Design of Carboxymethylcellulose/Poloxamer-Based Bioformulation Embedding Trichoderma afroharzianum for Agricultural Applications.

Microbial biological control agents are believed to be a potential alternative to classical fertilizers to increase the sustainability of agriculture. In this work, the formulation of Trichoderma afroharzianum T22 (T22) spores with carboxymethyl cellulose (CMC) and Pluronic F-127 (PF-127) solutions was investigated. Rheological and microscopical analysis were performed on T22-based systems at three different CMC/PF-127 concentrations, showing that polymer aggregates tend to surround T22 spores, without viscosity, and the viscoelastic properties of the formulations were affected. Contact angle measurements showed the ability of PF-127 to increase the wettability of the systems, and the effect of the formulations on the viability of the spores was evaluated. The viability of the spores was higher over 21 days in all the formulations, compared to the control in water, at 4 and 25 °C. Finally, the effectiveness of the formulations on sweet basil was estimated by greenhouse tests. The results revealed a beneficial effect of the CMC/PF-127 mixture, but none on the formulation with T22. The data show the potential of CMC/PF-127 mixtures for the future design of microorganism-based formulations.

Fungal metabolite 6-pentyl-α-pyrone reduces canine coronavirus infection.

Canine coronavirus (CCoV) can produce a self-limited enteric disease in dogs but, because of notable biological plasticity of coronaviruses (CoVs), numerous mutations as well as recombination events happen leading to the emergence of variants often more dangerous for both animals and humans. Indeed, the emergence of new canine-feline recombinant alphacoronaviruses, recently isolated from humans, highlight the cross-species transmission potential of CoVs. Consequently, new effective antiviral agents are required to treat CoV infections. Among the candidates for the development of drugs against CoVs infection, fungal secondary metabolites (SMs) represent an important source to investigate. Herein, antiviral ability of 6-pentyl-α-pyrone (6 PP), a SM obtained by Trichoderma atroviride, was assessed against CCoV. During in vitro infection, nontoxic concentration of 6 PP significantly increased cell viability, reduced morphological signs of cell death, and inhibited viral replication of CCoV. In addition, we found a noticeable lessening in the expression of aryl hydrocarbon receptor (AhR), a strategic modulator of CoVs infection. Overall, due to the variety of their chemical and biological properties, fungal SMs can decrease the replication of CoVs, thus identifying a suitable in vitro model to screen for potential drugs against CoVs, using a reference strain of CCoV (S/378), non-pathogenic for humans.

Biodegradable Mulch Films and Bioformulations Based on Trichoderma sp. and Seaweed Extract Differentially Affect the Metabolome of Industrial Tomato Plants.

The use of biostimulants and biofilms in agriculture is constantly increasing, as they may support plant growth and productivity by improving nutrient absorption, increasing stress resilience and providing sustainable alternatives to chemical management practices. In this work, two commercial products based on Trichoderma afroharzianum strain T22 (Trianum P®) and a seaweed extract from Ascophyllum nodosum (Phylgreen®) were tested on industrial tomato plants (Solanum lycopersicum var. Heinz 5108F1) in a field experiment. The effects of single and combined applications of microbial and plant biostimulants on plants grown on two different biodegradable mulch films were evaluated in terms of changes in the metabolic profiles of leaves and berries. Untargeted metabolomics analysis by LC-MS Q-TOF revealed the presence of several significantly accumulated compounds, depending on the biostimulant treatment, the mulch biofilm and the tissue examined. Among the differential compounds identified, some metabolites, belonging to alkaloids, flavonoids and their derivatives, were more abundant in tomato berries and leaves upon application of Trichoderma-based product. Interestingly, the biostimulants, when applied alone, similarly affected the plant metabolome compared to control or combined treatments, while significant differences were observed according to the mulch biofilm applied.

Metabolites from a Pleurotus tuber-regium collected in Madagascar.

