Industrial Crops & Products 193 (2023) 116175

Contents lists available at ScienceDirect

Industrial Crops & Products

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

Biopesticide and plant growth-promoting activity in maize distillers’ dried

grains with solubles
Jing Li a, Hoang Khai Trinh a, b, Seyed Mahyar Mirmajlessi c, Geert Haesaert c, Ramize Xhaferi d,
Ilse Delaere d, Monica Höfte d, Katrijn Raymaekers e, Bruno P.A. Cammue e, Wim Jonckheere f,
Thomas Van Leeuwen f, Lien De Smet g, Tina Kyndt g, Nathalie Bernaert h, Christof Van Poucke h,
Bart Van Droogenbroeck h, Emmy Pequeur i, Karel De Schamphelaere i, Gert Callebaut j,
Timoty De Cleyn j, Lin Xu a, Aldana Ramirez a, Thijs Van Gerrewey a, Danny Geelen a, *
a
HortiCell, Department Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
b
Institute of Food and Biotechnology, Can Tho University, 900000 Can Tho City, Vietnam
c
Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Diepestraat 1, 9820 Bottelare, Belgium
d
Laboratory of Phytopathology, Department Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
e
Centre of Microbial and Plant Genetics, KU Leuven Plant Institute, Kasteelpark Arenberg 20, 3001 Leuven, Belgium
f
Laboratory of Agrozoology, Department Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
g
Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
h
Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium
i
Laboratory of Environmental Toxicology and Aquatic Ecology, Department of Animal Sciences and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University,
Ghent University, 9000 Ghent, Belgium
j
S.A. Ajinomoto OmniChem NV, 9230 Wetteren, Belgium

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

Keywords: A collection of by-products was surveyed for novel biostimulant and biopesticide activity by establishing a
Bioactive compounds screening platform (Bio2Bio) that integrated a broad spectrum of in vitro and in planta bioassays. Here, we report
Bioassay plant growth-promoting and biopesticide activities obtained from four solvent extracts of maize distillers’ dried
Biostimulant
grains with solubles (DDGS), a bioethanol fermentation by-product. The hexane extract stimulated Arabidopsis
Biopesticide
Maize distillers’ dried grains with solubles
primary root growth, whereas the ethanol extract enhanced tomato shoot growth and fresh biomass. The water
(DDGS) extract showed fungicide, acaricide, nematicide, and protective activities, specifically conferring disease toler­
Screening platform ance by inducing the plant immune response. We demonstrate significant biopesticide effects of DDGS water
extract against the fungus Magnaporthe oryzae (32 % in vitro efficacy), oomycete Phytophthora infestans (73 % in
planta efficacy), and spider mite Tetranychus urticae (60 % in vitro efficacy), as well as plant-induced resistance
against the fungal pathogen Botrytis cinerea (30 % in planta efficacy) and root-knot nematode Meloidogyne gra­
minicola (91 % in vitro and 50 % in planta efficacy). The broad range of bioactivities suggests that DDGS is a
complex mixture composed of a wide variety of plant metabolites. DDGS represents a valuable source for
discovering bioactive ingredients with potential use as biostimulants and/or biopesticides.

1. Introduction
Agriculture faces a dual challenge, sustaining food security while
adopting more sustainable agricultural practices (FAO, 2014). Intensive
cropping systems to ensure food supply using large amounts of synthetic
chemicals raise public concern about pesticide residues causing soil and
water pollution (van der Werf et al., 2020). Various plant by-products
accumulate during crop production and food processing, increasing
environmental and health risks (Duque-Acevedo et al., 2020). Therefore,
to upcycle these underutilized biomass fractions with profitable

Abbreviations: DDGS, maize distillers’ dried grains with solubles; EBIC, European Biostimulants Industry Council; HO, water extract; EH, ethanol extract; EA, ethyl
acetate extract; HE, hexane extract; BBC, biostimulant and biopesticide characterization; PDF1.2a, Plant Defensin 1.2a; PR1, Pathogenesis-Related 1.
* Corresponding author.
E-mail address: Danny.Geelen@UGent.be (D. Geelen).

https://doi.org/10.1016/j.indcrop.2022.116175
Received 25 August 2022; Received in revised form 11 December 2022; Accepted 19 December 2022
Available online 5 January 2023
0926-6690/© 2022 Published by Elsevier B.V.
J. Li et al.
turnovers, a growing interest in biobased functional products is boosting
biorefinery technology development from abundant and cheap biomass
consisting of proteins, oligosaccharides, and bioactive chemicals
(Capanoglu et al., 2022). The value of organic biomass fractions as a
resource of bioactive molecules has recently attracted academic and
industry stakeholders to embark on screening and developing novel
biostimulants and biopesticides (Balasubramanian and Tyagi, 2017; Xu
and Geelen, 2018). The rush for discovering new molecules with bio­
stimulant and biopesticide activity is partly stimulated by a rapidly
growing market that by 2030 will reach a total value of $19.5 billion
(biostimulants 38 % and biopesticides 62 %) and an average annual
growth rate of over 10 % (Dent, 2021; Xie, 2021). Driven by the strong
economic returns and the need for better sustainability, Bio2Bio1 aims to
develop new biostimulants and biopesticides derived from underutilized
by-products and biowaste (Xu and Geelen, 2018). These above­
mentioned drivers align with the new "bioeconomy" and "zero waste"
trend (Ravindran and Jaiswal, 2016) and support the sustainable
farming goal to reduce 20 % of fertilizer and 50 % of chemical pesticide
usage by 2030 (European Commission, 2020).
The European Biostimulants Industry Council (EBIC) defines bio­
stimulants as bioactive substances including industrial by-products,
classified as fertilizing products, with the sole aim of improving plant
or plant rhizosphere through nutrient use efficiency, tolerance to abiotic
stress, quality traits, or availability of confined nutrients in the soil or
rhizosphere (EBIC, 2019; European Union, 2019). Under the regulatory
framework of plant protection products, biopesticides should specify at
least one agent active against pests or diseases, which could be any
compound or combination of compounds, including natural products
derived from plant by-products (European Commission, 2009). In order
to introduce biostimulants and biopesticides into the market, product
effectiveness and safety are essential to regulatory agents and consumers
(EBIC, 2021; Fenibo et al., 2021). These advantageous biological effects
are substantially attributed to the active ingredient(s) in natural sub­
stances. Taking advantage of drug discovery procedures, the first step in
cutting-edge strategic technologies is establishing a dedicated and
customized bioassay-based screening platform that allows the rapid
identification of relevant and significant bioactivity by conducting
multiplexed assays (Berg, 2021). In most discovery research targeting
biostimulant and biopesticide activity, screening involves only a single
or a few phenotypic parameters (Murgia et al., 2022; Povero, 2020). The
potential of bioactivity screens is strongly enhanced, however, by
monitoring a broad spectrum of biological traits by combining targeted
and untargeted phenotypic bioassays. This strategy was adopted in our
screening platform Bio2Bio compiling over 30 bioassays. The in vitro and
in planta bioassays assessed the impact on plant growth (stimulatory
activity), defense against important pests and diseases, plant-induced
resistance, and ecotoxicological effects on non-target species.
Plant primary and secondary metabolites largely contribute to the
diversity of natural chemicals and result from evolution and selection.
While phytochemicals are rarely utilized due to their low bioavail­
ability, bio-processing methods like bioethanol fermentation improve
the bioactive value by altering metabolite compositions via microor­
ganisms (Yeo and Ewe, 2015). In agreement with the development of
"green chemistry" practices for phytochemicals, classic extraction
methods, including liquid extraction and liquid-liquid extraction with
sustainable solvents, are commonly used (Tsiaka et al., 2017). Addi­
tionally, industrial extraction and processing are adopted to produce
enough biological extract to be screened in a wide range of bioassays
(Wen et al., 2020).
In this study, we used an agricultural by-product derived from in­
dustrial maize fermented to ethanol for biofuel production. After
fermentation and distillation, residues are co-produced, which consist of
approximately 30 % (w/v) of the original biomass. This residue is known
1 3
https://www.horticell.ugent.be/bio2bio/
2
Industrial Crops & Products 193 (2023) 116175
as maize distillers’ dried grains with solubles (DDGS) (US Grains
Council, 2012). DDGS from maize kernels for bioethanol production
contains around 25–35 % protein, 11 % lipids, 4 % remaining starch, 30
% non-starch carbohydrates (i.e., hemicellulose, cellulose), and small
quantities of betaine and phenolic compounds (Chatzifragkou and
Charalampopoulos, 2018; Kim et al., 2008). Because DDGS is continu­
ously available in high volumes it was evaluated for biostimulant and
biopesticide activity in the Bio2Bio screening platform. We demonstrate
here the Bio2Bio screening platform encompassing a series of in vitro and
in planta bioassays related to plant production and protection fields to
identify the bioactive properties of agricultural by-products. Bio­
stimulant, biofungicide, bioacaricide, bionematocide, and plant-induced
resistance-triggering activities were identified in DDGS extracts,
demonstrating a richness in bioactive ingredients in this agri-food res­
idue from fermented maize kernels. These findings encourage the
further exploration of DDGS for new biological products.
2. Materials and Methods
2.1. Raw material collection and extraction
DDGS, derived from maize (Zea mays L.) draff after bioethanol
fermentation and containing about 10 % moisture, was supplied in an
airtight bag by Alco Bio Fuel (Ghent, Belgium). Sustainable maize
certified by ISCC (International Sustainability & Carbon Certification)
was collected from the main European production regions for bioethanol
production. The chemical composition of DDGS is 28 % crude protein,
10.5 % crude fat, 7.0 % crude fiber, 4.3 % crude ash, and 4 % starch. The
raw material was stored at 14 ◦ C before milling in a vacuum cooking
cutter (Seydelmann K64AC8, Germany). The conditions for extraction
were based on previous experience with using a Stephan universal
extraction machine for the semi-industrial production of plant extracts
(Food Pilot, ILVO, Belgium). For homogenous mixing and hydrothermal
processing, the DDGS powder (5 Kg) was incubated in 25 L of water for
2 h at 80 ◦ C in a mixer cooker at 100 mbar (Stephan Universal Machine
UMSK 60 E, Germany). A mash of around 30 L was obtained and passed
through a vibrating screen machine (Finex 22, Belgium) equipped with
two successive sieves (1000 µm and 100 µm, respectively). The filtrate
was labeled as liquid water extract (HO) (dry matter content 75.39 mg
mL-1), while the solid residue was recycled for sequential solvent ex­
tractions with assistance from Ajinomoto OmniChem (Ghent, Belgium).
The solid phase (around 9 Kg) was mixed with ethanol (20 L) for 1 h at
60 ◦ C and then filtered to obtain an ethanol extract (EH). The solid left-
over (3.9 Kg) was extracted with ethyl acetate (18 L) for 1 h at 50 ◦ C and
filtered to generate an ethyl acetate extract (EA). The solid left-over (3.6
Kg) was extracted with hexane (17 L) for 1 h at 40 ◦ C to generate the
hexane extract (HE). The organic solvents were removed with a rota­
vapor (Büchi R-220, Switzerland) at 60 ◦ C followed by evaporation with
a high vacuum pump and yielded a total of 365 g EH, 148 g EA, 10 g HE,
and 1.6 Kg solid residue. The aqueous extract and three organic solvent
extracts were stored at − 20 ◦ C and aliquoted for bioassays.
2.2. Pesticide residue analysis
The raw material (DDGS powder) was analyzed for pesticide residues
after extraction with acidified acetonitrile (QuEChERS extraction)2 or
acetonitrile with 0.5 % acetic acid (v:v). The methods were multi-
residue GC-MS/MS and LC-MS/MS (Primoris, Belgium)3 (European
Commission, 2009, 2005).
2
https://www.sigmaaldrich.com/BE/en/applications/analytical-chemistry/
sample-preparation/quechers
https://www.primoris-lab.com/en
J. Li et al. Industrial Crops & Products 193 (2023) 116175

