Dual biocontrol and osmotic stress mitigation by endophytic Aspergillus micronesiensis and Penicillium momoi against fusarium pathogens

Ali Aligholizadeh; Mina Salehi; Seyed Bagher Mahmoudi; Naser Safaie

doi:10.1371/journal.pone.0353217

Abstract

Global challenges associated with crop diseases and abiotic stress necessitate sustainable agricultural solutions. This study investigated the biocontrol and osmotic-stress mitigation potential of two endophytic fungi, which were isolated from native plants and subsequently identified as Aspergillus micronesiensis and Penicillium momoi. Their antagonistic activities against Fusarium oxysporum f. sp. lycopersici (FOL), F. oxysporum f. sp. radicis-lycopersici (FORL), and F. pseudograminearum (FPS) were evaluated. Complementary antagonistic mechanisms were observed: P. momoi primarily inhibited pathogen growth through direct mycelial competition, while A. micronesiensis predominantly exerted effects through antibiosis. In dual-culture assays, A. micronesiensis inhibited FOL, FORL, and FPS by 39.05%, 39.93%, and 54.59%, respectively, whereas P. momoi caused inhibition rates of 51.84%, 64.74%, and 60.66%, respectively, demonstrating strong biocontrol potential. The salt tolerance assay revealed that both endophytes were able to grow on media containing up to 3 M NaCl. Although, optimal growth for both isolates occurred at 1 M NaCl, A. micronesiensis showed a greater increase in growth relative to the control (2.06 times). In contrast, P. momoi maintained more consistent growth across the 0–1 M NaCl range. However, growth of both isolates declined at higher salt concentrations. The developed wettable powder formulation maintained high spore viability for at least 27 months under room-temperature conditions. This research highlights A. micronesiensis and P. momoi as promising agents for managing Fusarium diseases and enhancing plant salinity tolerance. Further metabolomics investigations are recommended to clarify the functional roles of these endophytes in crop protection under stress conditions.

Citation: Aligholizadeh A, Salehi M, Mahmoudi SB, Safaie N (2026) Dual biocontrol and osmotic stress mitigation by endophytic Aspergillus micronesiensis and Penicillium momoi against fusarium pathogens. PLoS One 21(7): e0353217. https://doi.org/10.1371/journal.pone.0353217

Editor: Raed Abduljabbar Haleem, University of Duhok, IRAQ

Received: February 15, 2026; Accepted: June 19, 2026; Published: July 29, 2026

Copyright: © 2026 Aligholizadeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Iran National Science Foundation (INSF No. 4012798 to NS).

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Endophytic fungi are microorganisms colonizing internal plant tissues without causing disease symptoms, providing multiple benefits to their host plants through enhanced plant growth, improved nutrient acquisition, and increased tolerance to both biotic and abiotic stresses [1–5]. These symbionts have attracted attention in sustainable agriculture, particularly through seed-coating technologies that facilitate early microbial colonization and improve seedling establishment and vigor [6,7]. Such approaches reduce reliance on chemical inputs while promoting natural plant-microbe interactions and supporting sustainable agricultural practices [8,9].

One of the principal abiotic stresses limiting crop productivity worldwide is soil salinity. Saline soils reduce water availability and disrupt nutrient balance, adversely affecting plant growth and yield [10–12]. Endophytic fungi have demonstrated remarkable tolerance to salinity and can alleviate salt stress through physiological mechanisms such as osmolyte accumulation and antioxidant production [5,13,14]. Additionally, these fungi improve nutrient uptake and produce phytohormones that contribute to plant resilience under saline conditions [15,16].

Tomato Fusarium wilt, caused by Fusarium oxysporum f. sp. Lycopersici [17,18], and wheat crown rot, caused primarily by F. pseudograminearum and F. culmorum [19–21], are significant soil-borne diseases that threaten crop productivity globally. These pathogens not only reduce yield but also produce mycotoxins that pose a threat to food safety [22–24]. Therefore, integrated disease management strategies increasingly emphasize biological control approaches for sustainable crop protection. Biological control using microbial biological control agents (MBCAs) can suppress pathogens, reduce disease severity, and enhance plant tolerance to abiotic stresses, such as salinity [5]. The use of these beneficial fungi supports sustainable agriculture by reducing reliance on chemical fungicides and improving soil health.

Penicillium and Aspergillus are important MBCAs utilized in agriculture to suppress plant pathogens such as Fusarium [25]. These fungi produce antifungal metabolites that inhibit pathogen growth and reduce disease severity. Additionally, they enhance plant tolerance to abiotic stresses, such as salinity, by improving nutrient uptake and stimulating plant defense mechanisms [26,27].

Endophyte-based seed coatings have emerged as promising biocontrol tools by delivering beneficial microbes directly to seeds. This technology promotes early colonization by endophytic fungi that not only antagonize pathogen through multiple biocontrol mechanisms, but also enhance plant tolerance to abiotic stresses such as salinity [9,28–30]. Optimizing seed coating formulations that consider factors such as microbial viability, adhesive properties, and controlled release is crucial for ensuring prolonged, adequate protection during plant growth [31].

This study aimed to taxonomically identify the causal agents of Tomato Fusarium wilt and wheat crown rot, as well as fungal biocontrol agents “Penicillium sp. and Aspergillus sp.”, (2) evaluate their biocontrol potential against Fusarium oxysporum f. sp. lycopersici, F. o. f. sp. radicis-lycopersici, and F. pseudograminearum, and (3) develop optimized formulations ensuring long-term viability to support practical agricultural applications. This integrated approach combines antagonistic mechanisms and osmotic resilience to support sustainable crop management under stress conditions.

2. Materials and Methods

2.1. Isolation and identification of pathogenic fungi

Infected plant samples were collected in 2019−2024 from wheat fields in Shahin Dezh (West Azerbaijan), showing crown and root rot, and from tomato plants in East Azerbaijan with Fusarium wilt symptoms. The method of Ma et al. [32] was followed with slight modifications for the isolation of pathogenic fungi. First, the crown and root portions of the samples were washed under running tap water to remove excess soil. Tissues (3–5 mm) from the infected crown, stem, and root regions were surface-sterilized with 70% ethanol (v/v) for 2 min, followed by three rinses with sterile distilled water (2 min each), and subsequently immersed in 96% ethanol for 40 s. The surface-disinfected pieces were placed on potato dextrose agar (PDA) supplemented with 250 mg l^-1 Chloramphenicol and incubated at 27 ± 2 °C. Emerging hyphae were subcultured on PDA and purified using the hyphal-tip method on water agar (WA). Pathogenicity tests were performed on sterilized seeds of four wheat cultivars (Sardari, Homa, Sadra, and Hashtrood) and on tomato seedlings of cultivar Super chief in sterile soil inoculated with fungal material. Disease severity was assessed one month after inoculation by evaluating symptom development and growth reduction. The most virulent wheat isolates were identified by PCR amplification of the ITS region [33] and β-tubulin [34; S1 Table]. At the same time, universal primers (UniF and UniR [35]) and specific primers (sprlF and sprlR [35]) were used for identifying the tomato isolates (S1 Table). Genomic DNA was extracted from frozen mycelia (40–50 mg) as described previously [36]. The thermal program included initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for ITS, 58 °C for β-tubulin, and 62 °C for tomato-specific primers for 1 min, and extension at 72 °C for 2 min; followed by a final extension at 72 °C for 5 min. PCR products were separated using agarose gel electrophoresis and subsequently purified with a DNA gel extraction kit (Axygen Biotechnology Ltd., China). These purified fragments were then sequenced directly by Bioneer (Shanghai, China) using the same primers applied in the PCR reactions.

2.2. Morphological and Molecular identification of biocontrol fungi

Penicillium sp. (isolated from Astragalus squarrosus leaf) and Aspergillus sp. (isolated from Gypsophila mucronifolia leaf) were obtained from the Laboratory of Plant Diseases Bicontol and Management, Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.

For morphological characterization, both fungal isolates were cultured on six different agar media: Czapek Yeast Autolysate agar (CYA), Czapek’s agar (CZA), Malt Extract agar (MEA), Oatmeal agar (OA), Yeast Extract Sucrose agar (YESA), and PDA (S2 Table; [37]). Cultures were incubated at 27 ± 2 °C in the dark for 14 days in triplicate. Colony morphology, growth rate, and microscopic features (e.g., conidia, metulae, phialides, conidiophores, vesicles, and stipes) were analyzed. Microscopic observations were performed using Lactophenol-cotton blue-stained slides, and images were captured with an Olympus BX51 microscope equipped with a DP72 camera.

The PCR reaction mixtures (25 μl) consisted of 12.5 μl Premix Taq (TaKaRa Biotechnology Ltd., Japan), one μl of genomic DNA (~100 ng), one μl of forward and reverse primers (10 Pmol), and 10.5 μl PCR-grade water. The PCR reaction programs were an initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation (94 °C for 30 s), annealing (56 °C (ITS) [33], and 55 °C (TUB [34], RPB2 [38], and CMD [39]) for 30 s), extension (72 °C for 1 min) and a final extension at 72 °C for 5 min. The PCR products were confirmed by agarose gel electrophoresis and then purified using a DNA gel extraction kit (Axygen Biotechnology Ltd., China). The purified PCR product was directly sequenced using the same primers by Bioneer (Shanghai, China).

