Abstract
The increasing incidence of fungal phytopathogens poses a significant
challenge to agricultural sustainability, necessitating the development
of environmental alternatives to synthetic fungicides and mitigating
their ecological impact. This study explores the efficiency of
Nocardiopsis alba B57 to produce secondary metabolites with antifungal
and plant growth-promoting properties. Untargeted metabolomics using
ultra-high-performance liquid chromatography (UPLC-MS/MS) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified
key metabolites (e.g., carbapenem, menaquinone, and fumiquinazoline) in
the co-culture environment with fungal pathogens. Additionally,
principal component analysis and OPLS-DA differentiated monoculture and
co-culture metabolic profiles, revealed carbapenem biosynthesis as a
highly enriched pathway. The comprehensive metabolomics data and the
statistical analysis of the identified metabolites confirmed that
co-culturing of B57 and fungal strains showed upregulated metabolites
(e.g., carbapenem and menaquinone). However, other metabolites (e.g.,
mupirocin) were downregulated and significantly suppressed. These
changes in metabolic activity reflect the organism’s adaptive and
competitive responses during the co-culture conditions with fungal
pathogens and influence plant hormone signaling (e.g., auxin and
cytokinin), promoting plant growth and disease resistance. These
findings underscore B57’s adaptive biosynthetic response to co-culture
conditions, supporting its use as a sustainable biocontrol agent and
boosting crop productivity.
graphic file with name 41522_2025_796_Figa_HTML.jpg
Subject terms: Biological techniques, Microbiology
Introduction
Environmental sustainability has become a critical issue of the 21^st
century, necessitating a balance between economic growth, social
equity, and ecological preservation to mitigate the adverse impacts of
human activities on ecosystems^[40]1,[41]2. With the global population
projected to reach ~10 billion by 2050^[42]3, the agricultural sector
faces unprecedented challenges, including urban expansion, declining
soil quality, climate variability, and increasing pathogen resistance
to conventional treatments^[43]4. Plant diseases alone cause
substantial crop losses, exacerbating food insecurity. To address these
issues, many agricultural practices have relied heavily on chemical
pesticides and fungicides, such as dimetachlone^[44]5,[45]6 and
mancozeb^[46]7,[47]8. While effective in the short term, their overuse
presents significant environmental and health risks, including soil and
water contamination, reduced biodiversity, and chemical residues in
food^[48]9. Moreover, the reliance on these chemical agents undermines
sustainability goals by harming beneficial soil microorganisms and
contributing to environmental degradation.
Fungal pathogens (e.g., Fusarium spp. and Verticillium spp.) exemplify
the challenges to agricultural productivity and sustainability.
Fusarium species alone account for an estimated 14% reduction in annual
crop yields^[49]10, while Verticillium dahliae can cause yield losses
of up to 50% in susceptible crops such as potatoes and cotton^[50]11.
Managing these pathogens often necessitates increased use of chemical
fungicides, further exacerbating environmental issues. Effective
alternative strategies, including integrated pest management, crop
rotation, biopesticides, and the development of disease-resistant plant
varieties, are urgently needed to reduce chemical dependency and foster
sustainable agricultural practices^[51]12,[52]13. Recent research
emphasizes the use of biological antagonists, such as endophytic
bacteria, as eco-friendly biocontrol agents to combat plant diseases.
These bacteria establish symbiotic relationships with host plants and
produce bioactive compounds that enhance plant survival while
suppressing pathogens^[53]14. For instance, Thymus roseus, a medicinal
plant, has been identified as a source of endophytic actinobacteria
such as with broad-spectrum antimicrobial properties, offering
promising solutions for sustainable crop
protection^[54]1,[55]15,[56]16. Musa et al.^[57]15 found that
Streptomyces polyantibioticus isolated from T. roseus showed fungal
inhibition ratios of up to 67.1%, 64.2%, and 70.6% against Alternaria
solani, Valsa malicola, and Valsa mali, respectively. Additionally,
Abdelshafy Mohamad et al.^[58]1 successfully isolated Streptomyces
albidoflavus from T. roseus with antifungal activity against Fusarium
spp.
Emerging biotechnologies and metabolomics have revolutionized the
discovery of novel biocontrol agents. Metabolomics provides a
comprehensive framework for identifying and profiling metabolites,
offering insights into biochemical interactions between plants and
pathogens^[59]17. Metabolomics has facilitated the development of
biopesticides derived from bacterial strains (e.g., Streptomyces spp.),
which produce bioactive compounds and secondary metabolites with
antifungal properties^[60]15,[61]18. Commercial products (e.g.,
Actinovate, Mycostop, and Streptomycin) demonstrated the potential of
these biocontrol agents to manage soil-borne pathogens and promote
plant health^[62]19,[63]20. Moreover, advanced metabolomics techniques
enable the identification of novel bioactive compounds that enhance
plant resilience and suppress pathogens. By leveraging these tools,
researchers can pave the way for innovative, sustainable strategies to
control plant diseases, reduce chemical pesticide usage, and ensure
agricultural productivity in the face of growing global challenges.
Endophytic bacteria, such as Nocardiopsis alba B57, can produce
secondary metabolites as reported in our previous study^[64]15, which
possess the potential to control fungal pathogens and promote plant
growth. Therefore, this study aims to explore the ability and
underlying mechanisms of N. alba B57 to promote plant growth and
mitigate plant diseases. Specifically, this study sought to
comprehensively elucidate the metabolite composition of B57 and their
dynamic variations in response to antagonistic interactions with
specific fungal phytopathogens, employing cutting-edge untargeted
metabolomics analysis. Furthermore, the growth-promoting and biocontrol
efficacy of B57 crude extract was systematically evaluated. This
research provides the first scientific basis for an untargeted
metabolomics approach to exploring the antagonistic activity of crude
extracts from B57. In addition, this investigation is significant
because it represents a pioneering application of untargeted
metabolomics to unveil the antagonistic properties of B57, offering
novel insights into its potential as a sustainable biocontrol agent and
plant growth enhancer, contributing to sustainable agriculture, and
enhancing productivity within ecosystems.
Results and discussion
Comparative analysis of metabolomic profiles in N. alba B57
Microorganisms possess the ability to produce a wide range of
metabolites in response to varying ecological conditions^[65]1,[66]21.
In this study, B57 was subjected to metabolomics evaluation to detect
metabolic changes and provide a detailed understanding of the complex
interactions between N. alba and fungal pathogens, leading to the
development of effective, sustainable, and eco-friendly biocontrol
strategies. The interactions between B57 and plant pathogens (F1, F2,
F3, and F4) encoded as (B-Fungi) were analyzed using ultra-performance
liquid chromatography-mass spectrometry (UPLC-MS/MS), which employs
comprehensive specific metabolomics in negative and positive ionization
modes. The heatmap illustrates the Spearman rank correlation (SRC)
coefficients between B-Fungi compared to fungi groups as a control, as
shown in Fig. [67]1. The correlation values range from 0.5 to 1.0, with
darker blue shades indicating stronger positive monotonic
relationships. The diagonal values, which represent self-correlations,
are consistently 1.000.
Fig. 1.
[68]Fig. 1
[69]Open in a new tab
Spearman rank correlation coefficient analysis between Nocardiopsis
alba B57 and various plant fungal pathogens.
The significant strong correlations that occurred within the B-Fungi
showed notable correlation values, such as 0.652 between B57 and F1-57,
and 0.835 between F1-57 and F3-57, suggesting a significant
interdependency among these strains, as shown in Fig. [70]1. This
analysis indicates that the robust interactions between N. alba and
fungal strains. Conversely, the correlations between B-Fungi and the
control group were insignificant. Spearman’s rank correlation further
clarified B57’s antagonistic impact on the tested fungi, revealing a
strong positive association between B57 and F1, F2, F3, and F4 at
0.652, 0.674, 0.623, and 0.654, respectively (Fig. [71]1). Network
correlation analysis underscored specific, highly repeatable microbial
interactions with each fungal species.
Ghosh et al.^[72]22, stated that the metabolomics assay assists in the
monitoring of chemical variations in the biosystems’ metabolome,
presenting a deep understanding of the effect of fungal interactions on
the diverse metabolite profile of actinobacteria (e.g., N. alba). This
was aligned with Mohamed et al.^[73]16, who found that actinobacteria
can generate different plant growth-promoting (PGP) traits, including
phosphate solubilization (24%), auxin (88%), siderophore production
(94%), and ammonia (96%) with 45% and 26% inhibition effect against
Fusarium oxysporum and for Verticillium dahlia, respectively. This
could be because microbial interactions can activate antimicrobial
compound production as a defense strategy. In addition, activates
different biosynthetic gene clusters (BGCs) depending on the presence
of competing organisms^[74]23. Moreover, Rutledge and Challis^[75]24
highlighted the role of interspecies interactions in awakening silent
gene clusters, often via cross-species signaling or stress-induced
pathways.
