Endophytic Isolates Research Articles

Optimization of Antibacterial Metabolite Production and Antioxidant Activity of Endophytic Fungi Isolate from Kelakai (Stenochlaena palustris)

This research aims to optimize the production of antibacterial metabolites and test the antioxidant activity of endophytic isolates from kelakai (Stenochlaena palustris). The research results showed that the optimum growth profile of the isolate showed that exponential growth only started on day 1 and reached a maximum on day 17. Based on pH variations, it shows that pH 7 is the optimum pH in inhibiting bacterial growth, and based on variations in carbon sources, it shows that fructose is a better carbon source than glucose. Antimicrobial activity test using ethyl acetate extract from endophytic fungal fermentate based on differences in pH and carbon source showed antimicrobial activity against S. aureus and E. coli with the inhibition zone diameters being 13.50 mm and 11.35 mm, respectively.

Isolation, characterization and management of endophyte(s) prevailing during the in vitro establishment of endangered medicinal herb Himalayan Trillium (Trillium govanianum Wall. ex D. Don)

Isolation, characterization and management of endophyte(s) prevailing during the in vitro establishment of endangered medicinal herb Himalayan Trillium (Trillium govanianum Wall. ex D. Don)

Diversity of endophytic fungi isolated from tree spinach (Cnidoscolus aconitifolius) (Euphorbiaceae) and their bioactivities

Abstract. Praptiwi, Palupi KD, Ilyas M, Marlina L, Fathoni A. 2023. Diversity of endophytic fungi isolated from tree spinach Cnidoscolus aconitifolius (Euphorbiaceae) and their bioactivities. Biodiversitas 24: 6229-6239. Endophytic fungi associated with medicinal plants have been known as a reservoir for therapeutic compounds. Cnidoscolus aconitifolius I.M. Johns (Euphorbiaceae), which possesses several local names, including papaya jepang in Indonesia, has been historically used as traditional medicine. However, research on the bioactivity of endophytic fungi associated with it has yet to be conducted. The purposes of the study were to isolate endophytic fungi from C. aconitifolius and determine their bioactivity, i.e., antibacterial activity against Escherichia coli and S. aureus and free radical scavenging activity representing antioxidant activity. Identification of endophytic fungi was carried out based on their morphological characteristics. Total phenolic and flavonoid contents were determined by the spectrophotometric method. The qualitative antibacterial and antioxidant bioactivity screening was performed using the TLC-bioautography method. The quantitative analysis of antibacterial and antioxidant activity was conducted by microdilution to obtain IC50 and MIC values. In total, 14 endophytic fungal isolates were successfully isolated. Antioxidant analysis showed that one isolate had strong antioxidant activity, and one had moderate growth inhibition activity against E. coli. The total phenolic content strongly correlates with the AAI value as an indicator of antioxidant activity. The findings of this study reveal that Phomopsis PjBg-1, an endophytic fungus from C. aconitifolius, is identified as Phomopsis sp. LH243 possesses strong antioxidant activity and moderate antibacterial activity against E. coli. Endophytic fungi from C. aconitifolius have the prospect of being an alternative to antioxidants and antibacterials. The isolation of compounds responsible for bioactivities needs to be further studied.

Evaluation of Antibacterial Properties from Endophytic Fungi of Chrysanthemum indicum (L.) Flowers against Escherichia coli and Staphylococcus aureus

Uncovering the therapeutic potential of secondary metabolites produced by plants, animals, and microbes constitutes the foundation for the development of novel medications. The objective of this investigation is to discern the classes of secondary metabolites and assess the antibacterial properties of endophytic fungal extracts obtained from Chrysanthemum indicum L. flowers. Through the isolation process, five isolates designated as JEC1, JEC2, JEC3, JEC4, and JEC5 were identified. The cultivation of endophytic fungal isolates spanned a three-week period before undergoing extraction with ethyl acetate. The phytochemical tests revealed the presence of alkaloids, flavonoids, steroids, terpenoids, saponins, and tannins in the ethyl acetate extract. Antibacterial activity was determined using the agar well diffusion method, with ciprofloxacin serving as a positive control. Notably, all ethyl acetate extracts from endophytic fungi exhibited antibacterial activity. The most substantial inhibitory diameter against Staphylococcus aureus was recorded as 19.1±0.8 mm for the JEC3 endophytic fungi, while Escherichia coli exhibited an inhibitory diameter of 16±1.1 mm for the JEC2 endophytic fungi.