Pleurotus tuber-regium was isolated from a dead trunk of Raphia farinifera (Arecaceae) in a lowland moist forest in Antsohihy, Madagascar, and the species was confirmed by molecular analysis and morphological observations. The main bioactive metabolites of the mycelium extracts were identified by mass spectrometry techniques. Five structural diverse metabolites, tryptophol, pyroglutamic acid, prolyldiketopiperazine B, sporol and RKS-1778, were characterised by LC-MS qTOF analysis of the hydro-alcoholic extract. GC-MS analysis of both chloroform and ethyl acetate extracts revealed the presence of several saturated and -unsaturated fatty acids and their esters derivatives.

Chemopreventive effect of a milk whey by-product derived from Buffalo (Bubalus bubalis) in protecting from colorectal carcinogenesis.

BACKGROUND: Several studies show that natural foods are a source of compounds with anticancer properties that affect the gut microbiota and its metabolites. In the present study, we investigate the effect of a delactosed buffalo milk whey by-product (DMW) on colorectal carcinogenesis. METHODS: The effect of DMW on colorectal carcinoma (CRC) was investigated in the established mouse model of azoxymethane (AOM)-induced colon carcinoma, which closely resembles the human clinical condition of CRC. The effect of DMW on CRC immortalized cell lines was also evaluated to further identify the antineoplastic mechanism of action. RESULTS: Pretreatment of AOM-treated mice with DMW significantly (P < 0.05) reduced the percentage of mice bearing both aberrant crypt foci with more than four crypts (which are early precancerous lesions that progress to CRC) and tumors. In addition, DMW completely counteracted the effect of AOM on protein expression of caspase-9, cleaved caspase-3 and poly ADP-ribose polymerase in colonic tissue. Administration of DMW alone (i.e. without AOM) resulted in changes in the composition of the gut microbiota, leading to enrichment or depletion of genera associated with health and disease, respectively. DMW was also able to restore AOM-induced changes in specific genera of the gut microbiota. Specifically, DMW reduced the genera Atopobiaceae, Ruminococcus 1 and Lachnospiraceae XPB1014 and increased the genera Parabacteroides and Candidatus Saccharimonas, which were increased and reduced, respectively, by AOM. Blood levels of butyric acid and cancer diagnostic markers (5-methylcytidine and glycerophosphocholine), which were increased by AOM treatment, were reduced by DMW. Furthermore, DMW exerted cytotoxic effects on two human CRC cell lines (HCT116 and HT29) and these effects were associated with the induction of apoptotic signaling. CONCLUSIONS: Our results suggest that DMW exerts chemopreventive effects and restores the gut microbiota in AOM-induced CRC, and induces cytotoxic effect on CRC cells. DMW could be an important dietary supplement to support a healthy gut microbiota and reduce the prevalence of CRC in humans. Video Abstract.

Repression of autocrine pheromone signaling leads to fusaric acid over-production.

Fusaric acid (FA), a picolinic acid derivative, is a natural substance produced by a wide variety of fungal plant pathogens belonging to the Fusarium genus. As a metabolite, fusaric acid exerts several biological activities including metal chelation, electrolyte leakage, repression of ATP synthesis, and direct toxicity on plants, animals and bacteria. Prior studies on the structure of fusaric acid revealed a co-crystal dimeric adduct between FA and 9,10-dehydrofusaric acid. During an ongoing search for signaling genes differentially regulating FA production in the fungal pathogen Fusarium oxysporum (Fo), we found that mutants lacking pheromone expression have an increased production of FA compared to the wild type strain. Noteworthy, crystallographic analysis of FA extracted from Fo culture supernatants showed that crystals are formed by a dimeric form of two FA molecules (1:1 molar stoichiometry). Overall, our results suggest that pheromone signaling in Fo is required to regulate the synthesis of fusaric acid.

Interactions between plant-beneficial microorganisms in a consortium: Streptomyces microflavus and Trichoderma harzianum.