This study leverages metabolomics and bioinformatics to investigate the
links between microbial metabolites and their interactions with
phytopathogens. Metabolomics can track chemical variations within the
metabolome of B57 and provide insights into its fungal interaction
mechanism^[76]1.
Multivariate statistical analysis of metabolomic interactions between N. alba
B57 and fungal pathogens
The multivariate statistical analyses in Fig. [77]2 offer a detailed
exploration of the metabolic interactions between B57 and the tested
fungal pathogens. These analyses utilize principal component analysis
(PCA) and orthogonal partial least squares-discriminant analysis
(OPLS-DA) to uncover metabolic variations and relationships between N.
alba B57 and fungal strains. The PCA score plot revealed distinct
clustering patterns, with PC1 and PC2 explaining 20.61 and 18.43% of
the variance, respectively (Fig. [78]2A). A clear variation was
observed between the B-Fungi group (co-culture samples) and the fungi
group (monoculture samples). The B-Fungi group forms a compact cluster,
while the fungi group showed wider dispersion, suggesting high
variability in their metabolic profiles. Within the B-Fungi cluster,
samples F1-57, F2-57, F3-57, and F4-57 exhibited close clustering,
indicating metabolic consistency in their interactions with B57. B57 is
distinctly positioned along the PC2 axis, signifying a unique metabolic
profile that may reflect specialized biosynthetic activity. This
distinctiveness highlights the organism’s potential influence of
organisms on metabolite production during co-culture.
Fig. 2. Multivariate statistical analysis of B57 with the tested fungal
strains.
[79]Fig. 2
[80]Open in a new tab
PCA illustrates the clustering of B57 and the fungal pathogens based on
their metabolic profiles. The ellipses represent 95% confidence
intervals, with the B-Fungi subgroup in red and the fungi group in
green (A). OPLS-DA scores demonstrated strong separation between the
B-Fungi and fungi group as a control, indicating distinct metabolic
characteristics and relationships. The model performance metrics (R2X,
R2Y, Q2Y, and RMSEE) are displayed, indicating the robustness and
reliability of the analysis (B).
The PCA analysis for co-cultured and monoculture samples suggests that
B57 may produce specific metabolites (e.g., carbapenem, menaquinone,
and fumiquinazoline) against fungal pathogens. The general description
of compact clustering of the B-Fungi group (Fig. [81]2A) indicates a
consistent metabolic response when interacting with multiple fungal
strains, which could point to a robust defense mechanism that could be
harnessed for developing resistant plant varieties or natural
fungicides. The PCA results align with the data obtained by Ma et
al.^[82]25, who reported distinct clustering between co-culture and
monoculture samples, indicating that unique chemical compositions are
driven by microbial interactions. Similarly, Shi et al.^[83]26 observed
clear metabolite differentiation between co-culture and monoculture
groups, further supporting the role of chemically mediated interactions
in modulating biosynthetic pathways and enhancing secondary metabolite
production. The obtained data in this study collectively underscore the
influence of B57 in shaping metabolite profiles through its
interactions with fungal pathogens, emphasizing its potential
ecological and biotechnological relevance.
The OPLS-DA further substantiates these findings through a supervised
approach, enhancing the separation between predefined groups (Fig.
[84]2B). The OPLS-DA plot distinctly differentiates the B-Fungi group
(clustered in the bottom-left quadrant) from the fungi group (clustered
in the top-right quadrant), highlighting strong discriminatory
metabolites between these categories. The model demonstrates robust
reliability, as evidenced by high-performance metrics: R^2X = 0.323,
R^2Y = 0.999, and Q^2Y = 0.648. These values indicate excellent model
fitness and predictive power. Additionally, the tight clustering of
samples within each group suggested consistent metabolite profiles,
while the clear separation between B-Fungi and fungi emphasized unique
metabolic interactions with B57. Strong discriminatory metabolite
identification through OPLS-DA highlights potential targets for further
research into bioactive compounds produced by B57. Understanding how
microbial metabolites are produced in response to fungal pathogens
could lead to the development of biopesticides or other agricultural
products that mimic these natural defenses^[85]27 providing an
eco-friendly alternative to synthetic fungicides.
Collectively, the PCA and OPLS-DA analyses revealed that B57 exhibited
significant metabolic correlations with the co-culture (B-Fungi) group
compared to the fungi group (control group). This suggests a more
effective influence or interaction at the metabolite level. The
distinct positioning of B57 in the PCA plot underscores its unique
metabolomic profile, due to specialized metabolite production in
response to fungal pathogens. The successful discrimination of groups
achieved by the OPLS-DA model provides a solid foundation for exploring
specific metabolic pathways and compounds responsible for these
interactions. From a biological perspective, the obtained data
highlight the potential of B57 as a biocontrol agent against pathogenic
fungal strains. The clear metabolic distinctions and interactions
observed suggest promising applications in agricultural settings.
Further investigation into the specific pathways and metabolomic
profiles of B57 could uncover the mechanisms underlying its
specificity, potentially leading to the development of more effective
and targeted biocontrol strategies in agriculture.
An in-depth analysis of differential metabolites between monoculture
and co-culture reveals quantitative variations, highlights significant
metabolites, and visualizes their overall changes based on fold change
(FC) values, as illustrated in Fig. [86]3. The bar plot highlights the
top 10 upregulated and top 10 downregulated metabolites, expressed as
log2FC values. Among the upregulated metabolites, carbapenem
([87]C20821), menaquinone, and
(3S,4R)-3,4-dihydroxycyclohexa-1,5-diene-1,4-dicarboxylate showed the
highest FCs, with log2FC values of 6.7, 6.11, and 5.65, respectively,
as shown in Fig. [88]3. These metabolites may represent compounds whose
production is enhanced due to specific interactions in the co-culture.
In contrast, among the downregulated metabolites,
PS(18:1(11Z)/16:1(9Z)), ethylglyoxalbis(guanylhydrazone), and mupirocin
were the most significantly suppressed, with log2FC values of −4.24,
−3.74, and −3.46, respectively. These shifts suggest changes in
metabolic activity, reflecting the organism’s adaptive or competitive
responses during co-culture conditions.
Fig. 3. Comparative fold change analysis of the changes in the quantitative
data of the main upregulated and downregulated metabolites in N.alba B57 and
fungi co-culture system.
[89]Fig. 3
[90]Open in a new tab
The fold change highlights the differential expression of metabolites,
emphasizing the impact of co-cultivation on the metabolic profile of
B57. Each bar represents the magnitude of change, providing insights
into the specific metabolites that are significantly influenced by the
co-culture environment.
The variations in the expression of metabolites by plotting their log2
fold change (log2FC) values against their statistical significance
(-log10(p-value)), as shown in the volcano plot Fig. [91]4A. This
analysis identified unchanged metabolites (n = 3792) and key
metabolites that undergo significant changes in abundance during
co-culture. The upregulated metabolites (n = 19), such as
12-keto-leukotriene B4 and Lys-Val-Gly, are positioned prominently on
the right side of the plot, indicating substantial increases in their
abundance. These metabolites also display high variable importance in
projection (VIP > 1.4) values, emphasizing their strong contribution to
the metabolic distinctions between co-culture and monoculture
conditions. Conversely, downregulated metabolites, such as lubiprostone
(RU-0211) and phosphatidylserine (PS(16:1(9Z)/17:0)), appeared on the
left, reflecting significant decreases in abundance. Their elevated VIP
scores further validated their critical role in distinguishing
metabolic states. Most metabolites, represented in gray, cluster around
the center of the plot with log2FC values near zero and low VIP scores,
indicating their stability across the experimental conditions.
Together, the volcano plot highlights a subset of metabolites with both
high statistical significance and biological relevance.
Fig. 4. Metabolic profile variations in Nocardiopsis alba.
[92]Fig. 4
[93]Open in a new tab
A The volcano plot revealed variations in metabolites at (p < 0.01). B
Spider chart of the top ten metabolites ranked by log2FC values.