The antioxidant activity of endophytic fungal extract of nutmeg plant (Myristica fragrans Houtt.) from Cimande West Java, Indonesia

The bioproduction of bioactive metabolites from nutmeg endophytic fungi from Cimande, Wesh Java Indonesia was successful. In this research, bioactive antioxidant metabolites were extracted from nutmeg endophytic fungi. Endophytic fungi from nutmeg plant parts were grown on CMMA media. Each fungus was isolated by growing it on PDA media. A total of 21 endophytic fungi isolates were grown on PDB media before being extracted with ethyl acetate as a solvent. The morphology of 21 endophytic fungal isolates was identified based on morphological characteristics. There are 11 Phomopsis sp., 1 Fusarium oxysporum, 5 Colletotrichum sp., 1 Schizophyllum sp., 1 Dematiaceae sp., 1 Coelomycetes sp., and 1 Xylaria sp. The bioautographic TLC method and DPPH-free radical scavenging activity were used to determine antioxidant activity. The antioxidant activity analysis showed that 14 of the 21 endophytic fungus extracts were active. One nutmeg pericarp endophytic fungus showed strong antioxidant activity, with an IC50 of 30.0 g mL-1 and AAI of 1.04. Furthermore, the IC50 values for two fungal extracts from the leaf and pistil nutmeg were less than 100 g.mL-1. The three fungi with IC50 less than 100 g.mL-1 were identified as Phomopsis sp.

Diversity of endophytic bacteria isolated from mangrove Bruguiera gymnorrhiza (L.) Lam.) and Rhizopora mucronata Poir. originated from Enggano island

Endophytic bacteria are bacteria that live colonizing in plant tissues without causing harmful effects. Endophytic bacteria are found in the vascular tissues of plant, including mangroves (B. gymnorrhiza (L.) Lam. ) and R. mucronata Poir.). Mangroves have numerous advantages, one of them is as a medicinal plant. This study aims to obtain the diversity of endophytic bacteria isolated from Enggano Island mangroves (B. gymnorrhiza (L.) Lam. and R. mucronata Poir.). Endophytic bacteria were isolated using the grinding method through which the surface was sterilized with natrium hypochlorite and alcohol 70%. The obtained isolates were then identified in terms of morphological characters, Gram staining, and then biochemical tests. A total of 40 bacterial isolates were successfully isolated from the roots, stems, and leaves of both mangrove species. It was observed that 20 endophytic bacterial isolates were associated with mangrove B. gymnorrhiza (L.) Lam., while the remaining 20 isolates were from R. mucronata Poir. Based on morphological observation, Gram staining, and a series of biochemical tests, those 40 isolates were found to to be closely related with Bacilus, Micrococcus, and Pseudomonas. To conclude, B. gymnorrhiza (L.) Lam. and R. mucronata Poir. have a high diversity in endophytic bacteria.