2.3. Chemical characterization with tap water, and the three organic extracts, EH, EA, and HE, were
dissolved with 0.1 % DMSO (final concentrations indicated in the legend
Organic extracts (EH, EA, and HE) were dissolved in methanol at the of Fig. 2). Each treatment was performed in triplicates. Tomato plants
final concentration of 1 mg mL-1, and all the samples were pushed were cultivated in pots filled with standard commercial potting soil
through a 0.22 µm filter (Millix-GV, Millipore). Chemical profiling was (organic/mineral fertilizers NPK (nitrogen, phosphorus, potassium)
performed using ultra-high-performance liquid chromatography (Acq­ 14–16–18, pHH2 O 5–6.5, organic and dry matters of 25 % and 20 %,
uity UPLC) coupled to a Photodiode Array detector (PDA) (UPLC respectively) in a walk-in climatized greenhouse (18–20 ◦ C, 12 h
eLambda 800 nm) and a SYNAPT G2-S High-Resolution Mass Spec­ photoperiod). The growth rate was calculated from the plant height
trometer (Waters, Milford, MA, US) (HRMS) under positive and negative measured every week over three weeks. The above-ground fresh and dry
electrospray ionization modes (ESI+, ESI-) as described by Li et al. biomass weights of tomato plants were determined after three weeks,
(2022). and the dry matter percentage was calculated from the dry biomass to
fresh biomass ratio.
2.4. Biosafety assays to assess acute environmental toxicity on non-target
organisms
2.6. Screening bioassays for potential biopesticide activity
2.4.1. Algae growth inhibition assay
2.6.1. Biofungicide: in vitro antifungal activity
The algal species, Pseudokirchneriella subcapitata, was cultured ac­
The fungicidal in vitro assay was conducted in Petri dishes against
cording to OECD (Organization for Economic Cooperation and Devel­
Botrytis cinerea, Fusarium oxysporum f. sp. cubense, Magnaporthe oryzae
opment) guideline No. 201 (OECD, 2011). A 72 h P. subcapitata growth
(from the Ascomycota phylum), Rhizoctonia solani (from the Basidio­
inhibition test was conducted with DDGS extract additions (final con­
mycota phylum), and Phytophthora porri, Pythium splendens (from the
centrations indicated in the legend of Fig. 1) in culture media at 22 ±
Oomycota phylum) described in (Meng et al., 2020). The fungal strains
2 ◦ C with a 24 h light cycle. EH, EA, and HE were prepared in 1 %
that we used in this assay are B. cinerea isolate R16 (Faretra and Pol­
dimethyl sulfoxide (DMSO). Triplicates with an initial cell density of 104
lastro, 1991), F. oxysporum f. sp. cubense isolate FoxPDa15 (race 1)
cells mL-1 were performed for each treatment. Cell density changes were
(Deltour et al., 2018), M. oryzae isolate VT5M1 (Thuan et al., 2006),
monitored after exposure of 24, 48, and 72 h using a Coulter Counter
R. solani isolate CuHav-Rs18 (AG2–2) (Yaquelyn et al., 2010), P. porri
(Coulter Counter Z1, Beckman) and calculated as the average algae
isolate CBS127099 (Bertier et al., 2013), and P. splendens isolate 706
growth rate from 24, 48, and 72 h.
(Buysens et al., 1996). Mycelial plugs of each pathogen with 4 or 5 mm
in diameter were inoculated in the center of the Petri dish (90 mm
2.4.2. Daphnia magna immobility assay
diameter for HO and 35 mm diameter for the rest of DDGS extracts)
Juveniles of Daphnia magna within 24 h were sampled, and an acute
under laminar flow with potato dextrose agar (Becton, Dickinson, and
immobilization test was conducted according to OECD guideline No.
Company) or V8 agar (only for P. porri) with or without DDGS extract
202 (OECD, 2004). The test was conducted in modified COMBO media4
addition. HO was diluted with sterilized H2O, EH was made in 1 %
(Kilham et al., 1998) supplemented with DDGS extracts (final concen­
ethanol, EA and HE were prepared in 1 % DMSO (final concentrations
trations indicated in the legend of Fig. 1) at 20 ± 1 ◦ C with a 16: 8 h
indicated in the legend of Fig. 3). Each treatment included three indi­
light: dark cycle. The assay was performed with three replicates of 10
vidual plates. The radial growth diameter of mycelium was measured by
juveniles. Daphnia mobility ( %) was recorded 24 and 48 h after
perpendicular cross-sections (mm) in both x and y directions at 0, 1, 2, 3,
exposure.
and 4 days after inoculation. The area of fungal growth on day four of
treatment to blank was calculated and converted from diameters
2.5. Screening bioassays for plant growth-promoting activity
(Hendricks et al., 2017).
2.5.1. In vitro Arabidopsis root bioassay
2.6.2. Biofungicide: detached tomato and potato leaf assay
Arabidopsis (Arabidopsis thaliana) (Col-0) seeds were sterilized,
The detached leaf assay of 6 or 7-week-old tomato (Solanum lyco­
sown, vernalized, etiolated, and cultured in vitro for assaying root
persicum var. Dona hybrid F1) and potato (Solanum tuberosum var. Bintje)
development as described (Trinh et al., 2018). Briefly, three-day-old
plants were used to test the biocidal activity of DDGS extracts against
etiolated seedlings of similar size were transferred to Murashige and
Phytophthora infestans, Alternaria solani, and Botrytis cinerea, respectively
Skoog medium (1.5 g L-1 Murashige and Skoog Basal Salt Mixture,
(Najdabbasi et al., 2020). The fungal stains used in this assay are sup­
5.0 g L-1 D-sucrose, 0.5 g L-1 MES (2-Morpholinoethanesulforic acid),
plied in Supplementary Materials and Method. Foliar spraying of plants
8.0 g L-1 agar, pH 5.7) with or without DDGS extracts. HO and EH were
with the DDGS extracts was done in triplicates 24 h before inoculation
diluted with H2O, EA and HE were dissolved in ethyl acetate and hexane
(final concentrations indicated in the legend of Fig. 3). The DDGS
solutions, respectively (final concentrations indicated in the legend of
treatments were prepared the same way as the greenhouse assay on
Fig. 2). Each treatment consisted of three plates with ten seedlings. After
tomato plants (see Materials and Methods 2.5.2). After spraying, leaves
ten days of incubation in a growth room (21.4 ◦ C, 40–60 % room hu­
were removed (at least two compound leaves per replicate) and inocu­
midity, light intensity 140 μmol m-2 s-1, 14:10 h light:dark photoperiod),
lated with a single droplet (15 µL) of spore suspension of pathogens (105
root morphology was examined with a binocular microscope (Olympus,
spores mL-1). Leaflets in the floating system were kept in a growth
SZX9, Tokyo, Japan), for the number of adventitious roots, junction
chamber. Disease incidence was assessed seven days after inoculation
roots, and lateral roots. Primary root length and growth rate per day
with water or DDGS extracts according to an arbitrary grading scale of
after transfer during incubation were recorded and processed using
white flecks (disease severity level from 0 to 9, indicating least to most
ImageJ version 1.51 (Schneider et al., 2012).
serious infections) and converted to the disease severity index (Eq. 1).
∑
2.5.2. Greenhouse tomato bioassay Disease severity index = (Disease severity level(x)
All four DDGS extracts were foliar sprayed on 18-day-old tomato /
plants (Solanum lycopersicum var. Dona hybrid F1). The HO was prepared × Number of leaflets(x)) (Total number of leaflets
× 9) × 100%
4 (1)
https://utex.org/products/modified-combo-medium?
variant= 30991760031834

3
J. Li et al. Industrial Crops & Products 193 (2023) 116175

Fig. 1. Acute environmental toxicity assays of the four DDGS extracts on two non-target organisms. (a) Average growth inhibition of algae over 24, 48–72 h exposure
to DDGS extract. The concentrations used are: HO: 0.75 mg mL-1; EH(H): 20 mg L-1; EH(M): 0.2 mg L-1; EH(L): 0.02 mg L-1; EA(H): 100 mg L-1; EA(M): 10 mg L-1; EA
(L): 1 mg L-1; HE(H): 100 mg L-1; HE (M): 10 mg L-1; HE(L): 1 mg L-1. (b) Immobility of Daphnia magna 24 h and 48 h after incubation with DDGS extracts. The
concentrations were: HO(H): 7.5 mg mL-1; HO(L): 0.75 mg mL-1; EH(H): 20 mg L-1; EH(M): 2 mg L-1; EH(L): 0.2 mg L-1; EA(H): 100 mg L-1; EA(M): 10 mg L-1; EA(L):
1 mg mL-1; HE(H): 100 mg L-1; HE (M): 10 mg L-1; HE: 1 mg L-1. Negative or positive values of ecological effects indicate reductions or increases, respectively, of
growth rate or mobility compared to the negative control (’blank’). Asterisks indicate a statistical difference with blank (p < 0.05) (n = 3). DDGS: maize distillers’
dried grains with solubles; HO: water extract; EH: ethanol extract; EA: ethyl acetate extract; HE: hexane extract; H: high concentration; M: middle concentration; L:
low concentration.

Fig. 2. Biostimulant activity of four DDGS extracts on in vitro Arabidopsis root growth and greenhouse tomato cultivation. (a) In vitro root bioassay on Arabidopsis
(n = 30). The concentrations used were: HO(H): 1.51 mg mL-1; HO(M): 0.151 mg mL-1; HO(L): 0.0151 mg mL-1; EH(H): 4.17 mg mL-1; EH(M): 0.417 mg mL-1; EH(L):
0.0417 mg mL-1; EA(H): 1.5 mg mL-1; EA(M): 0.15 mg mL-1; EA(L): 0.015 mg mL-1; HE(H): 0.083 mg mL-1; HE (M): 0.0083 mg mL-1; HE(L): 0.00083 mg mL-1. (b)
Greenhouse tomato bioassay (n = 3). The concentrations used are: HO: 7.54 mg mL-1; EH: 10 mg mL-1; EA: 10 mg mL-1; HE: 10 mg mL-1. Asterisks indicate a sta­
tistical difference with blank (p < 0.05). BBC: biostimulant and biopesticide characterization; DDGS: maize distillers’ dried grains with solubles; HO: water extract;
EH: ethanol extract; EA: ethyl acetate extract; HE: hexane extract; H: high concentration; M: middle concentration; L: low concentration.