The alignments of combined ITS and TUB/ITS, TUB, CMD, and RPB2 sequences of the strains in this study and those obtained from GenBank (S3 and S4 Tables) were compared. Sequences were aligned using MUSCLE in MEGA v11 and manually refined in the MEGA software by deleting the headers and footers until the maximum number of shared sequences was reached. Hamigera avellanea and H. brevicompacta were added as outgroup taxa for Aspergillus sp. and Penicillium sp. [40]. Also, Fusarium nelsonii was designated as the outgroup taxon for F. pseudograminearum [41]. Analysis was performed based on Bayesian inference (BI). The phylogenetic analysis of Aspergillus sp. and Penicillium sp. was performed using the GTR + G + I model, which accounts for invariant sites. On the other hand, the phylogenetic tree for F. pseudograminearum was constructed using the SYM + G model. The Bayesian analyses were performed using MrBayes v3.1.2 [42]. with a random starting tree, and the chains were run for 5 million generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The Markov chain Monte Carlo (MCMC) method, within a Bayesian framework, was used to estimate the posterior probabilities of the phylogenetic trees [43] using the 50% majority rule. The convergence of model parameters and topology was assessed based on the average standard deviation of split frequencies and the potential scale reduction factor. A maximum likelihood (ML) tree was reconstructed with RaxmlGUI 1.1 [44] using the same nucleotide substitution model for BI, and 1000 bootstrap (BS) pseudo-replicates were generated. The output file of the phylogenetic program was visualized using Dendroscope V.3.2.8 [45] and annotated in PowerPoint.

2.3. Salt tolerance assay

The fungal growth was assessed on PDA supplemented with different NaCl concentrations (0, 1, 2, and 3 M). Cultures were incubated at 27 ± 2 °C in darkness [46] and radial growth was measured after 14 days. The experiment was designed as a factorial based on a randomized complete block design (RCBD) with five replicates.

2.4. Antagonistic activity assays

Antagonistic activity against pathogenic fungi was assessed using a dual culture assay. Biocontrol isolates of Penicillium sp. and Aspergillus sp. were first cultured on PDA plates for 7 days. Subsequently, mycelial discs (5 mm diameter) of the pathogens F. oxysporum f. sp. lycopersici, F. radicis-lycopersici, and F. pseudograminearum were placed on the opposite side of the same plate. Control plates contained only fungal pathogen discs. All plates were incubated at 27 ± 2 °C in the dark.

The percentage inhibition of radial growth (PIRG) was estimated using Eq. (1).

(1)

where R₁ denotes the pathogen colony radius in the control plate, and R₂ is the radius of the pathogen colony in dual culture.

The experiment was designed as a factorial RCBD with four replicates.

2.5. Formulation preparation

Wheat grains were hydrated either by boiling for 20–30 min, followed by autoclaving twice at 121 ºC for 40 min. The sterilized grains were inoculated with 2 agar discs (5 mm diameter) containing mycelia of biocontrol isolates, grown on PDA, and incubated at 27 ± 2 °C for 15 days with regular agitation to prevent clumping. Spores were harvested in sterile water, and the concentration was adjusted to 6 × 10⁵ (for Aspergillus sp.) and 3 × 10⁹ (for Penicillium sp.) spores ml^-1 using a hemocytometer.

Formulations were prepared by thoroughly mixing spore suspensions (48% v/v) with selected carriers (45% w/v; wheat bran, sodium alginate, sodium bentonite, and talc), stabilizers (0.04% w/v; sodium nitrate and dipotassium hydrogen phosphate), Tween 20 (3% v/v) as a wetting agent, and carboxymethyl cellulose (CMC) as a suspending agent. Detailed compositions of all formulations are presented in S5 Table.

The mixture was dried at 38.5 °C using a germinator, then ground into powder and stored in sealed zip-lock bags.

2.6. Optimal carrier selection and stability assessment

Eighteen different formulations (S5 Table) were evaluated by varying carriers and stabilizers while keeping Tween 20 and CMC constant.

Formulation stability was assessed by serial dilution plating (up to an eightfold dilution) at intervals ranging from 1 to 27 months post-production. Spore viability was determined by counting germinated colonies on PDA plates. Colony-forming units (CFU) were quantified via serial dilution plating on PDA. Two independent experiments were set up in an RCBD with three replicates.

2.7. Greenhouse evaluation of optimized formulation of biocontrol agents under combined drought and pathogen stress

To evaluate the performance of optimized formulation of biocontrol agents “Penicillium sp. and Aspergillus sp. under greenhouse conditions, a factorial experiment was conducted using a RCBD with four replications. The study assessed the interactive effects of wheat cultivar, drought stress, pathogen inoculation, and treatment type on plant growth and disease development.

The experimental factors included: (A) wheat cultivar (Hashtrood and Sadra), (B) drought stress (well-watered control and drought stress), (C) pathogen inoculation (non-inoculated and inoculated with F. pseudograminearum), and (D) treatment type with five levels: optimized formulation based on Aspergillus sp. (AspF), optimized formulation based on Penicillium sp. (PenF), empty formulation without biological agent (EmpF), EmpF combined with tebuconazole (EmpF + TEB), and tebuconazole alone (TEB). This structure allowed separation of biological agent effects, empty formulation effects, and chemical fungicide efficacy.

Field soil was sieved and sterilized twice at 121 °C for 24 h before being air-dried. Inoculum of F. pseudograminearum was prepared using autoclaved wheat seeds colonized with 5-mm mycelial plugs from 7-day-old PDA culture incubated at 27 ± 2 °C. After full colonization, excess moisture was removed aseptically. Colonized seeds were mixed with the upper soil layer at 1% (w/w) and transferred into 2-L pots. Wheat seeds of Hashtrood and Sadra cultivars were surface-sterilized with 0.6% sodium hypochlorite for 2 min, followed by 70% ethanol for 2 min, and rinsed three times with sterile distilled water. Seeds were then coated with the respective treatments prior to sowing. Two seeds were sown per pot and maintained under greenhouse conditions with uniform irrigation.

The soil moisture content was maintained within the ranges of 35–45% and 15–25% for the well-watered control and drought-stress treatments, respectively, using a modified method described by Imakumbili [47].

Disease score was recorded 45 days after inoculation using a 0–4 scaleas described previously [48]. Growth parameters, including plant height (measured from the soil surface to the tip of the main stem in cm), SPAD chlorophyll index (measured using a SPAD meter on the uppermost fully expanded leaf), and shoot and root fresh and dry weights (measured in grams after drying at 70 °C to constant weight), were also determined.

The experiments were conducted in the research greenhouses of the Department of Plant Pathology at Tarbiat Modares University (TMU), and no permission was required.

2.8. Statistical analysis

Normality of data and homogeneity of variances were verified. Following confirmation of data normality and homogeneity of variances (especially after data transformation for disease score), parametric tests were applied. Disease scores were initially converted to proportions by dividing the 0–4 scale by 4, yielding values ranging from 0 to 1. Subsequently, these proportions were transformed using the arcsin-square root transformation (arcsin(√(p))), where p represented the proportion of disease score. Analysis of variance (ANOVA) and mean comparisons using the least significant difference (LSD) test were performed using SAS and SPSS. GraphPad software was used to generate graphical representations of the data.

3. Results

3.1. Molecular identification of Fusarium pathogens

Using the specific primers (Uni and Spr1), two isolates of F. oxysporum, namely F. o. f. sp. lycopersici and F. o. f. sp. radicis-lycopersici, were distinguished from other specific F. oxysporum species (S1–S3 Figs). Amplification with the Uni primer produced DNA fragments of 670–672 bp for both isolates (S2 Fig). One of the isolates did not produce any amplicon when tested with the Sprl primer targeting F. o. f. sp. lycopersici races 1–3 (S3 Fig). However, one isolate obtained a 700 bp amplicon using the Spr1 primer, which is specific to F. o. f. sp. radicis-lycopersici (S3 Fig).

Molecular characterization of the wheat crown and root rot causal agent used two genetic loci: ITS and TUB. Phylogenetic assessment using Bayesian and maximum-likelihood methods placed this isolate within the F. pseudograminearum clade, exhibiting considerable sequence homology across all genetic markers (S3 Table and Fig 1).

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Fig 1. The phylogenetic tree, constructed using Bayesian 50% majority-rule consensus, employed the ITS and TUB gene regions from 50 nucleotide sequences under the SYM + G model.

The most recently identified species, F01. Fusarium nelsonii (CIB04) and F. nelsonii (NRRL 13338), were designated as outgroup taxa. Bayesian posterior probabilities (BPP) exceeding 50% are provided for relevant clades. Ex-type strains are denoted by a superscript T. Bootstrap percentages from the maximum likelihood (ML) analysis and Bayesian inference (BI) posterior probability (pp) values are illustrated at the nodes (ML/pp). Values less than 70% bootstrap support (ML) or less than 0.95 posterior probability (Bayesian analysis) are indicated with a hyphen or not shown. Asterisks indicate full support (100% bootstrap or 1.00 pp). The bar indicates the frequency of substitutions per site.

https://doi.org/10.1371/journal.pone.0353217.g001

3.2. Morphological and microscopic identification of Aspergillus sp. and Penicillium sp

The radial growth of the endophytic Aspergillus sp. and Penicillium sp. was measured on six culture media—CYA, CZA, YESA, OA, MEA, and PDA—over 14 days at 27 ± 2 °C. Growth rates of Aspergillus sp. on these culture media varied significantly, with YESA showing the largest average colony diameter (7.97 cm), followed by OA (4.23 cm) and PDA (4.13 cm, Fig 2). CYA (3.33 cm) and MEA (3.30 cm) supported moderate growth (Fig 2). In comparison, CZA produced the smallest colonies (3.00 cm, Fig 2). Radial growth of Aspergillus sp. followed this order: YESA > OA = PDA > CYA = MEA > CZA. Aspergillus sp. colony morphology differed clearly among media: YESA colonies appeared dense, velvety, and intensely orange-to-reddish brown, with deep orange-brown reverse pigmentation (Fig 3), indicating optimal nutrient use. OA colonies were sparse and floccose with faint yellow pigmentation (Fig 3), suggesting limited growth. MEA and PDA supported moderate, velvety to powdery colonies with reddish-brown hues (Fig 3). Colonies on CYA and CZA were powdery-floccose or velvety-granular with pale colors (Fig 3).