Insecticide activity from mangrove endophytic fungus isolate against larvae of fruit flies (Drosophila melanogaster)

The fruit fly Drosophila melanogaster (D. melanogaster) is one of the important pests for fruit and vegetable plants. However, pest control efforts are highly dependent on synthetic insecticides. This study aimed to determine the potential of mangrove endophytic fungi as bioinsecticides for D. melanogaster and to determine the insecticidal activity of secondary metabolites of mangrove endophytic fungi against D. melanogaster larvae. The study was conducted on five types of fungi extract isolated from R. mucronata, namely E. nidulans (BPPTCC 6035), E. nidulans (BPPTCC 6038), A. tamarii (BPPTCC 6037), A. versicolor (BPPTCC 6039), and A. oryzae (BPPTCC 6036) in 500 and 1000 ppm. The results showed that the A. versicolor (BPPTCC 6039) extract was the most toxic. Tukey’s HSD test showed that the A. versicolor (BPPTCC 6039) extract was most effectively used as a bioinsecticide for D. melanogaster larvae. All extracts do not have antifeedant compounds and showed activity to inhibit larvae growth. Therefore, in this study, the A. versicolor (BPPTCC 6039) extract had the most significant potential as a bioinsecticide for D. melanogaster larvae.

English

Salinity and drought are among the major abiotic stresses, leading to decreased crop productivity. Fungi are notorious for their detrimental effects on crop growth and development. In light of these circumstances, the primary objective of this project is to assemble a collection of endophytic fungi to develop new organic bio-stimulants that can effectively combat salinity and drought. To achieve this goal, diverse soil types, including cultivated, desert, and salt marshes, were sampled (five samples each) from different habitats and locations in the southern part of Sinai, the Saint Katherine Protectorate (SKP) is part of the upper Sinai massif, spanning specific coordinates (33°550 to 34°300 E and 28°300 to 28°350 N). Additionally, 80 samples of the predominant medicinal plant species from ten localities in the SKP were collected. The results of survey showed that the highest abundance of endophytic fungi was observed in Artemisia judaica L. and Alkanna orientalis (L.) Boiss., with both species exhibiting 61.1 % endophytic fungi. Five isolates of fungi labeled as SAF41, SAF46, SAF51, SAF57, and SAF63, were assessed for their tolerance to salinity stress by exposing them to different NaCl concentrations. Isolate SAF63 had the most substantial negative and significant correlation with salinity stress (R2=0.9565; p<0.001), followed by SAF51 (R2=0.9432; p<0.001) and SAF41 (R2=0.9351; p<0.001). The results indicate that different fungi isolates exhibited distinct responses to varying salinity levels and drought stress, as measured by their growth in different NaCl concentrations. Isolate SAF63 demonstrated the most significant negative correlation with salinity stress, followed by SAF51 and SAF41. These findings suggest that specific endophytic fungi isolates exhibit enhanced tolerance to both salinity and drought stress, underscoring their promising potential in mitigating metabolic and physiological damage in date palm plants, ultimately leading to increased growth and yields

DIVERSITY OF ENDOPHYTIC FUNGI FROM SHALLOTS AS A FUSARIUM OXYSPORUM BIOLOGICAL CONTROL AGENT

Fusarium oxysporum is the primary cause of Fusarium wilt, one of the major diseases that harm shallots. 50% of yield can be lost due to Fusarium wilt. It is anticipated that endophytic fungus will inhibit the growth of Fusarium wilt disease in shallots. The endophytic fungi from shallot plants that have the potential to act as biological control agents for the fungal disease F. oxysporum are suggested to be chosen in this study. Using Malt Extract Agar (MEA) medium, endophytic fungi were isolated from healthy shallot plant components (roots, tubers, stems, and leaves). Using dual culture and volatile techniques, the antagonistic potential of endophytic fungi against F. oxysporum was examined. Using shallot seeds, pathogenicity studies for endophytic fungus were conducted in vitro. Twelve endophytic fungal isolates were able to impede the growth of F. oxysporum using the dual culture method; the percentage of inhibition reached 72.52% at seven days post-inoculation. The endophytic fungus was also able to stop F. oxysporum mycelial growth using the volatile compound test, with a percentage inhibition ranging from 4.08 - 27.37%. The findings of the pathogenicity test demonstrated that the four isolates posed a risk to the shallot seeds. The most successful isolate in preventing the growth of F. oxysporum was the endophytic fungus AB4D1, which is a member of the Trichoderma asperellum species.

