A Metabolomic Study of Epichloë Endophytes for Screening Antifungal Metabolites

Epichloë endophytes, fungal endosymbionts of Pooidae grasses, are commonly utilized in forage and turf industries because they produce beneficial metabolites that enhance resistance against environmental stressors such as insect feeding and disease caused by phytopathogen infection. In pastoral agriculture, phytopathogenic diseases impact both pasture quality and animal production. Recently, bioactive endophyte strains have been reported to secrete compounds that significantly inhibit the growth of phytopathogenic fungi in vitro. A screen of previously described Epichloë-produced antifeedant and toxic alkaloids determined that the antifungal bioactivity observed is not due to the production of these known metabolites, and so there is a need for methods to identify new bioactive metabolites. The process described here is applicable more generally for the identification of antifungals in new endophytes. This study aims to characterize the fungicidal potential of novel, ‘animal friendly’ Epichloë endophyte strains NEA12 and NEA23 that exhibit strong antifungal activity using an in vitro assay. Bioassay-guided fractionation, followed by metabolite analysis, identified 61 metabolites that, either singly or in combination, are responsible for the observed bioactivity. Analysis of the perennial ryegrass-endophyte symbiota confirmed that NEA12 and NEA23 produce the prospective antifungal metabolites in symbiotic association and thus are candidates for compounds that promote disease resistance in planta. The “known unknown” suite of antifungal metabolites identified in this study are potential biomarkers for the selection of strains that enhance pasture and turf production through better disease control.

Metabolic Potential of Epichloë Endophytes for Host Grass Fungal Disease Resistance

Asexual species of the genus Epichloë (Clavicipitaceae, Ascomycota) form endosymbiotic associations with Pooidae grasses. This association is important both ecologically and to the pasture and turf industries, as the endophytic fungi confer a multitude of benefits to their host plant that improve competitive ability and performance such as growth promotion, abiotic stress tolerance, pest deterrence and increased host disease resistance. Biotic stress tolerance conferred by the production of bioprotective metabolites has a critical role in an industry context. While the known antimammalian and insecticidal toxins are well characterized due to their impact on livestock welfare, antimicrobial metabolites are less studied. Both pasture and turf grasses are challenged by many phytopathogenic diseases that result in significant economic losses and impact livestock health. Further investigations of Epichloë endophytes as natural biocontrol agents can be conducted on strains that are safe for animals. With the additional benefits of possessing host disease resistance, these strains would increase their commercial importance. Field reports have indicated that pasture grasses associated with Epichloë endophytes are superior in resisting fungal pathogens. However, only a few antifungal compounds have been identified and chemically characterized, and these from sexual (pathogenic) Epichloë species, rather than those utilized to enhance performance in turf and pasture industries. This review provides insight into the various strategies reported in identifying antifungal activity from Epichloë endophytes and, where described, the associated antifungal metabolites responsible for the activity.

Antifungal Metabolites as Food Bio-Preservative: Innovation, Outlook, and Challenges

Perishable food spoilage caused by fungi is a major cause of discomfort for food producers. Food sensory abnormalities range from aesthetic degeneration to significant aroma, color, or consistency alterations due to this spoilage. Bio-preservation is the use of natural or controlled bacteria or antimicrobials to enhance the quality and safety of food. It has the ability to harmonize and rationalize the required safety requirements with conventional preservation methods and food production safety and quality demands. Even though synthetic preservatives could fix such issues, there is indeed a significant social need for “clean label” foods. As a result, consumers are now seeking foods that are healthier, less processed, and safer. The implementation of antifungal compounds has gotten a lot of attention in recent decades. As a result, the identification and characterization of such antifungal agents has made promising advances. The present state of information on antifungal molecules, their modes of activity, connections with specific target fungi varieties, and uses in food production systems are summarized in this review.

Biocontrol Potential of Endophytic Actinobacteria against Fusarium solani, the Causal Agent of Sudden Decline Syndrome on Date Palm in the UAE