2.6.3. Biocontrol of plant disease: induced resistance in Arabidopsis
In planta assay for plant-induced resistance was conducted on Ara­
bidopsis against Botrytis cinerea B05.10 (Schoch et al., 2020) as
described by Mathys et al. (2012). Seven-day-old Arabidopsis seedlings
were transferred on plates containing half-strength Murashige and
Skoog media. Each assay was performed as four replicates with six plants
per treatment. After 22 days of growth, 2 µL of the DDGS extracts were
applied by pipetting onto four different spots on each root system (final
concentrations indicated in the legend of Fig. 4). Mevalone
(2.5 mg mL-1) was used as the positive control (Eden Research, 2019).
Three days later, two leaves from each plant were infected twice with
3 µL of B. cinerea spore suspension (5 × 105 spores mL-1 in 1/2 potato
dextrose broth solution). The disease severity was further evaluated on
the third day after infection by measuring the lesion diameter.
Moreover, as an initial survey of the main signaling pathways involved
in the observed induced resistance, gene expression analysis of Arabi­
dopsis Plant Defensin 1.2a (PDF1.2a) and Pathogenesis-Related 1 (PR1)
was performed using quantitative real-time PCR (qRT–PCR), as markers
of the salicylic acid- and jasmonate/ethylene-mediated signaling path­
ways, respectively (Thomma et al., 1998).
2.6.4. Bioacaricide: in vitro insecticidal assays against Tetranychus urticae
Toxicity bioassays on the two-spotted spider mite Tetranychus urticae
(Acari: Tetranychidae) were performed as described by Khajehali et al.
(2011). Briefly, 20–30 young adult female mites of the London strain
(Grbić et al., 2011) were transferred to the upper side of 9 cm2
square-cut bean (Phaseolus vulgaris, cv. ’Prelude’) leaf discs on wet
cotton wool, lined with tissue paper to avoid mite escape. The leaf discs

4
J. Li et al. Industrial Crops & Products 193 (2023) 116175

Fig. 3. Biofungicidal efficacy of four DDGS extracts from in vitro and in planta fungicidal activity on plant pathogens for potential biofungicide discovery. (a) In vitro
antifungal assay on Petri dishes against six phytopathogens based on fungal growth diameters (n = 3). The sample concentrations involved are, HO: 37.70 mg mL-1;
EH(H): 10 mg mL-1; EH(L): 5 mg mL-1; EA(H): 10 mg mL-1; EA(L): 5 mg mL-1; HE(H): 5 mg mL-1; HE(L): 1 mg mL-1. (b) In planta assay on detached potato and tomato
leaf against pathogens infecting based on disease severity index (n ≥ 2). The testing concentrations involved are, HO: 7.54 mg mL-1; EH: 10 mg mL-1; EA: 10 mg mL-1;
HE: 10 mg mL-1. Each data point indicates the BBC index of each extract under a certain concentration for each parameter from the bioassay. Asterisks indicate a
statistical difference with blank (p < 0.05). BBC: biostimulant and biopesticide characterization; DDGS: maize distillers’ dried grains with solubles; HO: water extract;
EH: ethanol extract; EA: ethyl acetate extract; HE: hexane extract; H: high concentration; L: low concentration.

with mites were sprayed with 0.8 mL spray fluid (DDGS extract solution)
at 1 bar pressure using a Cornelis spray tower (Van Laecke and
Degheele, 1993), resulting in an aqueous deposit of 2.1 ± 0.1 mg cm-2.
Following treatment, the leaf disks were transferred to a climatically
controlled room at 25 ◦ C, 60 % relative humidity, and 16:8 h light:dark
photoperiod. Mite mortality was assessed after 24 h. Mites were scored
"dead" if they could not move their body length within 10 s after prod­
ding with a fine brush. Mites drowned in the water barrier were
excluded from the analysis. The mite survival rate ( %) was calculated
using Eq. 2.
Survival rate(%) = (Number of alive mite(s)/Number of total mites) × 100%
(2)
For each extract, four replicates were treated. HO was sprayed as
such while spraying with water served as a control. EH, EA, and HE were
dissolved in a mixture of 3:1 (v:v) N, N-dimethylformamide (DMF) and
emulsifier W, which was subsequently diluted 100-fold in deionized
water. A blank formulation was used as the control. The final concen­
trations are indicated in the legend of Fig. 5.

2.6.5. Bioinsecticide: in vitro biocidal assays against Spodoptera littoralis

Fig. 4. Plant-induced resistance-induction assays on Arabidopsis. (a) In planta
induced resistance on Arabidopsis against Botrytis cinerea was evaluated by
analyzing the reduction of lesion diameter (n = 3). (b) Relative expression
levels of the plant defense marker genes PDF1.2a and PR1 (n = 3). PDF1.2a:
PLANT DEFENSIN 1.2a; PR1: Pathogenesis-Related 1. The testing concentrations
used are: Mevalone: 2.5 mg mL-1; HO: 0.75 mg mL-1; EH: 250 mg mL-1; EA:
20 mg mL-1; HE: 20 mg mL-1. Asterisks indicate a statistical difference with
Mevalone (p < 0.05). BBC: biostimulant and biopesticide characterization;
DDGS: maize distillers’ dried grains with solubles; HO: water extract; EH:
ethanol extract; EA: ethyl acetate extract; HE: hexane extract.
For Spodoptera littoralis (Lepidoptera: Noctuidea), DDGS extracts
were combined with an artificial diet, and the effect was evaluated on
larval survival rate and body weight. For this purpose, an agar-based
artificial diet was added to 5 × 5 well plates, and 0.15 mL of DDGS
extract solution was added on top of the diet. The test solutions were
prepared for the T. urticae, with the difference that the DMF:emulsifier
W mixture was diluted 50-fold. The final test concentrations are indi­
cated in the legend of Fig. 5. After absorption of the extract solution in
the diet, one S. littoralis L2 larva was added per well. For each extract,
three plates with 20 larvae were prepared. The plates were incubated at
25 ◦ C, 60 % relative humidity, and 16:8 h light: dark photoperiod for
eight days, after which the effect on larval survival rate (Eq. 2), as well

5
J. Li et al. Industrial Crops & Products 193 (2023) 116175

Pratylenchus zeae (Bridge et al., 2005). The M. graminicola suspension

Fig. 5. In vitro biocidal assays against Tetranychus urticae and Spodoptera lit­
toralis. (a) For the T. urticae assay based on mortality rate (n = 4), the test
concentrations are: HO: 75.39 mg mL-1; EH: 1 mg mL-1; EA: 1 mg mL-1; HE:
1 mg mL-1. (b) For the S. littoralis assay based on the effect on larval weight and
mortality rate (n = 3), the test concentrations were: HO: 75.39 mg mL-1; EH:
2 mg mL-1; EA: 2 mg mL-1; HE: 2 mg mL-1. Asterisks indicate a statistical dif­
ference with blank (p < 0.05). BBC: biostimulant and biopesticide character­
ization; DDGS: maize distillers’ dried grains with solubles; HO: water extract;
EH: ethanol extract; EA: ethyl acetate extract; HE: hexane extract.
as on relative weight gain, were evaluated.
2.6.6. Bionematocide: in vitro and in planta nematocidal efficacy
In vitro nematocidal assays were conducted against the root-knot
nematode Meloidogyne graminicola and the root-lesion nematode
was initially extracted from infected barnyardgrass (Echinochloa crus-­
galli L.) roots in pot soil and incubated in tap water for three days on a
200 µm mesh sieve. Nematodes that migrated through the sieve were
collected on a 20 µm sieve. About 50 s-stage juveniles (J2) of
M. graminicola were exposed to DDGS extracts (EH was made in 0.7 %
ethanol, EA, and HE in 1 % DMSO, final concentrations indicated in the
legend of Fig. 6), or 0.2 % Vertimec (Syngenta, 2021) as the positive
control, with three replicates in 12-well plates and incubated on a shaker
at room temperature. The nematode survival rate ( %) (similar calcu­
lation in Eq. 2) was evaluated after 48 h of incubation in each well.
In planta induced resistance against M. graminicola on rice was
executed as described by (Singh et al., 2020). The foliar spray of DDGS
extracts (final concentrations indicated in the legend of Fig. 6) formu­
lated for efficient uptake by adding 0.2 % Tween 20 was applied to 14
D-old rice (Oryza sativa cv Nipponbare). The rice plants were grown in
individual PVC tubes containing sand-absorbent polymer, a mixture of
fine silica sand and ultra-absorbent acrylic copolymer (AquaPerla, DCM,
Grobbendonk, Belgium) in a ratio of 1 Kg sand to 1.5 g copolymer. The
crops were irrigated three times per week with 8 mL of Hoagland so­
lution. One day after pretreatment, plants were inoculated in at least five
replicates next to roots with around 200 J2 M. graminicola and grown for
another 14 days. Then the root and shoot length were assessed with a
ruler. The number of root galls formed by M. graminicola was evaluated
using fuchsin staining and tissue clearing (Bybd et al., 1983), after which
they were counted using a binocular microscope.
2.7. Statistical analysis and data visualization
Bioassays performed in different batches were compared to the blank
or control from the same batch. For non-parametric comparisons, the
statistical differences among treatments were determined according to
Kruskal-Wallis tests with Dunn’s post hoc analysis (p < 0.05) applied to
count data and Mann-Whitney (p < 0.05) on proportions. For para­
metric statistics, Welch’s t-test or one-way ANOVA (analysis of variance)
with Dunnett’s post hoc analysis (p < 0.05) were used on continuous

Fig. 6. In vitro and in planta nematocidal efficacy. (a) In vitro nematocidal assay against Meloidogyne graminicola and Pratylenchus zeae based on nematocidal efficacy
(n = 3). The concentrations used are: Vertimec: 0.0036 mg mL-1; HO(H): 0.75 mg mL-1; HO(L): 0.075 mg mL-1; EH(H): 0.5 mg mL-1; EH(MH): 0.1 mg mL-1; EH(mL):
0.01 mg mL-1; EH(L): 0.001 mg mL-1; EA(H): 0.05 mg mL-1; EA(MH): 0.01 mg mL-1; EA(mL): 0.001 mg mL-1; EA(L): 0.0001 mg mL-1; HE(H): 0.01 mg mL-1; HE(M):
0.001 mg mL-1; HE(L): 0.0001 mg mL-1. (b) In planta induced resistance against M. graminicola on rice based on galls reduction as well as root and shoot growth
(n ≥ 5). The concentrations used were, HO: 75.39 mg mL-1; EH: 1 mg mL-1; EA: 0.1 mg mL-1; HE: 0.1 mg mL-1. Asterisks indicate a statistical difference with blank
(p < 0.05). BBC: biostimulant and biopesticide characterization; DDGS: maize distillers’ dried grains with solubles; HO: water extract; EH: ethanol extract; EA: ethyl
acetate extract; HE: hexane extract; H: high concentration; MH: higher middle concentration; M: middle concentration; mL: lower middle concentration; L: low
concentration.