Physico-Chemical Alternatives for Synthesis of Metal Nanoparticles Using Fusarium Chlamydosporum

Abstract Introduction/Objective Background: Synthesizing metal nanoparticles (NPs) by chemical or physical methods is difficult due to energy and time consumption, equipment costs, and chemical toxicity. Chemosynthesis-derived NPs are unstable, limiting their biological applications. Thus, identifying alternative green sources to address these drawbacks is difficult scientifically. The hardest part of green metal nanoparticle synthesis is finding a nontoxic natural product and an eco-friendly solvent. Objectives: Isolation of fungal endophytes from Eucalyptus sideroxylon leaves to synthesize ZnO and Au nanoparticles. Methods/Case Report Materials and methods: We used healthy Eucalyptus sideroxylon plant samples from the desert research center to separate endophytes. All fungi were grown on malt extract broth on a rotary shaker at 28 °C (120 rpm) for 96 hours after isolation. Biomass components were removed by filtration and washing in distilled water. Nanoparticles came from biomass cell filtrate. 0.1 M, 1 mL of Zn SO4, and HAuCl4 were added to 10 mL of fungal filtrate and agitated for 24 hours in the dark at room temperature to make Zn and Au nanoparticles separately. Characterization of nanoparticles shows a green synthesis of ZnONPs and AuNPs. Results (if a Case Study enter NA) Results: Fungal endophytes' filtrates were mixed with 1mM Zn SO4 and HAuCl4 solutions to test their extracellular biosynthesis of ZnO and Au NPs. The fungal filtrate became colorless and violet after mixing with Zn SO4 and HAuCl4. Only FI-03 is from 13 (FI-01 to FI-013). The most potent fungal strain for Zn O NP and Au NP nanoparticle synthesis was Fusarium Chlamydosporum. Pure, mostly spherical ZnO and Au NPs averaged 19.3 and 22.1 nm. UV-Vis absorption: Excitonic absorption peaks of ZnO NPs were between 300 and 400 nm. ZnO NPs' maximum absorption peak was 305 nm, while Au NPs' peak was 520 nm. One absorption peak indicates metallic gold nanoparticles. Au and ZnO NPs aggregate the most due to their hydrophilicity, HD of 34.88 and 15.77 nm, and PdI of 0.77, 1.00, and 0.952. Gold face-centered cubic fcc structure dominates Au NPs' XRD pattern. EDX shows 64.61 and 35.39 percent Zn and O, respectively, in both nanoparticles. The optical absorption band of gold nanocrystallites peaked at 2.2 keV in energy-dispersive X-rays. Conclusion Endophytic fungus biosynthesizes ZnO and Au NPs, which are bioactive compounds for medicine and food without toxic chemicals and are environmentally friendly.

Optimization of L-asparaginase production from endophytic bacteria isolated from the mangrove Rhizophora mucronata

Abstract. Nafisaturrahmah A, Susilowati A, Pangastuti A. 2023. Optimization of L-asparaginase production from endophytic bacteria isolated from the mangrove Rhizophora mucronata. Nusantara Bioscience 15: 279-287. L-asparaginase is an enzyme that hydrolyes L-asparaginase to L-aspartate and ammonia. L-asparaginase has the potential to treat acute lymphoblastic leukemia and other malignant cancers. So far, purified L-asparaginase from Escherichia coli and Erwinea chrysanthemi has been available and applied clinically in humans. However, this treatment has side effects such as allergy, cross-interaction, immune system stimulation, drug resistance, and nonspecific L-glutaminase activity. These side effects can be overcome by discovering new sources of L-asparaginase, which are serologically different but have similar therapeutic effects. This study aims to determine the optimal conditions of endophytic bacterial culture in producing L-asparaginase. Endophytic bacteria were screened using an M9 medium with asparagine as a substrate; the L-asparaginase-producing isolates showed pink zones around the colonies. Optimization of L-aparaginase production by endophytic bacteria is carried out by One Factor at A Time (OFAT). Optimization of enzyme production includes incubation time, temperature, pH, ammonium sulfate levels, and glucose concentration in the bacterial growth medium; determination of enzyme production by Nesslerization method. The results showed that 8 isolates could produce high L-asparaginase, 14 isolates had medium ability, 30 isolates had low ability, and 2 bacterial isolates did not produce L-asparaginase. Endophytic isolates were able to produce the highest L-Aparaginase under different optimal conditions. The optimal incubation time for endophytic isolates in this study was 60-84 hours, the optimal temperature was 37ºC, the optimal pH was 7, the nitrogen content was 0.25 mg/L, and the optimal glucose level was 3%.