Thirty-one endophytic streptomycete and non-streptomycete actinobacteria were isolated from healthy date palm root tissues. In vitro screening revealed that the antifungal action of isolate #16 was associated with the production of cell-wall degrading enzymes, whereas with diffusible antifungal metabolites in isolate #28, albeit their production of volatile antifungal compounds. According to the 16S rRNA gene sequencing, isolates #16 and #28 were identified as Streptomyces polychromogenes UAE2 (Sp; GenBank Accession #: OK560620) and Streptomyces coeruleoprunus UAE1 (Sc; OK560621), respectively. The two antagonists were recovered from root tissues until 12 weeks after inoculation, efficiently colonized root cortex and xylem vessels, indicating that the date palm roots are a suitable habitat for these endophytic isolates. At the end of the greenhouse experiments, the development of sudden decline syndrome (SDS) was markedly suppressed by 53% with the application of Sp and 86% with Sc, confirming their potential in disease management. Results showed that the estimated disease severity indices in diseased seedlings were significantly (p < 0.05) reduced from 4.75 (scale of 5) to 2.25 or 0.67 by either Sp or Sc, respectively. In addition, conidial numbers of the pathogen significantly (p < 0.05) dropped by 38% and 76% with Sp and Sc, respectively, compared to infected seedlings with F. solani (control). Thus, the suppression of disease symptoms was superior in seedlings pre-inoculated with S. coeruleoprunus, indicating that the diffusible antifungal metabolites were responsible for F. solani retardation in these plants. This is the first report of actinobacteria naturally existing in date palm tissues acting as microbial antagonists against SDS on date palm.

In Vitro Evaluation and Genome Mining of Bacillus subtilis Strain RS10 Reveals Its Biocontrol and Plant Growth-Promoting Potential

Recently, crop management has involved excessive use of chemical fertilizers and pesticides, compromising public health and environmental integrity. Rhizobacteria, which can enhance plant growth and protect plants from phytopathogen, are eco-friendly and have been attracting increasing attention. In the current study, Bacillus subtilis RS10 isolated from the rhizosphere region of Cynodon dactylon, inhibited the growth of indicator strains and exhibited in vitro plant growth-promoting traits. A whole-genome analysis identified numerous biosynthetic gene clusters encoding antibacterial and antifungal metabolites including bacillibactin, bogorol A, fengycin, bacteriocin, type III polyketides (PKs), and bacilysin. The plant growth-promoting conferring genes involved in nitrogen metabolism, phosphate solubilization, hydrogen sulfide, phytohormones, siderophore biosynthesis, chemotaxis and motility, plant root colonization, lytic enzymes, and biofilm formation were determined. Furthermore, genes associated with abiotic stresses such as high salinity and osmotic stress were identified. A comparative genome analysis indicated open pan-genome and the strain was identified as a novel sequence type (ST-176). In addition, several horizontal gene transfer events were found which putatively play a vital role in the evolution and new functionalities of a strain. In conclusion, the current study demonstrates the potential of RS10 antagonism against important pathogens and plant growth promotion, highlighting its application in sustainable agriculture.

Development of next-generation formulation against Fusarium oxysporum and unraveling bioactive antifungal metabolites of biocontrol agents

Biocontrol agents serve as a sustainable means of controlling wilt caused by the widespread plant pathogen, Fusarium oxysporum f. sp. lycopersici. The present study aimed to develop water dispersible granules (WDG) using response surface methodology (RSM) for Bacillus subtilis MTCC 2274 and Trichoderma harzianum MTCC 3928, and to compare their antifungal efficacy with other formulations. Further, characterization of the bioactive metabolites responsible for biocontrol was performed. A new microbial formulation, WDG, was developed in the present study with talcum powder (substrate), alginic acid (dispersing agent) and acacia gum (wetting agent) (suspensibility 82.23%; wetting time 2.5 min; dispersion time 10.08 min) that fulfilled the guidelines of Collaborative International Pesticides Analytical Council (CIPAC). In planta study demonstrated that WDG of B. subtilis showed maximum reduction in disease incidence (48%) followed by talc formulation of B. subtilis (44%) and WDG of T. harzianum (42%) with profound effect on plant growth promotion. B. subtilis and T. harzianum demonstrated protease (929 and 846 U ml−1 min−1), chitinase (33.69 and 154 U ml−1 min−1), and β-1,3-glucanase (12.69 and 21.47 U ml−1 min−1) activities. Culture filtrates of B. subtilis and T. harzianum exhibited significant inhibition against mycelial growth of pathogen. The compounds present in the culture filtrates were identified with GC–MS as fatty acids, alkanes, phenols, benzene, pyran derivatives etc. The major non-volatile compounds in bioactive antifungal fraction were identified as derivatives of morpholine and piperdine for T. harzianum and B. subtilis, respectively. The findings propose a multivariate biocontrol mechanism against phytopathogen by production of hydrolytic enzymes, volatile and non-volatile compounds, together with development of an efficient next-generation formulation.

