Trichoderma Zhang, J.D., Yang, Q., 2015. Optimization of solid-state fermentation conditions for
harzianum AS12-2, a Biocontrol Strain against Rice Sheath Blight Disease. Trichoderma harzianum using an orthogonal test. Genet. Mol. Res. 14 (1),
Agronomy 10, 1258. https://doi.org/10.3390/agronomy10091258. 1771–1781. https://doi.org/10.4238/2015.March.13.4.
Nawrocka, J., Małolepsza, U., 2013. Diversity in plant systemic resistance induced by
Trichoderma. Biol. 67, 149–156.

13