Antagonistic and antifungal activities of Kalanchoe daigremontiana endophytic fungi for potential biological control applications

The present study was intended to optimize the culture conditions for secondary metabolite production by endophytic fungi. Based on the morphology and phylogeny, the fungus was identified as Trichoderma longibrachiatum isolated from brinjal leaf based on morphological characterization. The antifungal activity was evaluated against phytopathogens such as Macrophomina phaseolina, Phytopthora infestans, Colletotrichum falcatum and Colletotrichum gloeosporioides through the overlapping method, culture filtrate and organic fraction from Potato dextrose both as a growth medium. The organic fraction exhibited a significant antifungal activity, while modifications in medium composition may possess a major impact on the quantity and quality of secondary metabolites production. To achieve maximum metabolite production, the growth of the culture was optimized with screening of basal media, carbon, nitrogen, pH, trace elements and incubation period. The final optimized fermentation conditions were Minimal ereavis broth as basal media; glucose and sucrose as carbon source; Peptone and Yeast extract as nitrogen source, sodium nitrate as precursor; pH as 6; and incubation period as 7 days at 28°C. This optimization resulted in antifungal activity of 47.19-60.67% against M. phaseolina, P. infestans, C. falcatum, and C. gloeosporioides which was higher than that before optimization (43.80%). GCMS revealed distinct metabolites of T. longibrachiatum, comprising antifungal metabolites and molecules with additional bioactivities. These results strengthen ongoing research on disease control in agriculture by emphasizing the biocontrol potential of T. longibrachiatum isolated from brinjal phyllosphere against plant pathogenic fungi.

Sclerotinia stem rot caused by necrotrophic plant pathogenic fungus Sclerotinia sclerotiorum (Lib) de Bary is a highly pervasive disease affecting a wide range of agricultural and horticultural crops. In this study, fungal endophytes recovered from inflorescence tissues of Aloe vera L. were examined for their antagonist activity against S. sclerotiorum and the presence of plant growth-promoting traits. Fungal endophytes were characterized by microscopic and molecular (ITS rDNA sequencing) methods. Chaetomium globosum was the predominant endophytic species followed by Trichoderma harzanium in this study. Trichoderma harzanium tested positive for the presence of plant growth-promoting traits, i.e. siderophore, indole acetic acid, hydrogen cyanide production and phosphate solubilization. Chaetomium globosum strain 2 and T. harzanium survived stressful conditions (high temperature, cold stress and UV-B exposure). In antagonism assay, T. harzanium exhibited the highest growth inhibition per cent of 81.01 ± 0.63 against S. sclerotiorum and also restricted sclerotia formation. The hexane extract of T. harzanium (TH hex) obtained from solid fermentation depicted best antifungal activity against phytopathogen having an IC50 value of 3.987 mg/ml which is moderately comparable with positive control carbendazim (50%, WP). The GC–MS analysis of TH hex detected abundant presence (39.4%) of hexadecanoic acid, 2, 3-bis [(trimethylsilyl) oxy] propyl ester and other unsaturated fatty acids (palmitic and linoleic acid) having antifungal activity. The study demonstrates antifungal activity and plant growth-promoting potential of fungal endophytes residing in the inflorescence of A. vera.

Effects of ambient pH and nutritional factors on antifungal activity of the mycoparasite Coniothyrium minitans

White mold Sclerotinia sclerotiorum (Lib.) de Bary is a potentially serious disease of pepper (Capsicum annuum L.) grown in unheated greenhouses. Control of Sclerotinia sclerotiorum is difficult due to lack of Capsicum spp. resistance towards this pathogen. Currently, among the registered fungicides there is none that can combat this pepper disease. The isolation and selection of local strains of antagonistic microbes is part of the strategy to combat pathogens by means of alternative methods. In vitro studies have been carried out on soil isolates of Trichoderma spp. regarding their antagonistic activity on the pathogen Sclerotinia sclerotiorum. All isolates were found to have a higher ability to compete with the phytopathogen for the food substrate. The competitive ability coefficient of Trichoderma spp. isolates varies widely – from 1.2 to 11. Among them, an isolate with pronounced antagonistic activity and a competitive ability coefficient of 10.8 stands out. The isolated fungus was identified as Trichoderma viride based on the cultural and morphological characteristics. The isolated strain was found to have high in vitro antagonistic activity not only against Sclerotinia sclerotiorum but also against various pathogens causing root and stem rot of pepper such as Botrytis cinerea, Verticillium dahliae and Rhizoctonia solani. The results of the current study showed that Trichoderma viride (Trv) has the potential to be used for production of biopreparation intended to control Sclerotinia sclerotiorum and other soil-borne pathogens in greenhouses. Keywords: antagonistic fungi, in vitro screening, antifungal activity, biological control, white mold, Capsicum annuum