Characterization of Endophytic Fungi in Robusta Coffee (Coffea canephora L.) Beans Through 18S rRNA Gene Sequencing and Evaluation of Antioxidant Activity and Chlorogenic Acid Content

Utilizing endophytic fungi to discover metabolites by characterizing the fungi produced from robusta coffee beans can be highly beneficial without causing harm to the plants. Additionally, this approach aids in identifying raw materials for medical drug development, specifically compounds like chlorogenic acid, which hold promise as potential treatments for diabetes mellitus. This study aimed at characterizing the endophytic fungi present in Robusta coffee (Coffea canephora L.) beans through 18S rRNA gene sequencing and evaluation of antioxidant activity and chlorogenic acid content found in these endophytic fungi. Green, unripe Robusta coffee beans were harvested from a coffee plantation in Tana Toraja and isolated using Potato Dextrose Agar medium. The isolation process yielded three isolates, designated RF1, RF2, and RF3. Subsequently, the isolates were subjected to testing for endophytic fungi activity, and the isolate that exhibited activity was further characterized using 18S rRNA gene sequencing. Following that, the isolates were fermented using a Potato Dextrose Broth medium, resulting in two phases: filtrate and biomass. The filtrate was then extracted using ethyl acetate as the solvent. The extracted compound was subsequently evaluated for antioxidant activity using ABTS and DPPH methods, and the concentration of chlorogenic acid was measured. From the three isolates of endophytic fungi obtained from green Robusta coffee beans with the codes RF1, RF2, and RF3, all of them exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. The 18S rRNA gene sequencing analysis revealed that isolate RF1 was identified as Fusarium sp. For the evaluation of antioxidant activity using the ABTS method, an IC50 value of 53.49 µg/mL was obtained, while the DPPH method resulted in an IC50 value of 54.03. Furthermore, the measurement of chlorogenic acid content in the extract of endophytic fungi from Robusta coffee beans indicated a concentration of 11.58%. The characterization result of the endophytic fungi isolates from green Robusta coffee beans revealed the presence of Fusarium sp. It exhibited high antioxidant activity and a chlorogenic acid content of 11.58%.

Potential Endophytic Fungi from the Leaves of Syzygium zeylanicum (L.) and Their Secondary Metabolite

Endophytic fungi coexist without harms in the host in some parts or all parts of its lifecycle. Endophytic fungi excrete specialized bioactive compounds beneficial for of its host; the compound itself can be different from what can be found on their host. Endophytic fungi are capable to excrete biologically significant secondary metabolites acting as antidiabetic, antioxidant, antimicrobial, and cytostatic agents. Endophytic fungi existence are common across different plants; including Syzygium zeylanicum that can be found in Indonesia. This study aims to isolate endophytic fungi found in leaves of Syzygium zeylanicum, L., to elucidate their characterized morphologically, and to examine its antimicrobial and antioxidant properties in addition to their chemical structures. Characterization of endophytic fungi was conducted by their macroscopic and microscopic features, followed by molecular characterization of highly bioactive metabolites. Antimicrobial activities were measured by disc diffusion method. Antioxidant properties were measured with DPPH. Secondary metabolites were chromatographically isolated and identified with spectroscopy techniques (NMR ID and 2D). Four endophytic fungi isolates were obtained: Penicillium citrinum (SZ1), Colletotrichum lindemuthianum (SZ2), Aspergillus nidulan (SZ3), Scopulariopsis asperula (SZ4). Penicillium citrinum (SZ1) showed antimicrobial activities against four different bacteria (71.3% against E. coli; 74.1% against S. aureus; 76.2% against S. typhi; and dan76.9% against B. subtilis). Antioxidant activity ini all ekstrak of endophytic fungi showed very activity (IC50 SZ3 extract = 3.85 µg/mL). Potential endophytic fungi SZ1 was molecularly identified as Penicillium citrinum. Extracts from SZ1 fungi contains bioactive 4-hydroxy-2-(4-hydroxyphenyl)-ɣ-butyrolactone-3-yl) methyl acetate. The newly obtained substance could be developed into antimicrobial and antioxidant agents in further studies.