6
J. Li et al.
data. The p-value was calculated with GraphPad Prism software version
8.0.2 (GraphPad, San Diego, USA) (see Table S1).
To determine the bioactivity of DDGS extracts in each biopesticide
assay, the efficacy for every parameter was calculated using Eq. 3
(Abbott, 1925; Kamaruzzaman et al., 2021). Likewise, the biostimulant
efficacy (focus on growth-promoting activity), or ecological effect of
each bioassay was adapted for individual measurement using Eq. 4.
Biopesticide efficacy(%)(X) = (xControl − xTreatment )/xControl × 100% (3)
Biostimulant efficacy or ecological effect(%)(X)
= (xTreatment − xControl )/xControl × 100% (4)
Where x indicates the mean of raw bioactivity data points (i.e., the
physiological traits of Arabidopsis root, tomato growth rate, etc.),
treatments represent bioactivity from DDGS extract applications. And
controls were conducted in the same batch, where solvent control is the
negative control. Vertimec and Mevalone were positive controls for in
vitro nematocidal assays and in planta induced resistance assays,
respectively. The efficacy of control will always be zero, and positive
results indicate beneficial biological effects and vice versa.
A comparison of the degree of performance across all bioassays was
made using a biostimulant or biopesticide characterization (BBC) index
inspired by Ugena et al. (2018). The BBC index was standardized to a
range between 0 and 1 via a linear (max-min) normalization method
among all the bioactivity data generated from the same bioassay (Glenn,
2020). The BBC index displays the normalized efficacy of biostimulant
or biopesticide activity following Eq. 5.
Biostimulant or biopesticide characterization(BBC)(Y)
= (X − Xmin )/(Xmax − Xmin ) (5)
Where X indicates the biostimulant or biopesticide efficacy. Xmax and
Xmin are denoted as the strongest and weakest bioactivity efficacy
involving all the treatments from one or multiple batches in each assay.
A larger BBC index displays more potent bioactivity when compared to
the control.
Bioactivity data representing the BBC index were visualized in circle
view charts using Tableau version 2021.4 (Batt et al., 2020). After
Min-Max scaling, the normalized BBC index maintains the relationships
of original data points but may cause bias due to possible outliers
(Loukas, 2020). Since our dataset in each bioassay is relatively small, we
kept all the raw data for more comprehensive bioactivity information
gathered from preliminary bioassays. For the potential users, both the
BBC index and the results of significant difference tests (p values) among
groups of treatments should be considered before further investigation
(Table S1).
3. Results and discussion
More than half of the commercialized agrochemicals on the market
derive from natural products (Sparks et al., 2017). To screen for putative
biostimulant and biopesticide activity in agri-food side streams, we have
compiled in vitro and in planta bioassays into a "biostimulant" and "bio­
pesticide" screening platform, called Bio2Bio. Here, we report on the
chemical analysis, ecotoxicity, and bioactivity of four extracts from
DGGS as a specific example of the results obtained from our screening
efforts.
3.1. DDGS metabolite profiling and ecotoxicity assessment
3.1.1. Pesticide residue detection in DDGS
DDGS raw material was screened for the presence of more than 500
existing pesticides. The pesticide anthraquinone was detected at a con­
centration of 0.023 mg Kg-1. Anthraquinone is used as an avian repel­
lent, but recently, natural anthraquinones have been shown to possess
insect repellent and insecticide activity (Yuan et al., 2020). The
7
Industrial Crops & Products 193 (2023) 116175
strongest activity was observed against the third instar larvae of Heli­
coverpa armigera with an LC50 (50 % Lethal Concentration) of
0.72 mg mL-1, which is far above the concentration we applied in our
insecticidal assays (see Fig. 5) (Gaudêncio and Pereira, 2015). The safety
requirements of active substances are covered by general chemical
legislation outlined in the EU (European Union, 2019). The low dose of
anthraquinone in DDGS does not pose a risk for application in agricul­
ture (Malfroy, 2020).
3.1.2. Ecotoxicity assessment of DDGS extracts
To assess the environmental hazard of DDGS extracts application,
standard acute ecotoxicity tests were performed on two non-target
aquatic species. Algae growth was inhibited by 17.10 % at
0.75 mg mL-1 of HO (Fig. 1a). Other extracts showed no inhibition or a
small positive effect. In another separate test, the mobility of the
D. magna was analyzed. High doses of EH showed moderate to high
toxicity from 24 h to 48 h after exposure, whereas EH was expected to
have a lower hazard since it was applied at more diluted doses in the
bioassays (Fig. 1b). EH and EA at a higher dose showed medium eco­
toxicity causing 23.86 % and 15.91 % of D. magna immobility after 48 h
of incubation (Table S1). As in none of the ecotoxicity tests, an EC50 (50
% Effective Concentration) of algae growth or D. magna mobility inhi­
bition was reached. DDGS extracts were then considered to display an
acceptable ecotoxicity hazard level. Natural-origin biologicals, i.e.,
biopesticides, are generally less persistent, less harmful to non-target
organisms, and show a lower pest resistance compared to synthetic
products (Pino-Otín et al., 2019). The low-environmental hazard of HO,
EA, and HE are encouraging and provide support to investigate these
extracts further.
3.1.3. Metabolite profiling of DDGS extracts
DDGS was extracted consecutively with solvents increasing hydro­
polarity, water, ethanol, ethyl acetate, and hexane. The water and
organic solvent extracted materials were analyzed using HRMS-guided
metabolite profiling (Table S2). A higher abundance of chemicals was
detected in the HRMS ESI+ mode in HE and HO extracts (Table 1).
Between 15.42 % and 60.76 % of detected features were tentatively
identified and classified into 12 groups of plant organic compounds. The
lipids and lipid-like molecules, organic acids and derivatives, and
organoheterocyclic compounds formed the bulk of the detected mole­
cules. The biological activities in natural mixtures are altered to a great
extent by synergistic, additive, or even antagonistic interactions among
single active constituents (Caesar and Cech, 2019). It was therefore
expected to detect a large body of bioactive compounds present in DDGS
and its derived extracts suggesting possible plant growth-promoting and
biopesticide activity. But in advance, to ensure consistency in the effi­
cacy across batches of raw materials (García-García et al., 2020) and to
be able to register the product as a biopesticide, which requires at least
one identified active ingredient (European Commission, 2009), the
causative active compound(s) in complex mixtures would need to be
in-depth identified.
3.2. Plant growth-promoting activity in DDGS extracts
As roots from in vitro grown Arabidopsis seedlings are highly sensi­
tive to environmental stimuli (Petricka et al., 2012), the biometrics of
root architecture was determined for plants grown in the presence and
absence of DDGS extracts. Application of HO, EH, and EA stimulated the
initiation of adventitious roots and junction roots, whereas clear signs of
inhibition were observed in lateral root formation and primary root
growth (Fig. 2a). Phytochemicals typically behave in a
concentration-dependent manner displaying desirable effects within a
non-toxic range (Jodynis-Liebert and Kujawska, 2020). For instance, HE
at the lower two concentrations significantly promoted primary root
growth and minimal lateral root suppression (Table S1). The contrasting
effect on adventitious root and junction root versus primary root and
J. Li et al. Industrial Crops & Products 193 (2023) 116175

Table 1
Metabolite profiling of four DDGS extracts by UHPLC-PDA-HRMS analysis under ESI+ and ESI- modes (for the complete list, see Table S2).
Peak (× 106)1 ESI+ mode ESI- mode
Superclass HO EH EA HE HO EH EA HE

Alkaloids and derivatives 5.51 0.96 0.62 1.13 1.08 0.33 1.71 7.51
Benzenoids 13.08 23.70 0.40 2.96 5.98 4.17 0.48 1.10
Hydrocarbons 0.11 ND 0.06 ND 0.10 ND ND ND
Lignans, neolignans, and related compounds 0.90 ND ND 1.67 0.50 0.10 ND ND
Lipids and lipid-like molecules 21.79 76.43 32.07 29.62 41.01 37.59 1.88 8.17
Nucleosides, nucleotides, and analogues 12.46 0.30 ND ND 4.74 0.08 ND ND
Organic acids and derivatives 30.43 18.70 3.61 23.06 34.62 1.93 0.48 0.76
Organic nitrogen compounds 4.56 3.48 0.91 1.85 0.90 ND ND ND
Organic oxygen compounds 9.06 12.18 0.42 5.10 11.09 1.84 0.62 0.09
Organoheterocyclic compounds 82.47 21.23 2.51 36.21 22.41 7.56 0.65 3.51
Organosulfur compounds 0.81 ND ND ND 0.26 ND ND ND
Phenylpropanoids and polyketides 10.36 10.15 0.21 0.61 11.78 3.66 ND 0.75
% identification/detection 35.80 60.76 40.43 16.08 55.14 50.42 15.54 54.67
Total 535.02 275.12 100.92 635.52 243.87 113.55 37.39 40.05

1 The peak density of total ion chromatograms was summed up for each superclass metabolite profiled in DDGS extracts (Zhang et al., 2019).
UHPLC: ultra-high-performance liquid chromatography; PDA: photodiode array detector; HRMS: high-resolution mass spectrometer; DDGS: maize distillers’ dried
grains with solubles; HO: water extract; EH: ethanol extract; EA: ethyl acetate extract; HE: hexane extract; ESI+ (-): positive (negative) electrospray ionization; ND: not
detected.

lateral root was recurrent in the root architecture assays, underlining the
differential regulation of roots emerging on the stem of Arabidopsis
versus roots emerging from root tissue. Remarkably, external signals,
such as the bioactive substance in HE, result in dynamic plant hormone
crosstalk networks among plant hormones, which coordinate similar
and different growth outcomes throughout primary and lateral root
development (Waidmann et al., 2020). The root system consists of
different root types that determine water and nutrient use efficiency and
assist crops in tackling climate challenges (Lynch, 2022).
Next, a growth assay was conducted with young tomato plants
cultured in the greenhouse. The growth rate and fresh/dry weight ratio
were analyzed as key crop yield factors (Liliane and Charles, 2020). EH,
EA, and HE increased fresh biomass with a relatively lower dry matter
percentage, in contrast to the HO treatment (Fig. 2b). EH application
showed the most robust stimulation of shoot growth rate (18 % higher
fresh weight) compared to HO, EA, and HE (Table S1). The high content
of lipids and lipid-like molecules in EH (Table 1) corroborates with a
recent study reporting that lipids-rich cyanobacteria extract from
wastewater promoted lettuce growth (Silambarasan et al., 2021). Lipids
and their monomers, fatty acids, are both energy sources and exocellular
and intracellular mediators in plant defense signaling (Lim et al., 2017).
As such, future studies on HE and EH to improve root and shoot
development will include bioactive fractionation and effectiveness
analysis under field conditions. Though we demonstrated plant
growth-promoting bioactivities in DDGS extracts, several criteria need
to be met before being considered as a biostimulant. Ricci et al. (2019)
proposed general principles of experimental data to justify biostimulant
claims following the European fertilizing product regulation. Together
with the justification and certification of safety and effectiveness, a
biostimulant is, therefore, qualified to enter the European single market
(European Commission, 2022).
3.3. Biopesticide activity in DDGS extracts
3.3.1. Fungicide and induced plant resistance
Activity against six economically important plant pathogens was
tested in vitro, Botrytis cinerea causing grey mold disease (Williamson
et al., 2007), Fusarium oxysporum causing vascular wilt diseases (Gor­
don, 2017), Rhizoctonia solani causing various root/stem diseases in
relation to the different anastomosis groups (Ajayi-Oyetunde et al.,
2018), Magnaporthe oryzae causing rice blast (Fernandez and Orth,
2018), Phytophthora porri causing white leek tip (Declercq et al., 2010),
and Pythium splendens causing blights and root rot (Caesar and Cech,
2019). HO and EH addition to the fungal growth medium exhibited
statistically significant in vitro fungicidal activity against M. oryzae
(Fig. 3a, Table S1). Previous studies reported that the organo­
heterocyclic compounds triterpenoid saponins and flavagline contribute
to the biocontrol of M. oryzae (Engelmeier et al., 2000; Ngo et al., 2019).
The relative abundance of organoheterocyclic compounds in HO and EH
suggests that similar functional molecules might be responsible for the
fungicidal activity observed (Table 1).
In addition to their direct fungicidal effects, the DDGS extracts were
also evaluated in planta for their potential to trigger a systemic induced
resistance playing a role as bioprotectant in the plant. In planta detached
leaf assays were used to test fungicidal activity against the widespread
plant pathogens Phytophthora infestans (late blight), Alternaria solani
(early blight), and Botrytis cinerea (grey mold) with their respective host
crops. HO and HE protected against P. infestans on potato, EH and HO
against A. solani, and only HO against B. cinerea on tomato but statisti­
cally significantly (Fig. 3b, Table S1). The host species specificity to
plant-pathogenic fungi would help narrow the targeted crop range (Li
et al., 2020). Additionally, the reason that HO exhibited the broadest
spectrum of fungicide activities is likely because it was extracted first,
resulting in maximal preservation of the aqueous phytochemical
mixture.
We also involve in planta induced resistance bioassay on roots of
Arabidopsis seedlings that were treated with DDGS extracts, and leaves
were infected three days later with Botrytis cinerea. The induced resis­
tance was evaluated by measuring the diameter of the leaf lesions in
DDGS extract-treated plants versus untreated ones. HO, EA, and HE
treatment decreased lesion diameter, albeit less pronounced than the
fungicide Mevalone (Fig. 4a, Table S1). Several studies have proven the
wide range of antifungal potency of direct botanic extracts from various
plant species (Santra et al., 2020), for example, seaweeds (Selim et al.,
2015) and bryophytes (Resour et al., 2022). Moreover, several phyto­
chemical priming agents exert plant-induce resistance to B. cinerea, such
as hexanoic acid (Finiti et al., 2014), isoleucine (Li et al., 2021), and
newly developed medicinal plant extract-based formulations (Hegedűs
et al., 2021). Bioactive molecules with similar functions in DDGS ex­
tracts should be further purified to confirm the responsible bioactivity.
To investigate the signaling pathways involved in plant-induced
resistance, the expression of PR1, a marker for salicylic acid (SA)
signaling, and PDF1.2a, a marker for jasmonate (JA) and ethylene (ET)
signaling, in Arabidopsis was determined using qRT–PCR (Thomma
et al., 1998). None of the tested extracts induced PR1 expression
(Fig. 4b, Table S1). However, strong induction of PDF1.2a expression
was observed for both EA and HE, suggesting that the decreased
B. cinerea lesion development was specific for