Endophytic microbes are hosted inside plants in a symbiotic and hugely benefitting relationship. In the present work, 60 asymptomatic fungi representing nine different genera were isolated from 286 plant tissues of Asparagus racemosus. Fungal endophytes were identified by ITS rDNA sequencing. Penicillium sp. was the most dominant fungus. Tissue specificity was observed by principal component analysis. Cluster analysis revealed correlation between fungal species abundance and mean temperature. Highest Shannon diversity was recorded in leaf tissues ( $${H^\prime }$$ = 1.279) from Delhi in 2010. Pielou’s evenness index was highest in stem tissues sampled from Hyderabad in the first survey. Of the total number of isolates examined, 12% of fungal endophytes demonstrated antifungal activity against the causal agents of four distinctive plant diseases (grey mould, stem rot, root rot and wilting, i.e. Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum, respectively) in dual-culture bioassay. Penicillium sp. (isolate ARDS-2.3) and Aspergillus oryzae (isolate ARHS-1.1) displayed most effective antifungal activity with IC50 value ranging from 0.381 to 0.955 mg/ml against the broad-spectrum phytopathogens investigated.

The present study was carried out to isolate endophytic fungi from Ocimum sanctum and Aloe vera and to assess their antifungal activity against Rhizoctonia solani, Fusarium oxysporum, Colletotrichum falcatum and Helminthosporium maydis. The isolated fungi were further evaluated for production of extracellular enzymes such as amylase, cellulase, chitinase, pectinase, laccase, lipase and urease. During the study 18 endophytic fungi were isolated from leaves, stem and roots of O. sanctum and Aloe vera which are different from each other morphologically. AVR1 and AVR3 show antagonistic activity towards the Fusarium oxysporum. T2S1 and AVL1 were found positive for amylase, TL1, AVR1, and AV2L1 were found positive in cellulase production while only AVR3 was found positive for chitinase production. AVR3, TL2, TL3, TS1, AV2R1, T2S1 and T2L1 were positive in pectinase production, none of the isolates were found positive in laccase production, only AVL1 was found positive in lipase production and TL2, T2R1, AVL1, AVL4, T2L1 and TL3 were positive in urease production. The results of the study suggest that endophytic fungi associated with O. sanctum and Aloe vera are potential agents for antimicrobial activity and a vast source of enzyme. Keywords: Endophytic fungi, antifungal activity, extracellular enzymes.

Endophytes are all microorganisms, culturable or not, that inhabit the interior of plant tissues, causing no harm to the host, and that do not develop external structures, excluding in this way, nodulating bacteria and mycorrhizal fungi. Theobroma cacao L. is important to food and cosmetic industry due the fact that it is used for production of cacao butter. Considering the importance of cacao and associated endophytic fungal community studies, the objectives of this work were to: i) study the diversity on endophytic community of cacao; ii) evaluate the potential of this community to biological control of Crinipellis perniciosa, causal agent of witches' broom disease and other phytopathogens and iii) study the interaction endophyte-pathogen and endophyte-host. The work began with isolation of the cacao endophytic fungi that was characterizated based on morphological and molecelar characters. The diversity was evaluated by rDNA (ITS1, ITS2 e 5.8 subunit regions) analyses (ARDRA and sequencing). The ARDRA analysis generated 64 haplotypes, which included at least 16 species. Following, antagonistic endophytic fungi from cacao were selected in vitro against C. perniciosa. It was observed that in 145 evaluated isolates, 38.6% was classified as antagonic, while 13.1% were classified as parasitic. After the selection, isolates were used for secondary metabolites extraction and further characterization. Also, detection of -(L--aminoadipil)-L-cisteinil-D-valin (ACV) gene, which is the precursor of -lactans, antibiotics like penicilins and cephalosporins, was carried out. The amplification of this gene generated a fragment of 600pb in 9.1% of the isolates evaluated. Some isolates were further evaluated against phytophathogenic fungi (Colletotrichum falcatum, C. sublineorum, Erythricium salmonicolor, Rhizoctonia solani, Ceratocystis paradoxa, Phytophthora sp., P. parasitica, P. palmivora and Fusarium moniliforme). Only C. sublineorum of sorghum and Ceratocystis paradoxa of sugarcane were in vitro inhibited by the endophytes 42.3 and 2, respectively. The investigation of the penetration and colonization ability of endophytic fungus 42.3 on susceptible cacao comum was carried out, using RAPD and SEM techniques. The penetration of the fungus was initiated between 3 and 6 hours after inoculation. The interaction between cacao endophyte 42.3 with C. perniciosa 281 (pathogenic), was evaluated by SDS-PAGE, aiming the detection of expressed proteins by pathogen and endophyte. The SDS-PAGE allowed the visualization of bands expressed in the interaction, when the microorganisms were grown together. In the presence of the endophyte growth substrate, C. perniciosa do not express visible proteins or, at least, distinguishable in SDS-PAGE, but the co-cultive of these fungi generated non observed bands in previously analysis.