Biotransformation of Sclareol by a Fungal Endophyte of Salvia sclarea.

Microbial endophytes are known as versatile producers of useful metabolites, which have extensive applications in pharmacy, fragrance, agriculture and food. This study aims to screen sclareol-biotransforming microorganisms from Salvia sclarea, an untapped source of diverse endophytes. In this study, 50 culturable endophytes were isolated from S. sclarea grown in Xinjiang using sclareol as the sole carbon source and screened for their potential to transform sclareol into analogues. A fungal endophyte, identified as the generally recognized as safe (GRAS) strain Aspergillus tubingensis, can produce labd-14-ene-3β,8α,13β-triol and 8α,13β-dihydroxylabd-14-en-3-one from sclareol, involving hydroxylation and carbonylation at the C3 site. Structures of the two metabolites were elucidated by HR-ESI-MS and NMR analysis. S. sclarea was proven to be a good source of endophytes that are prospective producers of secondary metabolites with valuable chemical and biological properties. This study is the first report regarding the isolation of endophytes from S. sclarea.

Efficacy of Bacillus licheniformis: a biocontrol agent against Colletotrichum gloeosporioides Penz. (Penz. & Sacc.) causing anthracnose in greater yam (Dioscorea alata L.)

BackgroundAnthracnose, caused by Colletotrichum gloeosporioides, is the most dangerous fungal disease of greater yam (Dioscorea alata L.), and leads to significant crop loss. Microbial technology in cropping systems is currently focused on biological control agents (BCAs), which include plant endophytes like bacteria, which contribute well to sustainable production. In the present study, the ability of an endophytic bacterium, Bacillus licheniformis (CTCRI EB12), isolated from Aloe vera was investigated to prevent the disease.ResultsThe bacterium, CTCRI EB12, was characterized as a member of the Bacillus genus by morphological and biochemical tests and confirmed as B. licheniformis via 16S ribosomal gene analysis. The effectiveness of the endophytic bacterium was affirmed through cell confrontation assays and scanning electron microscopy. In cell confrontation assay, the isolate inhibited all the three C. gloeosporioides isolates used in the study with more than 80% inhibition. The effect of culture filtrate of the isolate was also tested against pathogens that were compliant with the outcome of dual culture technique. The culture and culture filtrate could effectively restrain the spreading of C. gloeosporioides in greater yam leaf. The isolate could completely inhibit the growth of the pathogen through an antibiosis study, and the same could be explained through scanning electron microscopy results, in which the endophyte-treated C. gloeosporioides hyphae appeared longer and distorted compared to control and conidia possess disfigurement in endophyte-treated sample when compared to control.ConclusionsThe study showed that Bacillus licheniformis (CTCRI EB12), the endophytic bacterium that has the capacity to enhance the protection of greater yam plants, could be further developed as a microbial pesticide to reduce the effect of anthracnose in greater yam fields. Additional investigation will reveal the endophytic isolate's antifungal capabilities and assessment of their performance in the open field. The present study depicted that it might be a potent biological control agent against C. gloeosporioides causing greater yam anthracnose.