8
J. Li et al.
jasmonate/ethylene-mediated priming of plant resistance. The
well-characterized Arabidopsis plant defensin gene, PDF1.2a is
expressed upon pathogen attack by systematically activating jasmonate
and ethylene signaling pathways (De Coninck et al., 2010). In order to
further validate and unravel these results on signaling pathways medi­
ating DDGS extract-induced resistance, we plan to evaluate this activity
in Arabidopsis mutants impaired in these signaling pathways. Examples
are knock-out mutants in (i) the SID1(SALICYLIC ACID INDUCTION
DEFICIENT 1) and NPR1 (NONEXPRESSOR OF
PATHOGENESIS-RELATED PROTEINS 1) genes (Ding et al., 2018; Wil­
dermuth et al., 2001), that are impaired in SA biosynthesis and
perception, respectively, (ii) the MYC2 (JASMONATE INSENSITIVE 1)
encoding the master regulator of JA-signaling pathway (Wasternack and
Song, 2017), and (iii) the EIN2 (ETHYLENE INSENSITIVE 3) gene coding
for a positive regulator of the ET-signaling (Zheng and Zhu, 2016).
3.3.2. Acaricidal and insecticidal activity
The acaricidal activity was measured by determining the mortality
rate (Abbott, 1925) of Tetranychus urticae sprayed with DDGS extracts.
The insecticidal activity was measured by recording the reduction in
weight gain upon feeding Spodoptera littoralis with an
extract-supplemented artificial diet. Spraying of T. urticae with HO
extract caused severe mortality (60.31 %), while for HE, the effect was
small (9.55 %) but statistically significant compared to the blank control
(Fig. 5a, Table S1). Although EA addition to the artificial diet caused a
relative 12.98 % weight gain of S. littoralis larvae, this effect was not
significant (Fig. 5b, Table S1). Foliar contact and oral uptake treatment
with plant-origin biochemicals are the main strategies to biologically
control various invertebrate species, including arachnids and insects
(Kortbeek et al., 2018). The most-studied plant-specialized metabolites
that show insecticidal activity are isoprene-derived terpenoids, alka­
loids, and phenolic compounds (Boulogne et al., 2012). New insights to
expand the environmentally-benign bioacaricide and bioinsecticide
markets are required to reduce the risk level of resistance since only six
molecular target sites dominate the mechanisms of action of current
products (Jeschke, 2021).
3.3.3. Nematocidal activity
Root-knot and lesion nematodes rank first and third among the plant-
parasitic nematode species that cause significant crop yield losses
(Bernard et al., 2017). Nematocide activity was determined by in vitro
and in planta bioassays with the commercial nematocide Vertimec as the
positive control. HO with two higher doses showed significant in vitro
nematocidal activity against Meloidogyne graminicola and Pratylenchus
zeae (Fig. 6a, Table S1). In contrast, only the highest dose of EH and EA
exerted a considerable nematocidal effect against M. graminicola and
P. zeae, respectively, compared to the negative control. Linear dose
response in HO and EH was recorded, underlining the fact that subop­
timal doses were used. For the in planta induced resistance bioassay,
pre-treating with HO reduced 52.83 % of M. graminicola galls (Fig. 6b,
Table S1). HO showed the best nematocidal activity in both assays,
which was statistically different from the control. Naturally occurring
compounds of plant origin as bionematocides are more sustainable
(Desmedt et al., 2020). The observation that HO had no adverse effect on
the shoot and root development adheres to the concept that biocontrol
products typically show lower toxicity to plants. Similarly, four plant
extracts that were shown to suppress the root-knot nematode Meloido­
gyne incognita were found not to affect other soil organisms (D’Addabbo
et al., 2019). Regarding the dual use of bioactive substances, the
approval and authorization procedures should be applied to the Plant
Protection Products Regulation under the EU legislation regime (Clau­
dio, 2021; European Commission, 2009).
In preparation for the Bio2Bio screening platform, we speculated
that organic solvent extracts are likely to maintain more bioactivity in
line with the general conception that pesticide molecules tend to be on
the hydrophobic side of the hydrophobicity spectrum. Surprisingly,
9
Industrial Crops & Products 193 (2023) 116175
however, the water-based extracts were more bioactive in both bio­
stimulant and biopesticide assays (data not shown), and this was also the
case for DDGS. One of the reasons might be that we conducted a
sequential extraction on the by-products when water extraction was
carried out first and solubilized most of the compounds. Indeed, the HO
extract exhibited the most extensive range of bioactivities (fungicide,
acaricide, nematocide, and inducing plant resistance) and was the only
extract with a substantial effect on nematodes (M. graminicola and
P. zeae) and insects (T. urticae and S. littoralis), and fungi (R. solani and
B. cinerea) (Figs. 2–6). Compared to the organic DDGS extracts, HO was
richer in diverse compounds (Table S2). The high chemical complexity
of HO underscores the wide range of biopesticide activities observed.
Heterocyclic molecules are necessary agrochemical scaffolds accounting
for two-thirds of the crop protection agents (Lamberth, 2013). For
instance, piperidines and their derivatives, which we identified as the
most abundant in this superclass of HO, have been studied as natural
toxins affecting the viability of insects (Duke et al., 2010), arachnids,
and nematodes (Maienfisch et al., 2006). With ample evidence of effect,
biopesticides could be more competitive with synthetic pesticides due to
consumers’ and regulatory bodies’ push towards eco-friendly pest
management (Glare et al., 2012). However, low-abundant compounds
may be responsible for the bioactivities reported here. In this case,
fractionation of the DDGS extracts and bioactivity-guided chemical
identification will be required to determine the chemical structure of the
bioactive compound. It involves multiple rounds of bioassay-guided
fraction steps, in-depth chemical structural conformation, and
cross-validations to tackle this question (Nothias et al., 2018).
Knowing the dose-response relationship is crucial for the safe and
effective use of biologicals, including but not limited to biostimulants,
biopesticides, and bioprotectants (Calabrese, 2016). Many phytochem­
icals are reported to induce biphasic dose-response, namely, opposite
biological effects at different concentrations (Jodynis-Liebert and
Kujawska, 2020). As an example, auxin, the most studied phytohor­
mone, displays a typical bell-shaped dose-response curve when applied
in the maize coleoptile growth assay (Christian et al., 2008). Thus, we
included a series of dose treatments (three or four) in most Bio2Bio
bioassays to investigate the possible dose-responsive effects of the ex­
tracts. Next to the dose-response relationship, time-response is another
dimension required to comprehensively study the dynamic biological
activity of bioactive compounds (Gabrielsson et al., 2019). In addition, it
provides practical information for field dose and application timing.
4. Conclusions
The in-house developed Bio2Bio biostimulant and biopesticide
screening platform presented here demonstrates the advantages of
including a wide range of bioassays, pesticide residue analysis, chemical
profiling, and environmental safety tests (Graphic Abstract). The inte­
gration of the various datasets into a searchable database allowed for a
thorough comparison of the bioactivity of various organic waste-derived
extracts. Agricultural by-products turned out to be particularly active in
the screening bioassay platform and identified DDGS as a rich source of
plant growth promoting and biopesticide activity. The water extract
from DDGS consists of a complex mixture of metabolites and possesses
plant growth-promoting, fungicide, insecticide, and nematocide activity
against economically important pathogens and pests while displaying
limited ecotoxicity. In addition, we identified plant growth-promoting
activities in the hexane and ethanol DDGS extracts. The findings point
out that a by-product from a common staple crop such as maize is suf­
ficiently complex to harbor many bioactive molecules, suggesting that
DDGS is potentially a valuable source for developing novel bio­
stimulants and biopesticides that pave the way for circular agriculture.
Determining dose-repose activity following bioassay-guided fraction­
ation and isolation are key elements in future experiments to identify the
bioactive ingredients and optimize the application of these bioactive
substrates.
J. Li et al.
CRediT authorship contribution statement
Jing Li: Conceptualization, Methodology, Data curation, Formal
analysis, Visualization, Writing – original draft; Hoang Khai Trinh:
Investigation; Seyed Mahyar Mirmajlessi: Investigation, Writing – re­
view & editing; Geert Haesaert: Funding acquisition, Writing – review
& editing; Ramize Xhaferi: Investigation; Ilse Delaere: Investigation;
Monica Höfte: Funding acquisition, Writing – review & editing; Katrijn
Raymaekers: Investigation; Bruno P.A. Cammue: Funding acquisition,
Writing – review & editing; Wim Jonckheere: Investigation, Writing –
review & editing; Thomas Van Leeuwen: Funding acquisition; Lien De
Smet: Investigation; Tina Kyndt: Funding acquisition, Writing – review