The spider chart complements (Fig. [94]4B) the volcano plot by
showcasing the proportional abundance of the top 10 metabolites with
the top absolute log2FC values. Upregulated metabolites, such as
menaquinone and
(3S,4R)-3,4-dihydroxycyclohexa-1,5-diene-1,4-dicarboxylate, exhibit
significant positive log2FC values and high VIP values, suggesting
enhanced biosynthetic activity in secondary metabolism pathways. These
metabolites may contribute to increased antimicrobial or competitive
capabilities under co-culture conditions. Notably, carbapenem showed
strong upregulation, reinforcing its potential role in antibiotic
production. In contrast, downregulated metabolites such as mupirocin
and PS(18:1(11Z)/16:1(9Z)) (phosphatidylserine; C[40]H[74]NO[10]P)
display considerable suppression, which may reflect adaptive strategies
to reduce antagonistic interactions and promote co-culture synergy.
Kenis et al.^[95]28 stated that the phosphatidylserine upregulation at
the cell surface functions as an “eat me” flag toward phagocytes and is
a part of the membrane dynamics of apoptosis. Thus, in this study,
phosphatidylserine downregulation refers to the survival and
adaptability of the bacterial cells.
By providing a comparative view of metabolite abundance, the spider
chart underscores the dynamic regulation of specific metabolic pathways
in response to co-culture conditions. The results showed that
metabolites associated with B57 and their differential expression
during co-culture conditions play crucial roles in both antifungal
activity and plant growth promotion. One of the prominent metabolites,
carbapenem (β-lactam antibiotic), is recognized for its broad-spectrum
antimicrobial properties, particularly against Gram-negative bacteria
and certain fungi. The observed upregulation of carbapenem in
co-culture conditions indicates its potential to enhance the plant’s
defense mechanisms against fungal pathogens by effectively inhibiting
their growth and proliferation.
This characteristic positions carbapenem as a significant player in the
plant’s biochemical arsenal against infections. Therefore, the
metabolomic analysis, as shown in Fig. [96]5, illustrated the intricate
biosynthetic pathway of carbapenem antibiotics, emphasizing the pivotal
roles of amino acids such as arginine and proline in the synthesis
process. This pathway involves a cascade of enzymatic reactions and
metabolic intermediates, showcasing the biochemical complexity
underlying the production of carbapenems. Carbapenem biosynthesis is
initiated through metabolic precursors, particularly L-glutamate and
L-proline, which are derived from the metabolism of arginine and
proline. These amino acids serve as fundamental building blocks,
linking basic metabolic pathways to the specialized biosynthesis of
carbapenems. This reliance on essential amino acid pathways highlights
the evolutionary efficiency of microbial systems in repurposing primary
metabolites for secondary metabolite production.
Fig. 5.
[97]Fig. 5
[98]Open in a new tab
Different carbapenems biosynthesis pathways and the integrated enzymes
and genes in B57.
A series of specialized enzymes catalyze critical transformations
throughout the pathway. Key enzymes such as carboxymethylproline
synthase (CarDE, CarB), and thienamycin genes (ThnE) play essential
roles, particularly in modifying L-proline to generate
carbapenem-specific intermediates, such as (2S,
5S)-trans-carboxymethylproline. These enzymatic steps ensure the
structural specificity required for the bioactivity of carbapenems.
Additionally, the involvement of enzymes such as ThnF and ThnG, which
regulate subsequent modifications, demonstrates the complexity and
precision of this biosynthetic process. The pathway features a variety
of intermediates, including L-glutamyl-P, L-glutamate 5-semialdehyde,
and diverse thienamycin derivatives such as 2,3-dihydrothienamycin and
N-acetylthienamycin. These intermediates signify systematic assembly
and progressive modifications that culminate in the formation of the
carbapenem core structure. The presence of multiple thienamycin
derivatives underscores the substrate variability and the potential to
generate bioactive compounds with distinct pharmacological profiles.
Such modifications may influence the spectrum and potency of the
resulting antibiotics.
The enzymatic activities within this pathway are subject to
sophisticated regulatory mechanisms, including feedback inhibition and
environmental modulation. For instance, the activity of key enzymes may
be fine-tuned by the cellular metabolic state or external stimuli,
optimizing antifungal production under varying conditions.
Understanding these regulatory networks could provide insights into how
microbial systems adapt biosynthetic outputs to meet survival demands.
The detailed enzymatic and metabolic insights revealed by this pathway
pave the way for synthetic biology innovations. By engineering
microbial strains or manipulating biosynthetic enzymes, researchers can
potentially enhance antibiotic yields or create new antibiotics with
improved efficacy and stability.
Carbapenem antibiotics are among the most effective agents against
multidrug-resistant bacterial infections, making them indispensable in
different applications. Also, FDA-approved lipopeptides daptomycin and
dalbavancin, and novel carbapenems, such as thienamycin, are precursors
of antifungals such as nystatin and candicidin^[99]29. A deeper
understanding of their biosynthesis could facilitate bioengineering
approaches to enhance production efficiency or design novel derivatives
capable of overcoming different plant pathogens. Another metabolite,
menaquinone (Vitamin K2), is involved in various biological processes,
including electron transport in bacteria^[100]30. Menaquinones play a
significant role in the growth and development of plants. This vitamin
K2 variant is primarily synthesized by certain bacteria and has
implications for plant health and nutrient cycling. Menaquinone is
involved in the electron transport chain, which is crucial for cellular
respiration in plants to facilitate energy production by acting as an
electron carrier, thus enhancing the efficiency of photosynthesis and
respiration processes^[101]31. Menaquinones are known to regulate
calcium levels within plant cells. This regulation is vital for various
physiological processes, including cell division, elongation, and
overall growth^[102]31. Proper calcium levels contribute to the
structural integrity of plant cells and influence nutrient uptake.
Environmental stressors such as salinity or drought can negatively
impact plant growth. Menaquinone has been shown to enhance stress
tolerance by modulating metabolic pathways that help plants adapt to
adverse conditions. For instance, studies indicate that the synthesis
of MK-7 can be stimulated under stress conditions, leading to improved
resilience^[103]31,[104]32. Menaquinones are not produced by plants
themselves; instead, they are synthesized by certain bacteria that can
be found in the soil or associated with plant roots. The presence of
beneficial bacteria such as Bacillus subtilis can enhance the
availability of MK-7 to plants, thereby promoting their growth and
health^[105]31,[106]32.
Mupirocin, which is primarily recognized for its antibacterial
properties, showed downregulation in co-culture settings. This
reduction suggests a strategic adaptation that may minimize certain
defensive responses that could inhibit beneficial interactions with
co-cultured organisms. By downregulating mupirocin, plants may foster a
more synergistic relationship with other microbes, promoting overall
plant health and resilience against pathogens. However,
(3S,4 R)-3,4-dihydroxycyclohexa-1,5-diene-1,4-dicarboxylate has limited
specific studies available. However, similar metabolites are often
associated with antimicrobial properties. This compound may indicate
enhanced metabolic pathways that bolster antifungal activity or
facilitate plant defense responses during co-culture conditions. Its
role underscores the complexity of metabolic interactions that occur
within these environments. In addition to their antifungal roles,
several metabolites also contribute to plant growth promotion.
Siderophores chelate iron from the environment, increasing its
bioavailability to plants. This enhanced iron uptake supports plant
growth and improves resilience against environmental stressors by
ensuring a sufficient nutrient supply^[107]33. The upregulation of
metabolites such as menaquinone is indicative of enhanced secondary
metabolism pathways that can lead to increased production of
growth-promoting substances or antimicrobial compounds beneficial for
plants^[108]34. This dynamic regulation of metabolic pathways reflects
the adaptability and responsiveness during co-culture conditions.
The differential expression of metabolites during co-culture highlights
their dual roles in antifungal activity and plant growth promotion,
demonstrating the adaptive biosynthetic versatility of B57. The
upregulation of compounds such as menaquinone and carbapenem suggests
enhanced secondary metabolism, contributing to pathogen inhibition and
microbial interactions, while simultaneously supporting plant health
through growth-promoting activities. A deeper understanding of their
biosynthesis could facilitate bioengineering approaches to enhance
production efficiency or design novel derivatives capable of overcoming
different plant pathogens. Conversely, the downregulation of
metabolites (e.g., mupirocin) may reflect a strategy to minimize
antagonism, fostering a cooperative environment within the metabolic
interaction systems. These findings underscore the potential of B57 as
a bioinoculant for sustainable agriculture, offering innovative
solutions for disease management and crop productivity enhancement. The
identified metabolites, particularly those with high VIP and log2FC
values, could serve as biomarkers for metabolic shifts and as leads for
novel antibiotic discovery^[109]1. Moreover, the dynamic regulation of
these metabolites points to promising applications in microbial
consortia optimization, where metabolic engineering could amplify
desired traits, ultimately contributing to more resilient and efficient
agricultural systems capable of thriving under challenging conditions.
