Antagonistic effects of selected endophytic fungi against Armillaria spp. in vitro Bellis Kullman Endophytic fungi are microorganisms which colonize living, internal tissues of plants without causing any harm to their associated host for at least a part of their life. As asymptomatic mutualists, they are beneficial to their host, stimulating growth and providing protection from diseases by secreting bioactive secondary metabolites. Endophytic fungi have been recognized as an novel source of bioactive compounds including anti-diabetic, anticancer, and anti-fungal compounds. Some chemical substances produced by endophytes are already being exploited in industry, medicine, and agriculture, but they represent only a small amount of the possible huge amount still to be discovered. Plant associated endophytes play an important role in balancing the ecosystem (modified from Kusari and Kayser) The objective of the study was 1*- to evaluate the diversity of endophytic fungi isolated from different host plant 2*- to screen them as potential biocontrol agents against forest pathogens • The study focuses on isolation, morphlogical and phylogenetic characterization, diversity evaluation, and assessment of the biocontrol potential of endophytes harbored in tissues of the Timothy grass and medicinal plants • We performed dual culture tests in vitro with selected endophytes against Honey Fungi - Armillaria borealis, A. cepistipes and A. ostoyae Honey fungi as pathogens used for antagonistic assays Honey Fungi are a group of species previously named as Armillaria mellea • Honey Fungi are long lived, not as mushrooms but as mycelia, living within the soil and feeding on living or dead wood • Honey fungi are spread worldwide infecting many host species and causing serious economic loss to forests • The damage internally is usually so great that the tree is doomed • The mycelium is able to travel great distances between trees in the form of black rhizomorphs • The mycelium of Dark Honey Fungus - Armillaria ostoyae referred to as a 'humungus fungus' covered some 800 hectares in eastern Oregon, and was estimated to be about 2400 years old Obtaining of pathogens for antagonistic assays Pathogens were obtaind from our culture collection EPS Pathogens substrate accession number Armillaria borealis Betula sp. [145551] EPS 1330 Armillaria cepistipes tree stump [145838] EPS 1328 Armillaria ostoyae Betula sp. [145517] EPS 1203 Obtaining of endophytes for antagonistic assays: • For obtaining pure cultures of endophytes surface sterilized plant segments (ethanol, commercial bleach, sterile water) were planted on growth medium (MEA and/or PDA ). • Morphological and molecular methods were used for identification. • rDNA ITS sequences that were more than 97% identical were considered to be conspecific. • The sequences were deposited at the Gene-Bank and voucher strains in our Collection of fungal living cultures TFC. • The cultures were monitored every two day to check the growth of endophytes for selection of suitable strains. Obtaining of endophytes for antagonistic assays: 1. Endophytes fromTimothy (Phleum pratense L.) • Timothy is an important agricultural grass in Europe and North America • We studied 58 isolates obtained from blades of Timothy plants collected from Estonia • As a result, 45 strains from 10 different taxa were identified • All isolates belonged to the phylum Ascomycota • Five species were found to be new to Timothy (Varavas, Kasekamp, Kullman 2013) and two new to Estonia (Varvas, Kullman 2012). Endophytes isolated fromTimothy on potato dextrose agar (PDA), surface and reverse A- Alternaria arbusti B- Phaeosphaeria herpotrichoides C- Gibberella avenacea (Fusarium avenaceum D- Fusarium