The construction of microbial consortia is challenging due to many variables to be controlled, including the cross-compatibility of the selected strains and their additive or synergistic effects on plants. In this work, we investigated the interactions in vitro, in planta, and at the molecular level of two elite biological control agents (BCAs), that is Streptomyces microflavus strain AtB-42 and Trichoderma harzianum strain M10, to understand their attitude to cooperate in a consortium. In vitro, we observed a strong cross-antagonism between AtB-42 and M10 in agar plates due to diffusible metabolites and volatile organic compounds. In liquid co-cultures, M10 hindered the growth of AtB-42 very likely because of secondary metabolites and strong competition for the nutrients. The interaction in the co-culture induced extensive transcriptional reprogramming in both strains, especially in the pathways related to ribosomes, protein synthesis, and oxidoreductase activity, suggesting that each strain recognized the counterpart and activated its defence responses. The metabolome of both strains was also significantly affected. In contrast, in the soil, M10 growth was partially contrasted by AtB-42. The roots of tomato seedlings inoculated with the consortium appeared smaller than the control and single-strain-inoculated plants, indicating that plants diverted some energy from the development to defence activation, as evidenced by the leaf transcriptome. The consortium induced a stronger transcriptional change compared to the single inoculants, as demonstrated by a higher number of differentially expressed genes. Although the cross-antagonism observed in vitro, the two strains exerted a synergistic effect on tomato seedlings by inducing resistance responses stronger than the single inoculants. Our observations pose a question on the usefulness of the sole in vitro assays for selecting BCAs to construct a consortium. In vivo experiments should be preferred, and transcriptomics may greatly help to elucidate the activity of the BCAs beyond the phenotypic effects on the plant.

Harzianic Acid Activity against Staphylococcus aureus and Its Role in Calcium Regulation.

Staphylococcus aureus is a Gram-positive bacterium, which can be found, as a commensal microorganism, on the skin surface or in the nasal mucosa of the human population. However, S. aureus may become pathogenic and cause severe infections, especially in hospitalized patients. As an opportunistic pathogen, in fact, S. aureus interferes with the host Ca2+ signaling, favoring the spread of the infection and tissue destruction. The identification of novel strategies to restore calcium homeostasis and prevent the associated clinical outcomes is an emerging challenge. Here, we investigate whether harzianic acid, a bioactive metabolite derived from fungi of the genus Trichoderma, could control S. aureus-induced Ca2+ movements. First, we show the capability of harzianic acid to complex calcium divalent cations, using mass spectrometric, potentiometric, spectrophotometric, and nuclear magnetic resonance techniques. Then, we demonstrate that harzianic acid significantly modulates Ca2+ increase in HaCaT (human keratinocytes) cells incubated with S. aureus. In conclusion, this study suggests harzianic acid as a promising therapeutical alternative against diseases associated with Ca2+ homeostasis alteration.

Biological and phytochemical characterization of Phragmanthera regularis (Loranthaceae) from Ethiopia.

Phragmanthera regularis is a hemi-parasitic shrub. It is known for treating various health ailments. The aim of this study was to evaluate the antimicrobial activity, toxicity, and chemical characterization of the leaf extracts of P regularis collected from the Schinus molle host plant in Ethiopia. The antimicrobial properties of crude extracts obtained with chloroform, ethyl acetate, methanol, and water solvents were assayed against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The methanol extract significantly inhibited the growth of S. aureus, E. coli and P. aeruginosa were resistant to any of these solvent extracts. The methanol extract was tested at 175, 550, and 2000 mg/kg body weight doses in white mice and did not reveal any toxicity. The LC-MS qTOF analysis detected flavonoids, phenolic acids, and alkaloids in the crude methanol extract. Further study is needed to investigate the effectiveness of these compounds against S. aureus.

Biological activities of Viscum tuberculatum aqueous leaf extract.

CONTENT: Plant-based natural products have served as sources of remedies against pathogenic microorganisms. Although the biological activities of Viscum (Santalaceae) species are widely recognized, there is no scientific evidence for Viscum tuberculatum A. Rich. in Ethiopia. OBJECTIVE: To investigate the antimicrobial, acute toxicity, anti-inflammatory properties and phytochemical constituents of an aqueous extract of V. tuberculatum from Ethiopia. MATERIALS AND METHODS: The antibacterial activity of the aqueous leaf extract of V. tuberculatum was tested against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of this extract were determined using the broth macrodilution method. The acute toxicity and anti-inflammatory effects of the extract were investigated using standard procedures on female and male white albino mice, aged 8 and 10 weeks, respectively. The phytochemical constituents of V. tuberculatum were determined using LC-MS QTOF. RESULTS: The MIC and MBC values against S. aureus were found to be 6.25 and 100 mg/mL. The LD50 value was more than 2000 mg/kg body weight of the mouse. The 400 mg/kg dose exerts 87% inhibition after 5 h of carrageenan injection. Twenty-five different metabolites, mainly flavonoids, phenolic acids and alkaloids, were identified. CONCLUSIONS: These findings demonstrate the potential antimicrobial and anti-inflammatory potential of the aqueous extract of V. tuberculatum.