KEGG metabolic pathways
Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways,
metabolic pathway analysis was conducted to interpret the biological
relevance of significantly altered metabolites detected through
untargeted metabolomics. This approach enabled the identification of
enriched biosynthetic and signaling pathways involved in antifungal
activity and plant growth promotion. Different metabolic pathways and
plant hormone signal transduction were specifically explored using
MetaboAnalyst 5.0 and KEGG Mapper to visualize the functional roles of
B57-derived metabolites under monoculture and co-culture conditions.
The investigation of detailed metabolomic comparison of B-Fungi and
fungi was performed using a combination of Z-score analysis and
hierarchical clustering. The analysis highlights the metabolic
differences between these groups, identifying specific metabolites and
their relative abundances. The individual metabolites are plotted with
their corresponding Z-scores (Fig. [110]6A), differentiating the
metabolomic profiles of the B-Fungi (red) and fungi group (blue). A
clear distinction in metabolite abundance was observed. For example,
metabolites such as ethylglyoxalbis (guanylhydrazone), menaquinone, and
veratramine were more abundant in the B-Fungi group, with significantly
elevated Z-scores. Several other metabolites, including canthaxanthin
and butyric acid, were more abundant in the control group than in the
B-Fungi group. The Z-score distribution reflects significant metabolic
variation, indicating distinct biochemical processes or environmental
adaptations between the two groups. This visualization underscores the
role of specific metabolites in differentiating these two
fungal-related groups, potentially concerning their metabolic pathways
or ecological niches.
Fig. 6. Heatmap analysis of the detected compounds in B57 versus co-culture.
[111]Fig. 6
[112]Open in a new tab
Hierarchical clustering of the detected 48 metabolites (A). The
standardized quantitative value of the Z-score for each metabolite
classified all metabolic spectra (B). Ordered main matrix heatmap of
upregulated compounds across tested groups (C).
The heatmap in Fig. [113]6B provides a comprehensive visualization of
the metabolomic profiles of the two groups. Each column represents a
sample, and each row corresponds to a metabolite. The color spectrum
provides insights into the expression levels and clustering patterns.
Cluster analysis revealed a distinct grouping of B-Fungi and fungi,
emphasizing metabolic dissimilarities. The clear separation suggests
robust differences in metabolome composition. Metabolites are clustered
based on co-expression patterns, with some metabolites consistently
expressed at relatively high levels in B-Fungi, while others are
predominantly elevated in the fungi group. High variance in metabolite
levels reflects biochemical diversity, potentially influenced by
genetic, environmental, or functional differences between the two
fungal-related groups. The obtained data demonstrates the comparative
analysis of KEGG metabolic pathways in B57 under monoculture and
co-culture growth conditions, focusing on the top metabolites with the
highest number of differential annotations, which could influence plant
growth promotion and pathogen inhibition.
The ordered main matrix heatmap showed that B57 generates distinct
metabolites under different antagonistic conditions when co-cultured
with fungal pathogens, as shown in Fig. [114]6C. The levels of
metabolites vary significantly depending on the interaction type
(monoculture vs. co-culture with specific fungi). Many metabolites,
such as tryptophyl-Lysine, zeatin, 13,14-dihydro-15-keto-PGF2α, and
1,7-diphenyl-4-hepten-3-one, are present at significantly higher levels
in co-culture (e.g., F1-57, F2-57, F3-57, F4-57) compared to
monoculture of B57 and individual fungal strains. For instance,
tryptophan-lysine and 1,7-diphenyl-4-hepten-3-one were increased in
F4-57 co-culture. Also, zeatin drastically increased in F3-57. Certain
metabolites that are absent or present in negligible quantities in
monoculture conditions appear under co-culture. [115]C20821 (involved
in carbapenem biosynthesis), this metabolite showed a massive
upregulation in co-culture, particularly in F3-57, compared to
negligible levels in monoculture, showing its production is stimulated
by fungal interaction. Similarly, [116]C20823 (involved in carbapenem
biosynthesis), this compound is not detected in B57 monoculture but is
upregulated across co-culture conditions, indicating its inducible
production. Menaquinone, which contributes to plant growth and pathogen
defense, is highly produced in monoculture but decreases during
co-culture. However, its levels remain substantial, particularly in
F3-57, indicating its dual role. Some metabolites are downregulated in
co-culture, possibly to modulate antagonistic interactions or focus
resources on critical antifungal activities. Mupirocin was almost
absent in co-culture, suggesting that it may not be critical for fungal
suppression in the case of this study. Lumichrome was also
significantly reduced in co-culture compared to monoculture, as listed
in Supplementary data Table [117]S1 and Fig. [118]S1. The interactions
with specific fungi influence metabolite profiles. The
13,14-dihydro-15-keto-PGF2α peak in F4-57 indicates that this
metabolite is strongly induced by V. dahlia interactions. However, the
content of Tryptophyl-Lysine increased in F3-57, reflecting a tailored
metabolic response. B57 adapts its metabolic profile based on the
fungal species it interacts with, producing different bioactive
metabolites with antifungal or plant-growth-promoting properties. These
findings suggest that fungal interactions act as a trigger for the
biosynthesis of key secondary metabolites, such as carbapenem,
menaquinone, and tryptophyl-Lysine, enhancing the strain’s biocontrol
potential of the strain.
Several studies have highlighted the diverse roles of bacterial
metabolites as biofungicides, demonstrating their varying effectiveness
against different fungal pathogens in antagonistic
relationships^[119]12,[120]13,[121]35. For instance, Bacillus
velezensis produces lipopeptides (e.g., fengycin and iturin), which
have shown significant activity against pathogens such as Ralstonia
solanacearum and Fusarium oxysporum^[122]36. The mechanisms through
which these bacterial metabolites exert their antifungal effects
include competition for resources, the production of hydrolytic
enzymes, and the secretion of secondary metabolites that inhibit fungal
growth^[123]37,[124]38.
Studies have demonstrated that different fungal pathogens exhibit
different sensitivity to bacterial metabolites. For instance,
Kiesewalter et al.^[125]39 concentrated on differences in antifungal
metabolites (nonribosomal peptides) produced by B. subtilis isolated
from the soil against three phytopathogens (F. oxysporum, F.
graminearum, and Botrytis cinerea). The authors found that isolates
from the same soil showed different activities against the test fungi
and could generate distinct secondary metabolites with chemical
variations of B. subtilis in the environment. The results of
intraspecies interactions between isolates that coexist in nearby
microenvironments point to the influence of accessory biosynthesis gene
clusters on the sensitivity to secondary metabolites and their
inhibitory potential. The potential for using these bacterial
metabolites as biocontrol agents is significant because they inhibit
the growth of specific fungal pathogens and promote plant health by
enhancing resistance mechanisms within the host plants^[126]35,[127]40.
Hence, the effectiveness of bacterial metabolites as biofungicides
varies significantly depending on the specific fungal pathogens
involved, highlighting the need for tailored approaches in biocontrol
strategies.
Tryptophan and arginine metabolism pathways play a pivotal role in the
synthesis of secondary metabolites, including indole acetic acid and
polyamines, which are important for promoting plant growth and
improving stress resilience (Fig. [128]7). Additionally, bioactive
compounds, including alkaloids and flavonoids (8.12%), play roles in
plant hormone signaling and pathogen inhibition. The co-culture
environment seems to catalyze the production of these defense-related
metabolites due to interspecies microbial competition and plant-microbe
exchanges. For instance, Anjum et al.^[129]41 identified a total of 73
metabolites in resistant and susceptible tomato plant samples,
revealing extensive physiological re-modulations in response to F.
oxysporum infection. The functional categorization of these metabolites
indicated the activation of signaling pathways and the overproduction
of precursor molecules in various carbon cycles upon pathogen
perception. These precursor metabolites were predominantly redirected
into hormone biosynthesis, phenylpropanoid, and alkaloid biosynthesis
pathways. Consequently, the resistant tomato variety produced
significantly higher levels of defense-related compounds, including
phenolics, terpenoids, and alkaloids, upon pathogen attack. This
upregulation of defense-associated metabolic pathways was integral to
the tomato plant’s resistance against Fusarium wilt disease.
Fig. 7.
[130]Fig. 7
[131]Open in a new tab
Analysis of KEGG metabolic pathways in B57 showing top-generated
compounds with the maximum number of differential annotations.