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Fig 2. Radial growth of Aspergillus sp. and Penicillium sp. in Czapek yeast autolysate agar (CYA), Czapek’s agar (CZA), yeast extract sucrose agar (YESA), oatmeal agar (OA), malt extract agar (MEA), and potato dextrose agar (PDA) media.

Values represent the mean ± standard error of n = 3 independent biological replicates. Significant differences are shown by different letters above the bars (P < 0.05).

https://doi.org/10.1371/journal.pone.0353217.g002

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Fig 3. Aspergillus sp. on media cultures of Czapek Yeast Autolysate agar (CYA; A1 and A2), Czapek’s agar (CZA; B1 and B2), malt extract agar (MEA; C1 and C2), oatmeal agar (OA; D1 and D2), potato dextrose agar (PDA; E1 and E2), and yeast extract sucrose agar (YESA; F1 and F2).

Indices 1 and 2 correspond to the top and bottom views, respectively, of the cultures on the 14^th day.

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Penicillium sp. showed the most remarkable radial growth on YESA medium (7.63 cm), followed by CYA (7.2 cm, Fig 2). PDA (6.53 cm) and MEA (5.62 cm) supported moderate growth (Fig 2). In comparison, OA (4.8 cm) and CZA (3.86 cm) had the least expansion (Fig 2). Colonies on YESA were large, radially striated, dark green to bluish-green, surrounded by a yellow peripheral halo, with intense deep orange pigmentation on the reverse (Fig 4), indicating high metabolic activity. CYA supported dense, radially floccose colonies with dark green to grayish-green colonies. In contrast, CZA produced irregular, sparsely sporulating colonies with pale pigmentation (Fig 4). MEA and PDA promoted moderate growth, forming lobed colonies with distinct sporulation patterns. At the same time, OA resulted in thin, sparsely sporulating colonies (Fig 4). These observations highlighted the isolate’s preference for nutrient-rich environments and its potential for use in pigment-production studies related to biocontrol applications. Furthermore, the observed metabolic differences (Figs 2–4) in response to media composition provided significant taxonomic and ecological insights into the isolate’s adaptive strategies.

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Fig 4. Penicillium sp. on media cultures of Czapek Yeast Autolysate agar (CYA; A1, A2), Czapek’s agar (CZA; B1, B2), malt extract agar (MEA; C1, C2), oatmeal agar (OA; D1, D2), potato dextrose agar (PDA; E1, E2), and yeast extract sucrose agar (YESA; F1 and F2).

Indices 1 and 2 correspond to the top and bottom views, respectively, of the cultures on the 14^th day.

https://doi.org/10.1371/journal.pone.0353217.g004

3.3. Micromorphological characteristics of Aspergillus sp. and Penicillium sp

The conidia of Aspergillus sp. ranged from 2.02 to 3.04 μm in diameter (Table 1). The metulae measured between 4.14–7.32 μm in length and 1.75–4.71 μm in diameter (Table 1). Phialides displayed lengths ranging from 3.12 to 7.04 μm and diameters from 1.83 to 4.3 μm (Table 1). The vesicles were globose to subglobose, with lengths ranging from 9.19 to 23.92 μm and diameters from 9.12 to 20.89 μm (Table 1). The stipes were notably long, measuring 442–2092.5 μm in length and 6.57–11.7 μm in diameter (Table 1, Fig 5).

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Table 1. Dimensional measurements (μm) of fungal structures of Aspergillus sp. and Penicillium sp.

https://doi.org/10.1371/journal.pone.0353217.t001

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Fig 5. Fungal structures of Aspergillus sp.

Stipe (m), vesicle, metula and phialide (n), and vesicle and conidia (o).

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Micromorphological features of Penicillium sp. were characterized by conidia measuring 2.1 to 3.28 μm in diameter (Table 1). The metulae ranged from 7.26 to 15.42 μm in length and exhibited diameters between 2.03 and a broader range of 4.30 to 8.17 μm (Table 1). Phialides measured 5.20 to 8.59 μm in length with diameters varying from 1.23 to 3.99 μm (Table 1). The conidiophores exhibited considerable variability in size, ranging from 37.51 to 334 μm in length and 2.9 to 5.83 μm in diameter (Table 1, Fig 6).

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Fig 6. Fungal structures of Penicillium sp.

Conidiophore, phialide and conidia (m), conidiophore, metula, phialide and conidia (n-q).

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The observed macro- and micromorphological characteristics of both isolates were consistent with the diagnostic taxonomic criteria and polyphasic identification keys described for Aspergillus [37] and Penicillium [49] species in previous studies.

3.4. Molecular identification of Aspergillus sp. and Penicillium sp

Molecular characterization of the examined biocontrol isolates was performed by analyzing four genetic loci: ITS, TUB, CMD, and RPB2. Phylogenetic assessment using Bayesian and maximum-likelihood methodologies situated the Aspergillus isolate within A. micronesiensis clade, exhibiting considerable sequence homology across all genetic markers (S4 Table and Fig 7). In a similar vein, Penicillium isolate was found to cluster closely with P. momoi, thereby affirming its phylogenetic affiliation (S4 Table and Fig 7). The outgroup species Hamigera avellanea and H. brevicompacta provided substantial statistical validation (Bayesian posterior probabilities exceeding 50%, S4 Table and Fig 7). These results (Fig 7) reinforced the taxonomic classification of the isolates and suggested potential implications in the fields of plant pathology and biological control.

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Fig 7. The phylogenetic tree for Aspergillus and Penicillium species, constructed using Bayesian 50% majority-rule consensus, employed the ITS, TUB, CMD, and RBP2 gene regions, based on 50 nucleotide sequences under the GTR + G + I model.

This tree represents the main phylogenetic relationships for the biocontrol agents investigated in this study. The most recently identified species include GMEss5 and ASEss1. Hamigera avellanea (CBS 295 48) and H. brevicompacta (CBS 102661) were designated as outgroup taxa. Bayesian posterior probabilities (BPP) exceeding 50% are provided for relevant clades. Ex-type strains are denoted by a superscript T. The Bayesian inference (BI) posterior probability (pp) values and the bootstrap percentages obtained from the maximum likelihood (ML) analysis are illustrated at the nodes, with branches receiving full support being accentuated. Values below 70% bootstrap support (ML) or below 0.70 posterior probability (Bayesian analysis) are represented with a hyphen. The bar indicates the frequency of substitutions per site.”.

https://doi.org/10.1371/journal.pone.0353217.g007

The partial sequences of the ITS rDNA, TUB, CMD, and RPB2 obtained from A. micronesiensis (P. momoi) were deposited in GenBank (NCBI) under the accession numbers PX260295 (PX247873), PX283779 (PX307883), PX283778 (PX307883), and PX307882 (PX307885), respectively.

3.5. Antagonistic effects of A. micronesiensis and P. momoi against pathogenic Fusarium spp

Antagonistic effects against pathogens were evaluated using dual-culture assays to determine the biocontrol potential of A. micronesiensis and P. momoi isolates against F. oxysporum f. sp. lycopersici (FOL), F. oxysporum f. sp. radicis-lycopersici (FORL) (S4 Fig and Fig 8), and F. pseudograminearum (FPS, S5 Fig and Fig 8). On PDA, P. momoi demonstrated superior antagonistic activity, inhibiting FOL growth by 51.84% (3.30 cm growth compared to 6.85 cm in control) and FORL by 64.74% (2.45 cm versus 6.95 cm in control, Fig 8). A. micronesiensis showed moderate suppression of FOL (4.18 cm, 39.05% growth inhibition) and FORL (4.18 cm, 39.93% growth inhibition, Fig 8).

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Fig 8. Percentage inhibition of radial growth (PIRG) of the growth of Fusarium oxysporum f. sp.

Lycopersici (FOL), F. oxysporum f. sp. radicis-lycopersici (FORL), and F. pseudograminearum (FPS) on potato dextrose agar by Aspergillus micronesiensis and Penicillium momoi. Values represent the mean ± standard error of n = 4 independent biological replicates. Significant differences are shown by different letters above the bars (P < 0.05).

https://doi.org/10.1371/journal.pone.0353217.g008

Interestingly, against FPS, A. micronesiensis produced a larger inhibition halo (1.06 cm) relative to P. momoi (0.15 cm, S5 Fig), although P. momoi more effectively restricted FPS growth (3.10 cm, 60.66% growth inhibition, S5 Fig and Fig 8). Notably, A. micronesiensis inhibited FPS growth by 54.59% (3.58 cm growth compared to 7.89 cm in control). These observations (S5 Fig) suggested that P. momoi primarily exerted antagonism via direct mycelial growth inhibition, whereas A. micronesiensis appeared to rely on the production of volatile antifungal metabolites.