A model for microbial interactions and metabolomic alterations in Candida glabrata-Staphylococcus epidermidis dual-species biofilms.

The fungus Candida glabrata and the bacterium Staphylococcus epidermidis are important biofilm-forming microorganisms responsible of nosocomial infections in patients. In addition to causing single-species disease, these microorganisms are also involved in polymicrobial infections leading to an increased antimicrobial resistance. To expand knowledge about polymicrobial biofilms, in this study we investigate the formation of single- and dual-species biofilms of these two opportunistic pathogens employing several complementary approaches. First, biofilm biomass, biofilm metabolic activity and the microbial composition in single- and dual-species biofilms were assessed and compared. Then, the expression of three genes of C. glabrata and three genes of S. epidermidis positively related to the process of biofilm formation was evaluated. Although S. epidermidis is a stronger biofilm producer than C. glabrata, both biological and genetic data indicate that S. epidermidis growth is inhibited by C. glabrata which dominates the dual-species biofilms. To better understand the mechanisms of the interactions between the two microorganisms, a broad GC-MS metabolomic dataset of extracellular metabolites for planktonic, single- and dual-species biofilm cultures of C. glabrata and S. epidermidis was collected. As demonstrated by Partial Least Squares Discriminant Analysis (PLS-DA) of GC-MS metabolomic data, planktonic cultures, single- and dual-species biofilms can be sharply differentiated from each other by the nature and levels of an assortment of primary and secondary metabolites secreted in the culture medium. However, according to our data, 2-phenylethanol (secreted by C. glabrata) and the synergistically combined antifungal activity of 3-phenyllactic acid and of the cyclic dipeptide cyclo-(l-Pro-l-Trp) (secreted by S. epidermidis) play a major role in the race of the two microorganisms for predominance and survival.

The Suppressive Effects of Biochar on Above- and Belowground Plant Pathogens and Pests: A Review.

Soilborne pathogens and pests in agroecosystems are serious problems that limit crop yields. In line with the development of more ecologically sustainable agriculture, the possibility of using biochar to control pests has been increasingly investigated in recent years. This work provides a general overview of disease and pest suppression using biochar. We present an updated view of the literature from 2015 to 2022 based on 61 articles, including 117 experimental case studies. We evaluated how different biochar production feedstocks, pyrolysis temperatures, application rates, and the pathosystems studied affected disease and pest incidence. Fungal pathogens accounted for 55% of the case studies, followed by bacteria (15%), insects and nematodes (8%), oomycetes and viruses (6%), and only 2% parasitic plants. The most commonly studied belowground pathogen species were Fusarium oxysporum f. sp. radicis lycopersici in fungi, Ralstonia solanacearum in bacteria, and Phytophthora capisci in oomycetes, while the most commonly studied pest species were Meloidogyne incognita in nematodes, Epitrix fuscula in insects, and both Phelipanche aegyptiaca and Orobanche crenata in parasitic plants. Biochar showed suppression efficiencies of 86% for fungi, 100% for oomycetes, 100% for viruses, 96% for bacteria, and 50% for nematodes. Biochar was able to potentially control 20 fungal, 8 bacterial, and 2 viral plant pathogens covered by our review. Most studies used an application rate between 1% and 3%, a pyrolysis temperature between 500 °C and 600 °C, and a feedstock based on sawdust and wood waste. Several mechanisms have been proposed to explain disease suppression by biochar, including induction of systemic resistance, enhancement of rhizosphere competence of the microbial community, and sorption of phytotoxic compounds of plant and/or microbial origin. Overall, it is important to standardize biochar feedstock and the rate of application to improve the beneficial effects on plants in terms of disease control.