Additionally, tropane, piperidine, and pyridine biosynthesis (1.9%) was
a notable presence among the analyzed pathways (Fig. [132]7). These
compounds are known for their antibacterial and antifungal
properties^[133]42, indicating B57’s potential to bolster plant
immunity by suppressing pathogens in co-culture settings. Regarding
energy and lipid metabolism, arachidonic acid metabolism and methane
metabolism pathways (4.14%) were enriched, reflecting enhanced
energy-related processes. Arachidonic acid is a known precursor for
signaling molecules that mediate plant defense responses^[134]43. The
metabolic activity in these pathways signals energy allocation toward
plant protection and growth under co-culture conditions. However,
terpenoid and polyketide metabolism pathways, such as carotenoid
biosynthesis, Type II polyketide products, and diterpenoid biosynthesis
(7.9%) are significantly represented, reflecting the involvement of B57
in synthesizing biomolecules with antimicrobial and growth-enhancing
properties. These compounds support the ability of plants to suppress
pathogens and increase stress tolerance and plant developmental
processes^[135]44. With respect to the nucleotide and membrane
transport pathways, the purine metabolism (2.1%), and ABC transporter
(3.1%) pathways indicate that regulatory activity is essential for
microbial nutrient exchange and metabolite secretion between B57 and
plants. The nutrient exchange favors the production and transport of
bioactive secondary metabolites crucial for plant health.
Pathway enrichment analysis highlighted the metabolic versatility of
B57, demonstrating its capacity to produce metabolites involved in both
biocontrol and plant growth promotion. Key metabolites mapped to these
pathways exhibited significant FCs, underscoring the diverse roles of
B57’s metabolic activity (Fig. [136]8). KEGG pathway enrichment
analysis identified carbapenem biosynthesis as a highly enriched
pathway, exhibiting a highly rich factor and large marker size,
indicating its central role in the metabolic processes of B57, as shown
in Fig. [137]8A. Carbapenems are potent antimicrobial agents that
inhibit bacterial and fungal development. Their role in suppressing
pathogens is significant, demonstrating B57’s potential as a biocontrol
agent. However, enriched glutathione-related pathways indicate the
production of metabolites that detoxify reactive oxygen species (ROS)
in both bacteria and plants. This phenomenon enhances plant stress
resilience and promotes plant-pathogen defense capacity. The activity
of the butanoate metabolism pathway may be linked to the synthesis of
precursors for plant growth-enhancing metabolites and stress
regulators. This highlights B57’s involvement in optimizing conditions
for plant development.
Fig. 8. KEGG metabolic pathways analysis for monoculture and co-culture of
B57 and the tested fungal strains.
[138]Fig. 8
[139]Open in a new tab
KEGG pathway enrichment analysis (A); fold change (FC) of the top five
metabolites (B).
Additionally, pathways supporting plant growth and protection, and the
breakdown of fatty acids, support energy production and the
biosynthesis of cellular components, indirectly aiding plant growth.
Regarding Carbohydrate Digestion and Absorption, this enrichment
indicates metabolic adaptations to optimize nutrient utilization,
fostering symbiotic microbial and plant interactions. Additionally, the
plant hormone signal transduction pathway suggests possible modulation
of plant hormonal responses, such as those involving auxins and
cytokinins, which are critical for plant development and disease
resistance.
On the other hand, metabolite fold change analysis (FCA) revealed that
carbapenem biosynthesis was particularly active under co-culture
conditions, as evidenced by the upregulation of metabolites, such as
tabtoxin biosynthesis intermediate one (carbapenem biosynthesis
pathway), which exhibited a positive FC (Fig. [140]8B). This
upregulation supports the production of carbapenem antibiotics, a major
contributor to pathogen suppression. Also, in glutathione metabolism,
the metabolites Glutathionyl amino propylcadaverine and L-Ornithine
exhibit significant upregulation. These findings indicate that
increased antioxidant activity during stress-induced or
pathogen-exposed conditions helps plants mitigate oxidative damage.
However, during fatty acid degradation, palmitaldehyde demonstrated
downregulation, suggesting that it is consumed as a substrate to
support energy-demanding metabolic processes during co-culture.
In the butanoate metabolism pathway, the presence of up- and
downregulated metabolites, including
(R)-3-((R)-3-Hydroxybutanoyloxy)butanoate and butyric acid, suggests a
dynamic pathway activity that may be associated with providing
precursors for the biosynthesis of bioactive compounds. In carbohydrate
digestion and absorption, metabolites such as butyric acid are
upregulated, demonstrating the importance of energy metabolism in
co-culture conditions. Thus, differential changes in metabolite levels
reflect metabolic flexibility, enabling B57 to respond to the added
demands of co-culture or rhizospheric interactions, which include
nutrient competition, signaling, and pathogen suppression. Pathways
such as carbapenem biosynthesis and glutathione metabolism highlight
the production of antimicrobial and stress-resilience compounds.
Carbapenems inhibit critical cellular processes in pathogens^[141]45,
while glutathione supports plant immune responses^[142]46.
Additionally, enriched pathways such as plant hormone signal
transduction, carbohydrate metabolism, and fatty acid degradation
reinforce B57’s ability to enhance plant primary and secondary
metabolism. These pathways regulate the growth, stress tolerance, and
nutrient availability of plants. The differential expression of
metabolites in co-culture conditions reflects B57’s capacity to
dynamically modify its metabolism to support its role in promoting
plant health and suppressing pathogens. Consequently, the enrichment of
pathways linked to biocontrol (e.g., carbapenems and fatty acid
degradation) and growth promotion (e.g., glutathione and butanoate
metabolism) underscores B57’s potential as a bioinoculant. These
findings suggest that it can reduce the reliance on synthetic
agrochemicals while supporting plant productivity and health.
The enrichment of pathways such as carbapenem biosynthesis, glutathione
metabolism, and butanoate metabolism highlights the metabolic
flexibility and bioactive potential of B57, as depicted in Fig.
[143]8B, reflecting its dual role in both pathogen inhibition and plant
growth enhancement. The identification of differentially expressed
metabolites further highlights the ability of this organism to adapt to
co-culture conditions, demonstrating its promise as a sustainable and
environmentally friendly agricultural biocontrol agent. The data
indicated substantial differences in metabolic pathway expression. In
co-culture, B57 appears to enhance its metabolic potential by
redirecting resources toward pathways synthesizing antimicrobial
compounds, strengthening the plant’s ability to inhibit pathogens.
Boosting the biosynthesis of compounds involved in plant growth
promotion, such as plant hormones and siderophores. These findings
suggest that microbial interactions in co-culture promote physiological
adaptability and stimulate synergistic effects to protect plants and
promote sustainable growth. The KEGG pathway analysis underscores the
metabolic versatility of B57 under co-culture conditions. Its enhanced
activity in secondary metabolite biosynthesis, amino acid metabolism,
and terpenoid/polyketide biosynthesis pathways highlights its potential
as a powerful biocontrol agent against plant pathogens and a promoter
of plant growth. Several metabolic shifts in co-culture reflect B57’s
adaptability and its ability to interact beneficially to improve plant
health, making it a valuable candidate for sustainable agricultural
practices. These results align with recent studies by Ma et al.^[144]25
and Wang et al.^[145]47, further supporting the relevance of metabolic
pathway activation in microbial interactions.
Effects of N. alba B57 metabolites on plant pathogens
In this study, the inhibitory effects of B57 metabolites on four plant
fungal pathogens (designated as F1, F2, F3, and F4) were evaluated. The
obtained results revealed that the crude extracts of B57 exhibited
varying degrees of antifungal activity against the tested pathogens,
achieving an average growth inhibition of 30.27% compared with that of
the negative control (Fig. [146]9). Notably, the crude extracts
completely inhibited spore germination in all fungal pathogens and
induced significant morphological alterations in fungal conidia,
underscoring their potent antifungal properties. Similar findings were
reported by Cha et al.^[147]48, who found that Streptomyces S4-7
isolated from Korean soil showed suppressiveness antifungal activity
against Fusarium wilt disease. Metabolomic analysis revealed 35
biosynthetic-related gene clusters to produce putative antimicrobial
agents. Newitt^[148]49 found that 17 Streptomyces spp. isolated from
wheat roots produce specific metabolites that inhibit Gaeumannomyces
tritici (wheat take-all fungus). The authors reported that the genomes
of two Streptomyces strains with exceptionally potent antifungal
activity were sequenced and that putative antifungal gene clusters were
identified.
Fig. 9. Intelligent live digital imaging of the antifungal activity of
Nocardiopsis alba metabolites against four fungal pathogens.
[149]Fig. 9
[150]Open in a new tab
Fusarium oxysporum (A), Fusarium moniliforme (B), Fusarium graminearum
(C), and Verticillium dahliae Kleb (D) compared to control.