3.6. Salt tolerance of biocontrol fungi

Halotolerance of the isolates was assessed by culturing them on PDA supplemented with 0, 1, 2, and 3 M NaCl. A. micronesiensis exhibited maximum radial growth at 1 M NaCl (7.80 cm), exceeding that of the control (3.78 cm, Fig 9). However, growth markedly declined at higher concentrations (4.12 cm at 2 M and 1.82 cm at 3 M, S6 Fig and Fig 9). P. momoi demonstrated vigorous baseline growth at 0 M (7.98 cm), which slightly increased at 1 M (8.24 cm), but progressively decreased at 2 M (4.86 cm) and 3 M (1.6 cm, S7 Fig and Fig 9). These results indicated that moderate salinity enhanced growth in both fungal isolates (A. micronesiensis and P. momoi), likely due to osmoregulatory mechanisms. In contrast, elevated salinity imposed osmotic stress or ion toxicity. Such responses highlighted the potential of these fungi for applications in saline agroecosystems, although their efficacy diminishes under severe salt stress.

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Fig 9. Radial growth of Aspergillus micronesiensis and Penicillium momoi in culture media containing different concentrations (0, 1, 2, and 3 M) of NaCl.

Values represent the mean ± standard error of n = 5 independent biological replicates. Significant differences are shown by different letters above the bars (P < 0.05).

https://doi.org/10.1371/journal.pone.0353217.g009

3.7. Formulation development and spore germination performance testing

The development of carrier-based formulations was undertaken to maximize spore germination efficiency and ensure the long-term stability of fungal biocontrol isolates. In the case of A. micronesiensis, the formulation composed of “wheat bran, sodium alginate, and dipotassium hydrogen phosphate” as well as “wheat bran, sodium alginate, and sodium nitrate” exhibited superior germination performance, followed by the wheat bran–sodium bentonite–sodium nitrate combination (S8 Fig). For P. momoi, optimal spore germination was achieved with a formulation containing wheat bran, sodium alginate, and dipotassium hydrogen phosphate, followed by a wheat bran–sodium bentonite–dipotassium hydrogen phosphate formulation (S9 Fig). Based on these outcomes, the optimized carrier formulation (wheat bran, sodium alginate, and dipotassium hydrogen phosphate) was successfully developed for both A. micronesiensis and P. momoi (S10 Fig), which can be utilized as biofertilizers and biocontrol agents within sustainable agricultural practices.

3.8. Study of the stability of selected formulations

The optimized formulations were evaluated for their ability to maintain the population of existing antagonists over time. Stability evaluations of the formulations over a 27-month storage period demonstrated that “wheat bran, sodium alginate, and dipotassium hydrogen phosphate” formulation maintained high levels of viable spores for both species (A. micronesiensis and P. momoi, Fig 10). In A. micronesiensis, counts decreased from 6 × 10⁵ to 1 × 10³ spores g^-1 formulation, whereas in P. momoi, they declined from 3 × 10⁹ to 1 × 10⁶ spores g^-1 formulation. These findings confirmed that the selected carrier systems constituted the most suitable formulations, ensuring extended viability and stability of the fungal isolates for practical field applications.

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Fig 10. Colony-forming units (CFU) of Aspergillus micronesiensis and Penicillium momoi in wheat bran + sodium bentonite-based powder and tablet formulations over 27 months.

https://doi.org/10.1371/journal.pone.0353217.g010

3.9. Greenhouse evaluation of optimized formulation of biocontrol agents under combined drought and pathogen stress

Analysis of variance (ANOVA) revealed that cultivar, drought stress, pathogen inoculation, and biocontrol/chemical treatments significantly affected the evaluated traits, including SPAD value, plant height, shoot and root biomass, and disease score (S6 Table). For SPAD, all interaction effects were significant except the cultivar × drought stress × pathogen inoculation interaction. Plant height was significantly influenced by most interaction effects, whereas the cultivar × drought stress and drought stress × pathogen inoculation × biocontrol/chemical treatment interactions were not significant (S6 Table). Shoot fresh weight (SFW) and root dry weight (RDW) were significantly affected by most interaction effects, while the cultivar × pathogen inoculation interaction was not significant (S6 Table). Root fresh weight (RFW) and shoot dry weight (SDW) were significantly influenced by all interaction terms except for the four-way interaction effect observed for RFW (S6 Table). Disease score was significantly affected by the cultivar × pathogen inoculation, drought stress × pathogen inoculation, and pathogen inoculation × biocontrol/chemical treatment interactions (S6 Table), indicating differential disease responses depending on host genotype, irrigation regime (drought stress), and biological/chemical treatment type.

The highest SPAD values were recorded in drought-stressed, non-inoculated plants treated with the biological formulations, reaching 46.6 and 46.7 in Hashtrood-PenF and Sadra-AspF, respectively, representing increases of 11.2% and 28.2% compared with the corresponding controls (Table 2). Under F. pseudograminearum stress, PenF improved SPAD value in Hashtrood (26.7%) and Sadra (15.7%) cultivars compared with their respective controls (Table 2).

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Table 2. Effects of drought stress (-/+), Fusarium pseudograminearum (-/+), and biocontrol/chemical treatments on plant height (cm), shoot fresh weight (SFW, g), root fresh weight (RFW, g), shoot dry weight (SDW, g), root dry weight (RDW, g), and disease score of two wheat cultivars (Hashtrood and Sadra).

https://doi.org/10.1371/journal.pone.0353217.t002

The greatest plant height was observed in Sadra cultivar under non-stress conditions treated with AspF or PenF, reaching 51.0 cm, which was approximately 12.8% higher than the corresponding control (EmpF, Table 2). Under drought, F. pseudograminearum inoculation, and combined stress conditions, biological formulations (AspF or PenF) improved plant height in both cultivars compared with their respective controls (Table 2). Increases in plant height under combined drought and pathogen stress reached 34.3% in Hashtrood and 29.4% in Sadra (Table 2).

Drought stress and F. pseudograminearum inoculation markedly reduced shoot and root biomass traits (Table 2). However, application of the biological formulations generally alleviated these negative effects across both cultivars. Among the tested combinations, the highest SFW values were observed in Hashtrood-PenF and Sadra-AspF treatments (6.3 g; Table 2). Hashtrood cultivar also exhibited the greatest RFW under PenF and AspF treatments, reaching 6.0 and 5.8 g, respectively. Under F. pseudograminearum stress, RDW of Sadra cultivar treated with PenF reached 0.519 g, representing an increase of approximately 53.6% compared with the corresponding control (EmpF; Table 2), highlighting the positive effect of the biocontrol formulation on root development under disease pressure.

As shown in Table 2, biological formulations (AspF and PenF) alleviated disease score in F. pseudograminearum-inoculated plants compared with the corresponding controls. In the absence of pathogen inoculation, disease score was zero in all treatments (Table 2). In Hashtrood, the lowest disease score was observed under pathogen stress alone and combined drought–pathogen stress, with PenF (1.8) and AspF (1.8), respectively, representing a 43.8% reduction compared with the corresponding infected controls (Table 2). In Sadra, F. pseudograminearum-inoculated plants treated with biological formulations (AspF and PenF) under both non-drought and drought conditions showed the lowest disease score (0.8), which was 46.7% and 63.6% lower than the corresponding controls, respectively (Table 2).

Overall, the microbial formulations, AspF and PenF, were more effective than the control (EmpF) and chemical fungicide treatment (TEB) in mitigating the adverse effects of drought and F. pseudograminearum stress on plant growth and disease development (Table 2).

4. Discussion

This study systematically evaluated A. micronesiensis and P. momoi as endophytic biocontrol agents against Fusarium pathogens, integrating morphological, molecular, and functional analyses. The observed variability in colony development across different culture media (Figs 2-4) reflected metabolic plasticity and adaptive growth strategies, suggesting ecological versatility that may contribute to environmental persistence rather than serving solely as a taxonomic feature.

Micromorphological characterization, including conidial size, metulae, phialides, vesicles, and stipe measurements (Table 1 and Figs 5 and 6), provided additional taxonomic resolution. Integration of morphological identification with multilocus molecular analysis (ITS, β-tubulin, calmodulin, and RPB2; Fig 7) confirmed the phylogenetic positions of A. micronesiensis and P. momoi, supporting the reliability of combining morphological and molecular approaches for identification of cryptic fungal taxa.

Antagonistic assays indicated complementary mechanisms of pathogen suppression. P. momoi likely inhibited Fusarium growth through direct mycelial competition, whereas A. micronesiensis appeared to rely on diffusible metabolites (S4, S5 Fig, and Fig 8). Beyond direct mycelial inhibition and antibiosis, the observed complementary antagonistic activity may also involve the induction of systemic resistance in the host plant, as previously reported in endophyte-mediated biocontrol systems [50,51]. These functional differences suggest that co-application of both fungi may enhance biocontrol effectiveness [25,52].

Moderate salinity (1 M NaCl) enhanced fungal growth (S6, S7 Figs, and Fig 9), indicating the presence of adaptive osmoregulatory mechanisms, whereas higher salinity imposed physiological stress. Such tolerance patterns may be associated with fungal osmoregulatory mechanisms, including the accumulation of compatible solutes (e.g., glycerol, trehalose) and ion homeostasis regulation, highlighting the potential application of these isolates in saline-affected agroecosystems while suggesting careful consideration of environmental constraints [53].

Although salinity and drought are distinct environmental stresses, both impose osmotic constraints that limit water availability and trigger related adaptive responses. Therefore, the in vitro salinity assay was used as a preliminary indicator of the ability of the fungal isolates to tolerate osmotic stress. The greenhouse experiment demonstrated that drought stress and F. pseudograminearum infection resulted in a marked reduction in plant growth and physiological traits, including SPAD, plant height, and biomass production. However, application of the endophytic fungal formulations improved plant performance under stress conditions compared with the corresponding controls (Table 2). Overall, the results confirmed that the formulated endophytes were able to partially alleviate the negative effects of the abiotic and biotic stress under greenhouse conditions.