Synergistic Effects of Trichoderma harzianum, 1,3 Dichloropropene and Organic Matter in Controlling the Root-Knot Nematode Meloidogyne incognita on Tomato.

Environmental concerns raised by synthetic nematicides are encouraging integrated management strategies based on their combination with non-chemical control tools, such as biocontrol agents and/or organic amendments. In this study, the combination of the fumigant 1,3-dichloropropene (1,3-D) with a commercial formulation of the biocontrol agent Trichoderma harzianum (TH) and an organic fertilizer (OF) was investigated in two consecutive tomato crops for its effect on the root-knot nematode Meloidogyne incognita and plant growth and yield. The application of 1,3-D was only performed on the first crop, while TH and OF were provided to both crops. Almost all treatments significantly reduced nematode infestation in both crops, though the greatest nematicidal effect was caused by a combination of the three products. The treatment with 1,3-D limited its nematicidal efficacy to the first crop only. Fumigant integration with TH and OF also resulted in the greatest increases of plant growth and yield. Therefore, the integrated management of root-knot nematodes with a soil fumigant, a bionematicide as T. harzianum and a source of organic matter demonstrated effective nematode suppression though limiting the number of chemical applications.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (Pr3+, Eu3+, Ho3+, Tm3+).

Rare-earth elements (REEs) are in all respect a class of new contaminants that may have toxic effects on organisms and microorganisms and information on their interactions with natural ligands should be of value to predict and control their diffusion in natural environments. In the current study, we investigate interactions of tripositive cations of praseodymium, europium, holmium, and thulium with harzianic acid (H2L), a secondary metabolite produced by selected strains of fungi belonging to the Trichoderma genus. We applied the same techniques and workflow previously employed in an analogous study concerning lanthanum, neodymium, samarium, and gadolinium tripositive cations. Therefore, in the current study, HPLC-ESI-HRMS experiments, circular dichroism (CD), and UV-Vis spectrophotometric absorption data, as well as accurate pH measurements, were applied to characterize bonding interactions between harzianic acid and Pr3+, Eu3+, Ho3+, and Tm3+ cations. Problems connected to the low solubility of harzianic acid in water were overcome by employing a 0.1 M NaClO4/(CH3OH + H2O 50/50 w/w) mixed solvent. For Pr3+, Ho3+, and Tm3+, only the mono complexes PrL+, HoL+, and TmL+ were detected and their formation constant determined. Eu3+ forms almost exclusively the bis complex EuL2- for which the corresponding formation constant is reported; under our experimental conditions, the mono complex EuL+ is irrelevant. Combining the results of the present and previous studies, a picture of interactions of harzianic acid with rare-earth cations extending over 8 of the 17 REEs can be composed. In order to complement chemical information with toxicological information, a battery of bioassays was applied to evaluate the effects of praseodymium, europium, holmium, and thulium tripositive cations on a suite of bioindicators including Aliivibrio fischeri (Gram-negative bacterium), Raphidocelis subcapitata (green alga), and Daphnia magna (microcrustacean), and median effective concentration (EC50) values of Pr3+, Eu3+, Ho3+, and Tm3+ for the tested species were assessed.

Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer.

Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.

The SWC4 subunit of the SWR1 chromatin remodeling complex is involved in varying virulence of Metarhizium brunneum isolates offering role of epigenetic regulation of pathogenicity.

The host - pathogen interaction is a multifactorial process subject to a co-evolutionary arms race consisting of rapid changes in both host and pathogen, controlled at the genetic and epigenetic levels. Previously, we showed intra-species variation in disease progression and pathogenicity in aphids for Metarhizium brunneum isolates MbK and Mb7. Herein, we compared genomic, epigenetic, and metabolomic variations between these isolates and their effects on pathogenicity. Genomic variation could not completely explain the observed differences between the isolates. However, differential N6-adenine methylation (6 mA) and its correlation to reduced expression of the essential SWC4 subunit of SWR1 chromatin-remodelling complex (SWR1-C) led us to hypothesize a role for swc4 in the varying pathogenicity. Mutagenesis of the essential swc4 gene in MbKisolate resulted in reduction of secondary-metabolite (SM) secretion and impaired virulence in Galleria mellonella. Our results suggest the role of SWC4 in the regulation of SMs and the role of both SWC4 and SWR1-C in virulence of M. brunneum isolates. A better understanding of epigenetic regulation of SM production and secretion in entomopathogenic fungi may enable theirmanipulation for better biocontrol performance, and expand possibilities for environmentally friendly pest control.