The plant Chloranthus japonicus Sieb is known for its anticancer properties and mainly distributed in China, Japan, and Korea. In this study, we firstly investigated the diversity and antimicrobial activity of the culturable endophytic fungi from C. japonicus. A total of 332 fungal colonies were successfully isolated from 555 tissue segments of the medicinal plant C. japonicus collected from Qinling Mountains, China. One hundred and thirty representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, Mucoromycetes), and at least 30 genera. Colletotrichum (RA, 60.54%) was the most abundant genus, followed by Aspergillus (RA, 11.75%) and Diaporthe (RA, 9.34%). The Species Richness Index (S, 56) and the Shannon-Wiener Index (H′, 2.7076) indicated that C. japonicus harbored abundant fungal resources. Thirteen out of 130 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Among of these, F8158, which was identified as Trichoderma cf. harzianum, exhibited good antagonistic capacities (the percent inhibition of mycelial growth ranged from 47.72~88.18) for different pathogens and has a potential application in biological control. In addition, it is noteworthy that the strain F8157 (Thanatephorus cucumeris, an opportunistic pathogen) showed antibacterial and antifungal activity, which is reported firstly in this study, and should be investigated further. Taken together, these results indicated that the endophytic fungi from C. japonicus may be of potential interest in screening bio-control agents and discovering of new bioactive compounds.

Medicinal plants may become a favourable habitat for associative microorganisms, especially endophytic microbes inhabiting Zingiberaceaen species. Preliminary investigation has revealed an assemblage of endophytic fungi colonizing the healthy rhizome of Elettaria sp. collected from Sibayak Forest, North Sumatra based on molecular evidences. This study investigated the IAA-producing and antifungal activity from Elletaria endophytic fungi identified as Curvularia lunata, Pholiota multicingulata, Trichoderma atroviride, T. harzianum and Schizophyllum commune. Quantification of IAA was based on colorimetry method using Salkowsky reagent which produced pinkish to reddish solution indicating the presence of IAA. Antagonistic test was based on dual culture assay measured in colony growth inhibition (%). Statistical test to signify the differences were analysed using one-way ANOVA. C. lunata produced the highest IAA concentration of 45.17 µg.mL−1 followed by S. commune, and T. atroviride with concentration of 11.7 and 5.27 µg.mL−1, respectively. The strongest antagonistic activities were displayed by T. harzianum against both G. boninense (CGI>75%) and R. lignosus (CGI>50%). In addition, the strongest endophytic fungus against F. oxysporum was P. multicingulata (CGI>70%). Further investigations are being conducted to elaborate other plant growth promotion properties and even characterization of bioactive metabolites produced by Elettaria endophytic fungi.

Fungal endophytes use different strategies to protect host plants from abiotic and biotic stress. In this study, we isolated endophytic fungi from Pistacia vera and characterised their antifungal activity against Aspergillus flavus, Rhizoctonia solani and Sclerotinia sclerotiorum, and their release of some factors that can alter plant growth capability. Trichoderma harzianum TH 5-1-2, T. harzianum TH 10-2-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition percentages in dual culture assays against A. flavus, R. solani and S. sclerotiorum, respectively. Among the fungal endophyte cultures, ethyl acetate extracts of T. harzianum TH 10-2-2, T. harzianum TH 5-1-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition of S. sclerotiorum, R. solani and A. flavus, respectively. Phosphate solubilisation was induced by Byssochlamys nivea BN 1-1-1 in culture. Large amounts of siderophore production were observed with Quambalaria cyanescens QC 11-3-2 and Epicoccum nigrum EN1, but Trichoderma spp. also produced siderophore in lower amounts. Trichoderma harzianum TH 5-1-2 produced the highest chitinase activity (2.92 U/mL). In general, among the endophytes isolated, Trichoderma spp. appear to have the most promise for promoting healthy growth of P. vera.