Production of Extracellular Enzymes, Antimicrobial and other Agriculturally Important Metabolites by Fungal Endophytes of Litsea glutinosa (Lour.) C.B.Rob. a Medicinal Plant

The present paper deals with the endophytic fungi of Litsea glutinosa, a medicinal plant highly valued in Ayurvedic system. A total of 21 fungal endophytes were extracted from different parts of the plant. The following eight species viz, Aspergillus oryzae, A. terreus, Curvularia sp, Fusarium oxysporum, Gliocladium solani, Penicillium citrinum, Trichoderma viride and Verticillium dahliae were selected for testing the production of secondary metabolites including hydrolytic enzymes. All the eight endophytic fungal species produced amylase, cellulase, protease and lipase. Except for Curvularia species, others did not elaborate laccase. All the test fungi produced antibacterial and antifungal metabolites; however, the crude extracts exhibited poor antimicrobial activity. The present endophytic fungi also produced IAA, ammonia and phosphate solubilizing substances. They were also capable to produce IAA with or without adding the tryptophan. An affect was also made to assess the influence of crude extracts on the root growth of maize seedlings. These studies revealed the enhancement effect of extracts on root growth. Based on these observations, discussions and conclusions were made.

Effectiveness of Augmentative Biological Control of Streptomyces griseorubens UAE2 Depends on 1-Aminocyclopropane-1-Carboxylic Acid Deaminase Activity against Neoscytalidium dimidiatum

To manage stem canker disease on royal poinciana, actinobacterial isolates were used as biological control agents (BCAs) based on their strong in vitro inhibitory effects against Neoscytalidium dimidiatum. Streptomyces griseorubens UAE2 and Streptomyces wuyuanensis UAE1 had the ability to produce antifungal compounds and cell-wall-degrading enzymes (CWDEs). Only S. griseorubens, however, restored the activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD). In vivo apple fruit bioassay showed that lesion development was successfully constrained by either isolates on fruits inoculated with N. dimidiatum. In our greenhouse and container nursery experiments, S. griseorubens showed almost complete suppression of disease symptoms. This was evident when the preventive treatment of S. griseorubens significantly (p < 0.05) reduced the numbers of conidia of N. dimidiatum and defoliated leaves of royal poinciana seedlings to lesser levels than when S. wuyuanensis was applied, but comparable to control treatments (no pathogen). The disease management of stem canker was also associated with significant (p < 0.05) decreases in ACC levels in royal poinciana stems when S. griseorubens was applied compared to the non-ACCD-producing S. wuyuanensis. This study is the first to report the superiority of antagonistic actinobacteria to enhance their effectiveness as BCAs not only for producing antifungal metabolites and CWDEs but also for secreting ACCD.

PSX-A-4 Late-Breaking: Assessing the antifungal efficacy of EverWildTM D01 against Aspergillus flavus in semi-moist pet treats

Semi-moist pet treats contain moisture levels ranging from 20–30% making them ideal for mold growth and mycotoxin production. To control mold, synthetic mold inhibitors such as potassium sorbate or calcium propionate are used. Synthetic additives are effective, but there is a shift in the pet owner preferences for ‘natural’ products. Fermented and (or) cultured whey has been shown to contain antifungal metabolites and has been used to control fungal growth in bread. EverWildTM D01 is a commercially available cultured whey product. The objective of this study was to assess the efficacy of EverWildTM D01 against Aspergillus flavus in a semi-moist pet treat. A model semi-moist pet treat fortified to be nutritionally complete was produced with three levels of powdered whey fermentate, [1.0%, 3.0% and 5% (w/w)], a negative control with no antifungal and a “positive control” which contained potassium sorbate as a standard industrial mold inhibitor. Treats were produced by baking in 3cm x 3cm squares. Individual squares were inoculated with 0.1 mL of Aspergillus flavus (ATCC 204304) to achieve a final concentration of ~ 5.0 Log CFU/square. The inoculated squares were individually incubated at 250 C. Fungal analysis was performed at day 3, 6, 9, 12, 15, 18, 21, 24 and 28 by surface plating on Potato Dextrose Agar (PDA) followed by incubation at 250 C for 72h. The total log reductions were calculated by subtracting the initial inoculum with the colony counts on day 28. EverWildTM D01 at 1.0%, 3.0%, and 5% (w/w) had a 1.90, 3.89 and 4.58 Log CFU/square log reduction, respectively. The positive control had a 1.19 Log CFU/square log reduction. All treatments differed from the negative control (P &lt; 0.05). In conclusion, EverWildTM D01 may be effective as a natural alternative to synthetic mold inhibitors in semi-moist pet treats.