The metabolomic analysis of B57, as shown in Fig. [151]10, highlighted
the biosynthesis of secondary metabolites in B57. These secondary
metabolites contribute to essential agricultural processes (e.g.,
pathogen-inhibiting bioactive molecules). The dityrosine biosynthesis
pathway is initiated from D-fructose-6-phosphate and involves
intermediates leading to the final product, (-)-Dityrosylphenaline,
through the action of DtpA, DtpB, and DtpC enzymes. Ditryptophenaline
is an alkaloid metabolite that showed antifungal properties by
inhibiting the germination of fungal sclerotia^[152]50. Additionally,
the fumiquinazoline (antifungal agent) pathway incorporates
phenylalanine and tryptophan as precursors to complex molecules, such
as fumiquinazoline F, C, and D. Their mechanism involves disrupting key
fungal metabolic processes, such as inhibition of
Na^+/K^+-ATPase^[153]51, making them potential candidates for use in
agricultural settings to control fungal diseases^[154]52.
Fig. 10.
[155]Fig. 10
[156]Open in a new tab
The metabolomic analysis of the biosynthesis of various secondary
metabolites in B57.
Although paerucumarin was reported to be produced by different types of
bacterial strains, such as Pseudomonas aeruginosa^[157]53. The
metabolomic analysis showed that paerucumarin can also be generated by
B57 through a series of enzymatic processes originating from tyrosine
and transformed into intermediates by specific enzymes, eventually
leading to the formation of paerucumarin (Fig. [158]10). Paerucumarin
has antifungal properties and can promote plant growth by producing
various growth-promoting substances, such as indole-3-acetic acid (IAA)
and gibberellic acid, which increase root development and overall plant
vigor. Additionally, it exhibits antifungal activity against several
plant pathogens, improving crop yields through dual action^[159]54.
Staphyloferrins are synthesized via pathways involving precursors
including serine, glutamate, and ornithine. The conversion of these
compounds involves multiple enzymes such as L-2,3-diaminopropionate
synthase and various aminotransferases, which are modified by
hydroxylation and cyclization. These reactions produce siderophores
(iron-chelating compounds), such as L-2,3-diaminopropionate and
ultimately Staphyloferrin A and Staphyloferrin B. The biosynthesis of
Staphyloferrin A utilizes 2-oxoglutarate and glutamate and produces
NS-Citryl-D-ornithine, illustrating the strain’s continued emphasis on
iron acquisition and pathogen suppression. Staphyloferrin is a
siderophore that plays a crucial role in iron acquisition for bacteria.
While primarily known for its role in microbial iron transport, it may
also indirectly promote plant growth by increasing the availability of
iron in the soil, which is essential for many physiological processes
in plants^[160]55. Its direct antifungal properties are less well
documented but may contribute to overall soil health and plant
resilience against fungal pathogens.
Moreover, cyclooctatin is produced through cyclohexanone biosynthesis
pathways involving precursors (e.g., cyclooctat-9-en-7-ol).
Cyclooctatin is a cyclic peptide that is an important plant growth
regulator and antimicrobial agent, providing dual benefits of enhancing
plant development while suppressing pathogen activity^[161]56. However,
lovastatin biosynthesis showcases another aspect of B57’s antimicrobial
potential. The pathway utilizes malonyl-CoA to produce lovastatin, a
known polyketide that can inhibit fungal growth by targeting critical
metabolic processes. Lovastatin is a statin that acts by inhibiting
3-hydroxy-3-methylglutaryl-coenzyme A reductase (a rate-limiting enzyme
of the mevalonate pathway), as well as other statins have activity
towards several pathogens^[162]57. These findings further emphasize the
strain’s role in biological control.
The pathway for grixazone B biosynthesis incorporates aspartate and
leads to metabolites that possess antioxidant properties, as depicted
in Fig. [163]10. These compounds may help mitigate oxidative stress
during the fungal attack, bolstering plant resilience. Le Roes-Hill et
al.^[164]58 reported that the grixazones produced by Streptomyces
griseus subsp. griseus is divided into grixazone A, which has an
aldehyde at position 8, and grixazone B, which has a carboxyl group.
Few reports have discussed the bioactivity of grixazone in general.
However, grixazone B has antifungal, antiviral, antihelminthic, and
lipooxygenase inhibitory activities^[165]58. While there are no studies
on its role as a plant growth promoter, its effectiveness in
controlling fungal pathogens suggests that it could indirectly support
plant health by minimizing disease impact.
L-B-ethynylserine biosynthesis is another important pathway featured in
the metabolomic analysis of B57. This pathway uses lysine as a
precursor, leading to the production of 4-chloro-L-lysine before
resulting in L-B-ethynylserine. L-B-ethynylserine is noteworthy due to
its potential effects on microbial communication and stress responses.
LB-ethynylserine is a non-proteinogenic L-α-amino acid that is
L-propargylglycine, which carries a hydroxy group at the 3R position.
It has a role as an antimetabolite, an antimicrobial agent. It is a
terminal acetylenic compound and a non-proteinogenic L-α-amino acid. It
is functionally related to L-propargylglycine. It is a tautomer of an
LB-ethynylserine zwitterion^[166]59. However, more research is needed
to fully understand its mechanisms and efficacy as a plant growth
promoter. Lastly, the biosynthesis of aerobactin, which uses lysine as
a precursor, results in compounds that increase nutrient availability
while inhibiting competition from pathogens. Aerobactin is a
siderophore that facilitates iron uptake in bacteria and may improve
soil fertility by increasing the bioavailability of iron to
plants^[167]60. Its role as an antifungal agent is not well
established, but it could contribute to overall soil health and support
plant growth. Thus, these compounds exhibit significant potential both
as antifungal agents and as plant growth promoters through various
mechanisms, including nutrient solubilization, pathogen inhibition, and
the enhancement of beneficial microbial activity in the rhizosphere.
Further research into their specific mechanisms will help optimize
their use in agricultural practices.
The study highlighted the remarkable ability of N. alba B57 to produce
a diverse range of antibiotics (Fig. [168]11) through well-defined
biosynthetic pathways, showcasing its potential as a producer of
bioactive compounds for agricultural and antimicrobial applications.
These antibiotics are synthesized from various metabolic precursors,
including glucose, pyruvate, and amino acids such as tyrosine and
serine, reflecting the metabolic versatility of the strain. The study
emphasizes the strain’s capacity to combat bacterial, fungal, and
protozoal pathogens through its diverse antimicrobial arsenal. Among
the key biosynthetic pathways, kanosamine biosynthesis is initiated by
intermediates such as 3-dehydro-D-glucose, catalyzed by enzymes like
transaminase (EC 2.6.1.104) and phosphatase (EC 3.1.3.92). Kanosamine
is an effective antibiotic that inhibits fungal strains, including
Saccharomyces cerevisiae and human pathogenic fungi^[169]61. Similarly,
aurachins A-D, synthesized from D-fructose-6-phosphate through the
intermediate anthranilate, act as inhibitors of bacterial electron
transport chains by targeting cytochrome enzymes, significantly
disrupting energy metabolism. Aurachins also exhibit antifungal and
antiprotozoal activity, showcasing their broad-spectrum
potential^[170]62.
Fig. 11.
[171]Fig. 11
[172]Open in a new tab
The metabolomic analysis of the biosynthesis of antibiotics by B57.
Another critical pathway is bacilysin biosynthesis, which begins with
pyruvate and involves intermediates such as
4-hydroxy-3-hexaprenoyl-AMP. Bacilysin disrupts bacterial cell wall
synthesis, granting B57 a competitive advantage in microbial
environments^[173]63. Likewise, puromycin biosynthesis utilizes
tyrosine as a precursor to produce highly bioactive compounds such as
N-acetyl-O-methyl-puromycin^[174]64. Singh et al.^[175]65 reported that
puromycin disrupts microbial protein synthesis and is effective against
resistant strains, including Escherichia coli, Klebsiella pneumoniae,
and Staphylococcus spp. Dapdiamide biosynthesis is another notable
pathway in B57, producing dapdiamides A, B, and C from serine and
glutamate. These compounds, derived through catalytic steps involving
enzymes such as transferase (EC 2.5.1.140), exhibit antibacterial
activity, particularly against Erwinia amylovora^[176]66. Similarly,
fosfomycin biosynthesis starts with phosphoenolpyruvate and
glyceraldehyde-3-phosphate and involves enzymes like isomerase (EC
5.4.2.5). Fosfomycin irreversibly inhibits MurA, a critical enzyme in
bacterial cell wall synthesis, making it highly effective against
Gram-positive pathogens^[177]67. Additionally, the study identifies the
biosynthesis of cremomycin and pentalenolactone. Cremomycin is
synthesized through nitro intermediates, although its bioactivity
remains underexplored. Pentalenolactone, on the other hand, targets
microbial energy metabolism through its epoxylactone moiety,
demonstrating activity against bacteria, fungi, and protozoa^[178]68.