sporotrichioides E- Apiospora montagnei (Arthrinium arundis) F- Paraphaeosphaeria michotii G- Aureobasidium pullulans H- Monographella sp. I- Epicoccum nigrum J- Epicoccum nigrum Identified endophytes and their colonization frequency (CF%) inTimothy (CF%, the percentage of plants colonized = (Ncol/Nt) x 100, were Ncol is the number of segments colonized by each endophyte and Nt is the total number of segments observed (Hata, Futai 1995)) (Varavas, Kasekamp, Kullman 2013) Endophytes fromTimothy (Phleum pratense L.) • In terms of abundance, E. nigrum was the dominating species (colonizing frequency 67% among all isolates) • When one plant sample had more than one endophyte, E. nigrum was always present among them • Growth rates showed significant variation both between species (1-7 mm/day) and also within species • Growth rate within the E. nigrum group was 1-4 mm/day Great variability in the genome size of Epicoccum nigrum • In our study genome size of E. nigrum varied from 53 to 128 Mbp (measured using image analysis (Kullman & Teterin 2006)) We assume that the phenomena responsible for such high genetic variation are endoreduplication displayed as heteroploidy Fig. One of the most parsimonious trees inferred from the rDNA ITS sequences of 54 Epicoccum starains. Numbers above the branches indicate bootstrap values. Genome size is given in Mbp. - endopolyploidy Colony aspect of Epicoccum nigrum strains on MEA Antagonism within strain Is it posible that formation of sectors is caused by the parasexuality? Anastomosis between the own hyphae of E. nigrum culture H4-2-2. What can be the reason for the appearance of antagonistic effects within strain? self-anastomoses with formation of dikaryons and somatic diploids as well as haploidisation seems to support parasexuality of E. nigrum Endoreduplication and parasexuality of E. nigrum deserve further study, which can also offer opportunities for new biotechnological applications. – For example, parasexuality of Penicillum allows to obtain strains for new antibiotics Epicoccum nigrum • We selected for antagonistic assays E. nigrum culture which revealed an antagonistic effect alredy within strain • E. nigrum is a non-systemic fungal endophyte of grasses with a worldwide distribution which colonizes different types of soils and host plants • E. nigrum has also been known as a biological control agent for plant pathogens and has important biotechnological application (Fávaro et al., 2011) Obtaining of endophytes for antagonistic assay 2. Endophytes from medicinal plants In the study, some important medicinal plants were investigated for presence of endophytes. Medicinal plants are known to harbour endophytic fungi that are believed to be associated with the production of pharmaceutical products (Zhang et al., 2006). We hoped to find some endophytes from medicinal plants repelling plant parasitic fungi. Obtaining of endophytes for antagonistic assays 2. Endophytes from medicinal plants 241 isolates were obtained from six traditional medicinal plants from different sites of Estonia. Host species studied: • Ledum palustre (wild rosemary) • Arctostaphylos uva-ursi (bearberry) • Oxycoccus palustris (cranberry) • Alchemilla vulgaris (ladies mantle) • Plantago major (plantain) • Urtica dioica (nettle) Isolation rates (IR%) of endophytic fungi from medicinal plants (IR= (total no. of segments/segments with mycelium) x100%) 100,0 80,0 IR % 60,0 40,0 20,0 0,0 A. uva-ursi O. palustris L. palustre A. vulgaris P. major U. dioica Fungal endophytes were present in all host species, isolation rates varied between 60% in nettle - Urtica dioica and 95% in bearberry Arctostaphylos uva-ursi. To select the most active antagonists we selectid strains with