A systemic study of fungal endophytes associated with different plant parts of Cannabis sativa and their antifungal activity was investigated in the present study. A total of 281 plant segments, including 91 leaves, 93 stem and 97 petioles samples, were screened for the isolation of endophytic fungi. Totally, 212 (77.65%) segments were found colonised by different fungi. Highest colonisation frequency were observed in stem parts (84.94%), then leaves (82.41%) and lowest 59.79% in petiole. Total eight fungal genera belonging to 12 species were isolated. Aspergillus is recorded as the most frequently occurring genera with three species Aspergillus niger, Aspergillus flavus and Aspergillus nidulans followed by Penicillium with two species Penicillium chrysogenum and Penicillium citrinum, while Phoma, Rhizopus, Colletotrichum, Cladosporium and Curvularia with single species. The antifungal potential of A. niger and A. flavus – two most frequently isolated endophytic fungi – was evaluated against two common plant pathogen, Colletotrichum gloeosporioides and Curvularia lunata. Different plant and fungal extracts individually and in combinations showed variations in antifungal activity against both the pathogens. The primary results obtained on antifungal activity of endophytes show their possible role in plant defence mechanism but it is a preliminary approach and more extensive research is still required.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), significantly affects the productivity of the banana crop in the field. Currently, there are no effective control measures available, and therefore, there is an urgent need to develop novel strategies to control the spread of the disease. Biological control is a promising strategy for the management and control of the disease. The aim of this study was to identify fungal endophytes associated with Moringa (Moringa oleifera), Neem tree (Azadirachta indica) and Lavender (Lavandula angustifolia) and their antifungal activities against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). We isolated 69 fungal endophytes from different tissues of M. oleifera, A. indica and L. angustifolia and screened for antifungal activity against Foc TR4. Six fungal endophytes exhibited highest antagonistic activity against Foc TR4 based on dual-culture assays. Based on morphology and ITS gene sequence analysis, the selected six endophytes were identified to be related to Alternaria alternata (MB7 and NR3), Neofusicoccum parvum (LB1), Fusarium oxysporum (LR1), Talaromyces amestolkiae (MB14) and Alternaria tenuissima (NB6). The culture filtrates of the six fungal endophytes (LB1, LR1, MB7, MB14, NB6 and NR3) exhibited more than 50% inhibition of mycelial growth of Foc TR4 in vitro and were producers of β-1,3-glucanase. The six fungal endophytes showed biocontrol efficacy against Fusarium wilt in pot experiments. The findings from this study demonstrate that fungal endophytes LB1, LR1, MB7, MB14, NB6 and NR3 should be explored as biocontrol agents and biofertilizers in banana production.

Orchids form a symbiotic relationship with endophytic fungi throughout their life cycle, including their seed germination. Research on orchid endophytic fungi in Indonesia is still limited. This study aimed to isolate and identify endophytic fungi of Phalaenopsis amabilis, Dipodium brevilabium, Vanilla planifolia, and Aerides odorata orchids. Endophytic fungi were isolated from surface-sterilized orchid root pelotons. Fungal identification was based on morphological and molecular characteristics using ITS rDNA sequences, followed by BLAST and phylogenetic tree analysis. All orchids studied had peloton structures in root cortex tissues. From the four orchids studied, 22 fungal isolates were obtained. Five fungal isolates were obtained from the P. amabilis orchid, consisting of 1 Penicillium and 4 sterile mycelium isolates. The D. brevilabium had 7 fungal isolates, namely 1 Penicillium and 6 Fusarium isolates. The V. planifolia had 6 Fusarium isolates, whereas the A. odorata had 4 isolates, such as 1 Penicillium, 2 sterile mycelium, and 1 unidentified isolate. The genus Penicillium was found in 3 out of the 4 orchid species studied. One Penicillium isolate was molecularly identified as Penicillium citrinum. Endophytic fungi in P. amabilis were mostly sterile mycelium, while D. brevilabium, V. planifolia, and A. odorata were dominated by Fusarium.

Medicinal plants have been used in healthcare since time immemorial, as have their therapeutic activities and the production of plant-based medicines. This study aims to use gene-targeted molecular markers for genetic diversity analysis of 16 medicinal plants. Besides, phytochemical analysis antibacterial and antifungal activities of some medicinal plant extracts commonly used in Egypt are compared to major compounds. DNA-based classification of 16 medicinal species using Conserved DNA-Derived Polymorphism (CDDP) and Start Codon Targeted (SCoT) primers. Three species representing three orders (Pelargonium graveolens, Matricaria chamomilla, and Hyoscyamus muticus were analysed [high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS)] and evaluated for their antibacterial and antifungal activities against (Escherichia coli O157: H7 ATCC 93111, Salmonella typhimurium ATCC 14028, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Bacillus ceruse ATCC 33018, and Sclerotinia sclerotiorum in comparison with some of their antimicrobial components. Our results revealed 309 and 349 polymorphic bands with 100% polymorphism. Among them, 51 and 57 were unique loci for CDDP and SCoT, respectively. The 16 species were categorised into three groups depending on the similarity matrix. The results of antibacterial and antifungal activities revealed that Pelargonium oil showed significant antifungal and antibacterial activities against the tested pathogens. Gallic acid severely reduced all tested bacteria's growth, but atropine severely reduced the growth of the B. ceruse only. Molecular modelling revealed their activity against sclerotium development. The gene-targeted marker techniques were highly useful tools for the classification of the 16 medicinal plant species, despite displaying high similarities at morphological and phytochemical analyses but, have antifungal and antibacterial activities.

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.