Furthermore, roseoflavin production in B57 utilizes FMN as a precursor,
a compound previously reported only in Streptomyces davawensis and
Streptomyces cinnabarinus, with significant potential as a
broad-spectrum antibiotic^[179]69. Lastly, cycloserine biosynthesis
involves the conversion of arginine and ornithine into intermediates
that disrupt bacterial peptidoglycan synthesis^[180]70
These diverse biosynthetic pathways underscore B57’s metabolic
adaptability and its capacity to produce clinically and agriculturally
valuable antibiotics. Compounds such as kanosamine, fosfomycin, and
puromycin target critical bacterial processes such as the cell wall and
protein synthesis, whereas other compounds, including aurachins and
cremomycin, extend their activity to fungal and protozoal pathogens.
Its ability to upregulate multiple pathways in response to
environmental stress makes B57 a promising candidate for
biotechnological applications, particularly in addressing antibiotic
resistance and enhancing sustainable agricultural practices.
Effects of N. alba B57 metabolites on plant growth
To investigate the effects of N. alba B57 metabolites on plant growth,
this study evaluated their impact on Lolium perenne (monocot) and
Amaranthus retroflexus (dicot) at varying concentrations (100, 500,
1000, and 2000 µg/mL). Growth parameters, including root length (RL)
and shoot length (SL), were measured to assess species-specific and
concentration-dependent responses (Fig. [181]12).
Fig. 12. Effect of different concentrations of B57 metabolites on plant
growth.
[182]Fig. 12
[183]Open in a new tab
A Growth response of Lolium perenne. B Root and shoot length variations
in Amaranthus retroflexus compared to control (CK).
In L. perenne, B57 metabolites significantly enhanced RL and SL
compared to the control (p = 0.0262 and p = 0.0341, respectively). At
100 µg/mL, RL and SL increased to 4.13 cm and 3.75 cm, respectively,
compared to the control values of 3.62 cm (RL) and 3.17 cm (SL). The
highest growth stimulation was observed at 1000 µg/mL, with RL and SL
reaching 4.74 cm and 4.20 cm, respectively (Fig. [184]12A). However, at
2000 µg/mL, both RL and SL decreased slightly, suggesting a threshold
beyond which the stimulatory effects diminished, possibly due to
metabolite-induced phytotoxicity or osmotic stress. This dose-dependent
response aligns with the hormesis model, where low to moderate
concentrations promote growth, while higher concentrations may inhibit
it.
In contrast, A. retroflexus exhibited a different response pattern. At
100 µg/mL, RL and SL increased to 1.64 cm and 1.57 cm, respectively,
compared to the control values of 1.40 cm (RL) and 1.42 cm (SL). Growth
peaked at 500 µg/mL, with RL and SL reaching 2.11 cm and 1.88 cm, at
P = 0.0127 and P = 0.0256, respectively (Fig. [185]12B). However, at
higher concentrations (1000 and 2000 µg/mL), RL and SL declined,
indicating potential allelopathic inhibition due to toxic metabolite
accumulation or metabolic disruption. Comparative analysis revealed
that L. perenne exhibited greater tolerance to higher concentrations of
B57 metabolites than A. retroflexus. For instance, at 2000 µg/mL, L.
perenne maintained RL and SL values of 4.62 cm and 4.19 cm,
respectively, while A. retroflexus showed significantly lower values
(1.91 cm RL and 1.90 cm SL). These species-specific responses highlight
the varying tolerance levels of target plants and the specificity of
allelopathic interactions. The findings suggest that B57 metabolites
could serve as natural growth regulators with applications in
sustainable agriculture, such as weed control or crop enhancement.
Several pathways involved in plant hormone signal transduction in N.
alba B57 were illustrated in Fig. [186]13, providing a comprehensive
overview of multiple pathways that regulate plant growth, stress
response, and metabolic adaptations. Each pathway is associated with
specific plant hormones that initiate biochemical cascades, leading to
gene expression changes and physiological changes. Auxin is a plant
growth regulator and plays a role in almost every aspect of plant
growth and development. IAA is the most abundant natural auxin and can
execute the majority of auxin-related regulatory actions in
plants^[187]71. Metabonomic analysis showed that B57 auxin signal
transduction pathways can play a central role in regulating cell
enlargement and overall plant growth. B57’s auxin is synthesized from
tryptophan metabolism and is transported into cells via the AUX1
receptor. The downstream signaling steps involve interactions with TIR1
receptors and ARFs (auxin response factors), which activate the
transcription of auxin-related genes (e.g., GH3 and SAUR) necessary for
plant growth.
Fig. 13.
[188]Fig. 13
[189]Open in a new tab
The signaling pathways in B57 involved in plant hormone signal
transduction that regulate plant growth and metabolic adaptations.
Cytokinin signaling, as shown in Fig. [190]13, can regulate cell
division and shoot development through a two-component signaling
system. The cytokinin receptor (CRE1), upon ligand (zeatin) binding,
activates Arabidopsis histidine phosphotransfer protein (AHP), which is
translocated to the nucleus and activates B-ARR (type-B response
regulators). The transcription of target genes by A-ARR (type-A ARR)
controls critical developmental processes. Cytokinins also interact
antagonistically with auxins to maintain root-shoot balance. Also,
gibberellins (GAs) are vital for stem elongation and germination.
Additionally, gibberellin binds to the Gibberellin Insensitive Dwarf1
(GID1) receptor, resulting in the degradation of the DELLA (domain
family of proteins), which is a growth suppressor^[191]72. This
degradation releases transcription factors that promote plant
growth-related gene expression. Ubiquitin-mediated proteolysis ensures
the removal of DELLA when gibberellin levels rise, providing an
efficient regulatory mechanism to modulate plant height and
developmental transitions^[192]72. Similarly, the abscisic acid (ABA)
pathway, where ABA regulates stomatal closure and seed dormancy,
occurs, particularly under stress conditions (e.g., drought). ABA binds
to PYR/PYL receptors, leading to the inhibition of PP2C phosphatases.
This allows the activation of SnRK2 kinases, which phosphorylate
transcription factors, such as ABF (ABA-responsive element binding
factors), triggering the expression of stress-response genes. This
pathway plays a critical role in plant survival under adverse
conditions.
The metabolomic analysis showed that ethylene signaling involves ETR1
and downstream components such as EIN2 and ERF1/2, controlling
processes like fruit ripening, senescence, and environmental
adaptability^[193]73. The pathway also integrates environmental and
mechanical stress, emphasizing its role in environmental adaptability
and developmental regulation. Additionally, N. alba B57 metabolic
analysis showed that brassinosteroids influence cell elongation and
division through the receptor kinase proteins BRI1 and BAK1. Upon
ligand binding, a cascade involving BSU1 phosphatase and BIN2 kinase
leads to the dephosphorylation and activation of transcription factors,
such as BZR1/2. These transcription factors regulate genes responsible
for elongation and division, contributing to enhanced growth^[194]74.
Additionally, the metabolomic analysis (Fig. [195]13) showed that
jasmonic acid primarily mediates stress responses and senescence and
regulates secondary metabolite production. Jasmonoyl–isoleucine
(JA–Ile) conjugate binds to COI1 receptors, which target JAZ repressors
to facilitate stress responses, defense, and secondary metabolite
production^[196]75. Moreover, salicylic acid (SA), a key player in
plant immunity, regulates systemic acquired resistance (SAR) by
activating NPR1 and TGA transcription factors to induce
pathogenesis-related proteins. It also enhances photosynthesis and
redox homeostasis while modulating hormonal crosstalk with JA,
ethylene, and ABA to improve stress tolerance^[197]76,[198]77. These
pathways, triggered by pathogen invasion, drive SA synthesis and
subsequent conjugation into biologically active or inactive
forms^[199]78.
The findings highlight the critical role of N. alba B57 in regulating
plant hormone signaling pathways, which are essential for plant
development. Through intricate signaling networks, N. alba B57
facilitates plant bioprocesses to ensure a balanced and adaptive
response to environmental challenges while promoting healthy plant
development. The ability of N. alba B57 to modulate these hormone
pathways underscores its potential as a valuable tool in sustainable
agriculture. Thus, N. alba B57 can contribute to improving crop yield
and environmental sustainability. Further research into these pathways
can unlock innovative applications in agriculture, such as developing
stress-tolerant crops and optimizing plant performance under changing
climatic conditions.