Screening, Identification, and Growth Promotion of Antagonistic Endophytes Associated with Chenopodium quinoa Against Quinoa Pathogens.

Fungal disease is one of the important reasons for crop yield reduction. Isolation of important endophytes with biocontrol and growth-promoting effects is of great significance for the exploitation of beneficial microbial resources and the biological control of crop fungal diseases. In this study, endophytes from roots, stems, and leaves of quinoa at different growth and development stages were isolated and purified; then the antagonistic activity and growth-promoting characteristics of antagonistic endophytes were determined. Finally, the antagonistic endophytes were identified by morphological characteristics and ITS/16S rRNA sequence analysis. Our results showed that 122 endophytic fungi and 371 endophytic bacteria were isolated from quinoa, of which three endophytic fungi and seven endophytic bacteria were screened that had inhibitory activity against quinoa pathogenic fungi. Most of the antagonistic strains could produce indole-3 acetic acid and had the ability to dissolve organic phosphorus. In addition, the bacterial suspension of endophytic bacteria had the ability to promote the seed germination and plant growth of quinoa. The endophytic fungi with antagonistic activity were identified as Penicillium raperi and P. pulvillorum; the endophytic bacteria were identified as Bacillus paralicheniformis, B. tequilensis, and B. velezensis, respectively. The strains of quinoa endophytes in this study can provide rich microbial resources and a theoretical basis for biological control of plant fungal diseases and agricultural production.

Diversity of fungal endophytes isolated from the invasive plant Solanum rostratum

A culture-dependent method was used to isolate fungal endophytes from the leaves, stems, and roots of the invasive plant Solanum rostratum Dunal. growing in Xinjiang Province, China. All isolates were identified according to ITS (internal transcribed spacer) region of ribosomal DNA sequences and analyzed by Nucleotide BLAST according to NCBI GenBank and Mycobank database. Altogether 176 endophytic fungal isolates corresponding to 44 OTUs were identified, which were classified into 12 genera, with Penicillium (59.66%) and Aspergillus (23.29%) being the highly dominant genera. Ten endophytic isolates (OTU1, OTU15, OTU16, OTU21, OTU23, OTU25, OTU26, OTU30, OTU37 and OTU44) were identified as potential new species.

Untargeted metabolomic analyses support the main phylogenetic groups of the common plant-associated Alternaria fungi isolated from grapevine (Vitis vinifera)

Alternaria, a cosmopolitan fungal genus is a dominant member of the grapevine (Vitis vinifera) microbiome. Several Alternaria species are known to produce a variety of secondary metabolites, which are particularly relevant to plant protection and food safety in field crops. According to previous findings, the majority of Alternaria species inhabiting grapevine belong to Alternaria sect. Alternaria. However, the phylogenetic diversity and secondary metabolite production of the distinct Alternaria species has remained unclear. In this study, our aim was to examine the genetic and metabolic diversity of endophytic Alternaria isolates associated with the above-ground tissues of the grapevine. Altogether, 270 Alternaria isolates were collected from asymptomatic leaves and grape clusters of different grapevine varieties in the Eger wine region of Hungary. After analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and RNA polymerase second largest subunit (rpb2) sequences, 170 isolates were chosen for further analyses. Sequences of the Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), OPA10-2, and KOG1058 were also included in the phylogenetic analyses. Identification of secondary metabolites and metabolite profiling of the isolates were performed using high-performance liquid chromatography (HPLC)–high-resolution tandem mass spectrometry (HR-MS/MS). The multilocus phylogeny results revealed two distinct groups in grapevine, namely A. alternata and the A. arborescens species complex (AASC). Eight main metabolites were identified in all collected Alternaria isolates, regardless of their affiliation to the species and lineages. Multivariate analyses of untargeted metabolites found no clear separations; however, a partial least squares-discriminant analysis model was able to successfully discriminate between the metabolic datasets from isolates belonging to the AASC and A. alternata. By conducting univariate analysis based on the discriminant ability of the metabolites, we also identified several features exhibiting large and significant variation between A. alternata and the AASC. The separation of these groups may suggest functional differences, which may also play a role in the functioning of the plant microbiome.