& editing; Nathalie Bernaert: Funding acquisition, Writing – review &
editing; Christof Van Poucke: Investigation, Writing – review & edit­
ing; Bart Van Droogenbroeck: Funding acquisition; Emmy Pequeur:
Investigation; Karel De Schamphelaere: Funding acquisition, Writing –
review & editing; Gert Callebaut: Investigation; Timoty De Cleyn:
Investigation; Lin Xu: Investigation, Project administration, Writing –
review & editing; Aldana Ramirez: Conceptualization, Project admin­
istration, Writing – original Draft; Thijs Van Gerrewey: Project
administration, Writing – review & editing; Danny Geelen: Conceptu­
alization, Methodology, Supervision, Funding acquisition, Project
administration, Writing – review & editing.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
This research financially supported by Fonds Wetenschappelijk Onder­
zoek – Vlaanderen (FWO) under project Bio2Bio (S006017N). Jing Li
reports financial support by China Scholarship Council and Special
Research Fund of Ghent University (BOF). Gert Callebaut and Timoty De
Cleyn report a relationship with SA Ajinomoto OmniChem NV that in­
cludes: employment. Danny Geelen has patent NO. EP21152489.7
submitted to European Patent.
Data Availability
All data generated and analysed during this study are included in this
article and its supplementary materials.
Acknowledgment
We thank Pablo Vercruysse (Alco Bio Fuel, Belgium) for providing
DDGS raw material and Dr. Maaike Perneel (CropFit, Ghent University,
Belgium) for the strong support during the Bio2Bio project.
Data statement
All data generated and analyzed during this study are included in this
article and its supplementary materials.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.indcrop.2022.116175.
References
Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ.
Entomol. 18, 265–267. https://doi.org/10.1093/jee/18.2.265a.
Ajayi-Oyetunde, O.O., Bradley, C.A., Ajayi-Oyetunde, O.O., Bradley, C.A., 2018.
Rhizoctonia solani: taxonomy, population biology and management of rhizoctonia
seedling disease of soybean. Plant Pathol. 67, 3–17. https://doi.org/10.1111/
ppa.12733.
Balasubramanian, S., Tyagi, R.D., 2017. Biopesticide production from solid wastes. Curr.
Dev. Biotechnol. Bioeng.: Solid Waste Manag. 43–58. https://doi.org/10.1016/
B978-0-444-63664-5.00003-4.
10
Industrial Crops & Products 193 (2023) 116175
Batt, S., Grealis, T., Harmon, O., Tomolonis, P., 2020. Learning Tableau: a data
visualization tool. J. Econ. Educ. 51, 317–328. https://doi.org/10.1080/
00220485.2020.1804503.
Berg, E.L., 2021. The future of phenotypic drug discovery. Cell Chem. Biol. https://doi.
org/10.1016/j.chembiol.2021.01.010.
Bernard, G.C., Egnin, M., Bonsi, C., 2017. The impact of plant-parasitic nematodes on
agriculture and methods of control. Nematol. -Concepts, Diagn. Control 10. https://
doi.org/10.5772/intechopen.68958.
Bertier, L., Brouwer, H., de Cock, A.W.A.M., Cooke, D.E.L., Olsson, C.H.B., Höfte, M.,
2013. The expansion of Phytophthora clade 8b: three new species associated with
winter grown vegetable crops. Pers.: Mol. Phylogeny Evol. Fungi 31, 63–76. https://
doi.org/10.3767/003158513X668554.
Boulogne, I., Petit, P., Ozier-Lafontaine, H., Desfontaines, L., Loranger-Merciris, G., 2012.
Insecticidal and antifungal chemicals produced by plants: a review. Environ. Chem.
Lett. 2012 10:4 10, 325–347. https://doi.org/10.1007/S10311-012-0359-1.
Bridge, J., Luc, M., Plowright, R.A., 2005. Nematode parasites of rice. In: Sikora, R.,
Coyne, D., Hallmann, J, T.P. (Eds.), Plant Parasitic Nematodes in Subtropical and
Tropical Agriculture. CABI Publishing, Wallingford, UK, pp. 87–130.
Buysens, S., Heungens, K., Poppe, J., Hofte, M., 1996. Involvement of pyochelin and
pyoverdin in suppression of pythium-induced damping-off of tomato by
pseudomonas aeruginosa 7NSK2. Appl. Environ. Microbiol 62, 865–871. https://doi.
org/10.1128/AEM.62.3.865-871.1996.
Bybd, D.W., Kirkpatrick, T., Barker, K.R., 1983. An improved technique for clearing and
staining plant tissues for detection of nematodes. J. Nematol. 15, 142–143.
Caesar, L.K., Cech, N.B., 2019. Synergy and antagonism in natural product extracts:
when 1 + 1 does not equal 2. Nat. Prod. Rep. 36, 869–888. https://doi.org/10.1039/
C9NP00011A.
Calabrese, E.J., 2016. The Emergence of the Dose–Response Concept in Biology and
Medicine. Int. J. Mol. Sci. 17, 2034. https://doi.org/10.3390/IJMS17122034.
Capanoglu, E., Nemli, E., Tomas-Barberan, F., 2022. Novel approaches in the
valorization of agricultural wastes and their applications. J. Agric. Food Chem. 70,
6787–6804. https://doi.org/10.1021/acs.jafc.1c07104.
Chatzifragkou, A., Charalampopoulos, D., 2018. Distiller’s dried grains with solubles
(DDGS) and intermediate products as starting materials in biorefinery strategies, in:
Sustainable Recovery and Reutilization of Cereal Processing By-Products. Elsevier,
Cambridge, UK, pp. 63–86. https://doi.org/10.1016/B978–0-08–102162-
0.00003–4.
Christian, M., Hannah, W.B., Lüthen, H., Jones, A.M., 2008. Identification of auxins by a
chemical genomics approach. J. Exp. Bot. 59, 2757–2767. https://doi.org/10.1093/
JXB/ERN133.
Claudio, M., 2021. Biostimulants and Biopesticides Regulatory Landscape in light of
Regulation (EU) No 2019/1009 | Fieldfisher [WWW Document]. fieldfisher.com.
URL https://www.fieldfisher.com/en/locations/belgium/insights/plant-
biostimulant (accessed 10.25.22).
D’Addabbo, T., Laquale, S., Perniola, M., Candido, V., 2019. Biostimulants for plant
growth promotion and sustainable management of phytoparasitic nematodes in
vegetable crops, 616 9, 616 Agronomy 2019 Vol. 9. https://doi.org/10.3390/
AGRONOMY9100616.
De Coninck, B.M.A., Sels, J., Venmans, E., Thys, W., Goderis, I.J.W.M., Carron, D.,
Delauré, S.L., Cammue, B.P.A., De Bolle, M.F.C., Mathys, J., 2010. Arabidopsis
thaliana plant defensin AtPDF1.1 is involved in the plant response to biotic stress.
N. Phytol. 187, 1075–1088. https://doi.org/10.1111/J.1469-8137.2010.03326.X.
Declercq, B., Van Buyten, E., Claeys, S., Cap, N., De Nies, J., Pollet, S., Höfte, M., 2010.
Molecular characterization of Phytophthora porri and closely related species and
their pathogenicity on leek (Allium porrum. Eur. J. Plant Pathol. 127, 341–350.
https://doi.org/10.1007/s10658-010-9601-5.
Deltour, P., França, S.C., Heyman, L., Pereira, O.L., Höfte, M., 2018. Comparative
analysis of pathogenic and nonpathogenic Fusarium oxysporum populations
associated with banana on a farm in Minas Gerais, Brazil. Plant Pathol. 67, 707–718.
https://doi.org/10.1111/PPA.12757.
Dent, M., 2021. Biostimulants and Biopesticides 2021–2031: Technologies, Markets and
Forecasts [WWW Document]. IDTechEX Research. URL https://www.idtechex.com/
zh/research-report/biostimulants-and-biopesticides-2021–2031-technologies-
markets-and-forecasts/773 (accessed 12.29.21).
van der Werf, H.M.G., Knudsen, M.T., Cederberg, C., 2020. Towards better
representation of organic agriculture in life cycle assessment. Nat. Sustain. 2020 3 6
(3), 419–425. https://doi.org/10.1038/s41893-020-0489-6.
Desmedt, W., Mangelinckx, S., Kyndt, T., Vanholme, B., 2020. A phytochemical
perspective on plant defense against nematodes. Front Plant Sci. 11, 1765. https://
doi.org/10.3389/FPLS.2020.602079/BIBTEX.
Ding, Y., Sun, T., Ao, K., Peng, Y., Zhang, Yaxi, Li, X., Zhang, Yuelin, 2018. Opposite roles
of salicylic acid receptors NPR1 and NPR3/NPR4 in transcriptional regulation of
plant immunity. Cell 173, 1454–1467. https://doi.org/10.1016/J.
CELL.2018.03.044.
Duke, S.O., Cantrell, C.L., Meepagala, K.M., Wedge, D.E., Tabanca, N., Schrader, K.K.,
2010. Natural toxins for use in pest management. Toxins (Basel) 2, 1943–1962.
https://doi.org/10.3390/toxins2081943.
Duque-Acevedo, M., Belmonte-Ureña, L.J., Cortés-García, F.J., Camacho-Ferre, F., 2020.
Agricultural waste: Review of the evolution, approaches and perspectives on
alternative uses. Glob. Ecol. Conserv 22, e00902. https://doi.org/10.1016/J.
GECCO.2020.E00902.
EBIC, 2019. Industrial by-products used in plant biostimulants [WWW Document]. URL
https://biostimulants.eu/publications/industrial-by-products-used-in-plant-
biostimulants/ (accessed 1.6.22).
J. Li et al.
EBIC, 2021. EU Regulation Ensures that Biostimulants Are Safe and Effective [WWW
Document]. biostimulants.eu. URL https://biostimulants.eu/highlights/eu-
regulation-ensures-that-biostimulants-are-safe-and-effective/ (accessed 4.5.22).
Eden Research P.L.C., 2019. MEVALONE® is a unique biofungicide based on natural
plant chemistry [WWW Document]. URL https://www.edenresearch.com/products/
mevalone.aspx (accessed 1.5.21).
Engelmeier, D., Hadacek, F., Pacher, T., Vajrodaya, S., Greger, H., 2000. Cyclopenta[b]
benzofurans from Aglaia species with pronounced antifungal activity against rice
blast fungus (Pyricularia grisea). J. Agric. Food Chem. 48, 1400–1404. https://doi.
org/10.1021/JF990509H/ASSET/IMAGES/LARGE/JF990509HF00002.JPEG.
European Commission, 2005. Regulation (EC) No 396/2005 of the European Parliament
and of the Council of 23 February 2005 on maximum residue levels of pesticides in
or on food and feed of plant and animal origin and amending Council Directive 91/
414/EEC [WWW Document]. Official Journal of the European Union. URL https://
eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex %3A32005R0396 (accessed
11.10.21).
European Commission, 2009. Regulation (EC) No 1107/2009 of the European
Parliament and of the Council of 21 October 2009 concerning the placing of plant