Formulation studies indicated that wheat bran–sodium alginate–dipotassium hydrogen phosphate matrices supported spore viability and long-term storage stability for up to 27 months (S8–S10 Fig and Fig 10). These findings suggest improved shelf stability of biocontrol formulations; however, further field-scale validation is required to confirm their performance under practical agricultural conditions.

A key limitation of this study is the lack of mechanistic resolution at the metabolite and molecular levels. Although antagonistic activity and salt tolerance were demonstrated under controlled conditions, the specific bioactive compounds responsible for antifungal effects, as well as the osmolytes and metabolic pathways involved in salinity adaptation, were not characterized. In addition, while preliminary greenhouse evaluations provide partial in planta support, the findings remain primarily based on controlled experimental conditions. Therefore, extrapolation to field environments should be made cautiously, as complex plant–microbe–environment interactions may significantly influence the performance of these endophytic fungi. Future studies employing metabolomics profiling and targeted chemical analyses are essential to elucidate the underlying mechanisms and strengthen the functional interpretation of these biocontrol agents.

5. Conclusion

This study demonstrates the potential of A. micronesiensis and P. momoi as effective biocontrol agents against key Fusarium pathogens. Both fungi exhibited strong and complementary antagonistic activity through direct mycelial inhibition and metabolite-mediated suppression under in vitro conditions.

The isolates also showed moderate halotolerance in culture-based assays, indicating their ability to grow under saline conditions in vitro. Carrier-based formulations maintained high spore viability and long-term stability, supporting their potential for further development.

Preliminary greenhouse experiments further demonstrated that application of the fungal formulations improved plant growth performance and reduced disease score under stress conditions compared with controls (EmpF and TEB).

However, it is important to emphasize that fungal responses to salinity were evaluated only under in vitro conditions, and plant-level physiological mechanisms of salt stress mitigation were not directly investigated in this study. Therefore, future research should include field-scale validation, detailed physiological analyses, and metabolomics characterization to fully elucidate the biocontrol and stress-related effects of these endophytic fungi.

Collectively, these findings support the potential application of these endophytic fungi in sustainable disease management and integrated pest management strategies under stress-prone agricultural systems.

Highlights

Both species of Aspergillus micronesiensis and Penicillium momoi strongly inhibited key Fusarium pathogens.
These endophytic fungi had complementary biocontrol: direct inhibition and antibiosis.
A. micronesiensis and P. momoi tolerate high salinity, growing in up to 3 M NaCl.
Biological formulations maintain spore viability for over 27 months.
Dual biocontrol and osmotic stress mitigation support sustainable crop management.

Supporting information

S1 Table. Primers used for amplification and sequencing.

https://doi.org/10.1371/journal.pone.0353217.s001

(DOCX)

S2 Table. Media used for morphological characterization.

https://doi.org/10.1371/journal.pone.0353217.s002

(DOCX)

S3 Table. Accession numbers used for phylogenetic analysis of Fusarium pseudograminearum.

https://doi.org/10.1371/journal.pone.0353217.s003

(DOCX)

S4 Table. Accession numbers used for phylogenetic analysis of Aspergillus sp. and Penicillium sp.

https://doi.org/10.1371/journal.pone.0353217.s004

(DOCX)

S5 Table. Different compositions of carrier and stabilizers for maximum spore germination.

https://doi.org/10.1371/journal.pone.0353217.s005

(DOCX)

S6 Table. Analysis of variance for the effects of wheat cultivar, soil moisture content, pathogen and formulations on SPAD value, plant height, shoot and root biomass, and disease score.

https://doi.org/10.1371/journal.pone.0353217.s006

(DOCX)

S1 Fig. Fusarium oxysporum f. sp. lycopersici (FOL) and F. o. f. sp. radicis-lycopersici (FORL) on potato dextrose agar.

https://doi.org/10.1371/journal.pone.0353217.s007

(PNG)

S2 Fig. PCR analysis of polygalacturonase gene region of Fusarium oxysporum f. sp. lycopersici with uni primer and in 2% agarose gel.

Ladder DNA 1 Kbp (a), DNA fragments (670–672 bp) of two isolates amplified with primer pair uni (b and c).

https://doi.org/10.1371/journal.pone.0353217.s008

(TIF)

S3 Fig. PCR analysis of polygalacturonase gene region of Fusarium oxysporum f. sp. lycopersici with Spr1 primer and in 2% agarose gel.

Ladder DNA 1 Kbp (a), DNA fragments (700 bp) of one isolate amplified with primer pair Spr1 (b).

https://doi.org/10.1371/journal.pone.0353217.s009

(PNG)

S4 Fig. Dual culture assays of biocontrol isolates against pathogenic fungi.

Aspergillus micronesiensis against Fusarium oxysporum f. sp. lycopersici (FOL, a). Penicillium momoi against FOL (b). A. micronesiensis against F. oxysporum f. sp. radicis-lycopersici (FORL, c). P. momoi against FORL (d).

https://doi.org/10.1371/journal.pone.0353217.s010

(PNG)

S5 Fig. Fusarium pseudograminearum (FPS; A1 and A2), Dual cultures of biocontrol isolates with pathogenic fungi: Aspergillus micronesiensis against FPS (B1 and B2), Penicillium momoi against FPS (C1 and C2).

Indices 1 and 2 correspond to the top and bottom views, respectively.

https://doi.org/10.1371/journal.pone.0353217.s011

(PNG)

S6 Fig. Aspergillus micronesiensis in culture media containing different concentrations (0, 1, 2, and 3 M) of NaCl.

https://doi.org/10.1371/journal.pone.0353217.s012

(PNG)

S7 Fig. Penicillium momoi in culture media containing different concentrations (0, 1, 2, and 3 M) of NaCl.

https://doi.org/10.1371/journal.pone.0353217.s013

(PNG)

S8 Fig. Number of germinated spores in different formulations of Aspergillus micronesiensis.

The materials used in this study included wheat bran (Wb), sodium alginate (Sa), sodium bentonite (Sb), talc (Ta), sodium nitrate (Sn), and dipotassium hydrogen phosphate (Dhp).

https://doi.org/10.1371/journal.pone.0353217.s014

(TIF)

S9 Fig. Number of germinated spores in different formulations of Penicillium momoi.

The materials used in this study included wheat bran (Wb), sodium alginate (Sa), sodium bentonite (Sb), talc (Ta), sodium nitrate (Sn), and dipotassium hydrogen phosphate (Dhp).

https://doi.org/10.1371/journal.pone.0353217.s015

(TIF)

S10 Fig. Proposed formulation for Penicillium momoi and Aspergillus micronesiensis based on wheat bran + sodium alginate and the ratio of its components.

https://doi.org/10.1371/journal.pone.0353217.s016

(TIF)

Acknowledgments

The authors acknowledge Iran National Science Foundation and Research Deputy of Tarbiat Modares University, Tehran.