higher growh rate. NSm-3 NSm-5A NSm-5B NSm-3II 50 NSm-1B NSm-1A 45 NSm-6A NSm-2A NSm-6 40 NSm-5II NSm-5I 35 NSm-4III Diameter (mm) NSm-4II NSm-3I 30 NSm-2C NSm-4 NSm-6B 25 NSm-4I Growth rate of isolates from Urtica dioica 20 NSm-2 NSm-1 NS-1 NS-1I 15 NS-1II NS-2 10 NS-4I NV-5I NV-5II 5 NV-4II NV-6 0 0 5 10 15 Time (days) 20 25 30 35 50 TSm-3B TSm-4* TSm-2D TSm-1A TSm-4I TSm-4II TSm-1 TSm-3 TSm-1* TSm-3A TSm-2B TSm-2*C TS-4 TS-5 TS-3-2 TS-6 TS-3 TS-1 TS-2 TS-3-1A TV-1I TV-3II TV-1II TV-1III TV-3I TV-1B TV-1A TV-2A TV-2*B TV-2 TV-3B TV-3 45 40 Diameter (mm) 35 30 25 20 Growth rate of isolates from Plantago major 15 10 5 0 0 5 10 15 20 Day 25 30 35 50 KV-1* KV-1A 45 KV-2B KV-4 KV-3* 40 KV-6* KV-6 35 KV-3 Diameter (mm) KV-2* KV-1C 30 KV-5 KV-3 25 KV-* KV-I 20 KV-5I KV-3III KV-3II 15 Growth rate of isolates from Alchemilla vulgaris 10 KV-3I KS-2 KS-3 KS-4B KS-4A 5 KS-3A KS-2I 0 0 5 10 15 Day 20 25 30 35 60 VS-1 VS-2 VS-3 50 VS-4 VS-5 VS-6_1 VS-6_2 40 VS-I Diameter (mm) TS-1 TS-2 TS-4 30 TS-5 PS-1 PS-2 PS-3 20 PS-4 PS-5 PS-6 PS-7 10 PS-8 PS-9 Growth rate of isolates from Ledum palustre PS-10 PS-I 0 0 5 10 15 Day 20 25 30 PS-II VL-1 60 VL-2 VL-3 VL-4 VL-5 50 VL-6 VL-8 VL-9 Diameter (mm) 40 VL-10_1 TL-2 TL-3 30 TL-I TL-I PL-1 PL-2 20 PL-3 PL-4 PL-5 10 Growth rate of isolates from Arctostaphylos uva-ursi PL-5 PL-6 PL-6_1 0 PL-7 0 2 4 6 8 10 12 14 16 Day 18 20 22 24 26 28 30 PL-8 60 50 40 Diameter (mm) VJ-1 Growth rate of isolates from Oxycoccus palustris 30 VJ-2 VJ-I TJ-1 TJ-2 TJ-3 TJ-4_1 20 TJ-4_2 TJ-I 10 0 1 3 5 7 9 11 13 15 Day 17 19 21 23 25 27 29 The growth rate of some species identified by rDNA ITS 50 45 Phoma multirostrata 40 Phoma multirostrata 35 Hypoxylon howeanum Diameter (mm) Hypoxylon fuscum 30 Colletotrichum clavatum 25 Fusarium culmorum Apodus deciduus 20 Leptosphaeria sp 15 Epicoccum nigrum Pleosporales sp 10 Phoma sp 5 Alternaria rosae 0 0 5 10 15 20 Day 25 30 35 Identification of endophytic fungi from medicinal plants Conidial mean sizes of isolates 14 TSm-3B TSm-1* KS-4B TV-1A TV-2 TS-2 TV-1* KS-4A KV-5 TSm-2A NSm-1 KV-3* TV-1B NSm-1A NSm-2A NSm-1B NSm-1B TS-3 NSm-2C TV-2*B TSm-3A KV-2* NSm-5A KS-3 KV-1C NV-4I NV-4II NSm-4III KV-5I KV-3III 12 width of conidia in µm 10 8 6 4 2 0 0 5 10 length of conidia in µm 15 20 25 Both morphological and molecular investigations yielded similar identification of the species Identification of endophytic fungi from medicinal plants 36 isolates from 26 different taxa were identified from medicinal plants by rDNA ITS Species Alternaria rosae Annulohypoxylon multiforme Anthostomella Apodus deciduus Cladosporium cladosporioides Cladosporium tenuissimum Colletotrichum clavatum Coprinellus disseminatus Daldinia petriniae Diaporthe phaseolorum Engyodontium album Epicoccum nigrum Fusarium culmorum Gibberella avenacea Glomerella sp Hypholoma acutum Hypoxylon fuscum Hypoxylon howeanum Hypoxylon rubiginosum Leotiomycetes sp Leptosphaeria sp Phaeosphaeria pontiformis Phoma multirostrata Phoma poolensis var. verbascicola Phoma sp Physalospora vaccinii Nr of isolates 1 5 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 Endophytes isolated from the medicinal and grass host plants selected for testing against pathogens Endophytic fungi Hypoxylon howeanum Host plant Strain codes in TFC Oxycoccus palustris 2013-22 Alchemilla vulgaris 2013-54 Phleum pretense C1-7 Arctostaphylos uva-ursi 2013-29 Arctostaphylos uva-ursi 2013-18 Arctostaphylos uva-ursi 2013-44 Phleum pretense H4-2-3 Ledum palustre - Plantago major 2013-46 Fusarium culmorum Aureobasidium pullulans Cladosporium tenuissimum Phoma poolensis var. verbascicola Phoma multirostrata Epicoccum nigrum Annulohypoxylon multiforme Lewia infectoria Endophytic fungi tested against pathogens growing