protection products on the market and repealing Council Directives 79/117/EEC and
91/414/EEC [WWW Document]. Official Journal of the European Union. URL
https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=celex %3A32009R1107
(accessed 11.10.21).
European Commission, 2020. Farm to Fork targets - Progress [WWW Document].
Directorate-General for Health and Food Safety. URL https://ec.europa.eu/food/
plants/pesticides/sustainable-use-pesticides/farm-fork-targets-progress_nl (accessed
6.7.22).
European Commission, 2022. Safe and effective fertilisers on the EU market (from 2022)
[WWW Document]. European Union. URL https://eur-lex.europa.eu/EN/legal-
content/summary/safe-and-effective-fertilisers-on-the-eu-market-from-2022.html
(accessed 10.21.22).
European Union, 2019. Regulation (EU) 2019/1009 of the European Parliament and of
the Council of 5 June 2019 laying down rules on the making available on the market
of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC)
No 1107/2009 and repealing Regula [WWW Document]. Official Journal of the
European Union. URL https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?
uri=OJ:L:2019:170:FULL&from=NL (accessed 7.30.21).
FAO, 2014. Building a common vision for sustainable food and agriculture. Rome, Italy.
Faretra, F., Pollastro, S., 1991. Genetic basis of resistance to benzimidazole and
dicarboximide fungicides in Botryotinia fuckeliana (Botrytis cinerea. Mycol. Res 95,
943–951. https://doi.org/10.1016/S0953-7562(09)80091-9.
Fenibo, E.O., Ijoma, G.N., Matambo, T., 2021. Biopesticides in sustainable agriculture: a
critical sustainable development driver governed by green chemistry principles.
Front Sustain Food Syst. 5, 141. https://doi.org/10.3389/fsufs.2021.619058.
Fernandez, J., Orth, K., 2018. Rise of a cereal killer: the biology of Magnaporthe oryzae
biotrophic growth. Trends Microbiol 26, 582–597. https://doi.org/10.1016/j.
tim.2017.12.007.
Finiti, I., de la, O.Leyva, Vicedo, M., Gómez-Pastor, B., López-Cruz, R., García-Agustín, J.,
Real, P., González-Bosch, C, M.D., 2014. Hexanoic acid protects tomato plants
against Botrytis cinerea by priming defence responses and reducing oxidative stress.
Mol. Plant Pathol. 15, 550–562. https://doi.org/10.1111/MPP.12112/SUPPINFO.
Gabrielsson, J., Andersson, R., Jirstrand, M., Hjorth, S., 2019. Dose-response-time data
analysis: an underexploited trinity. Pharm. Rev. 71, 89–122. https://doi.org/
10.1124/PR.118.015750.
García-García, A.L., García-Machado, F.J., Borges, A.A., Morales-Sierra, S., Boto, A.,
Jiménez-Arias, D., 2020. Pure organic active compounds against abiotic stress: a
biostimulant overview. Front Plant Sci. 0, 1839. https://doi.org/10.3389/
FPLS.2020.575829.
Gaudêncio, S.P., Pereira, F., 2015. Dereplication: racing to speed up the natural products
discovery process. Nat. Prod. Rep. 32, 779–810. https://doi.org/10.1039/
c4np00134f.
Glare, T., Caradus, J., Gelernter, W., Jackson, T., Keyhani, N., Köhl, J., Marrone, P.,
Morin, L., Stewart, A., 2012. Have biopesticides come of age. Trends Biotechnol. 30,
250–258. https://doi.org/10.1016/j.tibtech.2012.01.003.
Glenn, N., 2020. Data Normalization Techniques: Easy to Advanced (& the Best) [WWW
Document]. Understand Data Analysis. URL https://developers.google.com/
machine-learning/data-prep/transform/normalization (accessed 1.27.22).
Gordon, T.R., 2017. Fusarium oxysporum and the Fusarium wilt syndrome. Annu Rev.
Phytopathol. 55, 23–39. https://doi.org/10.1146/annurev-phyto-080615-095919.
Grbić, M., van Leeuwen, T., Clark, R.M., Rombauts, S., Rouzé, P., Grbić, V., Osborne, E.J.,
Dermauw, W., Ngoc, P.C.T., Ortego, F., Hernández-Crespo, P., Diaz, I., Martinez, M.,
Navajas, M., Sucena, É., Magalhães, S., Nagy, L., Pace, R.M., Djuranović, S.,
Smagghe, G., Iga, M., Christiaens, O., Veenstra, J.A., Ewer, J., Villalobos, R.M.,
Hutter, J.L., Hudson, S.D., Velez, M., Yi, S. v, Zeng, J., Pires-Da Silva, A., Roch, F.,
Cazaux, M., Navarro, M., Zhurov, V., Acevedo, G., Bjelica, A., Fawcett, J.A.,
Bonnet, E., Martens, C., Baele, G., Wissler, L., Sanchez-Rodriguez, A., Tirry, L.,
Blais, C., Demeestere, K., Henz, S.R., Gregory, T.R., Mathieu, J., Verdon, L.,
Farinelli, L., Schmutz, J., Lindquist, E., Feyereisen, R., van de Peer, Y., 2011. The
genome of Tetranychus urticae reveals herbivorous pest adaptations. Nature 2011
479 7374 (479), 487–492. https://doi.org/10.1038/nature10640.
Hegedűs, G., Kiniczky, M., Nagy, Á., Pekker, P., Lang, B., Gracza, L., Pallos, J.P., Thomas-
Nyári, Z., Decsi, K., Kutasy, B., Székvári, K., Juhász, Á., Virág, E., 2021.
ELICE16INDURES ®: a plant immune-priming activator targeting jasmonate
metabolism via TIFY and RHOMBOID proteins in Hordeum vulgare. bioRxiv
2021.08.11.455979. https://doi.org/10.1101/2021.08.11.455979.
11
Industrial Crops & Products 193 (2023) 116175
Hendricks, K.E., Christman, M.C., Roberts, P.D., 2017. A statistical evaluation of methods
of in-vitro growth assessment for Phyllosticta citricarpa: average colony diameter vs.
area. PLoS One 12, e0170755. https://doi.org/10.1371/journal.pone.0170755.
Jeschke, P., 2021. Status and outlook for acaricide and insecticide discovery. Pest Manag
Sci. 77, 64–76. https://doi.org/10.1002/PS.6084.
Jodynis-Liebert, J., Kujawska, M., 2020. Biphasic dose-response induced by
phytochemicals: experimental evidence. J. Clin. Med 9, 718. https://doi.org/
10.3390/jcm9030718.
Kamaruzzaman, Md, Islam, Md.S., Mahmud, S., Polash, S.A., Sultana, R., Hasan, Md.A.,
Wang, C., Jiang, C., 2021. In vitro and in silico approach of fungal growth inhibition
by Trichoderma asperellum HbGT6-07 derived volatile organic compounds. Arab. J.
Chem. 14, 103290 https://doi.org/10.1016/j.arabjc.2021.103290.
Khajehali, J., Van Nieuwenhuyse, P., Demaeght, P., Tirry, L., Van Leeuwen, T., 2011.
Acaricide resistance and resistance mechanisms in Tetranychus urticae populations
from rose greenhouses in the Netherlands. Pest Manag Sci. 67, 1424–1433. https://
doi.org/10.1002/PS.2191.
Kilham, S.S., Kreeger, D.A., Lynn, S.G., Goulden, C.E., Herrera, L., 1998. COMBO: a
defined freshwater culture medium for algae and zooplankton. Hydrobiologia 377,
147–159. https://doi.org/10.1023/A:1003231628456.
Kim, Y., Mosier, N.S., Hendrickson, R., Ezeji, T., Blaschek, H., Dien, B., Cotta, M.,
Dale, B., Ladisch, M.R., 2008. Composition of corn dry-grind ethanol by-products:
DDGS, wet cake, and thin stillage. Bioresour. Technol. 99, 5165–5176. https://doi.
org/10.1016/J.BIORTECH.2007.09.028.
Kortbeek, Ruy W.J., Van Der Gragt, M., Bleeker, Petra, M., Kortbeek, R.W.J., Van Der
Gragt, M., Bleeker, P.M., 2018. Endogenous plant metabolites against insects. Eur. J.
Plant Pathol. 2018 154 1 (154), 67–90. https://doi.org/10.1007/S10658-018-1540-
6.
Lamberth, C., 2013. Heterocyclic chemistry in crop protection. Pest Manag Sci. 69,
1106–1114. https://doi.org/10.1002/ps.3615.
Li, J., Cornelissen, B., Rep, M., 2020. Host-specificity factors in plant pathogenic fungi.
Fungal Genet. Biol. 144, 103447 https://doi.org/10.1016/J.FGB.2020.103447.
Li, J., Evon, P., Ballas, S., Trinh, H.K., Xu, L., van Poucke, C., van Droogenbroeck, B.,
Motti, P., Mangelinckx, S., Ramirez, A., van Gerrewey, T., Geelen, D., 2022.
Sunflower Bark Extract as a Biostimulant Suppresses Reactive Oxygen Species in
Salt-Stressed Arabidopsis. Front Plant Sci. 13, 2083. https://doi.org/10.3389/
fpls.2022.837441.
Li, Y., Li, S., Du, R., Wang, J., Li, H., Xie, D., Yan, J., 2021. Isoleucine enhances plant
resistance against botrytis cinerea via jasmonate signaling pathway. Front Plant Sci.
12, 1738. https://doi.org/10.3389/FPLS.2021.628328/BIBTEX.
Liliane, T.N., Charles, M.S., 2020. Factors affecting yield of crops. Agron. -Clim. Change
Food Secur. 9. https://doi.org/10.5772/intechopen.90672.
Lim, G.H., Singhal, R., Kachroo, A., Kachroo, P., 2017. Fatty Acid– and Lipid-Mediated
Signaling in Plant Defense. https://doi.org/10.1146/annurev-phyto-
080516–035406 55, 505–536. https://doi.org/10.1146/ANNUREV-PHYTO-
080516–035406.
Loukas, S., 2020. Everything you need to know about Min-Max normalization: A Python
tutorial | by Serafeim Loukas | Towards Data Science [WWW Document]. medium.
com. URL https://towardsdatascience.com/everything-you-need-to-know-about-
min-max-normalization-in-python-b79592732b79 (accessed 8.16.22).
Lynch, J.P., 2022. Harnessing root architecture to address global challenges. Plant J. 109,
415–431. https://doi.org/10.1111/TPJ.15560.
Maienfisch, P., Molleyres, L., Cassayre, J., Cederbaum, F., Corsi, C., Pitterna, T., 2006.
WO2006003494-Piperidine derivatives and their use as insecticides, acaricides,
molluscicides or nematicides.
Malfroy, H., 2020. Biopesticides and biostimulants provide opportunity for sustainable
agriculture, but how are they regulated in the EU? [WWW Document]. Pen & Tec
Consulting. URL https://pentec-consulting.eu/biopesticides-and-biostimulants-
provide-opportunity-for-sustainable-agriculture-but-how-are-they-regulated-in-the-
eu/ (accessed 1.4.22).
Mathys, J., De Cremer, K., Timmermans, P., Van Kerckhove, S., Lievens, B.,
Vanhaecke, M., Cammue, B.P.A., De Coninck, B., 2012. Genome-wide
characterization of ISR induced in Arabidopsis thaliana by Trichoderma hamatum
T382 against Botrytis cinerea infection. Front Plant Sci. 3, 108. https://doi.org/
10.3389/FPLS.2012.00108/ABSTRACT.
Meng, L., Mestdagh, H., Ameye, M., Audenaert, K., Höfte, M., van Labeke, M.C., 2020.