References

1. Baron NC, Rigobelo EC. Endophytic fungi: a tool for plant growth promotion and sustainable agriculture. Mycology. 2021;13(1):39–55. pmid:35186412
- View Article
- PubMed/NCBI
- Google Scholar
2. Morales-Vargas AT, López-Ramírez V, Álvarez-Mejía C, Vázquez-Martínez J. Endophytic Fungi for Crops Adaptation to Abiotic Stresses. Microorganisms. 2024;12(7):1357. pmid:39065124
- View Article
- PubMed/NCBI
- Google Scholar
3. Nourian A, Salehi M, Safaie N, Khelghatibana F. Biocontrol of Diplodia bulgarica, the causal agent of apple canker, using Trichoderma zelobreve. Arch Microbiol. 2024;206(3):120. pmid:38396230
- View Article
- PubMed/NCBI
- Google Scholar
4. Ranjbar Z, Salehi M, Safaie N. An endophytic Trichoderma-based wettable powder formulation for biocontrol of apple stem cankers. J Phytopathol. 2024;172(2):e13266.
- View Article
- Google Scholar
5. Salehi M, Safaie N. Editorial: Endophytic fungi: secondary metabolites and plant biotic and abiotic stress management. Front Microbiol. 2024;15:1345210. pmid:38348191
- View Article
- PubMed/NCBI
- Google Scholar
6. Sujatha P, Madhavi M, Pallavi M, Bharathi Y, Jagan Mohan Rao P, Rajeswari B, et al. Biological Seed Coating Innovations for Sustainable Healthy Crop Growth in Tomato. In: Lops F, editors. Tomato Cultivation and Consumption - Innovation and Sustainability. IntechOpen; 2024.
7. Wang Y-L, Zhang H-B. Assembly and Function of Seed Endophytes in Response to Environmental Stress. J Microbiol Biotechnol. 2023;33(9):1119–29. pmid:37311706
- View Article
- PubMed/NCBI
- Google Scholar
8. Das D, Sharma PL, Paul P, Baruah NR, Choudhury J, Begum T, et al. Harnessing endophytes: innovative strategies for sustainable agricultural practices. Discov Bact. 2025;2(1):1.
- View Article
- Google Scholar
9. Rétif F, Kunz C, Calabro K, Duval C, Prado S, Bailly C, et al. Seed fungal endophytes as biostimulants and biocontrol agents to improve seed performance. Front Plant Sci. 2023;14:1260292. pmid:37941673
- View Article
- PubMed/NCBI
- Google Scholar
10. Munns R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002;25(2):239–50. pmid:11841667
- View Article
- PubMed/NCBI
- Google Scholar
11. Läuchli A, Grattan SR. Plant Growth And Development Under Salinity Stress. In: Jenks MA, Hasegawa PM, Jain SM, editors. Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops. Springer Netherlands; p. 1–32.
12. Chele KH, Tinte MM, Piater LA, Dubery IA, Tugizimana F. Soil Salinity, a Serious Environmental Issue and Plant Responses: A Metabolomics Perspective. Metabolites. 2021;11(11):724. pmid:34822381
- View Article
- PubMed/NCBI
- Google Scholar
13. Siddiqui ZS, Wei X, Umar M, Abideen Z, Zulfiqar F, Chen J, et al. Scrutinizing the Application of Saline Endophyte to Enhance Salt Tolerance in Rice and Maize Plants. Front Plant Sci. 2022;12:770084. pmid:35251059
- View Article
- PubMed/NCBI
- Google Scholar
14. Damankeshan B, Shamshiri MH, Alaei H. Endophytic fungi are able to induce tolerance to salt stress in date palm seedlings (Phoenix dactylifera L.). Braz J Microbiol. 2024;55(1):759–75. pmid:38157149
- View Article
- PubMed/NCBI
- Google Scholar
15. Khan AL, Hussain J, Al-Harrasi A, Al-Rawahi A, Lee I-J. Endophytic fungi: resource for gibberellins and crop abiotic stress resistance. Crit Rev Biotechnol. 2015;35(1):62–74. pmid:23984800
- View Article
- PubMed/NCBI
- Google Scholar
16. Rhouma A, Hajji-Hedfi L, Okon OG, Bassey HO. Investigating the effectiveness of endophytic fungi under biotic and abiotic agricultural stress conditions. J OASIS Agric Sustain Dev. 2024;6(01):111–26.
- View Article
- Google Scholar
17. Kanwal I, Iffat A, Shaukat MB, Shafique T, Majeed Y, Zafar MI, et al. Insights into Fusarium wilt of tomato (Fusarium oxysporum f. sp. lycopersici) and its management strategies. J Agric Biol. 2024;2:31–42.
- View Article
- Google Scholar
18. Haruna SG, Yahuza L, Tijjani I. Management of Fusarium Wilt of Tomato (Fusarium oxysporum f. sp. lycopersici) and Related Soil-borne Diseases using Eco-friendly Methods: A Review. AJRCS. 2024;9(1):154–68.
- View Article
- Google Scholar
19. Matny ON, Bates ST, Song Z. Original Article. Geographic distribution of Fusarium culmorum chemotypes associated with wheat crown rot in Iraq. J Plant Prot Res. 2016;57(1):43–9.
- View Article
- Google Scholar
20. Kazan K, Gardiner DM. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: recent progress and future prospects. Mol Plant Pathol. 2018;19(7):1547–62. pmid:29105256
- View Article
- PubMed/NCBI
- Google Scholar
21. Saad A, Christopher J, Martin A, McDonald S, Percy C. Fusarium pseudograminearum and F. culmorum affect the root system architecture of bread wheat. Crop J. 2023;11(1):316–21.
- View Article
- Google Scholar
22. Perincherry L, Lalak-Kańczugowska J, Stępień Ł. Fusarium-Produced Mycotoxins in Plant-Pathogen Interactions. Toxins (Basel). 2019;11(11):664. pmid:31739566
- View Article
- PubMed/NCBI
- Google Scholar
23. Ji X, Deng T, Xiao Y, Jin C, Lyu W, Wu Z, et al. Emerging Alternaria and Fusarium mycotoxins in tomatoes and derived tomato products from the China market: Occurrence, methods of determination, and risk evaluation. Food Control. 2023;145:109464.
- View Article
- Google Scholar
24. Qu Z, Ren X, Du Z, Hou J, Li Y, Yao Y, et al. Fusarium mycotoxins: The major food contaminants. mLife. 2024;3(2):176–206. pmid:38948146
- View Article
- PubMed/NCBI
- Google Scholar
25. Abdelrahem MMM, Abouelela ME, Abo-Dahab NF, Hassane AMA. Aspergillus-Penicillium co-culture: An investigation of bioagents for controlling Fusarium proliferatum-induced basal rot in onion. AIMS Microbiol. 2024;10(4):1024–51. pmid:39628715
- View Article
- PubMed/NCBI
- Google Scholar
26. Li X, Han S, Wang G, Liu X, Amombo E, Xie Y, et al. The Fungus Aspergillus aculeatus Enhances Salt-Stress Tolerance, Metabolite Accumulation, and Improves Forage Quality in Perennial Ryegrass. Front Microbiol. 2017;8:1664. pmid:28936200
- View Article
- PubMed/NCBI
- Google Scholar
27. Tarroum M, Romdhane WB, Al-Qurainy F, Ali AAM, Al-Doss A, Fki L, et al. A novel PGPF Penicillium olsonii isolated from the rhizosphere of Aeluropus littoralis promotes plant growth, enhances salt stress tolerance, and reduces chemical fertilizers inputs in hydroponic system. Front Microbiol. 2022;13:996054. pmid:36386667
- View Article
- PubMed/NCBI
- Google Scholar
28. Radhakrishnan R, Khan AL, Lee I-J. Endophytic fungal pre-treatments of seeds alleviates salinity stress effects in soybean plants. J Microbiol. 2013;51(6):850–7. pmid:24385364
- View Article
- PubMed/NCBI
- Google Scholar
29. Kim J, Ahn S-H, Yang JS, Choi S, Jung HW, Jeon J. Plant Protective and Growth Promoting Effects of Seed Endophytes in Soybean Plants. Plant Pathol J. 2023;39(5):513–21. pmid:37817497
- View Article
- PubMed/NCBI
- Google Scholar
30. Dargiri SA, Naeimi S, Nekouei MK. Enhancing wheat resilience to salinity: the role of endophytic Penicillium chrysogenum as a biological agent for improved crop performance. BMC Plant Biol. 2025;25(1):354. pmid:40102779
- View Article
- PubMed/NCBI
- Google Scholar
31. Rocha I, Ma Y, Souza-Alonso P, Vosátka M, Freitas H, Oliveira RS. Seed Coating: A Tool for Delivering Beneficial Microbes to Agricultural Crops. Front Plant Sci. 2019;10:1357. pmid:31781135
- View Article
- PubMed/NCBI
- Google Scholar
32. Ma G, Wang H, Qi K, Ma L, Zhang B, Zhang Y, et al. Isolation, characterization, and pathogenicity of Fusarium species causing crown rot of wheat. Front Microbiol. 2024;15:1405115. pmid:38873144
- View Article
- PubMed/NCBI
- Google Scholar
33. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego (CA): Academic Press; 1990. p. 315–22.
34. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol. 1995;61(4):1323–30. pmid:7747954
- View Article
- PubMed/NCBI
- Google Scholar
35. Hirano Y, Arie T. PCR-based differentiation of Fusarium oxysporum ff. sp. lycopersici and radicis-lycopersici and races of F. oxysporum f. sp. lycopersici. J Gen Plant Pathol. 2006;72(5):273–83.
- View Article
- Google Scholar
36. Salehi M, Moieni A, Safaie N. Elicitors Derived from Hazel (Corylus avellana L.) Cell Suspension Culture Enhance Growth and Paclitaxel Production of Epicoccum nigrum. Sci Rep. 2018;8(1):12053. pmid:30104672
- View Article
- PubMed/NCBI
- Google Scholar
37. Samson RA, Visagie CM, Houbraken J, Hong S-B, Hubka V, Klaassen CHW, et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol. 2014;78:141–73. pmid:25492982
- View Article
- PubMed/NCBI
- Google Scholar
38. Liu YJ, Whelen S, Hall BD. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit. Mol Biol Evol. 1999;16(12):1799–808. pmid:10605121
- View Article
- PubMed/NCBI
- Google Scholar
39. Hong S-B, Cho H-S, Shin H-D, Frisvad JC, Samson RA. Novel Neosartorya species isolated from soil in Korea. Int J Syst Evol Microbiol. 2006;56(Pt 2):477–86. pmid:16449461
- View Article
- PubMed/NCBI
- Google Scholar
40. Houbraken J, Kocsubé S, Visagie CM, Yilmaz N, Wang X-C, Meijer M, et al. Classification of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales): An overview of families, genera, subgenera, sections, series and species. Stud Mycol. 2020;95:5–169. pmid:32855739
- View Article
- PubMed/NCBI
- Google Scholar
41. Han SL, Wang MM, Ma ZY, Raza M, Zhao P, Liang JM, et al. Fusarium diversity associated with diseased cereals in China, with an updated phylogenomic assessment of the genus. Stud Mycol. 2023;104:87–148. pmid:37351543
- View Article
- PubMed/NCBI
- Google Scholar
42. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4. pmid:12912839
- View Article
- PubMed/NCBI
- Google Scholar
43. Larget B, Simon D. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Mol Biol Evol. 1999;16:750–9.
- View Article
- Google Scholar
44. Silvestro D, Michalak I. Raxmlgui: a graphical front-end for raxml. Org Divers Evol. 2012;12:335–7.
- View Article
- Google Scholar
45. Huson DH, Scornavacca C. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol. 2012;61(6):1061–7. pmid:22780991
- View Article
- PubMed/NCBI
- Google Scholar
46. Hosseyni Moghaddam MS, Safaie N, Soltani J, Hagh-Doust N. Desert-adapted fungal endophytes induce salinity and drought stress resistance in model crops. Plant Physiol Biochem. 2021;160:225–38. pmid:33517220
- View Article
- PubMed/NCBI
- Google Scholar
47. Imakumbili ML. Making water stress treatments in pot experiments: an illustrated step-by-step guide. Heliyon. 2019;4(11):e00935.
- View Article
- Google Scholar
48. Spagnoletti FN, Carmona M, Balestrasse K, Chiocchio V, Giacometti R, Lavado RS. The arbuscular mycorrhizal fungus Rhizophagus intraradices reduces the root rot caused by Fusarium pseudograminearum in wheat. Rhizosphere. 2021;19:100369.
- View Article
- Google Scholar
49. Visagie CM, Houbraken J, Frisvad JC, Hong S-B, Klaassen CHW, Perrone G, et al. Identification and nomenclature of the genus Penicillium. Stud Mycol. 2014;78:343–71. pmid:25505353
- View Article
- PubMed/NCBI
- Google Scholar
50. Shoresh M, Harman GE, Mastouri F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol. 2010;48:21–43. pmid:20192757
- View Article
- PubMed/NCBI
- Google Scholar
51. Pieterse CMJ, Zamioudis C, Berendsen RL, Weller DM, Van Wees SCM, Bakker PAHM. Induced systemic resistance by beneficial microbes. Annu Rev Phytopathol. 2014;52:347–75. pmid:24906124
- View Article
- PubMed/NCBI
- Google Scholar
52. Ayaz M, Li C-H, Ali Q, Zhao W, Chi Y-K, Shafiq M, et al. Bacterial and Fungal Biocontrol Agents for Plant Disease Protection: Journey from Lab to Field, Current Status, Challenges, and Global Perspectives. Molecules. 2023;28(18):6735. pmid:37764510
- View Article
- PubMed/NCBI
- Google Scholar
53. Jiménez-Gómez I, Valdés-Muñoz G, Moreno-Ulloa A, Pérez-Llano Y, Moreno-Perlín T, Silva-Jiménez H, et al. Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration. Front Microbiol. 2022;13:840408. pmid:35586858
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Baron NC, Rigobelo EC. Endophytic fungi: a tool for plant growth promotion and sustainable agriculture. Mycology. 2021;13(1):39–55. pmid:35186412
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Morales-Vargas AT, López-Ramírez V, Álvarez-Mejía C, Vázquez-Martínez J. Endophytic Fungi for Crops Adaptation to Abiotic Stresses. Microorganisms. 2024;12(7):1357. pmid:39065124
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Nourian A, Salehi M, Safaie N, Khelghatibana F. Biocontrol of Diplodia bulgarica, the causal agent of apple canker, using Trichoderma zelobreve. Arch Microbiol. 2024;206(3):120. pmid:38396230
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Ranjbar Z, Salehi M, Safaie N. An endophytic Trichoderma-based wettable powder formulation for biocontrol of apple stem cankers. J Phytopathol. 2024;172(2):e13266.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref5] 5. Salehi M, Safaie N. Editorial: Endophytic fungi: secondary metabolites and plant biotic and abiotic stress management. Front Microbiol. 2024;15:1345210. pmid:38348191
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Sujatha P, Madhavi M, Pallavi M, Bharathi Y, Jagan Mohan Rao P, Rajeswari B, et al. Biological Seed Coating Innovations for Sustainable Healthy Crop Growth in Tomato. In: Lops F, editors. Tomato Cultivation and Consumption - Innovation and Sustainability. IntechOpen; 2024.