Trichoderma Enzymes for Degradation of Aflatoxin B1 and Ochratoxin A.

The contamination of agricultural products with mycotoxins causes risks to animal and human health and severe economic losses. Mycotoxicoses can be reduced by preventing fungal infection using chemical and biological approaches. The chemical strategies can release toxic molecules; therefore, strategies for biological control are being evaluated, such as using nontoxic fungi and their metabolites. This work evaluated the effect of exoenzymes produced by the beneficial fungus Trichoderma afroharzianum strain T22 in degrading Aflatoxin B1 (AFB1) and Ochratoxin A (OTA). The ability of Trichoderma to produce hydrolases was stimulated by using different inducing substrates. The highest AFB1 and OTA degradation activity was obtained using a medium containing lyophilized mushrooms and crude fiber. The T. afroharzianum T22's ability to reduce mycotoxins may be attributed to peroxidase enzymes. This study showed that T.afroharzianum strain T22 or its peroxidase supplementation could represent a sustainable strategy for the degradation of AFB1 and OTA in feed and food products.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (La3+, Nd3+, Sm3+, Gd3+).

Rare-earth elements are emerging contaminants of soil and water bodies which destiny in the environment and effects on organisms is modulated by their interactions with natural ligands produced by bacteria, fungi and plants. Within this framework, coordination by harzianic acid (H2L), a Trichoderma secondary metabolite, of a selection of tripositive rare-earth cations Ln3+ (Ln3+ = La3+, Nd3+, Sm3+, and Gd3+) was investigated at 25 °C, and in a CH3OH/0.1 M NaClO4 (50/50 w/w) solvent, using mass spectrometry, circular dichroism, UV-Vis spectrophotometry, and pH measurements. Experimental data can be satisfactorily explained by assuming, for all investigated cations, the formation of a mono-complex (LnL+) and a bis-complex (LnL2-). Differences were found between the formation constants of complexes of different Ln3+ cations, which can be correlated with ionic radius. Since gadolinium is the element that raises the most concern among lanthanide elements, its effects on organisms at different levels of biological organization were explored, in the presence and absence of harzianic acid. Results of ecotoxicological tests suggest that harzianic acid can decrease gadolinium biotoxicity, presumably because of complex formation with Gd3+.

Metabolic Profile and Mycoherbicidal Activity of Three Alternaria alternata Isolates for the Control of Convolvulus arvensis, Sonchus oleraceus, and Xanthium strumarium.

Alternaria alternata isolates C1, S1, and X3 were isolated respectively from the weeds Convolvulus arvensis, Sonchus oleraceus, and Xanthium strumarium in Algiers during 2016 and identified by morphological and molecular analyses. The aim of this investigation was to chemically characterize the exometabolome of these fungi and to evaluate the myco-herbicidal potential of their culture filtrates, crude extracts, or fractions towards target weeds. Results revealed a great heterogeneity in the biochemical profiles of the exometabolome with the remarkable presence of two compounds: tenuazonic acid (TeA) and triprenyl phenol-7 (SMTP-7). To the best of our knowledge, SMTP-7-found in all the isolates-as well as 12-methoxycitromycin detected in the culture filtrate of isolate C1, have never been reported to be produced by A. alternata. Some fractions of isolates C1 and S1 showed symptoms (necrosis and chlorosis) on the detached leaves of C. arvensis and S. oleraceus, respectively with up to 100% phytotoxic effect at low concentration. In conclusion, biochemical characterization revealed great difference of C1, S1, and X3 exometabolome that is likely to explain the difference in their phytotoxic activity. Some fractions (d1, e1, h1, i1, a2, and f2) of isolates C1 and S1 of A. alternata caused severe necrosis and chlorosis on the injured detached leaves of C. arvensis and S. oleraceus, respectively.