Phenotypic Variation of Botrytis cinerea Isolates Is Influenced by Spectral Light
Quality. Front Plant Sci. 11, 1233. https://doi.org/10.3389/FPLS.2020.01233/
BIBTEX.
Murgia, M.V., Sharan, S., Kaur, J., Austin, W., Hagen, L., Wu, L., Chen, L., Scott, J.A.,
Flaherty, D.P., Scharf, M.E., Watts, V.J., Hill, C.A., 2022. High-content phenotypic
screening identifies novel chemistries that disrupt mosquito activity and
development. Pest. Biochem Physiol. 182, 105037 https://doi.org/10.1016/J.
PESTBP.2022.105037.
Najdabbasi, N., Mirmajlessi, S.M., Dewitte, K., Landschoot, S., Mänd, M., Audenaert, K.,
Ameye, M., Haesaert, G., 2020. Biocidal activity of plant-derived compounds against
Phytophthora infestans: An alternative approach to late blight management. Crop
Prot. 138, 105315 https://doi.org/10.1016/J.CROPRO.2020.105315.
Ngo, M.T., Han, J.W., Yoon, S., Bae, S., Kim, S.Y., Kim, H., Choi, G.J., 2019. Discovery of
new triterpenoid saponins isolated from maesa japonica with antifungal activity
against rice blast fungus magnaporthe oryzae. J. Agric. Food Chem. 67, 7706–7715.
https://doi.org/10.1021/ACS.JAFC.9B02236/ASSET/IMAGES/LARGE/JF-2019-
02236V_0003.JPEG.
Nothias, L.-F., Nothias-Esposito, M., da Silva, R., Wang, M., Protsyuk, I., Zhang, Z.,
Sarvepalli, A., Leyssen, P., Touboul, D., Costa, J., Paolini, J., Alexandrov, T.,
Litaudon, M., Dorrestein, P.C., 2018. Bioactivity-based molecular networking for the
J. Li et al.
discovery of drug leads in natural product bioassay-guided fractionation. J. Nat.
Prod. 81, 758–767. https://doi.org/10.1021/acs.jnatprod.7b00737.
OECD, 2004. Test No. 202: Daphnia sp. Acute Immobilisation Test. https://doi.org/
10.1787/9789264069947-en.
OECD, 2011. Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test.
https://doi.org/10.1787/20745761.
Petricka, J.J., Winter, C.M., Benfey, P.N., 2012. Control of Arabidopsis root
development. Annu Rev. Plant Biol. 63, 563–590. https://doi.org/10.1146/annurev-
arplant-042811-105501.
Pino-Otín, M.R., Ballestero, D., Navarro, E., González-Coloma, A., Val, J., Mainar, A.M.,
2019. Ecotoxicity of a novel biopesticide from Artemisia absinthium on non-target
aquatic organisms. Chemosphere 216, 131–146. https://doi.org/10.1016/J.
CHEMOSPHERE.2018.09.071.
Povero, G., 2020. Exploring Natural Resources for Biostimulants, in: Geelen, D., Xu, L.
(Eds.), The Chemical Biology of Plant Biostimulants. Wiley Online Library,
Chichester, UK, pp. 181–204. https://doi.org/10.1002/9781119357254.ch7.
Ravindran, R., Jaiswal, A.K., 2016. Exploitation of food industry waste for high-value
products. Trends Biotechnol. 34, 58–69. https://doi.org/10.1016/j.
tibtech.2015.10.008.
Resour, N., Joshi, S., Bhardwaj, P., Alam, A., 2022. Bryophytes as a safeguard of fruits
from postharvest fungal diseases: a review. Nat. Resour. Hum. Health 2, 327–334.
https://doi.org/10.53365/NRFHH/145476.
Ricci, M., Tilbury, L., Daridon, B., Sukalac, K., 2019. General principles to justify plant
biostimulant claims. Front Plant Sci. 10, 494. https://doi.org/10.3389/
fpls.2019.00494.
Santra, H.K., Banerjee, D., Santra, Kanti, Hiran, Banerjee, D., 2020. Natural products as
fungicide and their role in crop protection. Nat. Bioact. Prod. Sustain. Agric.
131–219. https://doi.org/10.1007/978-981-15-3024-1_9.
Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of
image analysis. Nat. Methods 9, 671–675. https://doi.org/10.1038/nmeth.2089.
Schoch, C.L., Ciufo, S., Domrachev, M., Hotton, C.L., Kannan, S., Khovanskaya, R.,
Leipe, D., McVeigh, R., O’Neill, K., Robbertse, B., Sharma, S., Soussov, V.,
Sullivan, J.P., Sun, L., Turner, S., Karsch-Mizrachi, I., 2020. NCBI Taxonomy: a
comprehensive update on curation, resources and tools. Database 2020. https://doi.
org/10.1093/DATABASE/BAAA062.
Selim, R., Ahmed, S.M., El-Zemity, S., Ramses, Moustafa, Y., 2015. Antifungal Activity
and Seasonal Variation of Green Alga (Ulva lactuca) Extracts. Asian J. Agric. Food
Sci. 3.
Silambarasan, S., Logeswari, P., Sivaramakrishnan, R., Kamaraj, B., Lan Chi, N.T.,
Cornejo, P., 2021. Cultivation of Nostoc sp. LS04 in municipal wastewater for
biodiesel production and their deoiled biomass cellular extracts as biostimulants for
Lactuca sativa growth improvement. Chemosphere 280, 130644. https://doi.org/
10.1016/J.CHEMOSPHERE.2021.130644.
Singh, R.R., Verstraeten, B., Siddique, S., Tegene, A.M., Tenhaken, R., Frei, M., Haeck, A.,
Demeestere, K., Pokhare, S., Gheysen, G., Kyndt, T., 2020. Ascorbate oxidation
activates systemic defence against root-knot nematode Meloidogyne graminicola in
rice. J. Exp. Bot. 71, 4271–4284. https://doi.org/10.1093/JXB/ERAA171.
Sparks, T.C., Hahn, D.R., Garizi, N.V., 2017. Natural products, their derivatives, mimics
and synthetic equivalents: role in agrochemical discovery. Pest Manag Sci. 73,
700–715. https://doi.org/10.1002/ps.4458.
Syngenta, 2021. Vertimec - Insecticide | Syngenta [WWW Document]. Syngenta. URL
https://www.syngenta.com.eg/product/crop-protection/vertimec (accessed 6.9.22).
Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A., Mauch-Mani, B., Vogelsang, R.,
Cammue, B.P.A., Broekaert, W.F., 1998. Separate jasmonate-dependent and
salicylate-dependent defense-response pathways in arabidopsis are essential for
resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA 95,
12
Industrial Crops & Products 193 (2023) 116175
15107–15111. https://doi.org/10.1073/PNAS.95.25.15107/ASSET/E332270E-
1444-40A9-ABD1-8951824DB019/ASSETS/GRAPHIC/PQ2481720005.JPEG.
Thuan, N.T.N., Bigirimana, J., Roumen, E., van der Straeten, D., Höfte, M., 2006.
Molecular and pathotype analysis of the rice blast fungus in North Vietnam. Eur. J.
Plant Pathol. 2006 114 4 (114), 381–396. https://doi.org/10.1007/S10658-006-
0002-8.
Trinh, H.K., Verstraeten, I., Geelen, D., 2018. In Vitro Assay for Induction of Adventitious
Rooting on Intact Arabidopsis Hypocotyls, in: Methods in Molecular Biology.
Humana Press Inc., pp. 95–102. https://doi.org/10.1007/978–1-4939–7747-5_7.
Tsiaka, T., Sinanoglou, V.J., Zoumpoulakis, P., 2017. Extracting bioactive compounds
from natural sources using green high-energy approaches: trends and opportunities
in lab-and large-scale applications, in: Grumezescu, A.M., Holban, A.M. (Eds.),
Ingredients Extraction by Physicochemical Methods in Food. Elsevier, Amsterdam,
pp. 307–365. https://doi.org/10.1016/B978–0-12–811521-3.00008–9.
Ugena, L., Hýlová, A., Podlešáková, K., Humplík, J.F., Doležal, K., Diego, N., De,
Spíchal, L., 2018. Characterization of biostimulant mode of action using novel multi-
trait high-throughput screening of arabidopsis germination and rosette growth.
Front Plant Sci. 9, 1327. https://doi.org/10.3389/fpls.2018.01327.
US Grains Council, 2012. A guide to distiller’s dried grains with solubles (DDGS).
Van Laecke, K., Degheele, D., 1993. Effect of insecticide—synergist combinations on the
survival of Spodoptera exigua. Pest. Sci. 37, 283–288. https://doi.org/10.1002/
PS.2780370308.
Waidmann, S., Sarkel, E., Kleine-Vehn, J., 2020. Same same, but different: growth
responses of primary and lateral roots. J. Exp. Bot. 71, 2397–2411. https://doi.org/
10.1093/JXB/ERAA027.
Wasternack, C., Song, S., 2017. Jasmonates: biosynthesis, metabolism, and signaling by
proteins activating and repressing transcription. J. Exp. Bot. 68, 1303–1321. https://
doi.org/10.1093/JXB/ERW443.
Wen, L., Zhang, Z., Sun, D.-W., Sivagnanam, S.P., Tiwari, B.K., 2020. Combination of
emerging technologies for the extraction of bioactive compounds. Crit. Rev. Food
Sci. Nutr. 60, 1826–1841. https://doi.org/10.1080/10408398.2019.1602823.
Wildermuth, M.C., Dewdney, J., Wu, G., Ausubel, F.M., 2001. Isochorismate synthase is
required to synthesize salicylic acid for plant defence. Nature 2001 414 6863 (414),
562–565. https://doi.org/10.1038/35107108.
Williamson, B., Tudzynski, B., Tudzynski, P., Van Kan, J.A.L., 2007. Botrytis cinerea: the
cause of grey mould disease. Mol. Plant Pathol. 8, 561–580. https://doi.org/
10.1111/j.1364-3703.2007.00417.x.
Xie, C., 2021. What do we find by comparing M&As in biostimulant and biopesticide
sectors? [WWW Document]. AgNews. URL https://news.agropages.com/News/
NewsDetail—37966.htm (accessed 1.12.22).
Xu, L., Geelen, D., 2018. Developing biostimulants from agro-food and industrial by-
products. Front Plant Sci. 9, 1567. https://doi.org/10.3389/fpls.2018.01567.
Yaquelyn, Y., Nerey Erey, N., Joke, J., Pannecoucque Annecoucque, P., Hector E ´ctor
P, H., Ablo H Hernandez Ernandez 2 2, P., Manuel, M., Diaz Iaz 2 2, D., Ray, R.,
Espinosa Spinosa 2, E., Tephanie, S., De, D., Vos Os, V., Arah V Van An B Beneden
Eneden 1 1, S., Lidcay, L., Herrera Errera, H., Monica, M., Hofte, H., 2010.
Rhizoctonia spp. Causing Root and Hypocotyl Rot in Phaseolus vulgaris in Cuba.
J. Phytopathol. 158, 236–243. https://doi.org/10.1111/J.1439-0434.2009.01609.X.
Yeo, S.K., Ewe, J.A., 2015. Effect of fermentation on the phytochemical contents and
antioxidant properties of plant foods. Adv. Ferment. Foods Beverage.: Improv. Qual.,
Technol. Health Benefits 107–122. https://doi.org/10.1016/B978-1-78242-015-
6.00005-0.
Zhang, X., Wang, X., Wang, M., Cao, J., Xiao, J., Wang, Q., 2019. Effects of different
pretreatments on flavonoids and antioxidant activity of Dryopteris erythrosora leave.
PLoS One 14, e0200174. https://doi.org/10.1371/journal.pone.0200174.
Zheng, Y., Zhu, Z., 2016. Relaying the ethylene signal: new roles for EIN2. Trends Plant
Sci. 21, 2–4. https://doi.org/10.1016/J.TPLANTS.2015.11.013.