[ref7] 7. Wang Y-L, Zhang H-B. Assembly and Function of Seed Endophytes in Response to Environmental Stress. J Microbiol Biotechnol. 2023;33(9):1119–29. pmid:37311706
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref8] 8. Das D, Sharma PL, Paul P, Baruah NR, Choudhury J, Begum T, et al. Harnessing endophytes: innovative strategies for sustainable agricultural practices. Discov Bact. 2025;2(1):1.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref9] 9. Rétif F, Kunz C, Calabro K, Duval C, Prado S, Bailly C, et al. Seed fungal endophytes as biostimulants and biocontrol agents to improve seed performance. Front Plant Sci. 2023;14:1260292. pmid:37941673
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref10] 10. Munns R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002;25(2):239–50. pmid:11841667
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref11] 11. Läuchli A, Grattan SR. Plant Growth And Development Under Salinity Stress. In: Jenks MA, Hasegawa PM, Jain SM, editors. Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops. Springer Netherlands; p. 1–32.

[ref12] 12. Chele KH, Tinte MM, Piater LA, Dubery IA, Tugizimana F. Soil Salinity, a Serious Environmental Issue and Plant Responses: A Metabolomics Perspective. Metabolites. 2021;11(11):724. pmid:34822381
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref13] 13. Siddiqui ZS, Wei X, Umar M, Abideen Z, Zulfiqar F, Chen J, et al. Scrutinizing the Application of Saline Endophyte to Enhance Salt Tolerance in Rice and Maize Plants. Front Plant Sci. 2022;12:770084. pmid:35251059
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref14] 14. Damankeshan B, Shamshiri MH, Alaei H. Endophytic fungi are able to induce tolerance to salt stress in date palm seedlings (Phoenix dactylifera L.). Braz J Microbiol. 2024;55(1):759–75. pmid:38157149
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref15] 15. Khan AL, Hussain J, Al-Harrasi A, Al-Rawahi A, Lee I-J. Endophytic fungi: resource for gibberellins and crop abiotic stress resistance. Crit Rev Biotechnol. 2015;35(1):62–74. pmid:23984800
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref16] 16. Rhouma A, Hajji-Hedfi L, Okon OG, Bassey HO. Investigating the effectiveness of endophytic fungi under biotic and abiotic agricultural stress conditions. J OASIS Agric Sustain Dev. 2024;6(01):111–26.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref17] 17. Kanwal I, Iffat A, Shaukat MB, Shafique T, Majeed Y, Zafar MI, et al. Insights into Fusarium wilt of tomato (Fusarium oxysporum f. sp. lycopersici) and its management strategies. J Agric Biol. 2024;2:31–42.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref18] 18. Haruna SG, Yahuza L, Tijjani I. Management of Fusarium Wilt of Tomato (Fusarium oxysporum f. sp. lycopersici) and Related Soil-borne Diseases using Eco-friendly Methods: A Review. AJRCS. 2024;9(1):154–68.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref19] 19. Matny ON, Bates ST, Song Z. Original Article. Geographic distribution of Fusarium culmorum chemotypes associated with wheat crown rot in Iraq. J Plant Prot Res. 2016;57(1):43–9.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref20] 20. Kazan K, Gardiner DM. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: recent progress and future prospects. Mol Plant Pathol. 2018;19(7):1547–62. pmid:29105256
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref21] 21. Saad A, Christopher J, Martin A, McDonald S, Percy C. Fusarium pseudograminearum and F. culmorum affect the root system architecture of bread wheat. Crop J. 2023;11(1):316–21.
View Article
Google Scholar

[70] View Article

[71] Google Scholar

[ref22] 22. Perincherry L, Lalak-Kańczugowska J, Stępień Ł. Fusarium-Produced Mycotoxins in Plant-Pathogen Interactions. Toxins (Basel). 2019;11(11):664. pmid:31739566
View Article
PubMed/NCBI
Google Scholar

[73] View Article

[74] PubMed/NCBI

[75] Google Scholar

[ref23] 23. Ji X, Deng T, Xiao Y, Jin C, Lyu W, Wu Z, et al. Emerging Alternaria and Fusarium mycotoxins in tomatoes and derived tomato products from the China market: Occurrence, methods of determination, and risk evaluation. Food Control. 2023;145:109464.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref24] 24. Qu Z, Ren X, Du Z, Hou J, Li Y, Yao Y, et al. Fusarium mycotoxins: The major food contaminants. mLife. 2024;3(2):176–206. pmid:38948146
View Article
PubMed/NCBI
Google Scholar

[80] View Article

[81] PubMed/NCBI

[82] Google Scholar

[ref25] 25. Abdelrahem MMM, Abouelela ME, Abo-Dahab NF, Hassane AMA. Aspergillus-Penicillium co-culture: An investigation of bioagents for controlling Fusarium proliferatum-induced basal rot in onion. AIMS Microbiol. 2024;10(4):1024–51. pmid:39628715
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref26] 26. Li X, Han S, Wang G, Liu X, Amombo E, Xie Y, et al. The Fungus Aspergillus aculeatus Enhances Salt-Stress Tolerance, Metabolite Accumulation, and Improves Forage Quality in Perennial Ryegrass. Front Microbiol. 2017;8:1664. pmid:28936200
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref27] 27. Tarroum M, Romdhane WB, Al-Qurainy F, Ali AAM, Al-Doss A, Fki L, et al. A novel PGPF Penicillium olsonii isolated from the rhizosphere of Aeluropus littoralis promotes plant growth, enhances salt stress tolerance, and reduces chemical fertilizers inputs in hydroponic system. Front Microbiol. 2022;13:996054. pmid:36386667
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

[ref28] 28. Radhakrishnan R, Khan AL, Lee I-J. Endophytic fungal pre-treatments of seeds alleviates salinity stress effects in soybean plants. J Microbiol. 2013;51(6):850–7. pmid:24385364
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref29] 29. Kim J, Ahn S-H, Yang JS, Choi S, Jung HW, Jeon J. Plant Protective and Growth Promoting Effects of Seed Endophytes in Soybean Plants. Plant Pathol J. 2023;39(5):513–21. pmid:37817497
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref30] 30. Dargiri SA, Naeimi S, Nekouei MK. Enhancing wheat resilience to salinity: the role of endophytic Penicillium chrysogenum as a biological agent for improved crop performance. BMC Plant Biol. 2025;25(1):354. pmid:40102779
View Article
PubMed/NCBI
Google Scholar

[104] View Article

[105] PubMed/NCBI

[106] Google Scholar

[ref31] 31. Rocha I, Ma Y, Souza-Alonso P, Vosátka M, Freitas H, Oliveira RS. Seed Coating: A Tool for Delivering Beneficial Microbes to Agricultural Crops. Front Plant Sci. 2019;10:1357. pmid:31781135
View Article
PubMed/NCBI
Google Scholar

[108] View Article

[109] PubMed/NCBI

[110] Google Scholar

[ref32] 32. Ma G, Wang H, Qi K, Ma L, Zhang B, Zhang Y, et al. Isolation, characterization, and pathogenicity of Fusarium species causing crown rot of wheat. Front Microbiol. 2024;15:1405115. pmid:38873144
View Article
PubMed/NCBI
Google Scholar

[112] View Article

[113] PubMed/NCBI

[114] Google Scholar

[ref33] 33. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego (CA): Academic Press; 1990. p. 315–22.