In conclusion, this study highlights the potential of Nocardiopsis alba
B57 as an effective biocontrol agent and plant growth promoter, capable
of producing antifungal and growth-enhancing metabolites. Metabolomic
analyses revealed its ability to modulate key pathways under co-culture
conditions, leading to the synthesis of bioactive compounds that
suppress pathogens and enhance plant resilience. These findings support
B57 as a sustainable alternative to chemical fungicides, with future
research needed to optimize its application and assess long-term
impacts on soil and crop health.
Methods
Nocardiopsis alba B57 crude extract preparation and fermentation
The endophytic actinobacterial strain N. alba B57 (Accession No.
[200]MN688677) was isolated and identified in our previous
study^[201]15 from T. roseus obtained from Xinjiang, China. To prepare
B57 for fermentation, a single colony was cultured in the International
Streptomyces Project-2 (ISP2) broth medium. The cultures were incubated
in 500 mL flasks under continuous shaking at 28 °C and 200 rpm for 21
days^[202]79. Similarly, fungal pathogens were cultured individually on
potato dextrose agar (PDA) for 6 days. A 5 mm mycelial disc from each
fungal culture was then transferred into a fresh ISP2 medium, where
they were incubated for 21 days under the same conditions. For
co-culture experiments, B57 was first pre-cultured in a modified liquid
ISP2 medium for 24 h. Subsequently, a 5 mm mycelial disc from each
fungal strain was introduced into 500 mL flasks containing the modified
ISP2 medium. These co-culture flasks were continuously incubated for 21
days at 200 rpm and 28 °C.
In large-scale fermentation setups, the fermentation broth was filtered
using Whatman No. 1 filter paper to separate the biomass, and the
filtrate was then centrifuged for 15 min at 10,000 rpm. To extract
bioactive metabolites, the supernatant was subjected to a three-step
liquid-liquid extraction process using ethyl acetate (1:1; v/v). The
produced extracts were filtered under reduced pressure using a rotary
vacuum evaporator (N-1300, EYELA, Ailang Instrument Co., Ltd.,
Shanghai, China) at 40 °C to be concentrated, following Zhang et
al.^[203]38. The crude extracts were subsequently stored for further
analysis.
Nocardiopsis alba B57 metabolite extraction
Nocardiopsis alba B57 metabolites were extracted and analyzed using a
Waters Acquity I-Class PLUS ultra-high-performance liquid
chromatography (UPLC) system coupled with a Waters Xevo G2-XS QTOF
high-resolution mass spectrometer. The separation process was achieved
using a Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) at
35 °C, with a 0.3 mL/min flow rate. For positive ion mode, mobile phase
A consisted of 0.1% formic acid in water, while mobile phase B was a
mixture of acetonitrile and 0.1% formic acid. The same mobile phases
were used for the negative ion mode. The gradient elution program was
designed as follows: the initial phase (30 s) consisted of 90% mobile
phase A. This condition was maintained until 7 min, after which phase A
was reduced to 0% between 7- and 8.5 min. Re-equilibration to 90% phase
occurred between 8.6 and 10 min. A volume of 1 μL as the injection
volume of each sample was set to ensure process accuracy^[204]47.
Nocardiopsis alba metabolites identification
The liquid chromatography with tandem mass spectrometry (LC-MS/MS)
analysis was conducted by a high-resolution mass spectrometer (Waters
Xevo G2-XS QTOF) operating in MSe mode. The low collision energy was
adjusted at 2 V, and the high collision energy was adjusted from 10 to
40 V. A scanning frequency of 0.2 s per mass spectrum ensured
comprehensive data collection. Data acquisition was managed and
performed via MassLynx v4.2 software provided by Waters Corporation.
The electrospray ionization (ESI) source parameters included a
capillary voltage of +2000 V for positive ion mode and −1500 V for
negative ion mode. The cone voltage was set at 30 V, with the ion
source temperature at 150 °C. For desolvation, the gas temperature was
held at 500 °C, with desolvation and backflush gas flow rates of 800
and 50 L/h, respectively. The obtained data were interpreted via
Progenesis QI software (v3.0). This software facilitated peak
extraction, alignment, and other data processing tasks. Metabolite
identification was conducted by referencing the METLIN database and
Biomark’s custom-built library. To ensure accuracy, theoretical
fragment matches and mass deviations were validated within a threshold
of 100 ppm. This rigorous approach ensured the reliable identification
of the metabolites present in the samples.
Metabolomic and pathway analyses
The original peak area data were normalized to the total peak area to
ensure consistent and comparable analysis. Subsequently, advanced
statistical methods, including PCA and SRC analysis, were applied to
evaluate sample repeatability within groups and the quality control
samples. The identified metabolites were classified and mapped to
metabolic pathways using the KEGG database. Grouping information was
utilized to calculate FC, and t-tests were carried out to detect the
statistical significance (p-value) of changes in compound abundance.
PCA was implemented following methodologies described in previous
studies^[205]21,[206]79,[207]80. Multivariate statistical analysis,
including PCA and hierarchical clustering, was carried out using
MetaboAnalyst 5.0. Heatmaps and SRC coefficient matrices were generated
to visualize the relationships among samples. For supervised analysis,
OPLS-DA modeling was applied using the R package ropls (v3.19). The
reliability of the OPLS-DA model was verified through 200 permutation
tests. VIP scores were determined using multiple cross-validation
methods. Differentially abundant metabolites were selected using a
comprehensive approach that incorporated fold changes (FC >1),
statistical significance (p < 0.01), and VIP scores (VIP >1).
Enriched pathways for these metabolites were identified using KEGG,
with significance determined via a hypergeometric distribution test.
Potential biomarkers were identified, and associated pathways were
analyzed using MetaboAnalyst 4.01 to further explore metabolic
insights. In addition, Spearman’s correlation analysis was employed to
investigate metabolite relationships, providing deeper insights into
the underlying metabolic networks^[208]81.
Antimicrobial activity of crude extracts against fungal pathogens
To evaluate the antifungal potential of crude extracts from B57, four
fungal pathogens served as representative organisms for evaluating
antifungal activity, as listed in Table [209]1. B57 crude extract
inhibitory effects on the spore germination of fungal pathogens were
tested using a modified protocol reported by Abdelshafy Mohamad et
al^[210]82. Briefly, the fungal strains were grown on PDA for 6 days to
ensure adequate mycelial development. A 5 mm mycelial disc was
positioned at the center of a fresh PDA plate (6 cm). Sterile filter
paper discs, 5 mm in diameter, were autoclaved at 121 °C for 30 min.
These discs were loaded with bioactive metabolites-containing crude
extracts and placed equally on each plate. The plates were sealed with
parafilm to maintain humidity and incubated at 26 ± 2 °C for 7 days.
The morphological response of Fusarium spp. and Verticillium dahliae
Kleb was observed under a laser microscope (Olympus SZX2-ILLT, Japan)
at different magnifications. Plates with pathogenic fungi alone served
as a control.
Table 1.
Plant fungal pathogens were used to assess the antifungal potential of
B57 crude extract in the present study
Strain name Identification number Code Source
Fusarium oxysporum ACCC37438 F1 China General Microbiological Culture
Collection Center (CGMCC)
Fusarium moniliforme CGMCC3.4269 F2
Fusarium graminearum CGMCC3.3488 F3
Verticillium dahliae Kleb ACCC30308 F4
[211]Open in a new tab
Fungal growth inhibition was observed and quantified by calculating the
inhibition zone (IZ) diameter according to the following Eq.
([212]1)^[213]83
[MATH: Inhibition percentage%=<
mrow>CD−CtCD−
D0X100 :MATH]
1
where CD is the diameter of the control fungal colony, Ct is the
diameter of the tested fungal colony, and D0 is the disc diameter
(5 mm).
Growth-promoting effects of N. alba B57 crude extracts
The allelopathic potential of B57 crude extracts was assessed on L.
perenne (monocot) and A. retroflexus (dicot) seedlings. Seeds were
surface sterilized with 0.5% HgCl[2] to eliminate contaminants. Crude
extracts of B57 were dissolved in ethyl acetate to prepare
concentrations of 100, 500, 1000, and 2000 μg/mL. Petri dishes lined
with filter paper were treated with these solutions, followed by
complete evaporation of the solvent. Subsequently, 2 mL of distilled
water was added to each dish, and 10 seeds of either L. perenne or A.
retroflexus were placed on the moistened filter paper. Controls were
treated with distilled water under identical conditions. All dishes
were incubated in darkness at 25 °C for 5 days to facilitate seedling
growth. Shoot and root lengths were measured to assess the
growth-promoting effects of the extracts. Each treatment was performed
in triplicate, analyzing 30 seeds per condition (n = 30), providing
valuable insights into the biostimulant potential of B57 extracts.
Supplementary information
[214]Supplementary Information^ (396.4KB, pdf)
Acknowledgements