on MEA Endophyte - pathogen interactions in dual culture antagonistic assay: • The medium used for activation of the fungi was malt extract agar (2% MEA) in 90 mm petri dishes. • 5 mm plugs of each endophyte and a pathogen were co-cultured. • The assay was performed in triplicate. • The pathogens alone were inoculated as the controls. Endophyte Annulohypoxylon multiforme inhibited the growth of Dark Honey Fungus A. ostoyae control assay On day 13 endophyte On day 33 pathogen Endophyte – A. ostoyae interactions in dual culture antagonistic assay endophytes are shown on the left and pathogen on the right of the Petri plates Endophyte - Armillaria borealis interactions in dual culture antagonistic assay Endophyte – A. cepistipes interactions in dual culture antagonistic assay Growth inhibition of the pathogen Dark Honey fungus A. ostoyae by fungal endophytes 2,5 HH FC Dark Honey fungus radial growth cm 2 AP 1,5 CT PV 1 PM EN 0,5 AM 0 0 5 10 15 Time (days) 20 25 30 35 LI Kontroll The percentages of antagonism against each pathogen were calculated using the formular Radial growth of pathogen in presence of endophyte (RG) Radial growth of pathogen in absence of endophyte (control) Kusari et al. (2013). Results • The results of the study give an overview of the potential of selected endophytes as a source of biocontrol agents against fungal forest pathogens Armillaria spp. • All selected endophytes inhibited the growth of Armillaria spp. • Among Armillaria spp. growth was inhibited most as follows: Fusarium culmorum inhibited the growth of A. cepistipes (67.3 ±6.8 %) Annulohypoxylon multiforme inhibited the growth of A. ostoyae (77.3 ±1.5 %) Phma poolensis var. verbascicola inhibited the growth of A. borealis (79.7 ±4.2 %) In each test A. multiforme inhibited the growth of A. ostoyae and A. borealis over 70%. Results This is the first report on assessment of the biocontrol potential of the studied endophyte against the fungal pathogens A. borealis, A. cepistipes and A. ostoyae Annulohypoxylon multiforme, Phoma poolensis var. verbascicola and Epicoccum nigrum are considered to be the best biocontrol agents against Armillaria spp. The endophytic fungus A. multiforme should also be tested against other Armillaria species such as A. mellea that causes economic loss to forests and to agriculture. Discussion Endophytic organisms have received considerable attention as they are found to protect their host against pathogens. They have been found to play an important role in the production of beneficial chemical compounds and considered to be a rich source of novel compounds which will be used as biocontrol agents for plant protection and as factories of bioactive products. Taxol, an antifungal and anticancer compound, is alredy used in medicine. In vitro antagonism assay for screening of endophytes as potential biocontrol agents We isolated and characterized endophytes harboured in grass and medicinal plants, which might provide favour to other host plants. In order to screen for the most promising endophytes, we estimated the potential of the endophytes by challenging them with fungal forest pathogens. In further research we plan to find bioactive compounds against plant invading pathogens as volatile compounds produced in vitro antagonism assay. Our studies will be aimed to elucidation of bioactive volatile organic compounds (VOC) correlating to endophyte - pathogen interactions. The study of these compounds would allow to discover new products for bioprospecting. Kertu Kais, Bellis Kullman, Kati Küngas, Tiina Poolak, Triin Varvas Thank you for the attention The research was supported from the project “Plant protection for sustainable crop production” SF0170057s09. View publication stats