[ref34] 34. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol. 1995;61(4):1323–30. pmid:7747954
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref35] 35. Hirano Y, Arie T. PCR-based differentiation of Fusarium oxysporum ff. sp. lycopersici and radicis-lycopersici and races of F. oxysporum f. sp. lycopersici. J Gen Plant Pathol. 2006;72(5):273–83.
View Article
Google Scholar

[121] View Article

[122] Google Scholar

[ref36] 36. Salehi M, Moieni A, Safaie N. Elicitors Derived from Hazel (Corylus avellana L.) Cell Suspension Culture Enhance Growth and Paclitaxel Production of Epicoccum nigrum. Sci Rep. 2018;8(1):12053. pmid:30104672
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref37] 37. Samson RA, Visagie CM, Houbraken J, Hong S-B, Hubka V, Klaassen CHW, et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol. 2014;78:141–73. pmid:25492982
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref38] 38. Liu YJ, Whelen S, Hall BD. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit. Mol Biol Evol. 1999;16(12):1799–808. pmid:10605121
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref39] 39. Hong S-B, Cho H-S, Shin H-D, Frisvad JC, Samson RA. Novel Neosartorya species isolated from soil in Korea. Int J Syst Evol Microbiol. 2006;56(Pt 2):477–86. pmid:16449461
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref40] 40. Houbraken J, Kocsubé S, Visagie CM, Yilmaz N, Wang X-C, Meijer M, et al. Classification of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales): An overview of families, genera, subgenera, sections, series and species. Stud Mycol. 2020;95:5–169. pmid:32855739
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref41] 41. Han SL, Wang MM, Ma ZY, Raza M, Zhao P, Liang JM, et al. Fusarium diversity associated with diseased cereals in China, with an updated phylogenomic assessment of the genus. Stud Mycol. 2023;104:87–148. pmid:37351543
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref42] 42. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4. pmid:12912839
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref43] 43. Larget B, Simon D. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Mol Biol Evol. 1999;16:750–9.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref44] 44. Silvestro D, Michalak I. Raxmlgui: a graphical front-end for raxml. Org Divers Evol. 2012;12:335–7.
View Article
Google Scholar

[155] View Article

[156] Google Scholar

[ref45] 45. Huson DH, Scornavacca C. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol. 2012;61(6):1061–7. pmid:22780991
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref46] 46. Hosseyni Moghaddam MS, Safaie N, Soltani J, Hagh-Doust N. Desert-adapted fungal endophytes induce salinity and drought stress resistance in model crops. Plant Physiol Biochem. 2021;160:225–38. pmid:33517220
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref47] 47. Imakumbili ML. Making water stress treatments in pot experiments: an illustrated step-by-step guide. Heliyon. 2019;4(11):e00935.
View Article
Google Scholar

[166] View Article

[167] Google Scholar

[ref48] 48. Spagnoletti FN, Carmona M, Balestrasse K, Chiocchio V, Giacometti R, Lavado RS. The arbuscular mycorrhizal fungus Rhizophagus intraradices reduces the root rot caused by Fusarium pseudograminearum in wheat. Rhizosphere. 2021;19:100369.
View Article
Google Scholar

[169] View Article

[170] Google Scholar

[ref49] 49. Visagie CM, Houbraken J, Frisvad JC, Hong S-B, Klaassen CHW, Perrone G, et al. Identification and nomenclature of the genus Penicillium. Stud Mycol. 2014;78:343–71. pmid:25505353
View Article
PubMed/NCBI
Google Scholar

[172] View Article

[173] PubMed/NCBI

[174] Google Scholar

[ref50] 50. Shoresh M, Harman GE, Mastouri F. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol. 2010;48:21–43. pmid:20192757
View Article
PubMed/NCBI
Google Scholar

[176] View Article

[177] PubMed/NCBI

[178] Google Scholar

[ref51] 51. Pieterse CMJ, Zamioudis C, Berendsen RL, Weller DM, Van Wees SCM, Bakker PAHM. Induced systemic resistance by beneficial microbes. Annu Rev Phytopathol. 2014;52:347–75. pmid:24906124
View Article
PubMed/NCBI
Google Scholar

[180] View Article

[181] PubMed/NCBI

[182] Google Scholar

[ref52] 52. Ayaz M, Li C-H, Ali Q, Zhao W, Chi Y-K, Shafiq M, et al. Bacterial and Fungal Biocontrol Agents for Plant Disease Protection: Journey from Lab to Field, Current Status, Challenges, and Global Perspectives. Molecules. 2023;28(18):6735. pmid:37764510
View Article
PubMed/NCBI
Google Scholar

[184] View Article

[185] PubMed/NCBI

[186] Google Scholar

[ref53] 53. Jiménez-Gómez I, Valdés-Muñoz G, Moreno-Ulloa A, Pérez-Llano Y, Moreno-Perlín T, Silva-Jiménez H, et al. Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration. Front Microbiol. 2022;13:840408. pmid:35586858
View Article
PubMed/NCBI
Google Scholar

[188] View Article

[189] PubMed/NCBI

[190] Google Scholar

Figures

Abstract

1. Introduction

2. Materials and Methods

2.1. Isolation and identification of pathogenic fungi

2.2. Morphological and Molecular identification of biocontrol fungi

2.3. Salt tolerance assay

2.4. Antagonistic activity assays

2.5. Formulation preparation

2.6. Optimal carrier selection and stability assessment

2.7. Greenhouse evaluation of optimized formulation of biocontrol agents under combined drought and pathogen stress

2.8. Statistical analysis

3. Results

3.1. Molecular identification of Fusarium pathogens

3.2. Morphological and microscopic identification of Aspergillus sp. and Penicillium sp

3.3. Micromorphological characteristics of Aspergillus sp. and Penicillium sp

3.4. Molecular identification of Aspergillus sp. and Penicillium sp

3.5. Antagonistic effects of A. micronesiensis and P. momoi against pathogenic Fusarium spp

3.6. Salt tolerance of biocontrol fungi

3.7. Formulation development and spore germination performance testing

3.8. Study of the stability of selected formulations

3.9. Greenhouse evaluation of optimized formulation of biocontrol agents under combined drought and pathogen stress

4. Discussion

5. Conclusion

Supporting information

S1 Table. Primers used for amplification and sequencing.

S2 Table. Media used for morphological characterization.

S3 Table. Accession numbers used for phylogenetic analysis of Fusarium pseudograminearum.

S4 Table. Accession numbers used for phylogenetic analysis of Aspergillus sp. and Penicillium sp.

S5 Table. Different compositions of carrier and stabilizers for maximum spore germination.

S6 Table. Analysis of variance for the effects of wheat cultivar, soil moisture content, pathogen and formulations on SPAD value, plant height, shoot and root biomass, and disease score.

S1 Fig. Fusarium oxysporum f. sp. lycopersici (FOL) and F. o. f. sp. radicis-lycopersici (FORL) on potato dextrose agar.

S2 Fig. PCR analysis of polygalacturonase gene region of Fusarium oxysporum f. sp. lycopersici with uni primer and in 2% agarose gel.

S3 Fig. PCR analysis of polygalacturonase gene region of Fusarium oxysporum f. sp. lycopersici with Spr1 primer and in 2% agarose gel.

S4 Fig. Dual culture assays of biocontrol isolates against pathogenic fungi.

S5 Fig. Fusarium pseudograminearum (FPS; A1 and A2), Dual cultures of biocontrol isolates with pathogenic fungi: Aspergillus micronesiensis against FPS (B1 and B2), Penicillium momoi against FPS (C1 and C2).

S6 Fig. Aspergillus micronesiensis in culture media containing different concentrations (0, 1, 2, and 3 M) of NaCl.

S7 Fig. Penicillium momoi in culture media containing different concentrations (0, 1, 2, and 3 M) of NaCl.

S8 Fig. Number of germinated spores in different formulations of Aspergillus micronesiensis.

S9 Fig. Number of germinated spores in different formulations of Penicillium momoi.

S10 Fig. Proposed formulation for Penicillium momoi and Aspergillus micronesiensis based on wheat bran + sodium alginate and the ratio of its components.

Acknowledgments

References