Isolation and Identification of Endophytic Bacterial Isolates from the Leaves, Roots, and Stems Parts of Artemisia annua, Moringa oleifera, and Ocimum lamiifolium Plants.

Medicinal plants are known to harbor diverse species of endophytic bacteria which are known for secretion of beneficial secondary metabolites, like enzymes and antimicrobial compounds. The present study aimed to isolate, characterize, and identify the endophytic bacteria isolates from Artemisia annua, Moringa oleifera, and Ocimum lamiifolium plants. Certain endophytic bacterial isolates were screened. Phosphate and Zinc solubilization were performed for newly obtained isolates. The 16S rRNA gene sequencing was performed for RPAAI-8 isolate. Data were analyzed. Our study showed that endophytic bacterial isolates were recognized to be Bacillus cereus, B. subtilis, Citrobacter freundii, Enterobacter asburiae, E. cloacae, E. kobei, E. ludwigii, Enterococcus faecium, and Pseudomonas monteilli. From among these differentiated endophytic bacterial isolates, Enterobacter species are the most frequently obtained isolates. These bacterial isolates were shown 99.77% sequence similarity to Enterobacter ludwigii EN-119T (JTLO01000001) using 16S rRNA gene sequencing. This isolate was designated as Enterobacter sp. RPAAI-8. This isolate was able to employ selected cheap and cost-effective agro wastes as a carbon source. This cheap agro waste utilization by these Enterobacter species could be the first report. In conclusion, the present isolates are found to be employed for plant growth promotion and solubilizing insoluble phosphate and zinc. Before this time, most of the recent isolates were not identified from these medicinal plants. The ethyl acetate extract of the isolates also showed inhibitory activity against selected test pathogens.

Plant Growth-Promoting Endophytic Bacteria Isolated from Miscanthus giganteus and Their Antifungal Activity.

Modern technologies can satisfy human needs only with the use of large quantities of fertilizers and pesticides that are harmful to the environment. For this reason, it is possible to develop new technologies for sustainable agriculture. The process could be carried out by using endophytic microorganisms with a (possible) positive effect on plant vitality. Bacterial endophytes have been reported as plant growth promoters in several kinds of plants under normal and stressful conditions. In this study, isolates of bacterial endophytes from the roots and leaves of Miscanthus giganteus plants were tested for the presence of plant growth-promoting properties and their ability to inhibit pathogens of fungal origin. Selected bacterial isolates were able to solubilize inorganic phosphorus, fix nitrogen, and produce phytohormones, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and siderophore. Leaf bacterial isolate Pantoea ananat is 50 OL 2 had high production of siderophores (zone ≥ 5 mm), and limited phytohormone production, and was the only one to show ACC deaminase activity. The root bacterial isolate of Pseudomonas libanensis 5 OK 7A showed the best results in phytohormone production (N6-(Δ2-isopentenyl)adenine and indole-3-acetic acid, 11.7 and 12.6 ng·mL-1, respectively). Four fungal cultures-Fusarium sporotrichioides DBM 4330, Sclerotinia sclerotiorum SS-1, Botrytis cinerea DS 90 and Sphaerodes fimicola DS 93-were used to test the antifungal activity of selected bacterial isolates. These fungal cultures represent pathogenic families, especially for crops. All selected root endophyte isolates inhibited the pathogenic growth of all tested fungi with inhibition percentages ranging from 30 to 60%. Antifungal activity was also tested in two forms of immobilization of selected bacterial isolates: one in agar and the other on dextrin-coated cellulose carriers. These results demonstrated that the endophytic Pseudomonas sp. could be used as biofertilizers for crops.